Methods, systems, compositions, and devices for nucleic acid extraction, ambient temperature amplification, and detection

Abstract

Quick, user-centric, equipment-independent, and cost-efficient nucleic acid processing systems, compositions and devices, as well as methods of use thereof, for isothermal nucleotide amplification of single-stranded amplicons and double-stranded ampliconsand inhibition of non¬ specific binding are described herein. The nucleic acid processing device is proficient in the seamless extraction, amplification, and detection of genetic material from biological or environmental samples. The methods, compositions, and systems herein do not require thermal melting and may be used in a point-of-need setting. The methods feature the amplification of a target region and the production of single -stranded amplicons including the target region, bound by an in-reaction probe.

Description

METHODS, SYSTEMS, COMPOSITIONS, AND DEVICES FOR NUCLEIC ACID EXTRACTION, AMBIENT TEMPERATURE AMPLIFICATION, AND DETECTIONCROSS-REFERENCE

[0001] This application claimsthebenefitof U.S. ProvisionalPatentApplicationNo. 63 / 584,130 filed on September 20, 2023, U.S. Provisional Patent Application No. 63 / 595,443 filed on November 2, 2023, U.S. Provisional Patent Application No. 63 / 563,142 filed on March 8, 2024, U.S. Provisional Patent Application No. 63 / 670,696 filed on July 12, 2024, and U.S. Provisional Patent Application No. 63 / 670,364 filed on July 12, 2024, each of which is incorporated by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure features systems, devices, kits and methods for nucleic acid extraction, amplification, and / or detection in a sample, e.g., biological, environmental, or other appropriate samples. The present disclosure also features amplifying and detecting target nucleic acids, without requiring any specific temperature control, with a probe present in -reaction that aids in detection or amplification or both. Also featured are compositions and systems for the amplification system, and approaches to further increase specificity and sensitivity by using an annexing probe, such as an approach termed Annexing Isothermal Nucleotide Amplification (ANINA).BACKGROUND

[0003] Nucleic acid detection is an extremely specific, sensitive, and accurate diagnostic technology that uses the specificity and sensitivity afforded by nucleic acid base pairing to detect different nucleic acid sequences, sometimes differingby a single nucleotide. It can help diagnose infectious diseases, cancer genetics, antibiotic-resistantbacteria, etc. Rapid and accurate molecular diagnosis is critical for timely detection and treatment of infectious human or agricultural pathogens. Molecular diagnostic technologies typically involve nucleic acid amplification and detection by expensive, sophisticated equipment such as quantitative PCR (qPCR), which cannot be deployed in field or true point-of-care (PoC) scenarios, thereby leading to delays in diagnosis and treatment. Recent pandemics have highlighted the necessity of instrument-free, cost-effective molecular diagnostics technologies that can be used by anyone, deployed anywhere and easily configured for any disease target. However, sample collection, processing, and equipment-free nucleic acid amplification and detection remain a roadblock in current point-of-care diagnostics.

[0004] A key component of molecular diagnostics technologies is nucleic acid extraction, the process of isolating and purifying DNA / RNA from a biological sample. Despite notable technological advances, nucleic acid extraction is the first step and one of the greatest impediments for PoC diagnostics. Typically, sample processing methods usually require laboratory -based procedures involving multiple steps and specific equipment (e.g., pipettors, centrifuge, incubator, vortex, magnetic beads) or hazardous reagents (e.g., phenol, chloroform, ethanol, etc.). Additionally, diagnostic samples can be a large variety of biological sample types (whole blood, saliva, urine, tissue, and body fluid swabs), each of which present significant problems for adaptable PoC technologies as each require sample -specific extraction processes and reagents. This requires reliance on skilled lab personnel and centralized laboratories which limits the ability to adapt nucleic acid detection assays to a point-of-care setting, especially in re source -limited settings.

[0005] The other key component of molecular diagnostics technologies is nucleic acid amplification which amplifies the target nucleic acid from the extracted sample to a detectable range. The gold standard nucleic acid amplification technology (NAAT) is quantitative polymerase chain reaction (qPCR) which incorporates both the amplification and detection technology in the same setup. However, qPCR is difficult to conduct in a point-of-need setting because of the requirement of specific cycling temperatures: for example, (i) a very high temperature (usually 95 °C) required for thermal melting of dsDNA / dsRNA (either the starting sequence or the amplicons after the first amplification cycle), (ii) annealingtemperature (usually between 50 -70°C) for the primers to bind to, and (iii) the extension temperature for the corresponding amplification by the common polymerases (e.g., Taq (72°C)). Additionally, PCR and real-time PCR are limited to the time required for each cycle, thereby increasing the total time of the reactions. Other isothermal NAATs, such as LAMP (loop -mediated isothermal amplification), HDA (helicasedependent isothermal DNA amplification), SDA (strand displacement amplification), NASBA (nucleic acid sequence-based amplification), RCA (rolling circle amplification), RPA (recombinase polymerase amplification) can amplify DNA or RNA in a simpler settingby avoiding the need for a cyclingmethod. However, all of the aforementioned methods still utilize an incubator for maintaining a constant elevated temperature. Additionally, most NAATs can amplify if there is contamination, or are prone to primer artefact generation, and therefore nested amplification strategies or primer modifications are utilized to make the amplicon results more specific, adding to assay design complexity . Thus, these methods have specific de sign constraints, are prone to nonspecific amplification which can inhibit specificity and sensitivity of detection and cannot easily differentiate single nucleotide mismatches in target sequences. Moreover, these methods requirean additional enzymatic technology for specific and sensitive detection at PoC, such as CRISPR / Cas-based SHERLOCK and DETECTR technologies or complex and limiting probe design strategies to avoid probe artefacts. Consequently, these methods increase total detection time, manufacturing costs, and assay design complexity.

[0006] Nucleic acid testing (NAT) biosensing technologies, such as those employing the use of fluorophores or redox sensors, or other physicochemical detectors, are currently used in numerous applications for the detection of nucleic acids (NA) of a particular pathogen or mutated genes in an organism. NATs use the specificity and sensitivity afforded by nucleic acid base pairing to detect differentNA sequences, sometimes differing by a single nucleotide. However, for an NAT to work effectively, the detectable target sequence for the probe sequence needs to be single - stranded DNA and / or RNA. This allows for a probe having a physicochemical detector (e.g., a genosensor probe) to hybridize to its complementary sequence, which is usually a single -stranded DNA and / or RNA target. Currently, the gold standard is to collect samples and send them to a testing facility where nucleic acid extraction, amplification, and detection can be carried out. Obtaining results can take days. Additionally, mostNAATs can amplify if there is contamination or are prone to primer artefact generation, and therefore nested amplification strategies or primer modifications are utilized to make the amplicon results more specific. Furthermore, mostNAATs utilize a dual-strand amplification strategy to increase the number of copies exponentially. However, this results in dsDNA amplicons, which may need to be denatured before being detected by a hybridization probe sequence. While asymmetric amplification strategies can be used to amplify mostly single-stranded DNA, it heavily limits the amplification rate to a linear rate instead of the exponential rate of the dual-strand approach. While there are additional probe design strategies which rely on specific modifications in the probe or the addition of another enzyme to facilitate a change in probe structure or sequence, most of these methods can complicate the amplification design strategy or result in probe artefact generation.

[0007] Currently, the commonly carried out process is to collect samples and send them to a testing facility where nucleic acid extraction, amplification, and detection can be carried out. Obtaining results can take days. Centralized laboratories dominate existing molecular diagnostic testing options and regularly cause delays and prohibit access for healthcare consumers. Recognized herein is a significant need of a nucleic acid detection platform technology that is rapid, cost-effective, easily adaptable for different target diseases or use cases, equipment-free and capable of long-term ambient temperature storage, and that can be performed easily without significant training. Ideally, this nucleic acid diagnostics platform should be capable of processing a variety of biological sample types without relying on any expensive equipment, it should be user-friendly and not require lab training, and it should be capable of integrating with a variety of detection technologies, such as lateral flow assay formats, fluorescence detection, colorimetric or electrochemical detection, nucleic acid sequencing, etc. depending on the use case scenario. This also necessitates an isothermal cost-effective nucleic acid amplification technology (NAAT) that specifically amplifies single-stranded and double-stranded nucleic acid target sequence already bound to detection probein reaction is needed. Furthermore, a methodthat does not require thermal melting and can be done in a point-of-need setting without requiring access to an incubator or power source would be ideal.SUMMARY

[0008] The present provides systems, devices, compositions, and methods that allow for nucleic acid extraction, detection in a biological sample, environmental sample, or other appropriate sample, and / or amplification of target double-stranded and single -stranded amplicons bound to an in-reaction probe, while also inhibiting non-specific binding. Embodiments of the present disclosure can be freely combined with each other if they are not mutually exclusive .

[0009] Any feature or combination of features described herein are included within the scope of the present disclosure providedthatthe features included in any such combination are notmutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present disclosure are apparent in the following detailed description and claims. Still other objects and advantages of the present disclosure will become readily apparent by those skilled in the art from the following detailed description, wherein it is shown and described only the preferred embodiments, simply by way of illustration of the best mode. As will be realized, the disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, without departing from the disclosure. Accordingly, the description is to be regarded as illustrative in nature and not as restrictive.

[0010] The present disclosure provides a device comprising an isolation system for isolating nucleic acid; an amplification system for amplifying a target nucleic acid; and a detection system for detecting the target nucleic acid (e.g., detecting a feature of a target nucleic acid, detecting a target sequence, detecting a probe linked to a target sequence, etc.). The present disclosure also features alternative configurations, e.g., systems and devices comprising an isolation system and an amplification system, an isolation and a detection system, or an amplification system and a detection system.

[0011] In some aspects, provided herein are methods of amplifying a target nucleic acid sequenceat an ambient temperature. In some embodiments, the method comprises binding a first oligonucleotide (Pl) primer to a strand of the target nucleic acid sequence. In some embodiments, the method comprises binding a second oligonucleotide (P2) primer to another strand of the target nucleic acid sequence. In some embodiments, the method comprises binding a third oligonucleotide to the target nucleic acid sequence. In some embodiments, the third oligonucleotide comprises a 3' end blocking feature that blocks extension of the third oligonucleotide by a polymerase. In some embodiments, the third oligonucleotide does not facilitate a localizing and a binding of the Pl or the P2 to the target nucleic acid sequence. In some embodiments, a 5 ' end of the third oligonucleotide and a 5 ' end of the Pl bind to the strand of the target nucleic acid sequence at a distance from about 3 nucleotides to about 25 nucleotides apart. In some embodiments, the method comprises providing a polymerase and a DNA processing enzyme to the target nucleic acid sequence. In some embodiments, the method comprises extending a 3 ' end of the Pl and a 3 ' end of the P2, thereby amplifying the target nucleic acid sequence at the ambient temperature. In some embodiments, the Pl localizes and binds to the strand of the target nucleic acid sequence independently of the third oligonucleotide. In some embodiments, the P2 localizes and binds to the another strand of the target nucleic acid sequence independently of the third oligonucleotide. In some embodiments, a 5' end of the third oligonucleotide binds to the strand of the target nucleic acid sequence. In some embodiments, the 5 ' end of the third oligonucleotide and a 5' end of the Pl bind to the strand of the target nucleic acid sequence at a distance from about 3 nucleotides to about 25 nucleotides apart. In some embodiments, the 5' end of the third oligonucleotide and the 5' end of the Pl bind to the strand of the target nucleic acid sequence at a distance of about 15 nucleotides apart. In some embodiments, the 5 ' end of the third oligonucleotide and the 5' end of the Pl bind to the strand of the target nucleic acid sequence at a distance of about 10 nucleotides apart. In some embodiments, the 5 ' end of the third oligonucleotide and the 5 ' end of the Pl bind to the strand of the target nucleic acid sequence at a distance of about 8 nucleotides apart. In some embodiments, the 5' end of the third oligonucleotide and the 3’ end of Pl bind to the strand of the target nucleic acid sequence with 1 base pair (bp) of overlap. In some embodiments, the 5 ' end of the third oligonucleotide and the 3 ’ end of Pl are adjacent and bind to the strand of the target nucleic acid sequence. In some embodiments, the 5' end of the third oligonucleotide and the 3’ end of Pl bind to the strand of the target nucleic acid sequence with a gap in contiguous sequence of the target nucleic acid sequence of between about 1 -9 bp. In some embodiments, the 3 ' end blocking feature that blocks extension of the third oligonucleotide by a DNA polymerase comprises an inverted nucleotide or a C3 -spacer. In some embodiments, the Pl comprises a sequence at least about 80% identical to a region on the strand of the target nucleicacid sequence. In some embodiments, the P2 comprises a sequence at least about 80% identical to a region on the another strand of the target nucleic acid sequence. In some embodiments, the third oligonucleotide comprises a sequence at least about 80% identical to a region of the target nucleic acid sequence. In some embodiments, the method further comprises providing the Pl, the P2, and the third oligonucleotide in a set of oligonucleotides prior to the binding of the Pl , the P2, and the third oligonucleotide to the target nucleic acid sequence. In some embodiments, the DNA processing enzyme is comprised in an enzyme mix. In some embodiments, the enzyme mix comprises a DNA polymerase or functional fragment thereof, the DNA processing enzyme, one or more nucleotides in an amount sufficient to support amplification of the target nucleic acid sequence, a single-strand binding protein, a crowding reagent, a magnesium salt or any combination thereof. In some embodiments, the DNA processing enzyme facilitates the binding of the Pl, P2, or the third oligonucleotide to the target region. In some embodiments, the DNA processing enzyme facilitates the binding of the third oligonucleotide to the target nucleic acid sequence. In some embodiments, the method of amplification results in an amplified target nucleic acid sequence. In some embodiments, the method of amplification results in target single -stranded (ss) amplicons. In some embodiments, the method of amplification results in target doublestranded (ds) amplicons. In some embodiments, the method of amplification results in target ds amplicons and target ss amplicons. In some embodiments, the Pl, the P2, or the third oligonucleotide comprises a label. In some embodiments, the third oligonucleotide comprises the label. In some embodiments, the label is located on a 5 ' end of the third oligonucleotide, a 3 ' end of the third oligonucleotide, or anywhere in between the 5’ end and the 3’ end . In some embodiments, the third oligonucleotide comprises a label at its 5 ’ end. In some embodiments, the label comprises a fluorescein or derivative thereof, a fluorophore, a chromophore, a redox molecule, a fluorescence quencher, a biotin, a digoxigenin, or any combination thereof. In some embodiments, the label comprises a xanthene core or a derivative thereof. In some embodiments, the label comprises a carboxyfluorescein (FAM). In some embodiments, the Pl ortheP2 comprise a label. In some embodiments, the label is located on a 5' end, a 3 ' end, or anywhere in between the 5’ end and the 3 ’ end of the Pl or the P2. In some embodiments, the label comprises a fluorescein or derivative thereof, a fluorophore, a chromophore, a redox molecule, a fluorescence quencher, a biotin, a digoxigenin, or any combination thereof. In some embodiments, the label comprises a biotin or digoxigenin. In some embodiments, the label allows for extension of an end of the Pl or the P2 by a DNA polymerase. In some embodiments, the third oligonucleotide can form a binding complex with the nucleic acid molecule. In some embodiments, the third oligonucleotide promotes the localizing and binding of the Pl to the strand of the target nucleicacid sequence. In some embodiments, the third oligonucleotide facilitates a localizing and a binding of P2 to the target nucleic acid sequence. In some embodiments, the third oligonucleotide promotes annealing specificity of Pl or P2 or both to the target nucleic acid sequence. In some embodiments, the third oligonucleotide promotes efficiency of the binding of Pl or P2 or both to the target nucleic acid sequence. In some embodiments, the third oligonucleotide binds between the Pl and the P2 bound to the target nucleic acid sequence. In some embodiments, the third oligonucleotide binds to a region at least partially overlapping with the Pl bound to the target nucleic acid sequence. In some embodiments, the Pl binds to a different region of the nucleic acid molecule than the third oligonucleotide. In some embodiments, the P2 binds to a different region of the target nucleic acid sequence than the third oligonucleotide. In some embodiments, the third oligonucleotide binds to a region at least partially overlapping with the P2 bound to the target nucleic acid sequence. In some embodiments, the method further comprises providing a fourth oligonucleotide, wherein the fourth oligonucleotide does not promote the localizing and binding of the Pl to the strand of the target nucleic acid sequence. In some embodiments, the fourth oligonucleotide binds to the target nucleic acid sequence between the Pl and the P2 that are bound to the target nucleic acid sequence. In some embodiments, the fourth oligonucleotide binds to the target nucleic acid sequence upstream or downstream of both the Pl and theP2 bound to the target nucleic acid sequence. In some embodiments, the DNA processing enzyme facilitates the binding of the fourth oligonucleotide to the target nucleic acid sequence. In some embodiments, the Pl binds to a different region of the target nucleic acid sequence than the fourth oligonucleotide. In some embodiments, the P2 binds to a different region of the target nucleic acid sequence than the fourth oligonucleotide. In some embodiments, the fourth oligonucleotide comprises a nucleotide sequence at least about 80% identical to a region on the strand or the another strand of the target nucleic acid sequence. In some embodiments, the fourth oligonucleotide comprises a label. In some embodiments, the label is located on a 5' end, a 3 ' end, or anywhere in between the 5’ end and the 3 ’ end of the fourth oligonucleotide. In some embodiments, the label comprises a fluorescein or derivative thereof, a fluorophore, a chromophore, a redox molecule, a fluorescence quencher, a biotin, a digoxigenin, or any combination thereof. In some embodiments, the method further comprises providing to the nucleic acid molecule a crowding reagent. In some embodiments, the crowding reagent comprises polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), Ficoll, Dextran, or any combination thereof. In some embodiments, the crowding reagent comprises PEG. In some embodiments, the polymerase comprises a DNA polymerase or functional fragment thereof. In some embodiments, the DNA polymerase or functional fragment thereof is a strand - displacing DNA polymerase. In some embodiments, the DNA polymerase comprises Bacillussubtilis DNA polymerase I (Bsu), Sau, Bst, or Klenow Fragment. In some embodiments, the DNA polymerase comprises Bacillus subtilis DNA polymerase I (Bsu). In some embodiments, the DNA processing enzyme comprises a recombinase or functional fragment thereof, a helicase or functional fragment thereof, a topoisomerase or functional fragment thereof, single -stranded binding protein or functional fragment thereof, or any combination thereof. In some embodiments, the DNA processing enzyme comprises a recombinase protein. In some embodiments, the DNA processing enzyme comprises UvsX DNA recombinase (UvsX). In some embodiments, the DNA processing enzyme comprises bacterial recombinase A (RecA). In some embodiments, the DNA processing enzyme comprises T4 GP32, E. coli SSB, HELB helicase, RTEL1 helicase, Pifl helicase, Topi topoisomerase, or topoisomerase Illa. In some embodiments, the DNA processing enzyme facilitates binding or annealing of one or more of the Pl, the P2, the third oligonucleotide, and the fourth oligonucleotide to the target nucleic acid sequence. In some embodiments, the DNA processing enzyme comprises a plurality of DNA processing enzymes. In some embodiments, the DNA processing enzyme comprises a first DNA processing enzyme, a second DNA processing enzyme, and a third DNA processing enzyme. In some embodiments, the first DNA processing enzyme is a single-stranded binding protein. In some embodiments, the single-stranded binding protein comprises T4 GP32. In some embodiments, the method further comprises providingto the target nucleic acid sequence a reverse transcriptase (RT) or a functional fragmentthereof to reverse transcribe an RNA template to a cDNA molecule, wherein the cDNA template comprises the target nucleic acid sequence. In some embodiments, the RT uses one or more of the Pl , the P2 and the fourth oligonucleotide as primers for a first strand cDNA synthesis. In some embodiments, the RT uses a set of random primers provided in a reaction mix as primers for a first strand cDNA synthesis. In some embodiments, the RT is M-MuLV RT, AMV RT, SuperScript II RT, SuperScript III RT, SuperScript IV RT, or any other RT capable of reverse transcribing in ambient temperatures. In some embodiments, the method further comprises providingto the target nucleic acid sequence an RnaseH enzyme or a functional fragmentthereof to promote reverse transcription. In some embodiments, the Pl, the P2, the third oligonucleotide, the fourth oligonucleotide or any combination thereof has a non -complementary 5' adaptor region. In some embodiments, the Pl, the P2, the third oligonucleotide, the fourth oligonucleotide or any combination thereof has a non- complementary 5' adaptor region. In some embodiments, the Pl, the P2, the third oligonucleotide, or the fourth oligonucleotide comprises a length of at least about 6 nucleotide s, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 60 nucleotides, at least about 80 nucleotides, or more. In some embodiments, the Pl or the P2 is a nucleotide length insufficient toproduce amplicons of the target nucleic acid sequence without the presence of the third oligonucleotide. In some embodiments, the Pl and the P2 are present in molar excess relative to the target nucleic acid molecule. In some embodiments, the method further comprises an incubation step for a length of time sufficient to replicate and amplify the target nucleic acid sequence. In some embodiments, an incubation temperature is within 15-45°C forthe length of time. In some embodiments, the incubation temperature is within 20-37°C forthe length of time. In some embodiments, the incubation temperature is maintained within 22-25°C forthe length of time. In some embodiments, the incubation temperature is room temp erature for the length of time. In some embodiments, the incubation temperature is maintained under isothermal conditions without previously denaturing the nucleic acid molecule. In some embodiments, maintaining isothermal conditions does not involve incubation in a temp erature -regulating or temperaturemaintaining device. In some embodiments, maintaining isothermal conditions does not involve incubation in an incubator. In some embodiments, the amplifying the target nucleic acid region is conducted in an absence of a thermocycler. In some embodiments, the incubation remains within 10% of the starting temperature until sufficient amplification of the target nucleic acid sequence to be detected is achieved. In some embodiments, the length of time is between about 1 -60 minutes. In some embodiments, the length of time is between about 10-40 minutes. In some embodiments, the length of time is between 20-30 minutes. In some embodiments, the length of time is about 20 minutes. In some embodiments, the length of time is about 30 minutes. In some embodiments, the method further comprises detecting an amplified target nucleic acid sequence. In some embodiments, a presence of a third oligonucleotide comprising a label increases production of the target ds amplicons. In some embodiments, a presence ofa third oligonucleotidecomprisinga label increases production of the target ss amplicons. In some embodiments, the target ss and ds amplicons or the two target ds amplicons are produced at least at a linear rate of amplification. In some embodiments, the target ds amplicons or the two target ds amplicons are produced at an exponential rate of amplification. In some embodiments, the target ss amplicons are produced at least at a linear rate of amplification. In some embodiments, the target ss amplicons are produced at an exponential rate of amplification. In some embodiments, the method further comprises detecting target ss amplicons, target ds amplicons, or target ss amplicons and target ds amplicons via a third oligonucleotide comprising a label. In some embodiments, a third oligonucleotide comprising a label can detect two target amplicons, and wherein the detection of the two target amplicons produces increased sensitivity of target nucleic acid sequence detection compared to detecting single target amplicons. In some embodiments, a 5' end of at least one of the Pl or the P2 is linked to the 5' or 3' end of the fourth oligonucleotide. In some embodiments, the methodcomprises detecting the target ds amplicons. In some embodiments, the method comprises detecting the target ss amplicons. In some embodiments, the detecting comprises analyzing reaction products using a molecular lateral flow assay (LFA). In some embodiments, the detecting comprises analyzing reaction products using an intercalating dye or fluorophore. In some embodiments, the method comprises use of FRET in the detecting. In some embodiments, the Pl or P2 or fourth oligonucleotide comprises a fluorophore label or fragment thereof for FRET detection of a labeled third oligonucleotide. In some embodiments, the Pl, the P2, or the fourth oligonucleotide comprises a quencher molecule label for fluorescence quenching detection of a labeled third oligonucleotide. In some embodiments, the detecting comprises analyzing reaction products using a nucleic acid hybridization technique. In some embodiments, the detecting comprises analyzing reaction products using a fluorescence detection technique. In some embodiments, the detecting comprises analyzing reaction products using a colorimetric technique. In some embodiments, the detecting comprises analyzing reaction products using nucleic acid sequencing. In some embodiments, the detecting comprises analyzing reaction products using DNA fragment size discrimination. In some embodiments, an oligonucleotide probe is added to ss amplicons or ds amplicons produced from the reaction to aid in the detecting. In some embodiments, a separate oligonucleotide probe comprising a quencher is added to ss amplicons or ds amplicons produced from the reaction to aid in the detecting. In some embodiments, the detecting comprises determining a quantitative measure of the target template in a sample. In some embodiments, the detecting comprises determining a quantitative measure of the target template present in a sample by using nucleic acid standards of known quantity. In some embodiments, the target ds amplicons are amplified and detected in real time. In some embodiments, the target ss amplicons and target ds amplicons are amplified and detected in real time. In some embodiments, target ds amplicons are amplified and detected in less than about 60 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 12 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, or 3 minutes. In some embodiments, target ss amplicons are amplified and detected in less than about 60 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 12 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, or 3 minutes. In some embodiments, the method further comprises detection of two different regions of the target nucleic acid sequence. In some embodiments, two or more distinct pathogenic variants are detected within a single amplification reaction. In some embodiments, two or more distinct disease mutations are detected within a single amplification reaction. In some embodiments, a single nucleotide polymorphism (SNP) within the target nucleicacid sequence is detected. In some embodiments, a nucleic acid molecule is extracted from a sample. In some embodiments, the nucleic acid molecule extracted from the sample comprises DNA or RNA. In some embodiments, the nucleic acid molecule extracted from the sample comprises the extracted nucleic acid molecule comprises dsDNA, ssDNA, circulating DNA, cell free DNA, cDNA, plasmid DNA, mRNA, miRNA, tRNA, viral RNA, siRNA, sgRNA, or any combination thereof. In some embodiments, the nucleic acid molecule extracted from the sample comprises DNA. In some embodiments, the nucleic acid molecule extracted from the sample comprises double -stranded DNA. In some embodiments, the nucleic acid molecule extracted from the sample comprises single-stranded DNA. In some embodiments, the nucleic acid molecule extracted from the sample comprises RNA. In some embodiments, a template to the target nucleic acid sequence is one or more copies of a target genome, and wherein the target nucleic acid sequence is amplified at about room temperature to a level of a detectable range. In some embodiments, the method comprises amplification and detection of presence of a target nucleic acid in the extracted sample. In some embodiments, the sample is obtained from a biological fluid, a tissue sample, a mammalian sample, an animal sample, a eukaryotic sample, a bacterial sample, a viral sample, a plant sample, a bacterial cell, a eukaryotic cell, an animal tissue, whole blood, saliva, urine, a nasal swab, a buccal swab, a tongue swab, a cervical fluid, a vaginal fluid, an animal cell, a muscle tissue, a spleen tissue, a skin tissue, or a liver tissue. In some embodiments, the sample comprises nucleic acid extracted from a subject. In some embodiments, the nucleic acid extracted from the subject comprises nucleic acid derived from a pathogen. In some embodiments, the pathogen is a virus. In some embodiments, the virus is listed in Table 1 . In some embodiments, the pathogen is a bacterium. In some embodiments, the bacterium is listed in Table 2. In some embodiments, the pathogen is a fungus. In some embodiments, the fungus is listed in Table 3. In some embodiments, the pathogen is an animal pathogen. In some embodiments, the animal pathogen is listed in Table 4. In some embodiments, the sample is obtained from an animal. In some embodiments, the animal is a livestock species. In some embodiments, the livestock species is a type of cattle, horse, goat, sheep, pig, swine, rabbit, donkey, mule, bison, llama, cat, dog, deer, camel, yak, turkey, alpaca, elk, chicken, duck, emu, ostrich, pheasant, goose, fish, shrimp, crustacean, mosquito, fly, or bee. In some embodiments, the animal is a human. In some embodiments, the sample is obtained from plant. In some embodiments, the target nucleic acid sequence is a selected region of a genomic DNA sequence from a pathogen. In some embodiments, the target nucleic acid sequence is a selected region of an RNA sequence from a pathogen. In some embodiments, the target nucleic acid sequence is from a pathogen listed in Table 1, Table 2, Table 3, Table 4, or Table 5. In some embodiments, the target nucleic acid sequence is amplified in thepresence of host genomic DNA.

[0012] In some aspects, provided herein are systems for amplifying a target nucleic acid sequence at an ambienttemperature. In some embodiments, the system comprises a first oligonucleotide (Pl) primer. In some embodiments, the system comprises a second oligonucleotide (P2) primer. In some embodiments, the system comprises a third oligonucleotide comprising a 3' end blocking feature that blocks extension of the third oligonucleotide by a polymerase. In some embodiments, the third oligonucleotide does not facilitate a localizing and a binding of the Pl or the P2 to the target nucleic acid sequence. In some embodiments, a 5' end of the third oligonucleotide and a 5' end of the Pl bind to the target nucleic acid sequence at a distance from about 3 nucleotides to about 25 nucleotides apart. In some embodiments, the system comprises providing a polymerase and a DNA processing enzyme. In some embodiments, the Pl localizes and binds to the strand of the target nucleic acid sequence independently of the third oligonucleotide. In some embodiments, the P2 localizes and binds to the another strand of the target nucleic acid sequence independently of the third oligonucleotide. In some embodiments, a 5’ end of the P3 binds to the strand of the target nucleic acid sequence. In some embodiments, the 5’ end of the third oligonucleotide and a 5' end of the Pl bind to the strand of the target nucleic acid sequence at a distance from about 3 nucleotides to about 25 nucleotides apart. In some embodiments, the 5' end of the third oligonucleotide and the 5' end of the Pl bind to the strand of the target nucleic acid sequence at a distance of about 15 nucleotides apart. In some embodiments, the 5 ' end of the third oligonucleotide and the 5 ' end of the Pl bind to the strand of the target nucleic acid sequence at a distance of about 10 nucleotides apart. In some embodiments, the 5' end of the third oligonucleotide and the 5' end of the Pl bind to the strand of the target nucleic acid sequence at a distance of about 8 nucleotides apart. In some embodiments, the 3 ' end blocking feature that blocks extension of the third oligonucleotide by a DNA polymerase comprises an inverted nucleotide or a C3 -spacer. In some embodiments, the Pl comprises a sequence at least about 80% identical to a region on the strand of the target nucleic acid sequence. In some embodiments, the P2 comprises a sequence at least about 80% identical to a region on the another strand of the target nucleic acid sequence. In some embodiments, the third oligonucleotide comprises a sequence at least about 80% identical to a region of the target nucleic acid sequence. In some embodiments, the system further comprises providing the Pl, the P2, and the third oligonucleotide in a set of oligonucleotides prior to the binding of the Pl , the P2, and the third oligonucleotide to the target nucleic acid sequence. In some embodiments, an enzyme mix comprises the DNA processing enzyme. In some embodiments, the enzyme mix comprises the polymerase or functional fragment thereof, the DNA processing enzyme, one or more nucleotides in an amount sufficient to support amplification of the targetnucleic acid sequence, a single-strand binding protein, a crowding reagent, a magnesium salt or any combination thereof. In some embodiments, the DNA processing enzyme facilitates the binding of the third oligonucleotide to the target nucleic acid sequence. In some embodiments, the system of amplification is configured to produce an amplified target nucleic acid sequence. In some embodiments, the system of amplification is configured to produce target single-stranded (ss) amplicons. In some embodiments, the system of amplification is configured to produce target double-stranded (ds) amplicons. In some embodiments, the system of amplification is configured to produce target ds amplicons and target ss amplicons. In some embodiments, the Pl , the P2, or the third oligonucleotide comprises a label. In some embodiments, the third oligonucleotide comprises the label. In some embodiments, the label is located on a 5' end of the third oligonucleotide, a 3' end of the third oligonucleotide, or anywhere in between the 5’ end and the 3 ’ end. In some embodiments, the third oligonucleotide comprises a label at its 5’ end. In some embodiments, the label comprises a fluorescein or derivative thereof, a fluorophore, a chromophore, a redox molecule, a fluorescence quencher, a biotin, a digoxigenin, or any combination thereof. In some embodiments, the label comprises a xanthene core or a derivative thereof. In some embodiments, the label comprises a carboxyfluorescein (FAM). In some embodiments, the Pl or the P2 comprise a label. In some embodiments, the label is located on a 5' end, a 3 ' end, or anywhere in between the 5’ end and the 3 ’ end of the Pl or the P2. In some embodiments, the label comprises a fluorescein or derivative thereof, a fluorophore, a chromophore, a redox molecule, a fluorescence quencher, a biotin, a digoxigenin, or any combination thereof. In some embodiments, the label comprises a xanthene core or a derivative thereof. In some embodiments, the label comprises a carb oxy fluorescein (FAM). In some embodiments, the system further comprises a crowding reagent. In some embodiments, the polymerase comprises a DNA polymerase or functional fragment thereof. In some embodiments, the DNA processing enzyme comprises a recombinase or functional fragment thereof, a helicase or functional fragment thereof, a topoisomerase or functional fragment thereof, single -stranded binding protein or functional fragment thereof, or any combination thereof. In some embodiments, the DNA processing enzyme comprises UvsX DNA recombinase (UvsX), bacterial recombinase A (RecA), T4 GP32, E. coli SSB, HELB helicase, RTEL1 helicase, Pifl helicase, Topi topoisomerase, topoisomerase Illa, or any combination thereof. In some embodiments, the system is configured to detectan amplifiedtargetnucleic acid sequence. In some embodiments, the system comprises a detection assay. In some embodiments, the detection assay comprises a lateral flow assay strip, a colorimetric strip, an electrochemical sensor, a colorimetric sensor, or a fluorometric sensor. In some embodiments, the system comprises: a first channel comprising a first openingwherein the first channel is configured to couple thereto a biological sample; a second channel comprising (i) a cellulose substrate coupled thereto and (ii) a second opening, wherein the second opening is configured to couple to the first opening, and wherein upon the coupling of the second openingto the first opening, the biological sample is transferred from the first channel to the second channel; and a third channel comprising (i) an elution solution and (ii) a third opening, wherein the third opening is configured to couple to the second opening, and wherein upon the coupling of the second openingto the third opening, the elution solution is transferred from the third channel to the second channel. In some embodiments, the first channel, the second channel, and the third channel comprise a beaker, a bottle, a burette, a container, a cylinder, a vial, a tube, a pipette, a syringe, and any other combination thereof. In some embodiments, the channel is configured for extraction of the target nucleic acid sequence. In some embodiments, the biological sample is a biological fluid, a tissue sample, a mammalian sample, an animal sample, a eukaryotic sample, a bacterial sample, a viral sample, or a plant sample. In some embodiments, the biological sample is a bacterial cell, a eukaryotic cell, an animal tissue, whole blood, saliva, urine, a nasal swab, a buccal swab, a tongue swab, a cervical fluid, a vaginal fluid, an animal cell, a muscle tissue, a spleen tissue, a skin tissue, or a liver tissue. In some embodiments, the biological sample is E. coli, N. gonorrhoeae, RSV, WSSV, EBV, or human genomic DNA. In some embodiments, the first channel further comprises a lysis solution. In some embodiments, the system comprises a processed biological sample. In some embodiments, the processed biological sample comprises an extracted nucleic acid molecule. In some embodiments, the nucleic acid molecule extracted from the sample comprises the extracted nucleic acid molecule comprises dsDNA, ssDNA, circulating DNA, cell free DNA, cDNA, plasmid DNA, mRNA, miRNA, tRNA, viral RNA, siRNA, sgRNA, or any combination thereof. In some embodiments, the extracted nucleic acid molecule comprises a DNA or an RNA. In some embodiments, the amplification of the nucleic acid molecule determines the presence or absence of the target nucleic acid sequence.

[0013] In some aspects, provided herein are systems comprising any of the systems disclosed herein and a detection system for detecting the target nucleic acid. In some embodiments, the second channel comprises an isolation chamber. In some embodiments, the device further comprises an amplification chamber. In some embodiments, the device further comprises a detection buffer chamber. In some embodiments, the i) isolation chamber and the amplification chamber, ii) the amplification chamber and the detection buffer chamber, and iii) the detection buffer chamber are fluidly connected via a plurality of channels. In some embodiments, the system further comprises an aliquot valve disposed between the isolation chamber, the amplification chamber, and the detection buffer chamber. In some embodiments, the system further comprises acoupler fluidly connected to the isolation chamber, wherein the coupler comprises an opening to the isolation chamber and is configured to engage at least a cap or a vial. In some embodiments, the system further comprises a coupler fluidly connected to the isolation chamber, wherein the coupler comprises an openingto the isolation chamber and is configured to engage at least a means for preventing access to the isolation chamber through the coupler. In some embodiments, the means for preventing access to the isolation chamber through the coupler comprises a cap, a seal, a plug, or a vial. In some embodiments, the system further comprises a first chamber fluidly connected to a first end of the isolation chamber. In some embodiments, the first chamber further comprises a first coupler, wherein the first coupler comprises an openingto the first chamber and is configured to engage at least a cap or a first vial. In some embodiments, the first coupler comprises an opening to the first chamber and is configured to engage at least a means for preventing access to the first chamber through the first coupler. In some embodiments, the means for preventing access to the first chamber through the first coupler comprises a cap, a seal, a plug or a vial. In some embodiments, the vial comprises a first vial. In some embodiments, the system further comprises an elution chamber fluidly connected to a first end of the isolation chamber. In some embodiments, the elution chamber further comprises a second coupler, wherein the second coupler comprises an openingto the elution chamber and is configured to engage at least a cap or a second vial. In some embodiments, the second coupler comprises an opening to the elution chamber and is configured to engage at least a means for preventing access to the elution chamber through the second coupler. In some embodiments, the means for preventing access to the elution chamber through the second coupler comprises a cap, a seal, a plug, or a vial. In some embodiments, the vial comprises a second vial. In some embodiments, a sample is either i) added directly to the isolation chamber or the first chamber or ii) is added to the isolation chamber or the first chamber via the use of the first vial. In some embodiments, the sample comprises a biological sample or an environmental sample. In some embodiments, the sample further comprises a lysis buffer. In some embodiments, the sample further comprises one or more actuators configured to move a fluid through each of the aforementioned chambers. In some embodiments, the means of detection comprises a detection unit. In some embodiments, the detection unit comprises a lateral flow assay strip, electrochemical sensor, or colorimetric sensor.

[0014] In some aspects, provided herein are kits comprising any of the systems, devices, and / or methods disclosed herein and instructions foruse. In some embodiments, the instructions designate a protocol foruse as a field kit. In some embodiments, the instructions designate a detection of a viral infection. In some embodiments, the instructions designate a detection of abacterial infection. In some embodiments, the instructions designate a detection of a specific antibiotic -resistantbacterial strain. In some embodiments, the instructions designate a detection of a DNA virus. In some embodiments, the instructions designate a detection of anRNA virus. In some embodiments, the instructions designate a detection ofWSSV, EBV, H1N1, H5N1, or any combination thereof.

[0015] In some aspects, provided herein are devices comprising: an isolation system for isolating nucleic acid; an amplification system for amplifying a target nucleic acid; and a detection system for detecting the target nucleic acid. In some aspects provided herein are devices comprising: an isolation system for isolating nucleic acid, the isolation system comprises an isolation chamber, wherein a binding component capable of binding nucleic acid is disposed therein; an amplification system for amplifying a target nucleic acid, the amplification system comprises an amplification chamber fluidly connected to the isolation chamber; and a detection system for detecting a target nucleic acid, the detection system comprises a means of detecting the target nucleic acid fluidly connected to the amplification chamber. In some aspects provided herein are devices comprising: an isolation system for isolating nucleic acid, the isolation system comprises an isolation chamber, wherein a binding component capable of binding nucleic acid is disposed therein; and a detection system for detecting a target nucleic acid, the detection system comprises a means of detecting the target nucleic acid fluidly connected to the isolation chamber. In some aspects provided herein are devices comprising: an isolation system for isolating nucleic acid, the isolation system comprises an isolation chamber, wherein a binding component capable of binding nucleic acid is disposed therein; an amplification system for amplifying a target nucleic acid, the amplification system comprises an amplification chamber fluidly connected to the isolation chamber. In some aspects provided herein are devices comprising: an amplification system for amplifying a target nucleic acid, the amplification system comprises an amplification chamber; and a detection system for detecting a target nucleic acid, the detection system comprises a means of detecting the target nucleic acid fluidly connected to the amplification chamber. In some embodiments, further comprising one or more components configured to regulate flow of fluid between one or more of: the isolation system and the amplification system, the isolation system and the detection system, or the amplification system and the detection system. In some embodiments, further comprising one or more components configured to prevent or allow flow of fluid between one or more of: the isolation system and the amplification system, the isolation system and the detection system, orthe amplification system and the detection system. In some embodiments, the device is configured to engage a sample vial. In some embodiments, the means of detecting the target nucleic acid comprises a lateral flow assay component. In some embodiments, the means of detecting the target nucleic acid provides a visual signal to indicate detection of the target nucleic acid. In some aspects provided herein are devices (1000) for extracting and detecting nucleic acids from a sample, thedevice comprising: an isolation chamber (1110) comprising a binding component (1115) capable of binding nucleic acid disposed therein, and a means of detection (1180) fluidly connected to a second end (1112) the isolation chamber (1110). In some embodiments, the isolation chamber (1110) and the means of detection (1180) are fluidly connected via a channel (1190). In some aspects provided herein are devices (1000) for extracting and detecting nucleic acids from a sample, the device comprising: an isolation chamber (1110) comprising a binding component (1115) capable of binding nucleic acid disposed therein, an amplification chamber (1140) fluidly connected to a second end (1112) of the isolation chamber (1110), and a means of detection (1180) fluidly connected to the amplification chamber (1140). In some embodiments, the amplification chamber (1140) is fluidly connected to the isolation chamber and the means of detection (1180) is fluidly connected to the amplification chamber (1140) via a plurality of channels (1190). In some embodiments, the device (1000) further comprises an aliquot valve (1170) disposed between the isolation chamber (1110) and the amplification chamber (1140). In some aspects provided herein are devices (1000) for extracting and detecting nucleic acids from a sample, the device comprising: an isolation chamber (1110) comprising a binding component (1115) capable of binding nucleic acid disposed therein, an amplification chamber (1140) fluidly connected to a second end (1112) of the isolation chamber (1110), a detection buffer chamber (1150) fluidly connected to the amplification chamber (1140), and a means of detection (1180) fluidly connected to the detection buffer chamber (1150). In some embodiments, the i) isolation chamber (1110) and the amplification chamber (1140), ii) the amplification chamber (1140) and the detection buffer chamber (1150), and iii) the detection buffer chamber (1150) are fluidly connected via a plurality of channels (1190). In some embodiments, further comprising an aliquot valve (1170) disposed between the isolation chamber (1110), the amplification chamber (1140), and the detection buffer chamber (1140). In some embodiments, further comprising a coupler (1210) fluidly connected to the isolation chamber (1110), wherein the coupler (1210) comprises an openingto the isolation chamber (1110) and is configured to engage at least a cap (1215) or a vial. In some embodiments, further comprising a coupler (1210) fluidly connected to the isolation chamber (1110), wherein the coupler (1210) comprises an openingto the isolation chamber (1110) and is configured to engage at least a means for preventing access to the isolation chamber (1110) through the coupler (1210). In some embodiments, the means for preventing access to the isolation chamber (1110) through the coupler (1210) comprises a cap (1215), a seal, a plug, or a vial. In some embodiments, further comprising a first chamber (1120) fluidly connected to a first end (1111) of the isolation chamber (1110). In some embodiments, the first chamber (1120) further comprises a first coupler (1211), wherein the first coupler (1211) comprises an openingto the first chamb er ( 1120) and is configuredto engage atleast a cap (1215) or a firstvial (1220). In some embodiments, thefirst coupler (1211) comprises an opening to the first chamber (1120) and is configured to engage at least a means for preventing access to the first chamber (1120) through the first coupler (1211). In some embodiments, the means for preventing access to the first chamber (1120) through the first coupler (1211) comprises a cap (1215), a seal, a plug, or a vial. In some embodiments, the vial comprises a first vial (1220). In some embodiments, comprising an elution chamber (1130) fluidly connected to a first end (1111) of the isolation chamber (1110). In some embodiments, the elution chamber (1130) further comprises a second coupler (1212), wherein the second coupler (1212) comprises an opening to the elution chamber (1130) and is configured to engage at least a cap (1215) or a second vial (1230). In some embodiments, the second coupler (1212) comprises an opening to the elution chamber (1130) and is configured to engage at least a means for preventing access to the elution chamber (1130) through the second coupler (1212). In some embodiments, the means for preventing access to the elution chamber (1130) through the second coupler (1212) comprises a cap (1215), a seal, a plug, or a vial. In some embodiments, the vial comprises a second vial (1230). In some embodiments, a sample is either i) added directly to the isolation chamber (1110) or the first chamber (1120) or ii) is added to the isolation chamber (1110) or the first chamber (1120) via the use of the first vial (1220). In some embodiments, the sample comprises a biological sample or an environmental sample. In some embodiments, the sample further comprises a lysis buffer. In some embodiments, further comprising one or more actuators (1160) configured to move a fluid through each of the aforementioned chambers. In some embodiments, the means of detection (1180) comprises a detection unit. In some embodiments, the detection unit comprises a lateral flow assay strip, electrochemical sensor, or colorimetric sensor.

[0016] In some aspects provided herein are methods of processing a nucleic acid from a biological sample, the method comprising: using a lysate buffer to prepare the biological sample into a lysate; providing the lysate to a binding unit configured to bind to the nucleic acid; and amplifying the nucleic acid of the lysate without a thermocycler or incubator. In some aspects provided herein are methods of processing a nucleic acid from a biological sample, the method comprising: usinga lysate bufferto prepare the biological sample into a lysate; providingthe lysate to a binding unit configured to bind to the nucleic acid; and amplifying the nucleic acid of the lysate using an annexing probe. In some embodiments, the lysate comprises a first portion, and wherein subsequent to (b) providingthe lysateto the binding unit configured to bind to the nucleic acid portion, the first portion of the lysate is discarded. In some embodiments, further comprising providing an elution buffer to the binding unit. In some embodiments, further comprising eluting the nucleic acid from the binding unit. In some embodiments, amplifying the nucleic acid of thelysate is performed usingtwo or more nucleic acid primers. In some embodiments, the nucleic acid is a viral nucleic acid. In some embodiments, the nucleic acid is an RSV nucleic acid. In some embodiments, the nucleic acid is a White Spot Syndrome Virus (WSSV) nucleic acid. In some embodiments, the nucleic acid is a bacterial nucleic acid. In some embodiments, the nucleic acid is a Neisseria gonorrhoeae nucleic acid. In some embodiments, the method occurs in 5 minutes or less. In some embodiments, further comprising sequencingthe nucleic acid. In some embodiments, the sequencing is nanopore sequencing. In some embodiments, the amplifying in (c) occurs at an ambient temperature of about 20°C to about40°C. In some embodiments, the amplifying in (c) occurs in about 30 minutes or less.

[0017] In some aspects, provided herein are systems of processing a nucleic acid from a biological sample, the system comprising: a binding unit configured to bind to the nucleic acid; and an amplification unit configured to amplify the nucleic acid of the lysate, wherein the amplification unit does not comprise a thermocycler or incubator. In some aspects provided herein are systems for processing a nucleic acid from a biological sample, the system comprising: a binding unit configured to bind to the nucleic acid; and an amplification unit configured to amplify the nucleic acid of the lysate, wherein the amplifying is performed using an annexing probe. In some embodiments, further comprising an elution unit comprising an elution buffer. In some embodiments, the amplifying is performed using two or more primers.

[0018] In some aspects, provided herein are kits comprising the systems disclosed herein and instructions for use. In some embodiments, the instructions designate a treatment of a subject in need thereof. In some embodiments, the instructions designate a treatment of a viral infection. In some embodiments, the instructions designate a treatment of RSV. In some embodiments, the instructions designate a treatment of White Spot Syndrome Virus (WSSV). In some embodiments, the instructions designate a treatment of a bacterial infection. In some embodiments, the instructions designate a treatment of a Neisseria gonorrhoeae infection.

[0019] In some aspects, provided herein are devices comprising: an isolation system for isolating nucleic acid; an amplification system for amplifying a target nucleic acid; and a detection system for detecting the target nucleic acid. In some aspects provided herein are devices comprising: an isolation system for isolating nucleic acid, the isolation system comprises an isolation chamber, wherein a binding component capable of binding nucleic acid is disposed therein; an amplification system for amplifying a target nucleic acid, the amplification system comprises an amplification chamber fluidly connected to the isolation chamber; and a detection system for detecting a target nucleic acid, the detection system comprises a means of detecting the target nucleic acid fluidly connected to the amplification chamber. In some aspects, provided herein are devices comprising:an isolation system for isolating nucleic acid, the isolation system comprises an isolation chamber, wherein a binding component capable of binding nucleic acid is disposed therein; and a detection system for detecting a target nucleic acid, the detection system comprises a means of detecting the target nucleic acid fluidly connected to the isolation chamber. In some aspects, provided herein are devices comprising: an isolation system for isolating nucleic acid, the isolation system comprises an isolation chamber, wherein a binding component capable of binding nucleic acid is disposed therein; an amplification system for amplifying a target nucleic acid, the amplification system comprises an amplification chamber fluidly connected to the isolation chamber. In some aspects, provided herein are devices comprising: an amplification system for amplifying a target nucleic acid, the amplification system comprises an amplification chamber; and a detection system for detecting a target nucleic acid, the detection system comprises a means of detecting the target nucleic acid fluidly connected to the amplification chamber. In some embodiments, further comprising one or more components configured to regulate flow of fluid between one or more of: the isolation system and the amplification system, the isolation system and the detection system, or the amplification system and the detection system. In some embodiments, further comprising one or more components configured to prevent or allow flow of fluid between one or more of: the isolation system and the amplification system, the isolation system and the detection system, orthe amplification system and the detection system. In some embodiments, the device is configured to engage a sample vial. In some embodiments, the means of detecting the target nucleic acid comprises a lateral flow assay component. In some embodiments, the means of detectingthe target nucleic acid provides a visual signal to indicate detection of the target nucleic acid. In some aspects, provided herein are devices (1000) for extracting and detecting nucleic acids from a sample, the device comprising: an isolation chamber (1110) comprising a binding component (1115) capable of binding nucleic acid disposed therein, and a means of detection (1180) fluidly connected to a second end (1112) the isolation chamber (1110). In some embodiments, the isolation chamber (1110) and the means of detection (1180) are fluidly connected via a channel (1190). In some aspects provided herein are devices (1000) for extracting and detecting nucleic acids from a sample, the device comprising: an isolation chamber (1110) comprising a binding component (1115) capable of binding nucleic acid disposed therein, an amplification chamber (1140) fluidly connected to a second end (1112) ofthe isolation chamber (1110), and a means of detection (1180) fluidly connected to the amplification chamber (1140). In some embodiments, the amplification chamber (1140) is fluidly connected to the isolation chamber and the means of detection (1180) is fluidly connected to the amplification chamber (1140) via a plurality of channels (1190). The device (1000) further comprises an aliquot valve (1170) disposed between the isolation chamber(1110) and the amplification chamber (1140). In some aspects provided herein are devices (1000) for extracting and detecting nucleic acids from a sample, the device comprising: an isolation chamber (1110) comprising a binding component (1115) capable of binding nucleic acid disposed therein, an amplification chamber (1140) fluidly connected to a second end (1112) of theisolation chamber(l 110), a detection buffer chamber (1150) fluidly connectedto the amplification chamber (1140), and a means of detection (1180) fluidly connectedto the detection buffer chamber (1150). In some embodiments, the i) isolation chamber (1110) and the amplification chamber (1140), ii) the amplification chamber (1140) and the detection buffer chamber (1150), and iii) the detection buffer chamber (1150) are fluidly connected via a plurality of channels (1190). In some embodiments, further comprising an aliquot valve (1170) disposed between the isolation chamber (1110), the amplification chamber (1140), and the detection buffer chamber (1140). In some embodiments, further comprising a coupler (1210) fluidly connected to the isolation chamber (1110), wherein the coupler (1210) comprises an opening to the isolation chamber (1110) and is configured to engage at least a cap (1215) or a vial. In some embodiments, further comprising a coupler (1210) fluidly connected to the isolation chamber (1110), wherein the coupler (1210) comprises an opening to the isolation chamber (1110) and is configured to engage at least a means for preventing access to the isolation chamber (1110) through the coupler (1210). In some embodiments, the means for preventing access to the isolation chamber (1110) through the coupler (1210) comprises a cap (1215), a seal, a plug, or a vial. In some embodiments, further comprising a first chamber (1120) fluidly connected to a first end (1111) of the isolation chamber (1110). In some embodiments, the first chamber (1120) further comprises a first coupler (1211), wherein the first coupler (1211) comprises an opening to the first chamber (1120) and is configured to engage at least a cap (1215) or a first vial ( 1220). In some emb odiments, the first coupler (1211) comprises an opening to the first chamber (1120) and is configured to engage at least a means for preventing access to the first chamber (1120) through the first coupler (1211). In some embodiments, the means for preventing access to the first chamber (1120) through the first coupler (1211) comprises a cap (1215), a seal, a plug, or a vial. In some embodiments, the vial comprises a first vial (1220). In some embodiments, comprising an elution chamber (1130) fluidly connected to a first end (1111) of the isolation chamber (1110). In some embodiments, the elution chamber (1130) further comprises a second coupler (1212), wherein the second coupler (1212) comprises an openingto the elution chamber (1130) and is configuredto engage at least a cap (1215) or a second vial (1230). In some embodiments, the second coupler (1212) comprises an openingto the elution chamber (1130) and is configured to engage at least a means for preventing access to the elution chamber (1130) through the second coupler (1212). In some embodiments, the means for preventing accessto the elution chamber (1130) through the second coupler (1212) comprises a cap (1215), a seal, a plug, or a vial. In some embodiments, the vial comprises a second vial (1230). In some embodiments, a sample is either i) added directly to the isolation chamber (1110) or the first chamber (1120) or ii) is added to the isolation chamber (1110) or the first chamber (1120) via the use of the first vial (1220). In some embodiments, the sample comprises a biological sample or an environmental sample. In some embodiments, the sample further comprises a lysis buffer. In some embodiments, further comprising one or more actuators (1160) configured to move a fluid through each of the aforementioned chambers. In some embodiments, the means of detection (1180) comprises a detection unit. In some embodiments, the detection unit comprises a lateral flow assay strip, electrochemical sensor, or colorimetric sensor.

[0020] In some aspects, provided herein are methods of processing a nucleic acid from a biological sample, the method comprising: using a lysate buffer to prepare the biological sample into a lysate; providing the lysate to a binding unit configured to bind to the nucleic acid; and amplifyingthe nucleic acid of the lysate without a thermocycler or incubator. In some aspects, provided herein are methods of processing a nucleic acid from a biological sample, the method comprising: using a lysate buffer to prepare the biological sample into a lysate; providingthe lysate to a binding unit configured to bind to the nucleic acid; and amplifyingthe nucleic acid of the lysate using an annexing probe. In some embodiments, the lysate comprises a first portion, and wherein subsequent to (b) providingthe lysate to the bindingunit configured to bind to the nucleic acid portion, the first portion of the lysate is discarded. In some embodiments, further comprising providing an elution buffer to the binding unit. In some embodiments, further comprising eluting the nucleic acid from the binding unit. In some embodiments, amplifyingthe nucleic acid of the lysate is performed usingtwo or more nucleic acid primers. In some embodiments, the nucleic acid is a viral nucleic acid. In some embodiments, the nucleic acid is an RSV nucleic acid. In some embodiments, the nucleic acid is a White Spot Syndrome Virus (WSSV) nucleic acid. In some embodiments, wherein the nucleic acid is a bacterial nucleic acid. In some embodiments, wherein the nucleic acid is a Neisseria gonorrhoeae nucleic acid. In some embodiments, wherein the method occurs in 5 minutes or less. In some embodiments, further comprising sequencing the nucleic acid. In some embodiments, the sequencingis nanopore sequencing. In some embodiments, the amplifying in (c) occurs at an ambient temperature of about 20°C to about 40°C. In some embodiments, the amplifyingin (c) occurs in about 30 minutes or less. In some aspects provided herein are methods for replicating and amplifying a target nucleic acid sequence, said method comprising: introducing to a sample comprising a template to the target nucleic acid sequence wherein the template is double-stranded or can be made double-stranded: a set of primerscomprising: a first oligonucleotide primer (Pl ) comprising a sequence at least about 90% identical to a region on the plus strand of the target nucleic acid sequence, a second oligonucleotide primer (P2) comprising a sequence at least about 90% identical to a region on the minus strand of the target nucleic acid sequence, wherein Pl and P2 are present in molar excess relative to the target nucleic acid sequence, and an in reaction probe (IrP) comprising: a nucleotide sequence at least about 90% identical to a region on the plus or minus strand of the target nucleic acid sequence, wherein the IrP nucleotide sequence is flanked by both Pl sequence and P2 sequence or at least partially overlaps with either Pl sequence or P2 sequence, and a blocking feature that blocks extension of a DNA polymerase; an enzyme mix comprising: a DNA polymerase or functional fragment thereof, a second enzyme having a DNA processing function, wherein the DNA processing function facilitates Pl and P2 annealing to the target region, nucleotides in an amount sufficient to support amplification of the target nucleic acid sequence, and a crowding reagent; reacting the sample with the set of primers, the IrP, and the enzyme mix for a length of time sufficient to replicate and amplify the target nucleic acid sequence, wherein the reacting is maintained under isothermal conditions without previously denaturing the template, wherein amplified target nucleic acid sequence comprises: target single -stranded (ss) amplicons, ii) target double-stranded (ds) amplicons, or iii) target ss amplicons and target ds amplicons, and wherein the IrP detects the target ss amplicons and / or target ds amplicons. In some embodiments, comprising: detecting target ss amplicons, target ds amplicons, or target ss amplicons and target ds amplicons via the IrP. In some aspects provided herein are methods for replicating and amplifying a target nucleic acid sequence, said method comprising: introducing to a sample comprising a template to the target nucleic acid sequence, wherein the template is double -stranded or can be made double-stranded: a set of primers comprising: a first oligonucleotide primer (Pl) comprising a sequence at least about 90% identical to a region on the plus strand of the target nucleic acid sequence, a second oligonucleotide primer (P2) comprising a sequence at least about 90% identical to a region on the minus strand of the target nucleic acid sequence, and a third oligonucleotide primer (P3) comprising a sequence at least about 90% identical to a region on the plus or minus strand of the target nucleic acid sequence; an annexing oligonucleotide (AO) comprising: a nucleotide sequence at least about 90% identical to a region on the plus or minus strand of the target nucleic acid sequence, wherein the AO nucleotide sequence is: flanked by Pl sequence, P2 sequence, and P3 sequence or at least partially overlaps with Pl sequence, P2 sequence, orP3 sequence, and a blocking feature that blocks extension of a DNA polymerase; and an enzyme mix comprising: a DNA polymerase or functional fragment thereof, (b) a second enzyme having a DNA processing function, wherein the DNA processing function facilitates oneor more of Pl, P2, P3, and AO annealingto the target region, nucleotides in an amount sufficient to support amplification of the target nucleic acid sequence, and a crowding reagent; and b) reacting the sample with the set of primers, the AO, and the enzyme mix for a length of time sufficient to replicate and amplify the target nucleic acid sequence, wherein the reacting is maintained under isothermal conditions without previously denaturing the template, wherein amplified target nucleic acid sequence comprises: target single-stranded (ss) amplicons, two target double-stranded (ds) amplicons, or target ss amplicons and two target ds amplicons, and wherein the AO improves amplification specificity and efficiency by annexing a target site location to allow improved annealing of either Pl, P2, or P3 within the template and / or within amplified target nucleic acid sequence. In some embodiments, the AO comprises a label allowing the labeled AO to function as an in-reaction probe (IrP). In some aspects provided herein are methods of amplifying a region of a double-stranded nucleic acid, the method comprising: providing a first oligonucleotide primer and an annexing oligonucleotide (AO) to a strand of the region of the double-stranded nucleic acid; binding a second oligonucleotide primer to another strand of the region of the double-stranded nucleic acid; providing a recombinaseto the target region; and using the recombinase to extend a 3 ’ end of the first oligonucleotide primer, wherein the first oligonucleotide primer is a substrate for the recombinase. In some aspects provided herein are methods of amplifying a region of a double-stranded nucleic acid, the method comprising: binding a first oligonucleotide primer and an annexing oligonucleotide (AO) to a strand of the region of the double-stranded nucleic acid, wherein a 5’ end of the AO comprises a biotin molecule; using the first oligonucleotide primer and the AO as substrates to bind the first oligonucleotide primer and the AO to the strand of the region; binding a second oligonucleotide primer to another strand of the region of the double-stranded nucleic acid; and extending a 3 ’ end of the first oligonucleotide primer and a 3 ’ end of the second oligonucleotide primer. In some aspects provided herein are methods of amplifying a region of a double-stranded nucleic acid, the method comprising: binding a first oligonucleotide primer and an annexing oligonucleotide (AO) to a strand of the region of the double-stranded nucleic acid, wherein the first oligonucleotide primer and the AO overlap to create an intervening region; binding a second oligonucleotide primer to another strand of the region of the double-stranded nucleic acid, wherein the second oligonucleotide primer and the AO do not overlap to create an intervening region; and extending a 3 ’ end of the first oligonucleotide primer and a 3’ end of the second oligonucleotide primer. In some embodiments, the doublestranded nucleic acid comprises a target nucleic acid sequence. In some embodiments, the first oligonucleotide primer (Pl) comprises a sequence at least about 90% identical to a region on the plus strand of the target nucleic acid sequence. In some embodiments, the second oligonucleotideprimer (P2) comprises a sequence at least about 90% identical to a region on the minus strand of the target nucleic acid sequence. In some embodiments, Pl and P2 are present in molar excess relative to the target nucleic acid sequence. In some embodiments, the method further comprises an in-reaction probe (IrP) comprising a nucleotide sequence at least about 90% id entical to a region on the plus or minus strand of the target nucleic acid sequence. In some embodiments, the IrP nucleotide sequence is flanked by both Pl sequence andP2 sequence or at least partially overlaps with either Pl sequence or P2 sequence. In some embodiments, the IRP further comprises a blocking feature that blocks extension of a DNA polymerase. In some embodiments, the method further comprises a third oligonucleotide primer (P3) comprising a sequence at least about 90% identical to a region on the plus or minus strand of the target nucleic acid sequence. In some embodiments, the annexing oligonucleotide (AO) comprises a nucleotide sequence at least about 90% identical to a region on the plus or minus strand of the target nucleic acid sequence. In some embodiments, the AO nucleotide sequence is flanked by Pl sequence, P2 sequence, and P3 sequence or at least partially overlaps with Pl sequence, P2 sequence, or P3 sequence. In some embodiments, the AO further comprises a blocking feature that blocks extension of a DNA polymerase. In some embodiments, the method further comprises an enzyme mix comprising a DNA polymerase or functional fragment thereof. In some embodiments, the enzyme mix further comprises a second enzyme having a DNA processing function, wherein the DNA processing function facilitates one or more of Pl , P2, P3 , and AO annealingto the target nucleic acid sequence. In some embodiments, the enzyme mix comprises nucleotides in an amount sufficient to support amplification of the target nucleic acid sequence. In some embodiments, the enzyme mix comprises a crowding reagent. In some embodiments, the method further comprises reacting a sample, the set of primers, the IrP, and the enzyme mix for a length of time sufficient to replicate and amplify the target nucleic acid sequence. In some embodiments, the reactingis maintainedunder isothermal conditions without previously denaturing the double-stranded nucleic acid. In some embodiments, an amplified target nucleic acid sequence comprises: i) target single-stranded (ss) amplicons, ii) target double-stranded (ds) amplicons, or iii) target ss amplicons and target ds amplicons. In some embodiments, the IrP detects the target ss amplicons and / or target ds amplicons. In some embodiments, the method further comprises detecting target ss amplicons, target ds amplicons, or target ss amplicons and target ds amplicons via the IrP. In some embodiments, the IrP can detect two target ds amplicons and the detection of the two target ds amplicons produces increased sensitivity of target region detection compared to detecting single target ds amplicons. In some embodiments, the AO improves amplification specificity and efficiency by annexing a target site location to allow improved annealing of either Pl , P2, or P3 within the template and / or withinamplified target nucleic acid sequence. In some embodiments, the second enzyme in the enzyme mix comprises a recombinase or functional fragment thereof, a helicase or functional fragment thereof, a topoisomerase or functional fragment thereof, or any combination thereof. In some embodiments, the enzyme mix comprises a single-strand binding protein such as T4 GP32. In some embodiments, the DNA polymerase of functional fragment thereof is a strand -displacing DNA polymerase. In some embodiments, the DNA polymerase comprises Bacillus subtilis DNA polymerase I (Bsu), Bst, or Klenow Fragment. In some embodiments, the DNA polymerase comprises Bst 3.0. In some embodiments, the DNA polymerase comprises Bacillus subtilis DNA polymerase I (Bsu). In some embodiments, the second enzyme having a DNA processing function comprises a bacterial or viral recombinase protein. In some embodiments, the second enzyme having a DNA processing function comprises UvsX DNA recombinase (UvsX). In some embodiments, the second enzyme having a DNA processing function comprises RAD51. In some embodiments, the second enzyme having a DNA processing function comprises HELB helicase, RTEL1 helicase, Pif 1 helicase, Top 1 topoisomerase, ortopoisomerase Illa. In some embodiments, the crowding reagent comprises polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), Ficoll, Dextran, or any combination thereof. In some embodiments, the crowding reagent comprises PEG. In some embodiments, the blocking feature comprises an inverted nucleotide or a C3 -spacer at the 3 ’ end of the in-reaction probe. In some embodiments, the sequence of any primer in the primer set and the IrP or AO sequence do not overlap. In some embodiments, the sequence of any one primer in the primer set and the IrP or AO sequence overlap. In some embodiments, at least one primer in the primer set is less than or equal to 15 nucleotides in length. In some embodiments, Pl, P2, P3, IrP and / or AO comprise a length of at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 60 nucleotides, at least about 80 nucleotides, or more. In some embodiments, a 5’ end of Pl, P2, or P3 and a 5’ end of the AO is separated by at least about 1 nucleotide. In some embodiments, a 5’ end of Pl, P2, orP3 and a 5’ end of the AO is separated by at least about 5 nucleotides. In some embodiments, a 5’ end of Pl, P2, or P3 and a 5’ end of the AO is separated by at least about 10 nucleotides. In some embodiments, at least one primer is a nucleotide length insufficient to produce amplicons of the target sequence without the presence of the IrP or AO. In some embodiments, the IrP is long enough (greater than or equal to 20 nts) to be an efficient substrate for the second enzyme having the DNA processing function and wherein the IrP becomes an annexing probe (AP) capable of invading the target dsDNA amplicon sequence and helping anneal at least one of the primers in the primer set to target region when that primer is less than or equal to 15 nucleotides in length. Insome embodiments, the AO comprises a label. In some embodiments, the AO comprising a label is configured to function as an in-reaction probe (IrP). In some embodiments, the IrP serves as an annexing oligo (AO). In some embodiments, the in-reaction probe comprises a label. In some embodiments, the label comprises a fluorophore, or a redox molecule, or biotin, or digoxigenin. In some embodiments, the label comprises an aromatic structure. In some embodiments, at least one primer comprises a label either on the 5 ’end or internally in the sequence so as not to prevent extension by polymerase but leading to amplification of labelled ss and ds amplicons. In some embodiments, the IrP bound to target labelled ds amplicons is detected via a lateral flow assay (LFA). In some embodiments, the in-reaction probe bound to target labelled ss amplicons is detected via a lateral flow assay (LFA). In some embodiments, a 5’ end of at least one of the primers in the primer set is linked to the 5’ or 3 ’ end ofthein-reaction probe. In some embodiments, at least one of the primers in the primer set is 6-21 nucleotides in length. In some embodiments, the label comprises a fluorophore or biotin or digoxigenin. In some embodiments, the label comprises a redox molecule. In some embodiments, the sample is incubated at a reaction temperature range maintained within 18-40°C for the length of time. In some embodiments, the sample is incubated at a reaction temperature range maintained within 20-37°C for the length of time. In some embodiments, the sample is incubated at a room temperature for the length of time. In some embodiments, the sample is incubated at a reaction temperature range maintained within 22-25°C for the length of time. In some embodiments, the reaction temperature range is an isothermal range. In some embodiments, maintaining isothermal conditions of the reaction does not involve incubating the reaction admixture in a temperature -regulating device. In some embodiments, maintaining isothermal conditions of the reaction does not involve incubating the reaction admixture in a thermocycler. In some embodiments, the reaction admixtureremains within a temperature range of + / - 10% of the starting reaction temperature until sufficient amplification of the target nucleic acid sequence to be detected is achieved. In some embodiments, the length of time is between about 1 -60 minutes. In some embodiments, the length of time is between about 20-40 minutes. In some embodiments, the length of time is about 30 minutes. In some embodiments, the target ss and ds amplicons and / or the two target ds amplicons are produced at least at a linear rate of amplification. In some embodiments, the target ds amplicons and / or the two target ds amplicons are produced at an exponential rate of amplification. In some embodiments, the target ss amplicons are produced at least at a linear rate of amplification. In some embodiments, the target ss amplicons are produced at an exponential rate of amplification. In some embodiments, the sample comprises nucleic acid extracted from a subject. In some embodiments, the target region of the nucleic acid comprises a continuous stretch of nucleotide sequence present within thesample. In some embodiments, the nucleic acid extracted from the subject comprises doublestranded DNA (dsDNA). In some embodiments, the nucleic acid extracted from the subject comprises single-stranded DNA (ssDNA). In some embodiments, the sample comprises RNA extracted from the subject. In some embodiments, reverse transcriptase andNTPs are added to the sample to reverse transcribe the RNA extracted from the subject to cDNA, wherein the cDNA becomes the template to the target region. In some embodiments, the sample comprising nucleic acid extracted from the subject comprises nucleic acid derived from a pathogen. In some embodiments, the sample comprising nucleic acid extracted from the subject comprises nucleic acid derived from a pathogen and nucleic acid derived from the subject. In some embodiments, the pathogen is a virus. In some embodiments, the virus is listed in Table 1 . In some embodiments, the pathogen is a bacterium. In some embodiments, the bacterium is listed in Table 2. In some embodiments, the pathogen is a fungus. In some embodiments, the fungus is listed in Table 3. In some embodiments, the pathogen is an animal pathogen. In some embodiments, the animal pathogen is listed in Table 4. In some embodiments, the sample is obtained from an animal. In some embodiments, the animal is a livestock species. In some embodiments, the livestock species is a type of cattle, horse, goat, sheep, pig, swine, rabbit, donkey, mule, bison, llama, cat, dog, deer, camel, yak, turkey, alpaca, elk, chicken, duck, emu, ostrich, pheasant, goose, fish, shrimp, crustacean, or bee. In some embodiments, the animal is a human. In some embodiments, the sample is obtained from plant. In some embodiments, ds amplicons having a sequence comprising at least the target region are produced at least at a linear rate of amplification. In some embodiments, ds amplicons having a sequence comprising at least the target region are produced at an exponential rate of amplification. In some embodiments, the presence of the IrP comprising the label increases production of target ds amplicons. In some embodiments, the presence of the in-reaction probe comprising the label increases production of target ss amplicons. In some embodiments, comprising detecting the target ds amplicons. In some embodiments, detecting the target ss amplicons. In some embodiments, the detecting comprises analyzing reaction products using a molecular lateral flow assay (LFA). In some embodiments, the detecting comprises analyzing reaction products using an intercalating dye or fluorophore. In some embodiments, the detecting comprises analyzing reaction products using a nucleic acid hybridization technique. In some embodiments, the detecting comprises analyzing reaction products using a fluorescence detection technique. In some embodiments, the detecting comprises analyzing reaction products using a colorimetric technique. In some embodiments, the detecting comprises analyzing reaction products using nucleic acid sequencing. In some embodiments, the detecting comprises analyzing reaction products using DNA fragment size discrimination. In some embodiments, anoligonucleotide probeis added to the reaction mix or to ss amplicons and / or ds amplicons produced from the reaction to aid in the detecting. In some embodiments, a separate oligonucleotide probe comprising a quencher is added to the reaction mix or to ss amplicons and / or ds amplicons produced from the reaction to aid in the detecting. In some embodiments, use of FRET in the detecting. In some embodiments, the detecting comprising determining a quantitative measure of an amplified reaction product comprising the target ds amplicons. In some embodiments, the detecting comprising determining a quantitative measure of an amplified reaction product comprising the target ss amplicons. In some embodiments, one of the primers has a non- complementary 5’ adaptor region. In some embodiments, one of the primers has a non- complementary 5 ’ adaptor region and has an additional primer that can bind to the adaptor region only. In some embodiments, the target nucleic acid sequence is a selected region of a genomic DNA sequence from a pathogen. In some embodiments, the target nucleic acid sequence is a selected region of an RNA sequence from a pathogen. In some embodiments, the target nucleic acid sequence is from a pathogen listed in Table 1, Table 2, Table 3, or Table 4. In some embodiments, the target nucleic acid sequence is amplified in the presence of human genomic DNA. In some embodiments, the template to the target nucleic acid sequence is a single copy of a viral genome, and wherein the amplified target nucleic acid sequence is amplified at about room temperature in less than about 30 minutes to a level of a detectable range. In some embodiments, the level of a detectable range is at least about 104 copies / pL, 105 copies / pL, 106 copies / pL, 107 copies / pL, or 108 copies / pL. In some embodiments, the target ds amplicons are amplified and detected in real time. In some embodiments, target ds amplicons are amplified and detected in less than about 15 minutes, 12 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, or 3 minutes. In some embodiments, at least one primer in the primer set is less than or equal to 14 nucleotides in length. In some embodiments, at least one primer in the primer set is less than or equal to 12 nucleotides in length. In some embodiments, at least one primer in the primer set is less than or equal to 10 nucleotides in length. In some embodiments, further comprising detection of two different regions of the target nucleic acid sequence. In some embodiments, two or more distinct pathogenic variants are detected within a single amplification reaction. In some embodiments, two ormore distinct disease mutations are detected within a single amplification reaction. In some embodiments, a single nucleotide polymorphism (SNP) within the target nucleic acid sequence is detected.

[0021] In some aspects, provided herein are systems of processing a nucleic acid from a biological sample, the system comprising: a binding unit configured to bind to the nucleic acid; and an amplification unit configured to amplify the nucleic acid of the lysate, wherein theamplification unit does not comprise a thermocycler or incub ator. In some aspects , provided herein are systems for processing a nucleic acid from a biological sample, the system comprising: a binding unit configured to bind to the nucleic acid; and an amplification unit configured to amplify the nucleic acid of the lysate, wherein the amplifyingis performed using an annexing probe. In some embodiments, further comprising an elution unit comprising an elution buffer. In some embodiments, the amplifyingis performed using two or more primers. In some aspects provided herein are systems of primers and probes comprising: a first oligonucleotide primer (Pl) comprising a sequence at least about 90% identical to a region on the plus strand of the target nucleic acid sequence, a second oligonucleotide primer (P2) comprising a sequence at least about 90% identical to a region on the minus strand of the target nucleic acid sequence, a third oligonucleotide primer (P3) comprising a sequence at least about 90% identical to a region on the plus or minus strand of the target nucleic acid sequence, and an annexing oligonucleotide (AO) comprising: a nucleotide sequence at least about 90% identical to a region on the plus or minus strand of the target nucleic acid sequence, wherein the AO nucleotide sequence is: flanked by Pl sequence, P2 sequence, and P3 sequence, or at least partially overlaps with Pl sequence, P2 sequence, or P3 sequence; and a blocking feature that blocks extension of a DNA polymerase; wherein the system is for replicating target nucleic acid sequence comprising: i) target singlestranded (ss) amplicons, ii) two target double-stranded (ds) amplicons, or iii) target ss amplicons and two target ds amplicons. In some aspects, provided herein are systems for amplifying a region of a double-stranded nucleic acid, the system comprising: an annexing oligonucleotide (AO), a first oligonucleotide primer, and a second oligonucleotide primer, wherein said AO and said first oligonucleotide primer are configured to invade the region of the double-stranded nucleic acid. In some aspects, provided herein are systems for amplifying a region of a double -stranded nucleic acid, the system comprising: an annexing oligonucleotide (AO), a first oligonucleotide primer, a second oligonucleotide primer, and a recombinase, wherein the first oligonucleotide primer is a substrate forthe recombinase duringthe amplifying of the first region. In some aspects, provided herein are systems for amplifying a region of a double -stranded nucleic acid, the system comprising: an annexing oligonucleotide (AO), a first oligonucleotide primer, and a second oligonucleotide primer, wherein a 5 ’ end of the AO comprises a biotin molecule. In some aspects, provided herein are systems for amplifying a region of a double-stranded nucleic acid, the system comprising: an annexing oligonucleotide (AO), a first oligonucleotide primer, and a second oligonucleotide primer, wherein the first oligonucleotide primer and the AO overlap to create an intervening region, and wherein the second oligonucleotide primer and the AO do not overlap to create an intervening region. In some embodiments, the double -stranded nucleic acid comprises atarget nucleic acid sequence. In some embodiments, the first oligonucleotide primer (Pl) comprises a sequence at least about 90% identical to a region on the plus strand of the target nucleic acid sequence. In some embodiments, the second oligonucleotide primer (P2) comprises a sequence at least about 90% identical to a region on the minus strand of the target nucleic acid sequence. In some embodiments, the system further comprises a third oligonucleotide primer (P3). In some embodiments, P3 comprises a sequence at least about 90% identical to a region on the plus or minus strand of the target nucleic acid sequence. In some embodiments, the AO comprises a nucleotide sequence at least about 90% identical to a region on the plus or minus strand of the target nucleic acid sequence. In some embodiments, the AO nucleotide sequence is flanked by Pl sequence, P2 sequence, and P3 sequence, or at least partially overlaps with Pl sequence, P2 sequence, orP3 sequence. In some embodiments, the AO comprises a blocking feature that blocks extension of a DNA polymerase. In some embodiments, the system is configured to amplify the target nucleic acid sequence. In some embodiments, an amplified target nucleic acid sequence comprises: i) target single-stranded (ss) amplicons, ii) two target double-stranded (ds) amplicons, or iii) target ss amplicons and two target ds amplicons. In some embodiments, the AO comprises a label allowing the labeled AO to function as an in-reaction probe (IrP). In some embodiments, the sequence of one of the primers and the AO sequence do not overlap. In some embodiments, the sequence of one of the primers and the AO sequence at least partially overlap. In some embodiments, one of the primers is about 15 -20 nucleotides in length. In some embodiments, a 3’ end of one of the primers is linked to the 5’ end of the in-reaction probe. In some embodiments, one of the primers is about 6-21 nucleotides in length. In some embodiments, the AO comprises a label. In some embodiments, the label is a fluorophore, redox molecule, or biotin. In some embodiments, the label comprises an aromatic group. In some embodiments, the system is prevents non-specific binding. In some embodiments, one of the primers is at least 25 nucleotides in length. In some embodiments, one of the primers is from about 10 to about 40 nucleotides in length. In some embodiments, the AO is from about 30 to about 75 nucleotides in length. In some embodiments, P1, P2, P3, IrP and / or AO comprise a length of at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 60 nucleotides, at least about 80 nucleotides, or more. In some embodiments, a 5’ end of Pl, P2, orP3 and a 5’ end of the AO is separated by at least about 1 nucleotide. In some embodiments, a 5’ end of Pl, P2, orP3 and a 5’ end of the AO is separated by at least about 5 nucleotides. In some embodiments, a 5’ end of Pl, P2, or P3 and a 5’ end of the AO is separated by at least about 10 nucleotides.

[0022] In some aspects, provided herein are kits comprising systems disclosed herein andinstructions for use. In some embodiments, the instructions designate a treatment of a subject in need thereof. In some embodiments, the instructions designate a treatment of a viral infection. In some embodiments, the instructions designate a treatment of RSV. In some embodiments, the instructions designate a treatment of White Spot Syndrome Virus (WSSV). In some embodiments, the instructions designate a treatment of a bacterial infection. In some embodiments, the instructions designate a treatment of a Neisseria gonorrhoeae infection.INCORPORATION BY REFERFENCE

[0023] All publications, patents, and patent applicationsmentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The features and advantages of the present disclosure will become apparent from a consideration of the following detailed descriptionpresented in connection with the accompanying drawings in which:

[0025] FIG. 1 shows a non-limiting embodiment of the present disclosure.

[0026] FIG. 2 shows a non -limiting configuration of the nucleic acid processing and detection devices as described herein.

[0027] FIG. 3 shows a non-limiting example of the different sections of a nucleic acid processing and detection device described herein and how itmay be used to process a sample (e.g., abiological or environmental sample).

[0028] FIG. 4 shows an alternate embodiment of the present disclosure.

[0029] FIG. 5 shows an alternate embodiment of the present disclosure.

[0030] FIG. 6 shows a non-limiting example of the aliquoting valve described herein.

[0031] FIG. 7 shows a non-limiting example of how a nucleic acid processing and detection device described herein may be used to process a sample (e.g., a biological or environmental sample).

[0032] FIG. 8 shows the front (top) and back (bottom) perspective views of the molded device shown in FIG. 4.

[0033] FIG. 9A- 9C show an alternate embodiment of the devices describedherein from variousperspective views. FIG. 9A shows a front perspective view. FIG. 9B shows a back perspective view. FIG. 9C shows an exploded perspective view.

[0034] FIG. 10 shows a non-limiting example of how a nucleic acid processing and detection device described herein may be used to process a sample (e.g., a biological or environmental sample).

[0035] FIG. 11A-11D shows various measurements of nucleic acid extraction using a nucleic acid extraction device (e.g., SeekIT Extraction device), such as sample processing and nucleic acid extraction time (FIG. 11 A), extracted nucleic acid concentration (FIG. 11B), extracted nucleic acid quality (FIG. 11C) and amount of target WSSV NA molecules (FIG. 11D) acquired from WSSV-infected shrimp tissue using the devices described herein compared to other devices or kits.

[0036] FIG. 12 shows an alternate embodiment of the devices described herein.

[0037] FIG. 13A-13C shows various views of an adapter that may be used in conjunction with the devices of the present disclosure. The adapter may be used to removably attach a vial (e.g., a first vial or a second vial) to a coupler (e.g., a first coupler or a second coupler). FIG. 13A shows a top front perspective view of an adaptor. FIG. 13B shows a front perspective view of an adaptor. FIG. 13C shows a top perspective view of an adaptor.

[0038] FIG. 14A depicts the average gNA (genomic nucleic acid) yield in ng / mg for mouse skeletal muscle tissue from a study on three frozen mouse skeletal muscle tissue samples processed by a Qiagen DNA extraction kit and an exemplary extraction device as disclosed herein (e.g., Seeklt extraction device; experimental).

[0039] FIG. 14B shows the average gNA quality (260 / 280) for the samples shown in FIG. 14 A.

[0040] FIG. 14C shows the average CQ on qPCR for a target sequence amplified in mouse skeletal muscle tissue samples shown in FIG. 14A and 14B.

[0041] FIG. 15A shows the average gNA yield (ng / mg) formouse skin tissue from a study on three frozen mouse skin tissue samples processed by a Qiagen DNA extraction kit and an exemplary extraction device as disclosed herein (e.g., Seeklt extraction device; experimental).

[0042] FIG. 15B shows the average gNA quality (260 / 280) for the mouse skin tissue samples in FIG. 15 A.

[0043] FIG. 15C shows the average CQ on qPCR for a target sequence amplified in the mouse skin tissue samples in FIG. 15 A and 15B.

[0044] FIG. 16A shows the average gNA concentration (ng / pL) for mouse spleen tissue from a study on three frozen mouse spleen tissue samples processed by a Qiagen DNA extraction kit and an exemplary extraction device as disclosed herein (e.g., Seeklt extraction device; experimental).

[0045] FIG. 16B shows the average gNA quality (260 / 280) for the mouse spleen tissue samples in FIG.16 A.

[0046] FIG. 16C shows the average CQ on qPCR for a target sequence amplifiedin mouse spleen tissue samples in FIG. 16A and 16B.

[0047] FIG. 17A shows the average gNA yield (ng / mg) for mouse liver tissue from a study on three frozen mouse liver tissue samples processed by a Qiagen DNA extraction kit and an exemplary extraction device as disclosed herein (e.g., Seeklt extraction device; experimental).

[0048] FIG. 17B shows the average gNA quality (260 / 280) for the mouse liver tissue samples in FIG. 17 A.

[0049] FIG.17C shows the average CQ on qPCR for a target sequence amplifiedin the mouse liver tissue samples in FIG. 17A and 17B.

[0050] FIG. 18A shows the average gNA concentration (ng / pL) for human skeletal tissue processed with an exemplary extraction device as disclosed herein (e.g., Seeklt extraction device) using different lysis buffers (LB-1, LB-2, LB-3, LB-4) and a Qiagen DNA extraction kit.

[0051] FIG. 18B shows the average gNA quality (260 / 280) for the human skeletal tissue samples in FIG. 18 A.

[0052] FIG. 18C shows the average CQ on qPCR for a target sequence amplified in the human skeletal tissue samples in FIG. 18A and 18B.

[0053] FIG. 19A shows the average gNA concentration (ng / pL) for embedded human tissue processed with an exemplary extraction device as disclosed herein (e.g., Seeklt extraction device) using different lysis buffers (Velox LB, A-PK LB, A-nPK) and a Qiagen DNA extraction kit.

[0054] FIG. 19B shows the average gNA quality (260 / 280) for the embedded human tissue samples in FIG. 19 A.

[0055] FIG. 19C shows the average gNA yield (ng / mg) for the embedded human tissue samples in FIG. 19A and 19B.

[0056] FIG. 19D shows the average CQ on qPCR for a target sequence amplified in the embedded human tissue samples in FIG. 19 A, 19B and 19C.

[0057] FIG. 19E shows an agarose gel run for amplified nucleic acids in the processed embedded human tissue samples in FIG. 19A-D.

[0058] FIG. 20A shows eluate concentrations (ng / pL) of 1 mL samples of human saliva spiked with increasing amounts (in uL) of RSV infected cells and processed with an exemplary extraction device as disclosed herein (e.g., Seeklt extraction device).

[0059] FIG. 20B shows the average lysate and eluate qualities (260 / 280) of 1 mL samples of human saliva spiked with RSV infected cells from FIG 20A.

[0060] FIG. 20C shows the average CQ for a target sequence of the RSV genome that was amplified from processed samples of 1 mL of human saliva spiked with RSV infected cells from FIG 20A and 20B.

[0061] FIG. 21 shows a few embodiments of the nucleic acid extraction, amplification and detection platforms disclosed herein, such as Seeklt platform.

[0062] FIG. 22 shows a field-deployable embodiment of the nucleic acid extraction, amplification and detection platforms disclosed herein, such as Seeklt platform for the processing of a biological sample using an extraction device (e.g., Seeklt extraction device or Seek extraction device), followed by amplification with an amplification device (e.g., Seeklt amplification or Seek amplification) comprising a lyophilized pellet and detecting of the amplified target nucleic acid using a molecular lateral flow assay (mLFA).

[0063] FIG. 23A shows a first channel receiving a lysis solution.

[0064] FIG. 23B shows a first channel receiving a biological sample.

[0065] FIG. 23C shows a first channel transferring a biological sample to a second channel.

[0066] FIG. 24A shows a first channel receiving a biological sample, followed by receiving a lysis solution and then transferring the lysed solution to a second channel .

[0067] FIG. 24B shows a housing receiving the lysed solution from the second channel in FIG. 24 A. The housing contains a binding membrane that binds to the nucleic acid in the lysate. The lysate is then transferred back to the second channel which is then discarded .

[0068] FIG. 24C shows the housing from FIG. 24B receiving an elution solution from a third channel, followed by transferring the eluate back to the third channel, which now has the analytes being extracted from a biological sample.

[0069] FIG. 25 shows a first channel containing an extracted nucleic acid sample (e.g. , the third channel containing the extracted analyte from FIG. 24C) being transferred to a second channel containing a lyophilized amplification pellet or bead and rehydrating the said pellet to start amplification of dual-labelled target DNA amplicons.

[0070] FIG. 26A shows aLateral Flow Analysis (LFA) detectingan ssDNA amplicon (e.g., duallabelled amplicon in second channel from FIG. 25).

[0071] FIG. 26B depicts a Lateral Flow Analysis being conducted on an amplified nucleic acid sample from a processed sample. It shows a first channel containing an amplified nucleic acid sample (e.g., dual-labelled amplicon solution in second channel from FIG. 25) being diluted with detection buffer added from buffer packet, followed by addition of an LFA strip, such as described in FIG. 26 A, into the diluted amplicon solution in the first channel and the LFA results are then interpreted to detect presence of target nucleotide sequence.

[0072] FIG. 27A - FIG. 27D shows the results of a nucleic acid extraction, amplification and detection system disclosed herein (e.g., Seeklt system or System A) for detection of WSSV infection in shrimp. In FIG. 27A, lyophilized amplification pellets are shown in Eppendorf tubes, where amplification enzymes were lyophilized with WSSV-specific primers and annexing probe designed according to specifications described in this disclosure (e.g., Seeklt Amplification). Amplification reaction buffer was used as elution buffer for extracting nucleic acid from a WSSV- infected shrimp tissue sample using an extraction device disclosed herein (e.g., Seeklt extraction device). The eluate was used to replace reaction buffer in the rehydration solution usedto rehydrate the lyophilized pellets. In FIG. 27B, reaction products seen on a Native PAGE gel following electrophoresis are seen, showing increase in amplification of target amplicons with increasing volume of eluate added. In FIG. 27C, amplicon identification is ob served on mLFA strips showing control line and test line. In FIG. 27D, a graph analysis of mLFA test line intensity per sample as a percentage of that of NTC is plotted showing increased detection with increasing volume of eluate added.

[0073] FIG. 28 shows a mean density analysis of a lateral flow analysis test line indicating an increase in mLFA detection compared to NTC with increasing concentration of reagents of reaction buffer in the elution buffer or solution.

[0074] FIG. 29 shows the results of a nucleic acid extraction, amplification and detection device disclosed herein (e.g., Seeklt system or System A) for detection of Epstein Barr virus (EBV) as described in this disclosure. Human saliva samples were spiked with EBV viral titers of increasing copies (10°-l 07copies / uL) and processedusing an extraction method / sy stem disclosed herein (e.g, Seeklt extraction), and these extracts were used as templates to amplify the lyophilized amplification beads that are shown in Eppendorf tubes, where amplification enzymes were lyophilized with EBV-specific primers and annexing probe designed according to specifications described in this disclosure (e.g. Seeklt Amplification). The Native PAGE gel image and the molecular lateral flow analysis show the increase in the EBV-specific target amplicons with increase in amount of spiked EBV.

[0075] FIG. 30 depicts a housing comprising a port and a substrate and is an embodiment of an extraction device as disclosed herein (e.g., Seeklt extraction device).

[0076] FIG. 31 depicts a workflow procedure for processing a biological sample using an extraction device as disclosed herein (e.g., Seeklt extraction device).

[0077] FIG. 32A shows the concentration (ng / pL) of nucleic acids extracted from various processed human biological samples using an extraction device as disclosed herein (e.g., Seeklt extraction device).

[0078] FIG. 32B shows the quality (260 / 280) of nucleic acids extracted from the various processed human biological samples in FIG. 32A.

[0079] FIG. 32C shows the average CQ for a target that was amplified from the variousprocessed human biological samples in FIG. 32A and FIG. 32B.

[0080] FIG. 33A shows a comparison of the quality (260 / 280) of DNA extracted from whole blood processed by a buffer containing sodium hydroxide and from whole blood processed by a buffer containing sodium acetate using an extraction device as disclosed herein (e.g., Seeklt extraction device).

[0081] FIG. 33B shows a comparison of the CQ values on qPCR for a target sequence of DNA extracted from whole blood processed by a buffer containing sodium hydroxide and from whole blood processed by a buffer containing sodium acetate using an extraction device as disclosed herein (e.g., Seeklt extraction device).

[0082] FIG. 34A shows the gNA yield (ng / mg) of WSSV extracted from shrimp muscle tissue and shrimp pleopod tissue using an extraction device as disclosed herein (e.g., Seeklt extraction device). Solid black bars depict Seeklt Extraction results. Shaded bars depict commercially available lab kit results.

[0083] FIG. 34B shows the gNA quality (260 / 280) of WSSV extracted from shrimp muscle tissue and shrimp pleopod tissue using an extraction device as disclosed herein (e.g., Seeklt extraction device). Solid black bars depict Seeklt Extraction results. Shaded bars depict commercially available lab kit results.

[0084] FIG. 34C shows the normalized qPCR results (WSSV copies / pL) for shrimp muscle tissue processed using an extraction device as disclosed herein (e.g., Seeklt extraction device) at various time points post-infection. Solid black bars depict Seeklt Extraction results. Shaded bars depict commercially available lab kit results.

[0085] FIG. 34D shows the normalized qPCR results (WSSV copies / pL) for shrimp pleopod tissue processed using an extraction device as disclosed herein (e.g., Seeklt extraction device) at various time points post-infection. Dark gray bars depict Seeklt Extraction results, Dark gray bars depict commercially available lab kit results.

[0086] FIG. 35A shows the time required to process a shrimp muscle tissue sample using an extraction device as disclosed herein (e.g., Seeklt extraction device) (System A), including grinding time compared to a commercial DNA extraction kit (Control).

[0087] FIG. 35B shows the average yield of gNA (ng / mg) of shrimp muscle tissue samples processed by using an extraction device as disclosedherein (e.g., Seeklt extraction device) (System A) and a commercial DNA extraction kit (Control).

[0088] FIG. 35C shows a comparison between the eluate quality (260 / 280) ratio of gNA extracted from processed shrimp muscle tissue samples using an extraction device as disclosed herein (e.g., Seeklt extraction device) (System A) and a commercial DNA extraction kit (Control).

[0089] FIG. 35D shows the qPCR detection accuracy of the presence of WSSV in a shrimp muscle tissue sample using an extraction device as disclosed herein (e.g., Seeklt extraction device) (System A) and a commercial DNA extraction kit (Control).

[0090] FIG. 36A shows the average qPCR analysis of shrimp 24 hours post-infection when processed using an extraction device as disclosed herein (e.g., Seeklt extraction device) (System A) and a commercial DNA extraction kit (Control).

[0091] FIG. 36B shows the gel result of an isothermal amplification and a molecular lateral flow analysis strip results for infected shrimp samples 24 -hours post infection processed using an extraction device as disclosed herein (e.g., Seeklt extraction device) (System A) and a commercial DNA extraction kit (Control).

[0092] FIG. 36C shows a mean density analysis of the lateral flow analysis test line in FIG 36B.

[0093] FIG. 37A shows an average DNA concentration (ng / pL) obtained from samples of whole blood spiked with a various % volume of bacterial suspension and processed using an extraction device as disclosed herein (e.g., Seeklt extraction device).

[0094] FIG. 37B shows an average quality (260 / 280) of DNA obtained from the samples of whole blood spiked with a various % volume of bacterial suspension in FIG 37A.

[0095] FIG. 37C shows a lateral flow analysis of DNA from samples of the whole human blood samples spiked with a various % volume of a bacterial suspension processed using an extraction device as disclosed herein (e.g., Seeklt extraction device) and after the DNA was amplified by isothermal amplification.

[0096] FIG. 38 A shows the average concentration (ng / pL) of extracted DNA from a simulated bodily fluid spiked with Neisseria gonorrhea processed using an extraction device as disclosed herein (e.g., Seeklt extraction device).

[0097] FIG. 38B shows the average quality (260 / 280) of extracted DNA from the simulated bodily fluid spiked with Neisseria gonorrhea processed using an extraction device as disclosed herein (e.g., Seeklt extraction device) in FIG. 38A.

[0098] FIG. 38C shows the average CQ for a target sequence that was amplified from processed samples of simulated bodily fluid spiked with Neisseria gonorrhea in FIG. 38A and 38B.

[0099] FIG. 39 depicts a workflow procedure for processing a biological sample using an extraction device as disclosed herein (e.g., Seeklt extraction device).

[0100] FIG. 40A shows an absorbance plot for plasmid samples grown in E. coh that wereprocessed by an extraction device as disclosed herein (e.g., the Seek Extraction kit).

[0101] FIG. 40B shows DNA purity results for pCRTOPO-4 DNA plasmids grown in E. coli postDNA extraction using e.g., Seek Extraction and Promega Wizard Extraction Kits, as in FIG. 40A.

[0102] FIG. 40C shows Full plasmid Nanopore sequencing from extracted materials without further cleanup after extraction from e.g., Seek Extraction, as in FIG. 40A and 40B.

[0103] FIG. 40D shows Full plasmid Nanopore sequencing from extracted materials without further cleanup after extraction from a control, as in FIG. 40A and 40B.

[0104] FIG. 41 shows Internal analysis for WSSV-infected shrimp processed using an extraction device as disclosed herein (e.g., Seeklt extraction device) and commercial DNA extraction kit (e.g Qiagen QiaAmp) showing (A) locations of shrimp samples collected, (B) time of extraction, (C) infection levels via qPCR, (D) UV-Vis 260 / 280 absorbance ratio, and (E) average yield.

[0105] FIG. 42 shows Animal Aquatic Health Lab (AAHL) analysis for WSSV-infected shrimp tissue processed using an extraction device as disclosed herein (e.g., Seeklt extraction device) and commercial DNA extraction kit (e.g. Promega) showing (A) total sample processingtime (B) DNA yield normalized per mg of tissue, (C) UV-Vis 260 / 280 absorbance ratio, and (D) percentage accuracy for specificity and sensitivity derived from qPCR results.

[0106] FIG. 43 shows results from samples processed using an extraction device as disclosed herein (e.g., Seeklt extraction device) (A) Total extracted nucleic acid concentration and (B) Cq values from qPCR GAPDH detection for mouse skeletal muscle, spleen, skin, and liver tissue samples.

[0107] FIG. 44 shows results from samples processed using an extraction device as disclosed herein (e.g., Seeklt extraction device) (A) Total extracted nucleic acid concentration and (B) Cq values for the qPCR detection of GAPDH for human whole blood, skeletal tissue, and FFPE tissue samples.

[0108] FIG. 45 shows results from samples processed using an extraction device as disclosed herein (e.g., Seeklt extraction device) (A) qRT-PCR detection of Respiratory Syncytial Virus (RSV) RNA extracted from 1 mL saliva samples spiked with 0, 5 or 10 uL of cultured cells containing active RSV inf ection . (B) qPCR detection of Neisseria gonorrhoeae (Ng) extracted from Simulated Cervical Mucus Fluid (SCMF) or Simulated Vaginal Fluid (SVF) spiked with serial dilutions of Ng bacterial culture.

[0109] FIG. 46 shows Native PAGE showing increasing amplification with eluate concentration of gNA template extracted from WSSV-infected shrimp using an extraction device as disclosed herein (e.g., Seeklt extraction device) and amplified using an amplification device as disclosedherein (e.g., Seeklt amplification device).

[0110] FIG. 47A show results from shrimp tissue processed using an extraction device as disclosed herein (e.g., Seek Extraction).

[0111] FIG. 47B show results from mouse tissue processed using an extraction device as disclosed herein (e.g., Seek Extraction).

[0112] FIG. 47C show results from human whole blood and human tissue processed using an extraction device as disclosed herein (e.g., Seek Extraction).

[0113] FIG. 48 shows Full plasmid Nanopore sequencing from extracted materials without further cleanup after extraction by e.g., Seek Extraction.

[0114] FIG. 49 shows a diagram of an amplification approach using two long primers and one in-reaction probe (5’ or internal labelled, polymerase non -extendible, may or may not be 3’ labelled). The reaction product can be directly detected on end point detection methods such as molecular LFA without requiring addition of another probe or enzyme. The in -reaction probe may form a partial triple helix structure with the target ds amplicon.

[0115] FIG. 50A - FIG. 50C shows a non -limiting example of the methods described herein using the primer systems described herein, as well as methods of detecting the single -stranded DNA and / or RNA target produced by the methods (e.g., molecular lateral flow Assay (MLFA)). As shown in the diagram of FIG. 50 A, the method may comprise 1. Elutingthe gNA (e.g., genomic DNA containing a target strand) in a rehydration buffer and transferring it to a lyophilized enzyme pellet (e.g., a pellet comprising enzymes, buffer, primers, and probes), mixing the solution thoroughly and incubating the solution at room temperature for a length of time (e.g., 30 minutes). In some embodiments, the final product, apart from gNA and unused reagents and primers, will be FITC labelled ssDNA and dsDNA amplicon already bound to a biotinylated probe. As shown in the diagram of FIG. 50B, the method may further comprise 2. Mixing the solution or part of the solution from the previous step (e.g., an ANINA reaction product) with a Lateral Flow Assay (LFA) hybridization solution. As shown in the diagram of FIG. 50C, in some embodiments, the LFA hybridization solution comprises gold nanoparticles linked to anti -FITC antibodies, which then bind to theFITC-ssDNA amplicons, as well as unused FITC-P2. To detectthe labeled ssDNA amplicons, the method may comprise transferring the solution from the aforementioned step (2) to a sample well of an LFA strip. In some embodiments, the gold nanoparticles linked to anti -FITC antibodies are lyophilized on the LFA strip in the region before the test and control lines. Addition of the ANINA solution mixed with LFA hybridization solution allows the anti -FITC antibodies to bind to the FITC-ssDNA amplicons. The capillary action of the paper moves the sample up the strip across the first test line. The gold nanoparticles (NP) bound by FITC-amplicons, which alsohave biotin probes attached to them, will bind to the test line (which contains biotin ligands, e.g., streptavidin). The gold NPs bound to the FITC-P2 or not bound to anything keep moving up the strip and bind to the control line. The gold NPs accumulation on the test and the control lines show up as pink bands. In the absence of a target DNA, only a control line shows up. The labels for primer and probe can be freely interchanged.

[0116] FIG. 51 shows the use of the LFA assay carried out with sample (target amplicons bound by in-reaction probe) detected at the Test Line for an analog readout. The initial target template (plasmid DNA) was diluted to show that detection is obtained even at lower target copies. The test line can be analyzed using image analysis software such as ImageJ, VisionWorks, etc. to conduct relative density analysis of the test lines (compared to negative controls) to convert to a digital readout.

[0117] FIG. 52 shows a 2% agarose gel and LFA detection of a 141 bp FP-RP amplicon made by recombinase polymerase amplification with and without nfo endonuclease IV with the reaction incubated at 39°C for 20 minute indicating that nfo (endonuclease IV) enzyme activity is not required for amplification or probe detection by LFA. Also, the gel lanes 3 and 6 and LFA strips 3 and 6 show that the inclusion of the in -reaction probe is necessary for the detection on LFA, but not for amplification of the 141 bp amplicon. Reaction was carried out using genomic DNA (Inf) isolated from White Spot Syndrome Virus (WSSV)-infected shrimp tissue using commercial DNA extraction kit.

[0118] FIG. 53A shows a 2% agarose gel indicating a 141 bp FP-RP amplicon usingtwo different in-reaction probes with the same target sequence but the probe sequence differing by one nucleotide. FAM-Pv3 -THF has one internal tetrahydro furan modification replacing a dTTP in the sequence of FAM-Pv3. The gel shows presence or absence of a single modification in the probe doesn’t affect amplification of the 141 bp amplicon. Reaction was carried out using genomic DNA (INF) isolated from WSSV-infected shrimp tissue using commercial DNA extraction kit. No template control (NTC) samples did not have any template DNA addition (however gel shows some template contamination).

[0119] FIG. 53B shows LFA detection of a 141 bp FP-RP amplicon from FIG. 53A with no cleaning up of samples. LFA was detected with probes with or without the internal THF residue, proving adding a THF internal residue plays no role in detection. LFA test line intensity with the FAM-Pv3 is higher than with the FAM-Pv3 -THF (vs respective NTC LFA strip test line) showing the sequence dependence of the probe’s detection. This also suggests that one base difference in target may be detectable by the in-reaction probe.

[0120] FIG. 53C shows LFA detection of a 141 bp FP-RP amplicon from FIG. 53A withproteinase K treatment and phenol chloroform extraction to clean up the reaction samples. The LFA with the infected samples shows specific detection (compared to NTC strips) showing that protein or enzyme interactions are not needed to stabilize the probe’s interaction with the target amplicon or for the detection step.

[0121] FIG. 54 shows a diagram of a method of target amplification using one long-to-medium length primer (Pl), one short primer (P2), and one annexing probe (AP). The annexing probe aids short primers in amplification and is also used for detection. Primer 1 (Pl) is long enough (>=20 bases) to be substrate for recombinase enzyme (UvsX), so it can invade the dsDNA sequence and anneal to target region, and strand displacing polymerase (Bsu) can extend its 3 ’ end. Pl can be labelled with biotin on 5’ end. The AP is long enough (>=20 bases) to be a UvsX substrate and can invade the target dsDNA sequence. AP has a 3 ’ modification such as an inverted nucleotide, C3 - spacer, etc. which blocks 3’ extension by polymerase. It can also have a 6-FAM orFITC or similar label on 5’ end. Primer 2 (P2) is too short (<= 15 b) to be an efficient UvsX substrate which means it cannot invade dsDNA by itself. The complementary sequence of P2 is adjacent to the complementary sequence of AP. The UvsX-mediated annealing of AP can open up the 5’ end the target region enough to allow the 3 ’ end of P2 to bind to the target sequence. This is termed as annexing of the short primer, and this process is termed as annexation. Once P2 is annexed, Bsu can bind to its 3 ’ end and start extending it, slowly displacing AP which can now invade another target dsDNA. Pl and annexed P2 can exponentially amplify biotinylated P1 -P2 dsDNA.

[0122] FIG. 55 shows a diagram of the method described in FIG. 54 using one labelled medium- to-long primer, one short primer, and one labelled annexing probe and a diagram of LFA to detect DNA amplicons.

[0123] FIG. 56 shows images of Gel 1, Gel 2 and LFA detection, and Gel 3 results using a method of amplification that uses an annexing probe. Reaction was carried out at 25°C 30 -minute incubation using genomic DNA (INF) isolated from WSSV-infected shrimp tissue using commercial DNA extraction kit. No template control (NTC) samples did not have any template DNA addition. The methods use the 1 short primer + 1 medium -long primer + Annexing probe Approach as described in FIG. 54, Gel 1 showing that amplification cannot occur without the annexing probe and Gel 2 and LFA image shows that a single Annexing probe can help annex and amplify short primers, and lead to LFA detection and increase specificity. Gel 3 shows that adding a 5’CCCCC extension on FAM-Pv3 doesn’t aid annexation process.

[0124] FIG. 57 shows a diagram of an ANINA method of target amplification using 2 long / medium / or short length primers + 1 shortprimer + 1 annexing probe. Primer 1 (Pl) is long enough (>20 bases) to be substrate for recombinase enzyme (UvsX), so it can invade the dsDNAsequence and anneal to target region, and strand displacingpolymerase (Bsu) can extendits 3 ’ end. Primer 2 (P2) may be long enough (>20 bases) to be substrate of recombinase by itself or it can be of medium sequence length (15-20 bases) where it is not an efficient recombinase substrate or it can be a short sequence (<=15 bases) where it is not at all a recombinase substrate. P2 can be labelled with biotin on 5’ end. Pl binds upstream of 5’ of P2. The Annexing Probe (AP) is long enough (>=20 bases) to be efficient UvsX substrate and can invade the target dsDNA sequence. AP has a 3 ’ modification such as an inverted nucleotide, C3 -spacer, etc. which blocks 3 ’ extension by polymerase. It can also have a 6 -FAM or FITC or similar label on 5’ end. Primer 3 (P3) is too short (<= 15 b) to be an efficient UvsX substrate. The UvsX -mediated annealing of AP can open up the 5’ end of the target region enough to allow the 3 ’ end of P3 to annex to the target sequence. Once P3 is annexed, Bsu can bind to its 3 ’ end and start extending it, slowly displacing AP which can now invade another target dsDNA. Pl and annexed P3 can exponentially amplify Pl -P3 dsDNA and displace biotinylated P2-ssDNA. This increases the template for P2- and P3 exponential amplification thereby making exponential amount of biotinylated P2-P3 dsDNA.

[0125] FIG. 58 shows a diagram of a method of target detection using 2 long / medium / or short length primers + 1 short primer + 1 annexing probe reaction products and detection using LFA to both Biotinylated P2-ssDNA and Biotinylated P2-P3 dsDNA can be detected on LFA thereby increasing specificity and efficiency.

[0126] FIG. 59A shows a diagram of an annexing probe approach of target amplification using 2 primers or 3 primers to amplify target regions in VP28 gene sequence of WSSV genome and an annexing probe and expected products of 176 bp and 108 bp.

[0127] FIG. 59B shows agarose gel and LFA detection results from the method described in FIG. 59A. Reaction was carried out at 25°C 30-minute incubation using genomic DNA (INF) isolated from WSSV-infected shrimp tissue using commercial DNA extraction kit. No template control (NTC) samples did not have any template DNA addition. Addition of upstream primer Rp 100 (24nt) increases the amount oftemplate available forFp91 :Rp20-Btn. Pv3 bind sand detects both biotinylated dsDNA (108 bp made by FP91 and Btn-RP20) as well as biotinylated ssDNA (108 b) displaced by the RPs.

[0128] FIG. 59C shows agarose gel and LFA detection results from the method described in FIG. 59Ausing reaction products for LFA detection directly (non-cleanup) vs cleaningup reaction products using phenol chloroform extraction before conducting LFA (cleanup). Both LFA approaches show sensitive detection of target amplicons (INF vs NTC). Reaction was carried out at 25°C 30 minute incubation using genomic DNA (INF) isolated from WSSV-infected shrimp tissue using commercial DNA extraction kit. No template control (NTC) samples did not have anytemplate DNA addition.

[0129] FIG. 60A shows a PAGE gel with VP28 target amplicon products using an ANINA method.

[0130] FIG. 60B shows a graph of quantified reaction products from the gel shown in FIG. 60A listing total density of 108 bp amplicon product to detect target sequence in the VP28 gene according to the illustrated products in FIG. 59A.

[0131] FIG. 60C shows a graph of quantified reaction products from the gel shown in FIG. 60A listing mean density of 108 bp amplicon product to detect target sequence in the VP28 gene according to the illustrated products in FIG. 59A.

[0132] FIG. 60D shows LFA results of the amplified products shown in FIG. 60A.

[0133] FIG. 60E shows a graph of quantified reaction products detected by LFA shown in FIG. 60D listing total density of 108 bp amplicon product to detect target sequence in the VP28 gene according to the illustrated products in FIG. 59A.

[0134] FIG. 60F shows a graph of quantified reaction products detected by LFA shown in FIG. 60D listing mean density of 108 bp amplicon product to detect target sequence in the VP28 gene according to the illustrated products in FIG. 59A.

[0135] FIG. 61A shows a diagram of an annexing probe method of target amplification using 2 or more primers either with or without an annexing probe. Expected reaction products using various primer combinations are 190 bp, 179 bp, 123 bp, and 97 bp . Target region is within the VP 19 gene of WSSV genomic DNA.

[0136] FIG. 61B shows three PAGE gels of amplification products to contrast not using an annexing probe to using an annexing probe approach. The primerand probe locations are described in FIG. 61 A. Reaction was carried out at 25°C 30 minute incubation using genomic DNA (INF) isolated from WSSV -infected shrimp tissue using commercial DNA extraction kit. The correct amplicon lengths were verified by using a commercial qPCR kit to amplify the target sequences using the same primers and running on the gel as a comparable product (Lanes 5 -8 and Lanes 13- 16). As observed in the leftmost gel (Lanes 1 -4), long forward primer (FP98-30 nt) was able to amplify with longer length reverse primers (RP258-30nt, RP276-30nt, RP220-24nt) but wasn’t able to amplify target region using shorter reverse primer (RP194-20nt) using e.g., Seek Amplification reaction mix not having an annexing probe in reaction. As observed in the middle gel (Lanes 9-12), short forward primer (FP98- 15 nt) was not able to properly amplify with either longer length reverse primers (RP258-30nt, RP276-30nt, RP220-24nt) or shorter reverse primer (RP194-20nt) using e.g., Seek Amplification reaction mix not having an annexing probe in reaction. As observed in the rightmost gel (Lanes 17-20), addition of annexing probe (Probel49)in the reaction mix allows the short forward primer (FP98-15 nt) to amplify correct dsDNA amplicon lengths with all four reverse primers irrespective of primer length or amplicon length. Additionally, the amplicons made with annexing probe approach (Lanes 17-20) shows as specific amplification as observed in comparison with the qPCR amplicons in the middle gel (Lanes 13 - 16). Specificity and efficiency are both highly improved usingthe annexingprobe approach (Lanes 17-20) compared to not using an annexing probe approach (Lanes 1 -4, Lanes 9-12).

[0137] FIG. 61C shows two isolated images of PAGE gels of amplification products shown in FIG 76B. Lanes 1-4 are the same as Lanes 9-12 in Fig 76B, and Lanes 5-8 are same as Lanes 17- 20 in FIG 76B

[0138] FIG. 61D shows a graph of quantification of the results in FIG. 61C using VisionWorks software. The band area of the 4 amplicons (190 bp, 179 bp, 123 bp, 97 bp) was analyzed to find mean density. Since the samples without and with addition of AnnexingProbe were run on separate gels, the band density of each amplicon region of interest (ROI) was normalized usingthe 100 bp ladder band on the respective gels. The bar graph shows that that no addition of annexing probe gives very low to no amplicon band density, whereas addition of annexing probe remarkably increases amplicon band density.

[0139] FIG. 62A shows a PAGE gel with VP19 target amplicon products using an annexing probe method using 2 or 3 primers and an annexing probe. Reaction was carried out at 25°C 30 minute incubation using genomic DNA (INF) isolated from WSSV-infected shrimp tissue using commercial DNA extraction kit. No template control (NTC) samples did not have any template DNA addition. Annexingprobe approach with FAM-Probe 149 and FP98-15nt can specifically amplify different lengths of VP19 amplicons using different length RPs including using multiple reverse primers. The addition of reverse primers (RP2) binding 5’ upstream of the first reverse primer (RP1) increases template for the FP98 -15nt:RPl primer set and results in increased yield as observed on gel especially for 97 bp amplicon. The intensity of the 97 bp amplicon in Lanes 8, 10, and 11 was analyzed using VisionWorks software to find total and mean density by doing background correction using respective NTC regions in Lanes 1 and 2, and the resulting graphs are shown in FIG. 62B and FIG. 62C respectively.

[0140] FIG. 62B shows a graph of quantified reaction products from the gel shown in FIG. 62A listing total density of 97 bp ampliconproductto detect target sequencein the VP19 gene according to the illustrated products in FIG. 62A.

[0141] FIG. 62C shows a graph of quantified reaction products from the gel shown in FIG. 62A listing mean density of 97 bp amplicon product to detect target sequence in the VP 19 gene according to the illustrated products in FIG. 62A.

[0142] FIG. 62D shows LFA results showing detection of the products from the reactions in FIG. 62A. Annexing probe approach with FAM-Probe 149 andFP98-15nt can specifically detect WSSV-Inf ection of shrimp gDNA (vs NTC) on LFA using all the biotinylated RPs.

[0143] FIG. 62E shows four graphs of quantified reaction products from the LFA results shown in FIG. 62D. The test line intensity of each LFA strip was analyzed using VisionWorks software to find mean density by doing background correction using LFA 5 and 6 (which didn’t have a biotinylated primer or probe). The first graph shows that the highest relative detection (mean density of test line on LFA strip) was obtained with FAM-Probe 149, FP98-15nt andBtn-RP194- 20nt on addition of RP220-24nt (LFA Strip 8). NTC showed no detection on LFA (LFA strip 1 -4) despite using 3 primers + Probe showing the high specificity of the reaction. The other graphs showed that adding an additional primer RP2 (3 primers annexing probe approach) gave significantly higher detection to using only 2 primers with annexing probe.

[0144] FIG. 63 shows two PAGE gels of amplification productsusingan annexingprobemethod using 2 primers + an annexing probe to show the importance of annexing probe proximity to the short primer in amplification efficiency.

[0145] FIG. 64A shows images of an agarose gel following electrophoresis of an annexing probe, 3 primer ANINA amplification strategy to produce amplicons of a VP19 target sequence. FIG. 64A also shows an image of LFA testing on the reaction products.

[0146] FIG. 64B shows a graph of image intensity band analysis for the 97 bp amplicon from test reactions shown in FIG. 64A.

[0147] FIG. 65 shows a non -limiting example of a primer system comprising a first primer (Pl) having a sequence comprising a 5’ adapter region and a target binding region downstream of the adapter region and a method of use therewith. First, using recombinase activity, two primers are inserted into a homologous region of a genome (e.g., white spot syndrome virus (W SSV) genome). The forward primer comprises a 5 ’ adapter region that is non -complementary to the target strand. DNA polymerase extends the primers and fills in the adaptor region resulting in a double -stranded amplicon with an adapter on one end. Next, again, using recombinase activity, an annexing probe (that does notact as a polymerase substrate) is inserted into the amplified dsDNA from step 1. Half of the annexing probe binds to the adapter and half to the target gene sequence (e.g., WSSV gene sequence). The binding of the annexing oligonucleotide opens the dsDNA and allows a short primer (e.g., P2) to bind entirely to the adapter region. DNA polymerase extends the short primer, exponentially amplifying dsDNA containing adaptor region as well as displacing an ssDNA containing adaptor region and the annexing probe can bind to both amplicons. This process can be repeated several times and can be detected using appropriate labels on the primers and probe.

[0148] FIG. 66 shows a non-limiting example of a primer system comprising primer (Pl ) having a sequence that is a set of nucleotides 5 ’ to the target region, a second primer (P2) having a sequence that is a set of nucleotides (a) 5’ to the target region or (b) 5’ to the target region and including a portion of the target region, a third primer (P3) having a sequence that is a set of nucleotides complementary to (a) at least a portion of the target region, or (b) a portion of the 3 ’ end of the target region and one or more nucleotides downstream of the 3 ’ end of the target region; or (c) an area 3 ’ to the target region, an annexing oligonucleotide and a method of use therewith. The figure shows an instance where an additional in-reaction probe can be used instead of the annexing oligo being the in-reaction probe.

[0149] FIG. 67 shows a non-limiting example of a primer system comprising primer (Pl) having a sequence that is a set of nucleotides 5 ’ to the target region, a second primer (P2) having a sequence that is a set of nucleotides (a) 5’ to the target region or (b) 5’ to the target region and including a portion of the target region, a third primer (P3) having a sequence that is a set of nucleotides complementary to (a) at least a portion of the target region, or (b) a portion of the 3 ’ end of the target region and one or more nucleotides downstream of the 3 ’ end of the target region; or (c) an area 3 ’ to the target region, an annexing oligonucleotide and a method of use therewith. The figure shows an instance where an additional primer can be used instead of the annexing oligo being the in-reaction probe. FIG. 67 shows methods of use in which the primers are labeled.

[0150] FIG. 68 shows a non-limiting example of a primer system comprising primer (Pl ) having a sequence that is a set of nucleotides 5’ to the target region, a second primer (P2) having a sequence that is a set of nucleotides (a) 5’ to the target region or (b) 5’ to the target region and including a portion of the target region, a third primer (P3) having a sequence that is a set of nucleotides complementary to (a) at least a portion of the target region, or (b) a portion of the 3 ’ end of the target region and one or more nucleotides downstream of the 3 ’ end of the target region; or (c) an area 3’ to the target region, an annexing probe and a method of use therewith. FIG. 68 shows methods of use in which P2 is connected to the annexing probe to increase efficiency and specificity. In some embodiments, the 5' end of P2 is linked to 5' end of or 3’ end of or internally in the annexing probe using either PEG linkers, inverted oligos, or locked-nucleic acids.

[0151] FIG. 69 shows a general schematic with operational steps of amplification methods / systems disclosed herein, such as Annexing Isothermal Nucleotide Amplification (ANINA).

[0152] FIG. 70A-FIG. 70B shows native PAGE results from amplification of human viral or bacterial pathogenic DNA target sequence via e.g., ANINA, in the presence of human gNA. FIG. 70 A shows EBV amplicons visualized on a Native PAGE gel (upper panel) and confirmation oftarget sequence detection of the Epstein-Barr Virus (EBV) amplicons on mLFA strips (bottom panel). Lane 1. No template control (NTC); Lane 2. EBV gNA (Positive Control); Lane 3. gNA from non-infected human saliva (Host gNA only); Lane 4. gNA from EBV-spiked human saliva. FIG. 70B shows Neisseria gonorrhoea (N. gonorrhoea or Ng) amplicons visualized on a Native PAGE gel (upper panel) and confirmation of target sequence detection of the N. gonorrhoea amplicons on mLFA strips (bottom panel). Lane 1 . N.gonorrhoeae gNA (Positive Control); Lane 2. gNA from N.gonorrhoeae-spiked human swab samples 1 :1 dilution; Lane 3. 1 / 10 dilution; Lane 4. 1 / 100 dilution; Lane 5. NTC.

[0153] FIG. 71 shows native PAGE results from amplification of human viral pathogenic RNA sequence via e.g., ANINA in the presence of human gNA. Respiratory Syncytial Virus (RSV) RNA target sequence was amplified in presence of reverse transcriptase (RT) enzyme. Lane 1. RSV RNA (no RT control) Lane 2. Human gNA (+RT); Lane 3. Human gNA (no RT); Lane 4. RSV cDNA (Positive Control); Lane 5. RNA from RSV -spiked human saliva; Lane 6. RSV RNA (+RT).

[0154] FIG. 72A-FIG. 72B show that amplification (e.g., Seek amplification using ANINA methods) is able to amplify a single copy of viral genome at 25°C within 30 minutes to LFA detectable range at room temperature without compromising specificity of amplification. Single copy detection of WSSV genome shown in FIG. 72A. Single copy detection of EBV genome shown in FIG. 72B.

[0155] FIG. 73 shows graphs of real-time or quantitative amplification (e.g., q ANINA using Seek ANINA methods) for WSSV viral loads at 39°C. Leftmost graph shows amplification plot showing increase in fluorescence of target amplicons in real time. Middle graph shows detection times for amplification (e.g., qANINA using Seek ANINA methods) as compared to qPCR. Rightmost graph shows standardization curve for real-time amplification (e.g., Seek Amplification or qANINA) which can be used for quantitative detection of WSSV infection .

[0156] FIG. 74A - FIG. 74C shows images of amplified products (using for example ANINA) with varying primer length as visualized following gel electrophoresis. FIG. 74A shows results from amplification with varyingthe forward primer (FP1) length from 10, 12, 14, 20, 25, and 35 nucleotides and correspondingintensity of Amplicon 1 . FIG. 74B shows results from amplification (using for example ANINA) with varyingthe reverseprimer (RPl) length from 15, 18, 20, 24, 30, and 35 and correspondingintensity of Amplicon 2. FIG. 74C shows gel images comparison Seek Amplification (using for example ANINA), polymerase chain reaction (PCR) amplification and traditional RPA using the same pairs of forward primer and reverse primer.

[0157] FIG. 75A-FIG. 75B show multiplexed amplification (using for example Seek amplification) and the effects of the annexing probe (AP) on decreasing non -specific amplification.FIG. 75A shows that Seek Amplification specifically amplifies and detects two different gene regions by using 2 reverse primers with different labels. Native PAGE and mLFA results showing detected amplicons using 5 '-biotin-labelled RP1 (Lane 1), 5'- digoxigenin (DIG) labelled RP2 (Lane 2), and both 5’-biotin-RPl and 5’-DIG-RP2 (Lane 3). FIG. 75B Lanes 1 -6 shows nonspecific amplification in reactions lacking AP (left gel image) with multiple amplified and smeared bands followinggel electrophoresis using forward primer 1 (FP1) lengths of 10, 12, 15, 20, 25, and 35 nucleotides) in the reaction (using for example ANINA). In contrast, FIG. 75B Lanes 7-12 shows highly specific amplification in reactionswith annexingprobe (AP) present (right gel image) using the same primers.

[0158] FIG. 76 shows images of native PAGE analy sis showing amplification (using f or example Seek amplification) to discriminate single nucleotide differences in target sequences with two different FPs, each with a single nucleotide mutation on the 3 ’ end. (Top gel image) FP-15nt with and without 3 ’ mismatches (C-> G, C-> T, C-> A) with RPs of 2 different lengths: RP1 -24nt and RP2-3 Ont. (Botton gel image) FP-15 nt with and without a 3 ’ mismatch (C-> G) tested against RP1 - 24nt with and without 3’ mismatches (G->A, G->C, G->T).

[0159] FIG. 77 shows a non-limiting example of a WSSV field kit contents (e.g., SeekIT Field kit).

[0160] FIG. 78 shows a non-limiting example of WSSV field kit process (e.g., SeekIT Field kit).

[0161] FIG. 79 shows a non-limiting example of an extraction protocol for field kit process (e.g, SeekIT Field kit).

[0162] FIG. 80 shows a non-limiting example of an amplification and detection protocol for WSSV field kit process (e.g., SeekIT Field kit).

[0163] FIG. 81 shows WSSV amplicons from WSSV field kit process (e.g., SeekIT Field kit) visualized on mLFA strips.

[0164] FIG. 82 shows graphs comparingthe results of WSSV field kitprocess (e.g., SeekITField kit) and a AAHL lab process for detection of WSSV.

[0165] FIG. 83A - FIG. 83C shows the results of WSSV field kit process (e.g., SeekIT Field kit) where amplification reaction buffer was used as elution buffer for extracting nucleic acid from WSSV-various quantities of infected or non-infected shrimp tissue sample using an extraction device disclosed herein (e.g., Seeklt extraction device), and the eluate was used to rehydrate lyophilized amplification pellets (e.g. Seeklt Amplification), followed by detection of WSSV amplicons on mLFA strips. In FIG. 83A, lyophilized amplification pellets are seen. In FIG. 83B, reaction products seen on a Native PAGE gel following electrophoresis are seen, showing WSSV amplicons only in samples from WSSV-infected shrimp. In FIG. 83C, a graph analysis of mLFAtest line intensity per infected shrimp sample as a percentage of that of non -infected shrimp sample is plotted showing specific detection with a large variety of sample quantity or type .

[0166] FIG. 84 shows a graph of quantified reaction products detected by LFA of EB V using an embodiment of a device disclosed herein (e.g., Seeklt sample-to-answer device).

[0167] FIG. 85 shows a non-limiting example of a sample collection and an extraction kit contents for a field kit disclosed herein (e.g., Seeklt Field Diagnostic kit, Seek Extraction kit).

[0168] FIG. 86 shows a non-limiting example of a lysis protocol for various samples for a field kit disclosed herein (e.g., Seeklt Field Diagnostic kit, Seek Extraction kit) as shown in FIG. 85.

[0169] FIG. 87 shows a non-limiting example of an extraction and elution protocol for a field kit disclosed herein (e.g., Seeklt Field Diagnostic kit, Seek Extraction kit) as shown in FIG. 85.

[0170] FIG. 88 shows a non-limiting example of an amplification and detection protocol for a field kit disclosed herein (e.g., Seeklt Field Diagnostic kit, Seek Amplification kit).

[0171] FIG. 89 shows a non-limiting example of using a sample-to-answer device described herein (e.g., Seeklt sample-to-answer device) to perform extraction, and amplification of a target nucleic acid sequence.

[0172] FIG. 90 shows a non-limiting example of using a sample-to-answer device described herein (e.g., Seeklt sample-to-answer device) to perform detection ofatargetnucleic acid sequence using the mobile application to read the results after the steps shown in FIG. 89.

[0173] FIG. 91 shows an exemplary test results and interpretation guide for a field kit disclosed herein (e.g., Seeklt Field Diagnostic kit).

[0174] FIG. 92 shows a non-limiting embodiment of an enclosed device (e.g., Seeklt Device) that has all components of a Molecular Diagnostics system from sample input to test result output (Nucleic Acid extraction, amplification and detection) that can be integrated with a mobile app for relaying of test results.

[0175] FIG. 93 shows a non-limiting embodiment of a device (e.g., Seeklt Device: Sample to Answer Molecular Diagnostics system).

[0176] FIG. 94 shows a non-limiting embodiment of a multiplex device (e.g., Seeklt Multiplex Device: Sample to Answer for up to 10 nucleic acid test targets, as described herein used for the systems, methods, and / or kits disclosed herein; right panel).

[0177] FIG. 95A shows a graphical depiction of the 5' end binding location of the primer (FP2) relative to the 5' end binding location of the annexing probe (AP).

[0178] FIG. 95B shows the effect of the distance between the 5 ' end of the primer (FP2) where it binds to a strand of the target nucleic acid sequence and the 5 ' end of the annexing probe (AP) where it binds to the strand of the target nucleic acid sequence.

[0179] FIG. 96A shows a Native PAGE gel image of the effect on amplification as a result of decreasing annexing probe length and decreasing primer (e.g., reverse primer) length.

[0180] FIG. 96B shows graphical analysis from mLFA test band results of the effect on amplification as a result of decreasing annexing probe length and decreasing primer (e.g., reverse primer) length.

[0181] FIG. 97A shows a Native PAGE gel image of the effect on amplification as a result of decreasing annexing probe length and decreasing primer (e.g., forward primer) length.

[0182] FIG. 97B shows graphical analysis from mLFA test band results of the effect on amplification as a result of decreasing annexing probe length and decreasing primer (e.g., forward primer) length.

[0183] FIG. 98 depicts Native PAGE gel images showing that the disclosed amplification method can differentiate one or more 3 ’ nucleotide mismatches in primer (e.g., FP) of lengths <= 15 nt. Shorter primers can amplify with single nucleotide specificity. This method can be used to differentiate template variations or mutations and even single nucleotide polymorphisms (SNPs).

[0184] FIG. 99 shows a non-limiting graphical image ofRNA Amplification using amplification methods disclosed herein.

[0185] FIG. 100A shows eluate concentrations (ng / pL) of 1 mL samples of human saliva spiked with RSV infected cells.

[0186] FIG. 100B shows the average lysate and eluate qualities (260 / 280) of 1 mL samples of human saliva spiked with RSV infected cells.

[0187] FIG. 100C shows the average CQ for a target sequence of the RSV genome that was amplified from processed samples of 1 mL of human saliva spiked with RSV infected cells.

[0188] FIG. 101A shows amplified nucleic acids of a human saliva sample spike with RSV infected cells run on a 2% agarose gel.

[0189] FIG. 101B shows amplified nucleic acids of a human saliva sample spike with RSV infected cells run on a 2% agarose gel.

[0190] FIG. 102A shows eluate concentrations (ng / pL) of 1 mL samples of human saliva spiked with RSV infected cells that were lysed with a lysis buffer containing SDS.

[0191] FIG. 102B shows the average lysate and eluate qualities (260 / 280) of 1 mL samples of human saliva spiked with RSV infected cells that were lysed with a lysis buffer containing SDS.

[0192] FIG. 102C shows the average CQ for a target sequence of the RSV genome that was amplified from processed samples of 1 mL of human saliva spiked with RSV infected cells that were lysed with a lysis buffer containing SDS.

[0193] FIG. 103A shows eluate concentrations (ng / pL) of 1 mL samples of human saliva spikedwith RSV infected cells that were lysed with a lysis buffer that was free of SDS.

[0194] FIG. 103B shows the average lysate and eluate qualities (260 / 280) of 1 mL samples of human saliva spiked with RSV infected cells that were lysed with a lysis buffer that was free of SDS.

[0195] FIG. 103C shows the average CQ for a target sequence of the RSV genome that was amplified from processed samples of 1 mL of human saliva spiked with RSV infected cells that were lysed with a lysis buffer free of SDS.

[0196] FIG. 104A shows eluate concentrations (ng / pL) of 1 mL samples of human saliva spiked with RSV infected cells that were lysed with an alkaline lysis buffer.

[0197] FIG. 104B shows the average lysate and eluate qualities (260 / 280) of 1 mL samples of human saliva spiked with RSV infected cells that were lysed with an alkaline lysis buffer.

[0198] FIG. 104C shows the average CQ for a target sequence of the RSV genome that was amplified from processed samples of 1 mL of human saliva spiked with RSV infected cells that were lysed with an alkaline lysis buffer.

[0199] FIG. 105A shows eluate concentrations (ng / pL) of 1 mL samples of human saliva spiked with RSV infected cells that were lysed with a lysis buffer that had its components doubled.

[0200] FIG. 105B shows the average lysate and eluate qualities (260 / 280) of 1 mL samples of human saliva spiked with RSV infected cells that were lysed with a lysis buffer that had its components doubled.

[0201] FIG. 105C shows the average CQ for a target sequence of the RSV genome that was amplified from processed samples of 1 mL of human saliva spiked with RSV infected cells that were lysed with a lysis buffer that had its components doubled.

[0202] FIG. 105D shows amplified nucleic acids of a human saliva sample spike with RSV infected cells that was lysed by a lysis buffer that had its components doubled run on a 2% agarose gel.

[0203] FIG. 106A shows eluate RNA concentrations (ng / pL) of 1 mL samples of human saliva spiked with RSV infected cells.

[0204] FIG. 106B shows the average RNA quality (260 / 280) of 1 mL samples of human saliva spiked with RSV infected cells.

[0205] FIG. 106C shows the average CQ for a target RNA sequence of the RSV genome that was amplified from processed samples of 1 mL of human saliva spiked with RSV infected cells.

[0206] FIG. 106D shows amplified RNA of a human saliva sample spike with RSV infected cells run on a 2% agarose gel.

[0207] FIG. 107A shows eluate RNA concentrations (ng / pL) of 1 mL samples of human salivaspiked with RSV infected cells lysed with buffers comprising various concentrations of guanidine HC1.

[0208] FIG. 107B shows the average RNA quality (260 / 280) of 1 mL samples of human saliva spiked with RSV infected cells lysed with buffers comprising various concentrations of guanidine HC1.

[0209] FIG. 107C shows the average CQ for a target RNA sequence of the RSV genome that was amplified from processed samples of 1 mL of human saliva spiked with RSV infected cells lysed with buffers comprising various concentrations of guanidine HC1.

[0210] FIG. 107D shows amplified RNA of a human saliva sample spike with RSV infected cells that was lysed with buffers comprising various concentrations of guanidine HC1 run on a 2% agarose gel.

[0211] FIG. 108A shows eluate RNA concentrations (ng / pL) of 1 mL samples of human saliva spiked with RSV infected cells lysed with buffers comprising various concentrations of Tween 20.

[0212] FIG. 108B shows the average RNA quality (260 / 280) of 1 mL samples of human saliva spiked with RSV infected cells lysed with buffers comprising various concentrations of Tween 20.

[0213] FIG. 108C shows the average CQ for a target RNA sequence of the RSV genome that was amplified from processed samples of 1 mL of human saliva spiked with RSV infected cells lysed with buffers comprising various concentrations of Tween 20.

[0214] FIG. 108D shows amplified RNA of a human saliva sample spike with RSV infected cells that was lysed with buffers comprising various concentrations of Tween 20 run on a 2% agarose gel.

[0215] FIG. 109A shows eluate RNA concentrations (ng / pL) of 1 mL samples of human saliva spiked with RSV infected cells lysed with buffers comprising various concentrations of Triton X.

[0216] FIG. 109B shows the average RNA quality (260 / 280) of 1 mL samples of human saliva spiked with RSV infected cells lysed with buffers comprising various concentrations of Triton X.

[0217] FIG. 109C shows the average CQ for a target RNA sequence of the RSV genome that was amplified from processed samples of 1 mL of human saliva spiked with RSV infected cells lysed with buffers comprising various concentrations of Triton X.

[0218] FIG. 109D shows amplified RNA of a human saliva sample spike with RSV infected cells that was lysed with buffers comprising various concentrations of Triton X run on a 2% agarose gel.

[0219] FIG. 110A shows eluate RNA concentrations (ng / pL) of 1 mL samples of human saliva spiked with RSV infected cells lysed with buffers comprising various concentrations of NaCl.

[0220] FIG. HOB shows the average RNA quality (260 / 280) of 1 mL samples of human salivaspiked with RSV infected cells lysed with buffers comprising various concentrations of NaCl.

[0221] FIG. HOC shows the average CQ for a target RNA sequence of the RSV genome that was amplified from processed samples of 1 mL of human saliva spiked with RSV infected cells lysed with buffers comprising various concentrations of NaCl.

[0222] FIG. HOD shows amplified RNA of a human saliva sample spike with RSV infected cells that was lysed with buffers comprising various concentrations of NaCl run on a 2% agarose gel.

[0223] FIG. 111 A shows eluate RNA concentrations (n9g / pL) of 1 mL samples of human saliva spiked with RSV infected cells lysed with buffers comprising various concentrations of guanidine thiocyanate.

[0224] FIG. 11 IB shows the average RNA quality (260 / 280) of 1 mL samples of human saliva spiked with RSV infected cells lysed with buffers comprising various concentrations of guanidine thiocyanate.

[0225] FIG. 1HC shows the average CQ for a target RNA sequence of the RSV genome that was amplified from processed samples of 1 mL of human saliva spiked with RSV infected cells lysed with buffers comprising various concentrations of guanidine thiocyanate.

[0226] FIG. 112A shows eluate RNA concentrations (n9g / pL) of 1 mL samples of human saliva spiked with RSV infected cells lysed with buffers comprising various concentrations of guanidine thiocyanate.

[0227] FIG. 112B shows the average RNA quality (260 / 280) of 1 mL samples of human saliva spiked with RSV infected cells lysed with buffers comprising various concentrations of guanidine thiocyanate.

[0228] FIG. 112C shows the average CQ for a target RNA sequence of the RSV genome that was amplified from processed samples of 1 mL of human saliva spiked with RSV infected cells lysed with buffers comprising various concentrations of guanidine thiocyanate.

[0229] FIG. 113A shows eluate RNA concentrations (n9g / pL) of 1 mL samples of human saliva spiked with RSV infected cells lysed with buffers comprising various concentrations of DTT.

[0230] FIG. 113B shows the average RNA quality (260 / 280) of 1 mL samples of human saliva spiked with RSV infected cells lysed with buffers comprising various concentrations of DTT.

[0231] FIG. 113C shows the average CQ for a target RNA sequence of the RSV genome that was amplified from processed samples of 1 mL of human saliva spiked with RSV infected cells lysed with buffers comprising various concentrations of DTT.

[0232] FIG. 114A shows eluate RNA concentrations (9g / pL) of 1 mL samples of human saliva spiked with RSV infected cells lysed with various buffers.

[0233] FIG. 114B shows the average RNA quality (260 / 280) of 1 mL samples of human salivaspiked with RSV infected cells lysed with various buffers.

[0234] FIG. 114C shows the average CQ for a target RNA sequence of the RSV genome that was amplified from processed samples of 1 mL of human saliva spiked with RSV infected cells lysed with various buffers.DETAILED DESCRIPTIONOverview

[0235] The present disclosure provides systems and methods for extraction, isolation, amplification, and / or detection of a target nucleic acid sequence. The present disclosure provides simplified and streamlined sample processing, such as extraction, isolation, amplification, and detection of a target nucleic acid sequence. The methods, devices, systems, and kits disclosed herein can be useful as molecular diagnostic technologies configured for deployment in field or true point-of-care (PoC) scenarios, instrument-free scenarios, use by anyone, deployed anywhere, and / or easily configured for any disease target.

[0236] The present disclosure also provides a simplified and streamlined sample processing methods, devices, systems, and / or kits capable of extracting nucleic acids, such as genomic DNA (gDNA), in high concentration while minimizing protein interference and other contaminants that could disrupt amplification and detection processes. The sample processing can also incorporate isothermal (e.g., ambient temperature) amplification and detection of target nucleic acids. This disclosed sample processing can refer to Seek Extraction or Seeklt Extraction, among other extraction methods, systems, devices and / or kits disclosed herein. Similarly, Seek Extraction, Seeklt Extraction, and similar language can refer to any of the extraction methods, devices, systems, and / or kits disclosed herein.

[0237] This disclosure further provides details of an ambient temperature nucleic acid amplification platform that can include a detection probe in reaction (e.g., in-reaction detection probe). This isothermal amplification platform described in this disclosure can refer to Seek Amplification or Seeklt Amplification, among other amplification methods, systems, devices and / or kits disclosed herein. Similarly, Seek Amplification, Seeklt Amplification, and similar language can refer to any of the extraction methods, devices, systems, and / or kits disclosed herein. In some embodiments in the present disclosure, the in -reaction detection probe also decreases nonspecific amplification by increasing specific primer annealing or annexation, thereby becoming an annexing probe (e.g., a labelled annexing oligonucleotide). In such cases, the isothermal amplification system described in the disclosure can refer to Annexing Isothermal Nucleotide Amplification (ANINA). In some embodiments, methods and systems for amplification to furtherincrease specificity and sensitivity by using an annexing probe approach can refer to Annexing Isothermal Nucleotide Amplification (ANINA)

[0238] This disclosure provides methods, devices, systems, and / or kits for detection. The present disclosure also provides an innovative sample -to-answer molecular detection platform that includesDNA extraction, amplification and detection platform which overcomes the limitations of existing molecular Diagnostics: equipment requirements, reliance on trained technicians, temperature controls, complex experiment design, incompatibility with developing technology, etc. The sample-to-answer system described herein, can refer to Seeklt system or Seeklt molecular diagnostics platform, among other extraction / amplification / detection methods, systems, devices and / or kits disclosed herein. Similarly, Seeklt system or Seeklt molecular diagnostics platform, and similar language can referto any of the methods, devices, systems, and / or kits disclosed herein. The sample-to-answer systems disclosed herein incorporate a sample processing system, as described in this disclosure (e.g., Seeklt Extraction) with isothermal (e.g., ambient temperature) amplification described in this disclosure (e.g., Seeklt Amplification), and incorporate detection of target nucleic acids as disclosed herein. This system is capable of working across a variety of sample types and can be easily adapted to detect or screen a wide variety of diseases or conditions. Its versatility make it particularly suited for Point of Care, or non -lab oratory applications, and readily deployable to wherever the user is located.

[0239] The present disclosure features devices, systems, compositions, and methods for processing a nucleic acid molecule. A nucleic acid molecule can be isolated, amplified, and / or detected. Methods as described herein may comprise isolating single -stranded and / or doublestranded nucleic acid molecules, such as the exemplary devices, systems, compositions, and methods depicted in the figures disclosed herein. Methods as described herein may comprise amplifying single-stranded and / or double-stranded amplicons comprising a target sequence, such as the exemplary devices, systems, compositions, and methods depicted in the figures disclosed herein. Methods as described herein may comprise detecting single -stranded and / or doublestranded amplicons comprising a target sequence, such as the exemplary devices, systems, compositions, and methods depicted in the figures disclosed herein. In aspects described herein, are composition, primer systems, and methods useful for isothermal (e.g., ambient temperature) nucleotide amplification.

[0240] In some embodiments, the present disclosure provides systems for amplifying a target nucleic acid sequence at an ambient temperature. The target nucleic acid sequence can be isolated and processed from a biological sample. In some embodiments, the system comprises a first oligonucleotide (Pl). In some embodiments, the Pl binds to a strand of the target nucleic acidsequence. In some embodiments, the system comprises a second oligonucleotide (P2). In some embodiments, the P2 binds to another strand of the target nucleic acid sequence. In some embodiments, the system comprises a third oligonucleotide. In some embodiments, the third oligonucleotide binds to the target nucleic acid sequence. In some embodiments, the third oligonucleotide comprises a 3' end blocking feature that blocks extension of the third oligonucleotide by a polymerase. In some embodiments, the third oligonucleotide does not facilitate a localizing and a binding of Pl or P2 to the target nucleic acid sequence . In some embodiments, the system comprises a polymerase. In some embodiments, the system comprises a DNA processing enzyme to the target nucleic acid sequence. In some embodiments, the system comprises nucleotides in an amount sufficientto support amplification of the target region. In some embodiments, the system of amplification is conducted in an absence of a denaturing of the target nucleic acid. In some embodiments, the Pl localizes and binds to the strand of the target nucleic acid sequence independently of the third oligonucleotide. In some embodiments, the P2 localizes and binds to the other strand of the target nucleic acid sequence independently of the third oligonucleotide. In some embodiments, a 5’ end of a third primer (P3) binds to the strand of the target nucleic acid sequence. In some embodiments, the 5’ end of the third oligonucleotide and a 5' end of the Pl bind to the strand of the target nucleic acid sequence at a distance from about 3 nucleotides to about 25 nucleotides apart.

[0241] In some embodiments, the present disclosure provides methods for amplifying a target nucleic acid sequence at an ambient temperature. The target nucleic acid sequence can be isolated and processed from a biological sample. In some embodiments, the method comprises a first oligonucleotide (Pl). In some embodiments, the Pl binds to a strand of the target nucleic acid sequence. In some embodiments, the method comprises a second oligonucleotide (P2). In some embodiments, the P2 binds to another strand of the target nucleic acid sequence. In some embodiments, the method comprises a third oligonucleotide. In some embodiments, the third oligonucleotide binds to the target nucleic acid sequence. In some embodiments, the third oligonucleotide comprises a 3 ' end blocking feature that blocks extension of the third oligonucleotide by a polymerase. In some embodiments, the third oligonucleotide does not facilitate a localizing and a binding of Pl or P2 to the target nucleic acid sequence. In some embodiments, the method comprises a polymerase. In some embodiments, the method comprises a DNA processing enzyme to the target nucleic acid sequence. In some embodiments, the method comprises nucleotides in an amount sufficientto support amplification of the target region. In some embodiments, the method of amplification is conducted in an absence of a denaturing of the target nucleic acid. In some embodiments, the Pl localizes and binds to the strand of the target nucleicacid sequence independently of the third oligonucleotide. In some embodiments, the P2 localizes and binds to the other strand of the target nucleic acid sequence independently of the third oligonucleotide. In some embodiments, a 5’ end of a third primer (P3) binds to the strand of the target nucleic acid sequence. In some embodiments, the 5’ end of the third oligonucleotide and a 5' end of the Pl bind to the strand of the target nucleic acid sequence at a distance from about 3 nucleotides to about 25 nucleotides apart.

[0242] The present disclosure provides systems, methods, devices, and kits for amplification, isolation, and / or detection of a biological sample. In some embodiments, the device comprises a system for isolating nucleic acid. In some embodiments, the device comprises a system for amplifying a target region of a nucleic acid molecule, such as any one of the systems described herein. In some embodiments, the device comprises a detection system for detecting the target nucleic acid. In some embodiments, the kit comprises an isolation / extraction system for isolating nucleic acid. In some embodiments, the kit comprises a system for amplifying a target region of a nucleic acid molecule, such as any one of the systems described herein. In some embodiments, the kit comprises a detection system for detecting the target nucleic acid.

[0243] Non-limiting examples of methods, systems, and / or devices for nucleic acid extraction, amplification and detection platforms disclosed herein, such as Seeklt platform, are depicted in FIG.21. FIG.21 shows an appropriate biological sample from a target source (e.g., as non -limiting examples: an animal, an insect, or a plant) that is collected for a diagnostic test. In some instances, the target source is a human. In embodiments in which the target source is a human subject, one or more biological samples are collected from the human subject in which the one or more biological samples are appropriate for a target diagnostic test. The sample collection system may comprise one or more flocked swabs, one or more sample vials, one or more pestles, one or more capillary tubes, one or more syringes with needles, one or more devices, or any combination thereof. The one or more flocked swabs may be used for swab samples such as buccal, throat, nasal, nasopharyngeal, cervical, or vaginal samples. The one or more sample vials may be used for collectingtissue or solid organ samples, such as spleen, skin; and body fluid samples such as saliva, urine, or whole blood. The one or more pestles may be used for solid tissue grinding. The one or more capillary tubes may be used for whole blood collection, for instance, ata point-of-care setting The one or more syringes with needles may be used for whole blood collection, for instance, at a point-of-care setting. The one or more devices may be used for blood collection, for instance, at a home setting or in a point-of-care setting. Following sample collection, the one or more biological samples may be processed as appropriate (e.g., tissue grinding) or may proceed without further processing. The one or more biological samples may be processed for nucleic acid extraction withapplication of an appropriate lysis buffer from a lysis buffer packet (a lysis buffer packet can comprise buffers as disclosed herein). The one or more biological samples plus lysis buffer may be transferred to a lysate vial. Nucleic acid (NA) extraction products from the one or more biological samples can be eluted to an eluate comprising the eluted NA sample which may then be transferred to one or more elution vials. Biological material remaining outside of the one or more elution vials may be transferred to a waste disposal bag. In field kit versions of the platform, NA extraction, NA amplification, and detection of amplified target nucleic acid sequence proceeds to achieve affordable true Point-of-care molecular diagnostics. In some embodiments, one Seek Extraction unit (such as extraction methods, systems, kits or devices disclosed herein)can be combined with one or more Seek Amplification units (such as amplification methods, systems, kits or devices disclosed herein) plus MLFA strip to detect one or more target DNA / RNA. In some embodiments, the Seek Extraction Unit connects to the lysate vial and the lysed solution of biological material is applied to the DNA / RNA binding membrane housed within the Seek Extraction Unit to bind nucleic acids from the biological sample . Following binding of the nucleic acids to the binding membrane, the lysate vial and remaininglysisbuffer is removed from the Seek Extraction Unit. In some embodiments, the Seek Extraction Unit connects to the Elute vial containing an appropriate elution buffer (e.g., a SeekIT Elution buffer described herein). The elution buffer is applied to the DNA / RNA bindingmembrane comprisingbound nucleic acids from the biological sample. Contact of the elution buffer to the DNA / RNA binding membrane results in elution of extracted nucleic acids bound to the DNA / RNA binding membrane into the elution buffer. The elution buffer comprising extracted nucleic acids from the biological sample is then moved to the Elute Vial that now comprises the eluate to be further processed and analyzed. In some embodiments, Seek Amplification reagents (e.g., a lyophilized Seek Amplification Pellet) is added to the Elute Vial comprising the eluate. In some embodiments, the eluate comprises a template for a target nucleic acid sequence to be amplified. In some embodiments, Seek Amplification reagents comprise an enzyme mix which is sufficient to initiate amplification of the target nucleic acid sequence at an ambient temperature. In some embodiments, following a period of time for sufficient amplification of the target nucleic acid sequence, a rehydrating transfer tube or similar device is used to apply an amount of solution comprising amplified target nucleic acid sequence to a sample pad of one or more molecular lateral flow assay (MLFA) test step s configured for specific detection of the amplified target nucleic acid sequence. In some embodiments, a detection buffer container, which contains a detection buffer solution, is applied to the one or more MLFA test strips to allow the flow of the nucleic acids within the solution comprising amplified target nucleic acid sequence through the oneor more MLFAtest strips. Presence of amplified targetnucleic acid sequence is indicated by the appearance of a visible test line (T) near a defined test line position that binds to dual -labeled amplicons of the amplified target nucleic acid sequence. In some embodiments, the one or more MLFA test strips contain a control line (C) that demonstrates test validity by the appearance of a visible control line near a defined control line position following application of the detection buffer solution to the sample pad and a flow of the detection buffer solution containing nucleic acids obtained from Seek amplification. In some embodiments, visible detection of both (i) a positive control line near the defined control line position and (ii) a positive test line near the defined test line position indicates the presence of amplified target nucleic sequence within the amplified sample. In some embodiments, detection of both (i) a positive control line near the defined control line position and (ii) a positive test line near the defined test line position indicates the presence of the a template for the target nucleic acid sequence within the NA sample extracted from the subject. In some embodiments, detection of (i) a positive control line near the defined control line position, butno detection of (ii) a positive test line near the defined test line position indicates absence of the a template for the target nucleic acid sequence within the NA sample extracted from the subject. In some embodiments, absence of detection of the template for the target nucleic acid sequence indicates that the biological sample does not comprise a template for the target nucleic acid sequence. In some embodiments, absence of template for the target nucleic acid sequencein the biological sample indicates thatthe subject do nothave presence of a pathogen for which the system is configured to detect at the time in which the biological sample was collected. In some embodiments, detection of target nucleic acid sequence allows for disease mapping. In some embodiments, detection of target nucleic acid sequence in allows for disease monitoring. In some embodiments, detection of target nucleic acid sequence in allows for disease transmission control. In some embodiments, detection of target nucleic acid sequence allows for confirmation of the presence of a pathogen selected for detection using a system described herein within the subject that was the source of the biological sample. In some embodiments, the system of sample collection, NA extraction, target sequence amplification and detection allows for affordable, true point-of-care molecular diagnostics. In some embodiments of SeekIT Device and SeekIT App fully -enclosed sample-to-result systems described herein, the biological sample plus lysis buffer may be transferred to a SeekIT Device by connecting the lysate vial to the SeekIT Device and transferring the lysate. In some embodiments, the lysate is moved to DNA / RNA extraction chamber comprising a binding membrane within the SeekIT Device by one of various means (e.g., by gravity). Following binding of the nucleic acids to the binding membrane, a NA sample extracted from the subject is eluted from the binding membrane via application of an elution buffer. In some embodiments, the eluantis passed through the DNA / RNAextraction chamberto one ormore amplification chambers. In some embodiments, the one ormore amplification chambers comprise one or more lyophilized Seek Amplification pellets comprising an enzyme mix which is sufficient to initiate amplification of the target nucleic acid sequence at an ambient temperature. In some embodiments, the eluate is transferred to the one or more amplification chambers via opening of an aliquoting valve. In some embodiments, there may be 1, 2, 3, 4, 5 or more amplification chambers. In some embodiments, following a period of time for sufficient amplification of the target nucleic acid sequence, the amplifiedNA sample products from the eluant is passed to one or more detection buffer chambers containing detection buffer. In some embodiments, following the passing of the amplified NA sample products to the one or more detection buffer chambers, the amplified NA sample is further passed to one or more MLFA test strips via opening of an LFA chamber valve. The detection buffer containing the amplified NA sample products flows through the one or more MLFA test strips housed within a detection chamber. In some embodiments, there may be 1, 2, 3, 4, 5 or more MLFA test strips within the detection chamber. Presence of amplified target nucleic acid sequence is indicated by the appearance of a visible test line (T) near a defined test line position that binds to dual -labeled amplicons of the amplified target nucleic acid sequence. In some embodiments, the MLFA test strips also contain a control line. In some embodiments, visible detection of both (i) a positive control line near the defined control line position and (ii) a positive test line near the defined test line position indicates the presence of amplified target nucleic sequence within the amplified sample. In some embodiments, detection of (i) a positive control line near the defined control line position, but no detection of (ii) a positive test line near the defined test line position indicates absence of the a template for the target nucleic acid sequence within the NA sample extracted from the subj ect. In some embodiments, the SeekIT device is configuredto be used with the SeekIT App in which a user may conduct all-in-one at-home (or in the field) molecular diagnostics and have test results available for distribution to a selected health care provider. This provides for timely diagnosis, and identification of correct treatment for the subject, both of which can aid in decisions to initiate appropriate health care for the subject.

[0244] The present invention also features a system or device comprising an isolation system (e.g., isolation chamber) having a binding component capable of binding nucleic acid disposed therein, an amplification system (e.g., amplification chamber), and a detection system (e.g., detection chamber, detection component, means of detection, a detection unit, etc.). The amplification system(e.g., amplification chamber) may be fluidly connectedto the isolation system (e.g., isolation chamber), e.g., via a channel, a plurality of channels, etc. The detection system (e.g, detection chamber, detection component) may be fluidly connected to the amplification system(e.g., amplification chamber), e.g., via a channel, a plurality of channels, etc.

[0245] For example, the present invention features a system or device comprising an isolation system (e.g., isolation chamber) having a binding component capable of binding nucleic acid disposed therein and a detection system (e.g., detection chamber, detection component, means of detection, a detection unit, etc.). In some embodiments, the detection system is fluidly connected to the isolation system (e.g., isolation chamber), e.g., via a channel, a plurality of channels, a tube, a plurality of tubes, other appropriate connection components, etc .

[0246] The present invention also features a system or device comprising an isolation system (e.g., isolation chamber) having a binding component capable of binding nucleic acid disposed therein, an amplification system (e.g., amplification chamber), and a detection system (e.g., detection chamber, detection component). The system may further comprise a detection buffer chamber fluidly connected to the amplification system (e.g., amplification chamber), e.g. via one or more channels. The detection buffer chamber may also be fluidly connected to the detection system (e.g., detection chamber, detection component), e.g. via one or more channels.

[0247] The systems and devices herein may further comprise at least one means for aliquoting a fluid, e.g., an aliquoting valve or the like. As an example, the aliquoting valve may separate the isolation chamber from either one or both of the amplification chamber or the detection buffer chamber.

[0248] In some embodiments, the amplification chamber may comprise one or more amplification reaction components, which may comprise enzymes and other materials to amplify the DNA / RNA target. The amplification reaction components may comprise oneor more enzymes, such as a DNA / RNA polymerase, a recombinase, a single-strand binding enzyme, etc. The amplification mix may also comprise oligonucleotides, such as primers and probes. In some embodiments, the oligonucleotides aid or are required for efficient amplification of the DNA / RNA target. The amplification mix may be preloaded in the amplification chamber or can be added by the user via the aliquoting chamber. The amplification mix may comprise a liquid component, lyophilized component, or a combination thereof. The elution buffer from the previous section can include an amplification reaction buffer, which can perform the dual role of eluting DNA / RNA from the binding membrane and then mixing with separate amplification components and starting an amplification reaction. In some embodiments, the amplification reaction buffer may comprise magnesium. In other embodiments, the amplification reaction buffer contains no magnesium. In some embodiments, the elution buffer comprises an amplification reaction buffer with or without magnesium, acetate, or other amplification reagents that cannot be lyophilized.

[0249] The amplification reaction buffer can also be added by a separate chamber, if needed,which can be kept separate from the amplification chamber until needed. In case the amplification mix is a lyophilized component, the amplification reaction buffer can also perform an additional function of a rehydration buffer, which can hydrate the lyophilized amplification components in the amplification chamber and start the amplification reaction.

[0250] The systems or devices herein may further comprise a coupler fluidly connected to the isolation system (e.g., isolation chamber). The coupler may comprise an openingto the isolation system (e.g., isolation chamber) and is configured to engage a means for preventing access to the isolation system (e.g., isolation chamber). For example, the coupler may be configured to engage a cap, a seal, a plug, or a vial so as to prevent access to the isolation system (e.g., an isolation chamber).

[0251] The systems and devices described herein may further comprise either one or both of: a sample chamber (e.g., a first chamber) or an elution chamber (e.g., a second chamber). Both chambers may be fluidly connected to the first end of the isolation chamber, e.g., via a channel (e.g., a first channel and a second channel). In some embodiments, both chambers may be fluidly connected to the first end of the isolation chamber via a single channel. In other embodiments, both chambers may be fluidly connected to the first end of the isolation chamber via two channels. In embodiments where the device comprises both a sample chamber and an elution chamber, the chambers are disposed on opposite sides of the first end of the isolation chamber. The sample chamber may further comprise a first coupler comprising an openingto the sample chamber and configured to engage a means for preventing access to the sample chamber through the first coupler, e.g., a cap, a seal, a plug, or a vial. Likewise, the elution chamber may further comprise a second coupler comprising an opening to the elution chamber and configured to engage at least a means for preventing access to the elution chamber through the second coupler, e.g., a cap, a seal, a plug, or a vial.

[0252] In some embodiments, the sample comprises a biological or an environmental sample and / or a lysis buffer. The sample may be added either directly or indirectly to the isolation chamber or the first chamber. In embodiments in which the sample is indirectly added to said chambers, a first vial may be used.

[0253] In some embodiments, the first vial can be made with a material, e.g., a flexible material, such that the first vial may be used as an actuator itself to transfer liquid into or out of the device. In such cases, there might not be an actuator inside the device itself, e.g., an internal actuator. The first vial can attach fluidly to the device either directly or by using an adaptor cap or unit. In some embodiments, the first vial is configured to house a lysis buffer before the addition of the biological sample. The first vial may or may not house the lysis buffer before the addition of a biologicalsample. The first vial can also be part of an acquisition system, e.g., a vial to collect a saliva or blood sample.

[0254] In some embodiments, the first vial may be used to lyse the sample (e.g., a biological or environmental sample) outside of the device by mechanical or chemical methods or some combination thereof. Then, the lysate may be transferred into the device by squeezing it (e.g., with a drip tube). In some embodiments, the vial can have a cap or adaptor unit to fluidly connect to the device. In some embodiments, the adaptor may further comprise a filter element to filter out specific components of the lysate from entering the device.

[0255] In some embodiments, after the binding step, the first vial may be used as a waste vial to get the lysate out of the device. In some embodiments, the waste vial can be a separate vial from the first vial. The vials can attach fluidly to the device either directly or by using an adaptor cap or unit.

[0256] In some embodiments, an additional second or third vial may be used and may contain an elution buffer (e.g., an elution vial). In some embodiments, the elution buffer may comprise an amplification reaction buffer, which can perform the dual role of eluting DNA / RNA from the binding membrane and then mixing with separate amplification components and starting an amplification reaction. The elution vial can attach fluidly with the device either directly or using a cap or adaptor. The elution vial may be made with a material, e.g., a flexible material, such that the elution vial may be used as an actuator itself to transfer liquid (e.g., the elution buffer) into or out of the device so it can elute the DNA / RNA from the binding component.

[0257] The devices mentioned above may further comprise one or more actuators inside or outside (e.g., an internal actuator or an external actuator), the device configured to move a fluid through each of the aforementioned chambers.

[0258] In some embodiments, the devices and systems described herein may comprise multiple aliquoting systems. Each aliquoting system may be fluidly connected to an amplification chamber and / or detection chamber (e.g., detection units). In other embodiments, the devices and systems described herein may comprise a single aliquoting system thatis attached to multiple amplification chambers and / or detection chambers (e.g., detection units).

[0259] In some embodiments, the detection system, e.g., means of detection, detection component, or detection unit, comprises a lateral flow assay strip, electrochemical sensor, a fluorometric sensor, colorimetric sensor, or the like. In some embodiments, the detection system or unit may be housed inside the detection system, or it can be added by the user separately. In some embodiments, the detection system may further comprise a detection assay buffer, which may be preloaded inside the detection buffer vial, or it can be added by the user separately beforestarting the detection step.

[0260] In some embodiments, the detection system may be housed in a detection chamber fluidly connected to the detection buffer chamber. The connection may contain a valve mechanism or a push or pull-tab mechanism, or the device may need to be rotated at a specific angle to allow the detection buffer to move from the detection buffer chamber to the detection chamber or detection system. This can also keep the detection system away from the assay buffer until the user needs it. In some embodiments, the detection chamber may also contain design elements to aid the flow of the assay buffer in a specific direction, or only through the detection system, and prevent the fluid from coming in contact with the detection system in manners that may affect its performance.

[0261] Although the disclosure herein describes specific embodiments and configurations of the systems and devices for isolating, amplifying, and / or detecting nucleic acid, one of ordinary skill in the art would recognize that changes or alternative configurations of the systems and devices can still teach an equivalent and effective system, and said changes and alternative configurations fall within the scope of the present invention.

[0262] Without wishing to limit the present invention to any theory or mechanism, it is believed that aspects of the present invention are advantageous. For example, the configuration described herein may provide rapid (e.g., less than 20 minutes), user-friendly, affordable, portable, enclosed, equipment-free (e.g., no centrifuge) devices and systems that are able to process multiple sample types and perform multiple test types. Further, configurations of the present invention can provide for physical separation of contaminants (e.g., lysis buffer debris). For example, the systems and devices may be configured to use combinations of filtration components to physically separate contaminants from the sample instead of reagents. Additionally, the use of open channels (e.g., tubes) between chamber and / or vials described herein advantageously allows for less force to be required to move liquid around the system or device. Furthermore, the present invention provides both positive and negative pressure within the systems and devices described herein to allow for fluid to flow in a continuous circuit. None of the presently known prior references or work has the unique inventive technical feature of the present invention.

[0263] The present disclosure provides a method of processing a nucleic acid from a biological sample comprising using a lysate buffer to prepare the biological sample into a lysate. In some embodiments, the method comprises providing the lysate to a binding unit configured to bind to the nucleic acid. In some embodiments, the method comprises amplifying the nucleic acid of the lysate without a thermocycler or incubator. In some embodiments, the method comprises amplifyingthe nucleic acid of the lysate using an annexingprobe. In some embodiments, the lysate comprises a first portion. In some embodiments, subsequent to (b) providing the lysate to thebinding unit configured to bind to the nucleic acid portion, the first portion of the lysate is discarded. In some embodiments, the method further comprises providing an elution buffer to the binding unit. In some embodiments, the method further comprises eluting the nucleic acid from the bindingunit. In some embodiments, (c) amplifyingthe nucleic acid of the lysate is performed using two or more nucleic acid primers. In some embodiments, the method further comprises the nucleic acid is a viral nucleic acid. In some embodiments, the nucleic acid is an RSV nucleic acid. In some embodiments, the nucleic acid is a White Spot Syndrome Virus (WSSV) nucleic acid. In some embodiments, the nucleic acid is a bacterial nucleic acid. In some embodiments, the nucleic acid is a Neisseria gonorrhoeae nucleic acid. In some embodiments, the method occurs in 5 minutes or less. In some embodiments, the method further comprises sequencing the nucleic acid. In some embodiments, the sequencing is nanopore sequencing. In some embodiments, the amplifying in (c) occurs at an ambient temperature of about 20°C to about 40°C. In some embodiments, the amplifying in (c) occurs in about 30 minutes or less.

[0264] The present disclosure provides a system of processing a nucleic acid from a biological sample. In some embodiments, the system comprises bindingunit configured to bind to the nucleic acid. In some embodiments, the system comprises an amplification unit configured to amplify the nucleic acid of the lysate. In some embodiments, the amplification unit does not comprise a thermocycler or incubator. In some embodiments, the amplifying is performed using an annexing probe. In some embodiments, the system further comprises an elution unit comprising an elution buffer. In some embodiments, the amplifying is performed using two or more primers.

[0265] The present disclosure provides a kit comprising (i) any one of the systems as disclosed herein and (ii) instructions for use. In some embodiments, the instructions designate a treatment of a subject in need thereof. In some embodiments, the instructions designate a treatment of a viral infection. In some embodiments, the instructions designate a treatment of RSV. In some embodiments, the instructions designate a treatment of White Spot Syndrome Virus (WSSV). In some embodiments, the instructions designate a treatment of a bacterial infection. In some embodiments, the instructions designate a treatment of a Neisseria gonorrhoeae infection.

[0266] Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skills in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.

[0267] In some aspects, described herein are methods for replicating and amplifying a target nucleic acid sequence, the methods of which produce double -stranded (ds) and / or single -stranded(ss) amplicons of a target region of nucleic acid. In some embodiments, the methods produce double-stranded (ds) amplicons of a target nucleic acid sequence. In some embodiments, the methods produce single-stranded (ss) amplicons of a target nucleic acid sequence. In some embodiments, the methods produce ss amplicons and ds amplicons of a target nucleic acid sequence. In some embodiments, ss amplicons are generated corresponding to at least a portion of the target nucleic acid sequence. In some embodiments, ds amplicons are generated corresponding to at least a portion of the target nucleic acid sequence. In some embodiments, the methodsproduce double-stranded (ds) and single-stranded (ss) amplicons of a target region of nucleic acid. In an aspect described herein, are methods for replicating and amplifying a target nucleic acid sequence, the methods comprising: a) introducing to a sample comprising a template to the target nucleic acid sequence wherein the template is double-stranded or can be made double-stranded: i) a set of primers comprising: a first oligonucleotide primer (Pl) comprising a sequence at least about 90% identical to a region on the plus strand of the target nucleic acid sequence, a second oligonucleotide primer (P2) comprising a sequence at least about 90% identical to a region on the minus strand of the target nucleic acid sequence, wherein Pl and P2 are present in molar excess relative to the target nucleic acid sequence, and ii) an in reaction probe (IrP) comprising: a nucleotide sequence at least about 90% identical to a region on the plus or minus strand of the target nucleic acid sequence, wherein the IrP nucleotide sequence is: flanked by both Pl sequence and P2 sequence or at least partially overlaps with either Pl sequence or P2 sequence, a blocking feature that blocks extension of a DNA polymerase; iii) an enzyme mix comprising: a DNA polymerase or functional fragment thereof, a second enzyme having a DNA processing function, wherein the DNA processing function facilitates Pl and P2 annealing to the target region, nucleotides in an amount sufficient to support amplification of the target nucleic acid sequence, and a crowding reagent; b) reacting the sample with the set of primers, the IrP, and the enzyme mix for a length of time sufficient to replicate and amplify the target nucleic acid sequence, wherein the reacting is maintained under isothermal conditions without previously denaturing the template, wherein amplified target nucleic acid sequence comprises: i) target single-stranded (ss) amplicons, ii) target double-stranded (ds) amplicons, or iii) target ss amplicons and target ds amplicons, and wherein the IrP detects the target ss amplicons and / or target ds amplicons. In some embodiments, the method comprises: c) detectingtarget ss amplicons, target ds amplicons, ortarget ss amplicons and target ds amplicons via the IrP. In an aspect described herein, are methods for replicating and amplifying a target nucleic acid sequence, the methods comprising: a) introducing to a sample comprising a template to the target nucleic acid sequence wherein the template is double -stranded or can be made double -stranded: i) a set of primers comprising: a first oligonucleotide primer (Pl)comprising a sequence at least about 90% identical to a region on the plus strand of the target nucleic acid sequence, a second oligonucleotide primer (P2) comprising a sequence at least about 90% identical to a region on the minus strand of the target nucleic acid sequence, a third oligonucleotide primer (P3) comprising a sequence at least about 90% identical to a region on the plus or minus strand of the target nucleic acid sequence ; and ii) an annexing oligonucleotide (AO) comprising: a nucleotide sequence at least about 90% identical to a region on the plus or minus strand of the target nucleic acid sequence, wherein the AO nucleotide sequence is: flanked by Pl sequence, P2 sequence, and P3 sequence or at least partially overlaps with Pl sequence, P2 sequence, orP3 sequence, a blocking feature that blocks extension of a DNA polymerase; iii) an enzyme mix comprising: a DNA polymerase or functional fragment thereof, a second enzyme having a DNA processing function, wherein the DNA processing function facilitates one or more of Pl , P2, P3 , and AO annealingto the target region, nucleotides in an amount sufficientto support amplification of the target nucleic acid sequence, and a crowding reagent; b) reacting the sample with the set of primers, the AO, and the enzyme mix for a length of time sufficientto replicate and amplify the target nucleic acid sequence, wherein the reacting is maintained under isothermal conditions without previously denaturing the template, wherein amplified target nucleic acid sequence comprises: i) target single-stranded (ss) amplicons, ii) two target double-stranded (ds) amplicons, or iii) target ss amplicons and two target ds amplicons, and wherein the AO improves amplification specificity and efficiency by annexing a target site location to allow improved annealing of either Pl, P2, or P3 within the template and / or within amplified target nucleic acid sequence. In some embodiments, the AO comprises a label allowing the labeled AO to function as an in-reaction probe (IrP). In some embodiments, the AO comprises a label allowing the labeled AO to function as an in-reaction probe (IrP). In some embodiments, the IrP can detect two target ds amplicons and the detection of the two target ds amplicons produces increased sensitivity of target region detection compared to detecting single target ds amplicons. In some embodiments, the second enzyme in the enzyme mix comprises a recombinase or functional fragment thereof, a helicase or functional fragment thereof, a topoisomerase or functional fragment thereof, or any combination thereof. In some embodiments, the enzyme mix comprises a single-strand binding protein such as T4 GP32. In some embodiments, the DNA polymerase of functional fragment thereof is a strand-displacing DNA polymerase. In some embodiments, the DNA polymerase comprises Bacillus subtilis DNA polymerase I (Bsu), Bst, or Klenow Fragment. In some embodiments, the DNA polymerase comprises Bst 3.0. In some embodiments, the DNA polymerase comprises Bacillus subtilis DNA polymerase I (Bsu). In some embodiments, the second enzyme having a DNA processing function comprises a bacterial or viral recombinaseprotein. In some embodiments, the second enzyme having a DNA processing function comprises UvsX DNA recombinase (UvsX). In some embodiments, the second enzyme having a DNA processing function comprises RAD51. In some embodiments, the second enzyme having a DNA processing function comprises HELB helicase, RTEL1 helicase, Pifl helicase, Topi topoisomerase, or topoisomerase Illa. In some embodiments, the crowding reagent comprises polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), Ficoll, Dextran, or any combination thereof. In some embodiments, the crowding reagent comprises PEG. In some embodiments, the blocking feature comprises an inverted nucleotide or a C3 -spacer at the 3 ’ end of the in -reaction probe. In some embodiments, the sequence of any primer in the primer set and the IrP or AO sequence do not overlap. In some embodiments, the sequence of any one primer in the primer set and the IrP or AO sequence overlap. In some embodiments, at least one primer in the primer set is less than or equal to 15 nucleotides in length. In some embodiments, at least one primer is a nucleotide length insufficient to produce amplicons of the target sequence without the presence of the IrP or AO. In some embodiments, the IrP is long enough (greater than or equal to 20 nts) to be an efficient substrate for the second enzyme having the DNA processing function and wherein the IrP becomes an annexingprobe (AP) capable of invadingthe target dsDNA amplicon sequence and helping anneal at least one of the primers in the primer set to target region when that primer is less than or equal to 15 nucleotides in length. In some embodiments, the IrP serves as an annexing oligo (AO). In some embodiments, the in -reaction probe comprises a label. In some embodiments, the label comprises a fluorophore, or a redox molecule, or biotin, or digoxigenin. In some embodiments, atleast one primer comprises a label either on the 5 ’end or internally in the sequence so as not to prevent extension by polymerase but leading to amplification of labelled ss and ds amplicons. In some embodiments, the IrP bound to target labelled ds amplicons is detected via a lateral flow assay (LFA). In some embodiments, the in-reaction probe bound to target labelled ss amplicons is detected via a lateral flow assay (LFA). In some embodiments, a 5’ end of at least one of the primers in the primer set is linked to the 5’ or 3 ’ end of the in -reaction probe. In some embodiments, at least one of the primers in the primer set is 6-21 nucleotides in length. In some embodiments, the label comprises a fluorophore or biotin or digoxigenin. In some embodiments, the label comprises a redox molecule. In some embodiments, the sample is incubated at a reaction temperature range maintained within 18-40°C for the length of time. In some embodiments, the sample is incubated at a reaction temperature range maintained within 20-37°C for the length of time. In some embodiments, the sample is incubated at a room temperature for the length of time. In some embodiments, the sample is incubated at a reaction temperature range maintained within 22-25°C for the length of time. In some embodiments, the reaction temperature range is anisothermal range. In some embodiments, maintaining isothermal conditions of the reaction does not involve incubating the reaction admixture in a temperature -regulating device. In some embodiments, maintaining isothermal conditions of the reaction does not involve incubating the reaction admixture in a thermocycler. In some embodiments, the reaction admixture remains within a temperature range of + / - 10% of the starting reaction temperature until sufficient amplification of the target nucleic acid sequence to be detected is achieved. In some embodiments, the length of time is between about 1-60 minutes. In some embodiments, the length of time is between about 20-40 minutes. In some embodiments, the length of time is about 30 minutes. In some embodiments, the target ss and ds amplicons and / or the two target ds amplicons are produced at least at a linear rate of amplification. In some embodiments, the target ds amplicons and / or the two target ds amplicons are produced at an exponential rate of amplification. In some embodiments, the target ss amplicons are produced at least at a linear rate of amplification. In some embodiments, the target ss amplicons are produced at an exponential rate of amplification. In some embodiments, the sample comprises nucleic acid extracted from a subject. In some embodiments, the target region of the nucleic acid comprises a continuous stretch of nucleotide sequence present within the sample. In some embodiments, the nucleic acid extracted from the subject comprises doublestranded DNA (dsDNA). In some embodiments, the nucleic acid extracted from the subject comprises single-stranded DNA (ssDNA). In some embodiments, the sample comprises RNA extracted from the subject. In some embodiments, reverse transcriptase andNTPs are added to the sample to reverse transcribe the RNA extracted from the subject to cDNA, wherein the cDNA becomes the template to the target region. In some embodiments, the sample comprising nucleic acid extracted from the subject comprises nucleic acid derived from a pathogen. In some embodiments, the sample comprising nucleic acid extracted from the subject comprises nucleic acid derived from a pathogen and nucleic acid derived from the subject. In some embodiments, the pathogen is a virus. In some embodiments, the virus is listed in Table 1. In some embodiments, the pathogen is a bacterium. In some embodiments, the bacterium is listed in Table 2. In some embodiments, the pathogen is a fungus. In some embodiments, the fungus is listed in Table 3. In some embodiments, the pathogen is an animal pathogen. In some embodiments, the animal pathogen is listed in Table 4. In some embodiments, the sample is obtained from an animal. In some embodiments, the animal is a livestock species. In some embodiments, the livestock species is a type of cattle, horse, goat, sheep, pig, swine, rabbit, donkey, mule, bison, llama, cat, dog, deer, camel, yak, turkey, alpaca, elk, chicken, duck, emu, ostrich, pheasant, goose, fish, shrimp, crustacean, orbee. In some embodiments, the animal is a human. In some embodiments, the sample is obtained from plant. In some embodiments, ds amplicons having a sequence comprising at least- 10 -the target region are produced at least at a linear rate of amplification. In some embodiments, ds amplicons having a sequence comprising at least the target region are produced at an exponential rate of amplification. In some embodiments, the presence of the IrP comprising the label increases production of target ds amplicons. In some embodiments, the presence of the in -reaction probe comprising the label increases production of target ss amplicons. In some embodiments, detecting the target ds amplicons. In some embodiments, the method comprises detecting the target ss amplicons. In some embodiments, the detecting comprises analyzing reaction products using a molecular lateral flow assay (LFA). In some embodiments, the detecting comprises analyzing reaction products using an intercalating dye or fluorophore. In some embodiments, the detecting comprises analyzing reaction products using a nucleic acid hybridization technique. In some embodiments, the detecting comprises analyzing reaction products using a fluorescence detection technique. In some embodiments, the detecting comprises analyzing reaction products using a colorimetric technique. In some embodiments, the detectingcomprisesanalyzing reaction products using nucleic acid sequencing. In some embodiments, the detecting comprises analyzing reaction products usingDNA fragment size discrimination. In some embodiments, an oligonucleotide probe is added to the reaction mix or to ss amplicons and / or ds amplicons produced from the reaction to aid in the detecting. In some embodiments, a separate oligonucleotide probe comprising a quencher is added to the reaction mix or to ss amplicons and / or ds amplicons produced from the reaction to aid in the detecting. In some embodiments, the method comprises use of FRET in the detecting In some embodiments, the detecting comprising determining a quantitative measure of an amplified reaction product comprising the target ds amplicons. In some embodiments, the detecting comprising determining a quantitative measure of an amplified reaction product comprising the target ss amplicons. In some embodiments, oneof the primers has a non -complementary 5’ adaptor region. In some embodiments, one of the primers has a non-complementary 5 ’ adaptor region and has an additional primer that can bind to the adaptor region only . In some embodiments, the target nucleic acid sequence is a selected region of a genomic DNA sequence from a pathogen. In some embodiments, the target nucleic acid sequence is a selected region of an RNA sequence from a pathogen. In some embodiments, the target nucleic acid sequence is from a pathogen listed in Table 1, Table 2, Table 3 , or Table 4. In some embodiments, the target nucleic acid sequence is amplified in the presence of human genomic DNA. In some embodiments, the template to the target nucleic acid sequence is a single copy of a viral genome, and wherein the amplified target nucleic acid sequence is amplified at about room temperature in less than about 30 minutes to a level of a detectable range. In some embodiments, the level of a detectable range is at least about 104copies / pL, 105copies / pL, 106copies / pL, 107copies / pL, or 108copies / pL. In someembodiments, the target ds amplicons are amplified and detected in real time. In some embodiments, target ds amplicons are amplified and detected in less than about 60 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 12 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, or 3 minutes. In some embodiments, at least one primer in the primer set is less than or equal to 14 nucleotides in length. In some embodiments, at least one primer in the primer set is less than or equal to 12 nucleotides in length. In some embodiments, at least one primer in the primer set is less than or equal to 10 nucleotides in length. In some embodiments, the method further comprises detection of two different regions of the target nucleic acid sequence. In some embodiments, two or more distinct pathogenic variants are detected within a single amplification reaction. In some embodiments, two or more distinct disease mutations are detected within a single amplification reaction. In some embodiments, a single nucleotide polymorphism (SNP) within the target nucleic acid sequence is detected.

[0268] In an aspect described herein are systems of primers and probes, the systems comprising: i) a first oligonucleotide primer (Pl) comprising a sequence at least about 90% identical to a region on the plus strand of the target nucleic acid sequence, ii) a second oligonucleotide primer (P2) comprising a sequence at least about 90% identical to a region on the minus strand of the target nucleic acid sequence, iii) a third oligonucleotide primer (P3) comprising a sequence at least about 90% identical to a region on the plus or minus strand of the target nucleic acid sequence, and iv) an annexing oligonucleotide (AO) comprising: a nucleotide sequence at least about 90% identical to a region on the plus or minus strand of the target nucleic acid sequence, wherein the AO nucleotide sequence is: flankedby Pl sequence, P2 sequence, andP3 sequence or at least partially overlaps with Pl sequence, P2 sequence, orP3 sequence, a blocking feature that blocks extension of a DNA polymerase; wherein the sy stemis for replicatingtargetnucleic acid sequence comprises: i) target single-stranded (ss) amplicons, ii) two target double-stranded (ds) amplicons, or iii) target ss amplicons and two target ds amplicons. In some embodiments, the AO comprises a label allowing the labeled AO to function as an in -reaction probe (IrP). In some embodiments, sequence of one of the primers and the AO sequence do not overlap. In some embodiments, the sequence of one of the primers and the AO sequence at least partially overlap. In some embodiments, one of the primers is 15-20 nucleotides in length. In some embodiments, a 3 ’ end of one of the primers is linked to the 5’ end of the in-reaction probe. In some embodiments, one of the primers is 6-21 nucleotides in length. In some embodiments, the AO is labeled. In some embodiments, the AO is labeled with a fluorophore, redox molecule, orbiotin. In some embodiments, the system is prevents non-specific binding. In some embodiments, one of the primers is at least 25 nucleotides in length.In some embodiments, one of the primers is 10 to 40 nucleotides in length. In some embodiments, the AO is 30 to 75 nucleotides in length.SYSTEMS

[0269] Provided herein are systems for amplification, isolation, extraction, and / or detection of a biological sample. In some embodiments, the systems as described herein are for amplification, isolation, extraction, and / or detection of a target region of a nucleic acid molecule. In some embodiments, the method comprises amplifying a target nucleic acid sequence located within the target region. In some embodiments, the method comprises amplifying a target nucleic acid sequence atan ambienttemperature. In some embodiments, the target region is on a single -stranded DNA. In some embodiments, the target region is on a double-stranded nucleic acid (dsDNA). In some embodiments, the target region is on an RNA. The present disclosure provides system for amplifying a target nucleic acid sequence. The systems disclosed herein can utilize any components (e.g., oligonucleotides, labels, enzymes, samples, etc.), methods, devices, and / or kits disclosed herein. In some embodiments, the present disclosure provides systems for amplifying a target nucleic acid sequence at an ambient temperature. The target nucleic acid sequence can be isolated and processed from a biological sample. In some embodiments, the system comprises a first oligonucleotide (Pl). In some embodiments, the Pl binds to a strand of the target nucleic acid sequence. In some embodiments, the system comprises a second oligonucleotide (P2). In some embodiments, the P2 binds to another strand of the target nucleic acid sequence. In some embodiments, the system comprises a third oligonucleotide. In some embodiments, the third oligonucleotide binds to the target nucleic acid sequence. In some embodiments, the third oligonucleotide comprises a 3 ' end blocking feature that blocks extension of the third oligonucleotide by a polymerase. In some embodiments, the third oligonucleotide does not facilitate a localizing and abindingof P2 to the target nucleic acid sequence. In some embodiments, a 5 ' end of the third oligonucleotide and a 5 ' end of the Pl bind to the strand of the target nucleic acid sequence at a distance from about 3 nucleotides to about 25 nucleotides apart. In some embodiments, the system comprises a polymerase. In some embodiments, the system comprises a DNA processing enzyme to the target nucleic acid sequence. In some embodiments, the system comprises nucleotides in an amount sufficientto support amplification of the target region. In some embodiments, the system of amplification is conducted in an absence of a denaturing of the target nucleic acid. In some embodiments, the Pl localizes and binds to the strand of the target nucleic acid sequence independently of the third oligonucleotide. In some embodiments, the P2 localizes and binds to the other strand of the target nucleic acid sequence independently of the third oligonucleotide. In some embodiments, a 5’ end of a third primer (P3) binds to the strand of thetarget nucleic acid sequence.

[0270] Provided in some embodiments herein is a system for processing a biological sample. In some embodiments, the processing comprises isolating a nucleic acid from a biological sample. In some embodiments, the system comprises a channel. In some embodiments, the system comprises a first channel. In some embodiments, the first channel comprises a first opening. In some embodiments, the first channel is configured to receive a biological sample. In some embodiments, the first channel is configured to receive, via the first opening, a biological sample. In some embodiments, the system comprises a second channel. In some embodiments, the second channel comprises a substrate. In some embodiments, the second channel comprises a cellulose substrate. In some embodiments, the second channel comprises a second opening. In some embodiments, the second channel comprises a cellulose substrate coupled to the first channel and a second opening. In some embodiments, the system comprises a second opening configured to couple to the first opening. In some embodiments, the system transfers a biological sample from the first channel to the second channel. For instance, the system may transfer a biological sample from the first channel to the second channel upon the coupling of the second opening to the first opening. In some embodiments, the system comprises a third channel. In some embodiments, the third channel comprises a third opening. In some embodiments, the system comprises an elution solution. For instance, in some embodiments, the third channel of the system comprises an elution solution. In some embodiments, the third channel comprises a third opening and an elution solution. In some embodiments, the third channel comprises a third opening configured to couple to the second opening. In some embodiments, the system transfers the elution solution from the third channel to the second channel. For instance, the system may transfer a biological sample from the second channel to the third channel upon coupling of the third opening to the second opening In some embodiments, the second channel is configured to transfer elution solution received from the second channel to the third channel, such as upon coupling of the third opening and the second opening. In some embodiments, the first channel receives a lysis solution (e.g., see FIG. 23 A). In some embodiments, the firstchannel already contains a lysis solution (e.g., see FIG. 23B). In some embodiments, the first channel receives a biological sample (e.g., see FIG. 23B). In some embodiments, the first channel transfers the biological sample to a second channel (e.g., see FIG. 23C). In some embodiments, the first channel receives a biological sample, followed by receiving a lysis solution and then transferring the lysed biological solution to a second channel, (e.g., see FIG. 24A). In some embodiments, a housing receives a lysed biological solution from a channel and transfers the lysate from the housing back to the channel which is then discarded (e.g., see FIG. 24B) In some embodiments, the housing receives an elution solution from a channel,followed by transferring the eluate back to the channel which now has analytes extracted from the biological sample (e.g., see FIG. 23C). In some embodiments, the first channel transfers an extracted nucleic acid sample into a second channel containing a lyophilized amplification pellet or bead and rehydrating the said pellet to start amplification of a dual-labelled amplified target nucleic acid sequence (e.g., dual-labelled WSSV target DNA amplicons) (e.g., see FIG. 25). In some embodiments, detection (e.g., detecting a DNA amplicon) comprises a Lateral Flow Analysis (LFA) (e.g., see FIG. 26A). In some embodiments, a first channel containing an amplified nucleic acid (e.g. dual-labelled amplicon solution) from a processed biological sample is diluted with detection buffer added from a buffer packet, an LFA strip is added into the diluted amplicon solution in the first channel and the LFA results are then interpreted to detect presence of target nucleotide sequence (e.g., see FIG. 26B). In some embodiments, Lateral Flow Analysis (LFA) results are quantified by mean density analysis (e.g., see FIG. 28). In some embodiments, molecular lateral flow analysis is performed on the processed biological sample (e.g., molecular lateral flow analysis for human saliva samples spiked with EBV) (e.g., see FIG. 29).

[0271] Provided in some embodiments herein is a system for the preparation of nucleic acid from a biological sample. In some embodiments, the system comprises a housing. In some embodiments, the housing comprises a port. In some embodiments, the port comprises an opening In some embodiments, the housing comprises a substrate. In some embodiments, the substrate is a cellulose substrate. In some embodiments, the substrate is affixed to the housing. In some embodiments, the system comprises a housing comprising a port and a substrate. In some embodiments, theportis configured to receive abiological sample. In some embodiments, theport is configured to provide the biological sample to the cellulose substrate. In some embodiments, the housing comprises one or more channels as disclosed herein.Extraction

[0272] Provided in some embodiments herein is a system for the preparation of nucleic acid from a biological sample. For example, nucleic acids canby extracted and / or isolated as shown in FIGs. 86-87. In some embodiments, the system comprises a housing. In some embodiments, the housing comprises a port. In some embodiments, the port comprises an opening. In some embodiments, the housing comprises a substrate. In some embodiments, the substrate is a cellulose substrate. In some embodiments, the substrate is affixed to the housing. In some embodiments, the system comprises a housing comprising a port and a substrate. In some embodiments, the port is configured to receive a biological sample. In some embodiments, the port is configured to provide the biological sample to the cellulose substrate. In some embodiments, the housing comprises one or more channels as disclosed herein.

[0273] For instance, as depicted in FIG. 31, a workflow process, hereinafter (“System A”) may comprise: inserting a biological sample into a tube 3101 mechanically lysing (e.g., pestling) the biological sample for about 30 seconds 3102, adding a lysis solution as described herein to the tube and mixingthe mechanically ly sedbiological sample and lysis elution solution 3103 to further lyse the biological sample, coupling a dropper cap onto the tube and rotating the tube over a housing, comprising a cellulose substrate, to transfer the lysed biological sample through the dropper cap into the housing 3104, laying the housing flat for a period of about 0 to about 30 seconds such as to allow the binding of nucleic acids of the biological sample to the cellulose substrate of the housing 3105, inverting the housing to discard unbound biological sample and lysate from the housing 3106, coupling a connector to the second housing such as to couple the housingto a vial comprising an elution buffer 3107, rotating the vial 180° to transfer the elution buffer into the housing 3108, layingthe housing flatfor a time period of about Oto about 30 seconds 3109, inverting the housing over the vial to transfer eluate from the housing to the vial 3110, discarding the housing 3111, and storing the extracted nucleic acids 3112.

[0274] In some embodiments, samples are processed according to the workflow, which is depicted in FIG. 39 (e.g., “Seek Extraction”). In some embodiments, Seek Extraction comprises: collecting a biological or environmental sample, mixingthe biological sample with a lysis solution, transferring the lysate to a lysate vial, pouring the contents of the lysate vial into an extraction unit, pouring the lysate solution back into the lysate vial, discarding the lysate vial, pouring an elution solution into the extraction unit to produce an eluate, and pouring the eluate into an elution vial, thereby extracting and purifying analytes from the biological sample.

[0275] In some embodiments, a first channel receives a lysis solution. As described herein, a lysis solution provided herein can lyse any biological samples as described herein. A lysis solution may include any solution which can lyse a biological sample. As described herein, a lysis solution provided herein can lyse biological samples described herein to provide analytes of the biological sample for binding to a cellulose substrate described herein.

[0276] In some embodiments, a lysis solution has a neutral pH. In some embodiments, a lysis solution has an acidic pH. In some embodiments, the lysis solution has a pH of about 1 to about 7. In some embodiments, the lysis solution has a pH of about 1 to about 2, about 1 to about 3, about 1 to about 4, about 1 to about 5, about 1 to about 6, about 1 to about 7, about2 to about 3, about 2 to about 4, about 2 to about 5, about 2 to about 6, about 2 to about 7, about 3 to about 4, about 3 to about 5, about 3 to about 6, about 3 to about 7, about 4 to about 5, about 4 to about 6, about 4 to about 7, about 5 to about 6, about 5 to about 7, or about 6 to about 7. In some embodiments, the lysis solution has a pH of about 1, about 2, about 3, about 4, about 5, about 6, or about 7. In someembodiments, the lysis solution has a pH of at least about 1, about 2, about 3, about 4, about 5, or about 6. In some embodiments, a lysis solution has a pH of at most about 2, about 3 , about 4, about 5, about 6, or about 7. In some embodiments, a lysis solution has a pH of about 7.5.

[0277] In some embodiments, a lysis solution has a basic pH. In some embodiments, the lysis solution has a pH of about 7 to about 14. In some embodiments, the lysis solution has a pH of about 7 to about 8, about 7 to about 9, about 7 to about 10, about 7 to about 11, about 7 to about 12, about 7 to about 13, about 7 to about 14, about 8 to about 9, about 8 to about 10, about 8 to about 11, ab out 8 to ab out 12 , ab out 8 to ab out 13 , ab out 8 to ab out 14, ab out 9 to ab out 10 , about 9 to about11 , about 9 to about 12, about 9 to about 13 , about 9 to about 14, about 10 to about 11, about 10 to about 12, about 10 to about 13, about 10 to about 14, about 11 to about 12, about 11 to about 13, about 11 to about 14, about 12 to about 13, about 12 to about 14, or about 13 to about 14. In some embodiments, the lysis solution has a pH of about 7, about 8, ab out 9, about 10, about 11 , about12, about 13, or about 14. In some embodiments, the lysis solution has a pH of at least about 7, about 8, about 9, about 10, about 11, about 12, or about 13. In some embodiments, the lysis solution has a pH of at most about 8, about 9, about 10, about 11, about 12, about 13, or about 14. In some embodiments, a lysis solution has a pH of about 8.

[0278] An additional aspect of the present disclosure provides systems and methods provides for a second channel. Suitable channels include, but are not limited to, beakers, bottles, burettes, containers, cylinders, vials, tubes, pipettes, syringes, adapters or connectors, and any other channel.

[0279] In some embodiments, a second channel comprises a second opening. In some embodiments, a second channel receives a biological sample as described herein from a first channel as described herein. In some embodiments, a second channel, by a second opening receives a biological sample from a first channel. In some embodiments, a second channel receives, by a second opening, a lysis solution from the first channel. In some embodiments, a second channel can receive, by a second opening, an elution solution from the third channel. In some embodiments a second channel can discard, by a second opening, lysate. In some embodiments a second channel can transfer, by the second opening, eluate to the third channel.

[0280] In some embodiments, a second channel comprises a substrate. In some embodiments, a substrate is a cellulose substrate. In some embodiments, a substrate is within a chamber of a second channel. In some embodiments, analytes of a lysed biological samplebind to the substrate upon transfer of the biological sample from a first channel to a second channel comprising the substrate. In some embodiments, a portion of thebiological sample comprises sample (e.g., nucleic acids, analytes, or other target materials) which binds to a substrate. In some embodiments, a substrate is permanently affixed to a second channel. In some embodiments, a substrate (e.g.,- 11 -cellulose beads) wraps around a chamber of a second channel. The substrate can retain particles of a biological sample, as described herein, such as to increase the quality and / or concentration of extracted analytes from the biological sample.

[0281] In some embodiments, a housing comprises the second channel. In some embodiments, the housing comprises a port. In some embodiments, the port comprises a second opening. For instance, as depicted in FIG. 30, housing 3000 may comprise a port 3001 and a binding chamber 3002 which comprises a cellulose substrate 3003. The port can also comprise a connector 3004, such as to connect the housing to a channel.

[0282] In some embodiments, a first channel transfers, via a first opening, a biological sample to a second channel through a second opening. In some embodiments, an opening of a first channel couples to a second opening of a second channel, such as to transfer a biological sample and / or a lysis solution from the first channel to the second channel. In some embodiments, a first channel comprises an adapter. In some embodiments, a second channel comprises an adapter. In some embodiments, an adapter couples a first and second openings. In some embodiments, an adapter is an external thread on a channel. In some embodiments, a first channel comprises internal tread. In some embodiments, a second channel comprises internal tread. In some embodiments, an internal thread couples a first channel to a second channel via a first and second op ening. In some embodiments, a first channel comprises flexible tubing. In some embodiments, a second channel comprises flexible tubing. In some embodiments, a flexible tubing couples the first and second channel. In some embodiments, an adapter is a Luer locking mechanism between the first and second channel. In some embodiments, the first channel is a female Luer lock connector, and the second channel is a male Luer lock connector. In some embodiments, the first channel is a male Luer lock connector, and the second channel is a female Luer lock connector. In some embodiments, the pathway between the first and second channel comprises a particulate filter.

[0283] In some embodiments, a dropper cap is positioned between a first and second channel. In some embodiments, a first channel transfers a biological sample as described herein through a dropper cap into a second channel. In some embodiments, a dropper cap comprises a particulate filter as described herein.

[0284] In some embodiments, a first opening of a first channel is inverted over a second channel, such as to transfer a biological sample as described herein from the first channel to the second channel. In some embodiments, a first opening of a first channel is rotated 180°, such as to invert the first opening of the first channel over a second channel. In some embodiments, gravity transfers a biological sample from a first channel to a second channel. In some embodiments, a force exerted onto a first channel transfers from the first channel to a second channel.

[0285] In some embodiments, a second channel disposes of lysate. In some embodiments, disposed lysate comprises lysis solution. In some embodiments, disposed lysate comprises a portion of the biological sample which does not bind to a substrate of a second channel. In some embodiments, a second channel transfers, as described herein, unbound lysate to a first channel, thereby disposing the unbound lysate.

[0286] In some embodiments, the systems and methods described herein comprise an actuator. In some embodiments an actuator exerts a force onto a biological sample to transfer the biological sample between channels as described herein. In some embodiments, an actuator is a pump, abulb, a syringe, or any other actuator, such as a pipette. In some embodiments, the systems and methods described herein do not comprise an actuator. In some embodiments, systems as described herein use gravity to exert force on the biological sample. For instance, in some embodiments, systems as described herein use gravity to transfer the biological subject between channels.

[0287] An additional aspect of the present disclosure provides systems and methods provides for a third channel. Suitable channels include, but are not limited to, pipettes, beakers, bottles, burettes, containers, cylinders, vials, tubes, syringes, adaptors, connectors, or any other channel.

[0288] The third channel can comprise a third opening. The third channel can couple to, via the third opening, the second channel. In some embodiments, the third channel comprises a connector. In some embodiments, the second channel comprises a connector. In some embodiments, a connector couples the second and third channels.

[0289] In some embodiments, a third channel comprises an elution solution. In some embodiments, a third channel couples to a second channel, as described herein, such as to transfer an elution solution from the third channel to the second channel. In some embodiments, a third channel is inverted over a second channel, such as to transfer an elution solution from the third channel to the second channel. In some embodiments, a third opening of a third channel is rotated 180°, such as to invert the third opening of a third channel over a second channel.

[0290] In some embodiments, an elution solution elutes analytesbound to a substrate. In some embodiments, an elution solution elutes a portion of the analytes bound to a substrate. In some embodiments, the elution solution elutes at least a portion of the analytes bound to a substrate. In some embodiments, the elution solution elutes all analytes bound to a substrate. In some embodiments, an eluate comprises analytes of a biological sample.

[0291] In some embodiments, eluate is transferred from a second channel to a third channel. In some embodiments, a second channel is coupled to a third channel, as described herein, to transfer eluate from the second channel to the third channel. In some embodiments, a second channel is inverted over a third channel to transfer, via gravity, eluate from the second channel tothe third channel. In some embodiments, a second opening of a second channel is rotated 180°, such as to invert the second opening of the second channel over a third channel.

[0292] An additional aspect of the present disclosure provides systems and methods which extract nucleic acids from a biological sample as described herein. Examples of protocols used for extraction of nucleic acids with systems and methods as described herein may be f ound at, for instance, Schnepp, Bruce C., et al., Characterization of Adeno-Associated Virus Genomes Isolated from Human Tissues. J. 79 Virology 23 (December, 2005) (DOI: 10.1128 / JVI.79.23.14793- 14803.2005) and Jackson, D. P., et al., Tissue Extraction of DNA and RNA and analysis by polymerase chain reaction, 43 J. Clin. Pathol.499- 504 (1990) (DOI: 10. 1136 / jcp .43.6.499), which are hereby incorporated by reference in its entirety. In some embodiments, nucleic acids extracted from a biological sample are amplified. Suitable amplification methods include, but are notlimited to, polymerase chain reaction (PCR), quantitative PCR (qPCR), isothermal amplification, or any other amplification technique. In some embodiments, an isothermal amplification comprises recombinase polymerase amplification (RPA). In some embodiments, an isothermal (e.g., ambient temperature) amplification comprises ANINA. In some embodiments, an isothermal (e.g., ambient temperature) amplification comprises Seek Amplification.

[0293] In some embodiments, a lysed biological sample is not incubated in a second channel before immediately moving to the next step. In some embodiments, a lysed biological sample is incubated in a second channel. In some embodiments, incubation can increase the amount of nucleic acids which bind to the cellulose substrate. In some embodiments, a second channel is rotated to lie flat to incubate a lysed biological sample. In some embodiments, a second channel is rested at room temperature to incubate a lysed biological sample. In some embodiments, a second channel is rotated to lie flat and rested at room temperature to incubate a lysed sample. In some embodiments, a lysed sample is incubated for a period of time of about 1 second to about 1 minute. In some embodiments, a lysed biological sample is incubated in the second chamber for a period of time of less than about 30 seconds.

[0294] In some embodiments, an elution solution and analytes bound to a substrate are not incubated before immediately moving to the next step. In some embodiments, an elution solution and analytes bound to a substrate are incubated. In some embodiments, incubating an elution solution and analytes bound to a substrate can increase the amount of analytes eluted. In some embodiments, a second channel is rotated to lie flat to incubate an elution solution and analytes bound to a substrate. In some embodiments, the second channel is rested at room temperature to incubate an elution solution and analytes bound to a substrate. In some embodiments, a second channel is rotated to lay flat and rested at room temperature to incubate an elution solution andanalytes bound to a substrate. In some embodiments, an elution solution and analytes are incubated foraperiod of time of about 1 second to about 1 minute. In some embodiments, the elution solution and analytes are incubated for a period of time of less than about 30 seconds.

[0295] For instance, as depicted in FIG. 31, a workflow process, hereinafter (“System A”) may comprise: inserting a biological sample into a tube 3101 mechanically lysing (e.g., pestling) the biological sample for about 30 seconds 3102, adding a lysis solution as described herein to the tube and mixingthe mechanically lysedbiological sample and lysis elution solution 3103 to further lyse the biological sample, coupling a dropper cap onto the tube and rotating the tube over a housing, comprising a cellulose substrate, to transfer the lysed biological sample through the dropper cap into the housing 3104, laying the housing flat for a period of about 0 to about 30 seconds such as to allow the binding of nucleic acids of the biological sample to the cellulose substrate of the housing 3105, inverting the housing to discard unbound biological sample and lysate from the housing 3106, coupling a connector to the second housing such as to couple the housingto a vial comprising an elution buffer 3107, rotating the vial 180° to transfer the elution buffer into the housing 3108, lay ing the housing flat f or a time period of about 0 to about 30 seconds 3109, inverting the housing over the vial to transfer eluate from the housing to the vial 3110, discarding the housing 3111, and storing the extracted nucleic acids 3112.

[0296] In some embodiments, samples are processed according to the workflow, which is depicted in FIG. 39 (“Seek Extraction” can refer to any extraction method, system, etc. disclosed herein). In some embodiments, Seek Extraction comprises: collecting a biological or environmental sample, mixingthe biological sample with a lysis solution, transferring the lysate to a lysate vial, pouring the contents of the lysate vial into an extraction unit, pouring the lysate solution back into the lysate vial, discarding the lysate vial, pouring an elution solution into the extraction unit to produce an eluate, and pouring the eluate into an elution vial, thereby extracting and purifying analytes from the biological sample.

[0297] In some embodiments, samples are processed according to the workflow, examples of which are depicted in FIGs. 24A-C, FIG. 79 or in FIG. 87 (“Seek Extraction” can refer to any extraction method, system, etc. disclosed herein). In some embodiments, Seek Extraction comprises attaching a Seek Extraction unit (comprising a binding substrate) vertically to a lysate vial containing a lysis solution of a biological or environmental sample, inverting the connected system to a position where the extraction unit is on the bottom, letting the lysate solution drop into the extraction unit via gravity and bind the analyte to the substrate, inverting the connected system again (by 180°) such that the lysate vial is on the bottom, letting the lysate solution drop back into the lysate vial via gravity, detaching the Seek Extraction Unit from the lysate vial, attaching theSeek Extraction unit vertically to an elute vial comprising an elution solution, inverting the connected system to a position where the extraction unit is on the bottom, letting the elution solution to drop into the extraction unit via gravity and eluting the analyte from the substrate, creating an eluate, inverting the connected system again (by 180°) such that the elute vial is on the bottom, letting the elution solution to drop back into the elute vial via gravity, thereby extracting and purifying analytes from the biological or environmental sample into the eluate in the elute vial.

[0298] In some embodiments, samples are pre-processed according to the workflow, which is depicted in FIG. 86 (Step l(i)) before moving to the sample processing workflow, depicted in FIGS. 24A-C, FIG. 79 or in FIG. 87 (“Seek Extraction” can refer to any extraction method, system, etc. disclosed herein). In some embodiments, Seeklt Extraction comprises: collecting a solid biological or environmental sample in a sample vial, mechanically lysing the sample with a pestle or metal bead, mixing the biological sample with a lysis solution, adding a cap containing a filtration substrate to the sample vial, transferringthe lysate to a lysate vial by squeezingthe sample vial. In some embodiments, the sample vial is discardedin an appropriatemanner after transferring the contents to a lysate vial.

[0299] In some embodiments, samples are pre-processed according to the workflow, which is depicted in FIG 23B or FIG. 86 (Step 1 (ii)) before moving to the sample processing workflow, depicted in FIGS. 24A-C, FIG. 79 or in FIG. 87 (“Seek Extraction” can refer to any extraction method, system, etc. disclosed herein). In some embodiments, Seeklt Extraction comprises collecting a swab sample from a biological or environmental specimen and mixingthe swab sample with a lysis solution contained in a lysate vial and then discarding the swab in an appropriate manner. In some embodiments, the sample vial is discarded in an appropriate manner after transferring the contents to a lysate vial.

[0300] In some embodiments, samples are pre-processed according to the workflow, which is depicted in FIG 23C or FIG. 86 (Step l(iii)) before moving to the sample processing workflow, depicted in FIGS. 24A-C, FIG. 79 or in FIG. 87 (“Seek Extraction” can refer to any extraction method, system, etc. disclosed herein). In some embodiments, Seeklt Extraction comprises collecting a fluid biological or environmental sample in a sample vial and transferring the sample to a lysate vial containing a lysis solution. In some embodiments, Seeklt Extraction comprises collecting a fluid biological or environmental sample in a sample vial containing a lysis solution, adding a cap containing a filtration substrate to the sample vial, and transferringthe lysate to a lysate vial by squeezingthe sample vial. In some embodiments, the sample vial is discarded in an appropriate manner after transferring the contents to a lysate vial.Oligonucleotides

[0301] The present disclosure provides oligonucleotides for amplification, isolation, extraction, and / or detection of a biological sample. In some embodiments, oligonucleotides as described herein can be used in methods for amplification as described herein. For instance, an oligonucleotide as described herein can be a primer, a target nucleic acid sequence, DNA, RNA, a single-stranded oligonucleotide, and / or a double-stranded oligonucleotide. In some embodiments, the systems or methods described herein comprise one or more oligonucleotides. For example, the systems or methods comprise a first oligonucleotide (Pl); a second oligonucleotide (P2); a third oligonucleotide; and / or a fourth oligonucleotide. In some embodiments, oligonucleotides can be primers. In some embodiments, Pl is a primer. In some embodiments, P2 is a primer. In some embodiments, the third oligonucleotide is an annexing oligonucleotide. In some embodiments, the third oligonucleotide is an in -reaction probe. In some embodiments, the fourth oligonucleotide is an in-reaction probe. In some embodiments, the fourth oligonucleotide is a primer (P3). In some embodiments, oligonucleotides can bind a target nucleic acid sequence. In some embodiments, oligonucleotides can be complementary to a target nucleic acid sequence. In some embodiments, the oligonucleotides comprise a sequence at least about 70% identical to a region on the strand of the target nucleic acid sequence. In some embodiments, the oligonucleotides comprise a sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more identical to a region of the target nucleic acid sequence. In some embodiments, the oligonucleotides comprise a length from about 10 nucleotides to about 100 nucleotides. In some embodiments, the oligonucleotides comprise a length of about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In some embodiments, the oligonucleotides comprise a label. In some embodiments, the oligonucleotides can be a probe (e.g., an in-reaction probe) for detection of the target nucleic acid sequence. In some embodiments, the oligonucleotides can aid in on-target annealing of a different oligonucleotide (e.g., annexing oligonucleotides).

[0302] In some embodiments, the first oligonucleotide (Pl) comprises a at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more identical to a region of the target nucleic acid sequence. In some embodiments, the second oligonucleotide (P2) comprises a atleast about 70%, atleast about 75%, atleast about 80%, atleast about 85%, atleast about 90%, atleast about 95%, or more identical to a region ofthe target nucleic acid sequence. In some embodiments, the third oligonucleotide comprises a at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more identical to a region of the target nucleic acid sequence. In some embodiments, the fourth oligonucleotide comprises a at least about 70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, atleast about 95%, or more identical to a region ofthe target nucleicacid sequence.

[0303] In some embodiments, the first oligonucleotide (Pl), the second oligonucleotide (P2), and / or the third oligonucleotide localize and bind to a strand of the target nucleic acid sequence. In some embodiments, the Pl localizes and binds to the strand of the target nucleic acid sequence independently of the third oligonucleotide. In some embodiments, the P2 localizes and binds to the other strand of the target nucleic acid sequence independently of the third oligonucleotide. In some embodiments, a 5' end of the third oligonucleotide binds to the strand of the target nucleic acid sequence. In some embodiments, the 5' end of the third oligonucleotide and a 5' end of the Pl bind to a strand of the target nucleic acid sequence at a distance from about 3 nucleotides to about 25 nucleotides apart. In some embodiments, the 5' end of the third oligonucleotide and a 5' end of the Pl bind to a strand of the target nucleic acid sequenceat a distance from about 15 nucleotides apart. In some embodiments, the 5’ end of the third oligonucleotide and a 5 'end of the Pl bind to a strand of the target nucleic acid sequence at a distance from about 10 nucleotides apart. In some embodiments, the 5’ end of the third oligonucleotide and a 5 ' end of the Pl bind to a strand of the target nucleic acid sequence at a distance from about 8 nucleotides apart. As described in Example 23, and shown in FIG. 95A and FIG. 95B, when a 5' end of the third oligonucleotide and a 5' end of a Pl (or P2) bind to a strand of the target nucleic acid sequence at a distance from about 3 nucleotides to about 25 nucleotides apart, amplification efficiency may be increased (e.g., increased amplification product, decreased off -target amplicons, etc.) compared to amplification efficiency when a 5' end of the third oligonucleotide and a 5' end of a Pl (or P2) bind to a strand of the target nucleic acid sequence at a distance greater than from about 3 nucleotides to about 25 nucleotides apart. In some embodiments, the Pl, the P2, the third oligonucleotide, the fourth oligonucleotide or any combination thereof has a non -complementary 5' adaptor region. In some embodiments, the Pl, the P2, the third oligonucleotide, the fourth oligonucleotide or any combination thereof has a non -complementary 5' adaptor region. In some embodiments, the Pl, the P2, the third oligonucleotide, or the fourth oligonucleotide comprises a length of at least about 6 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 60 nucleotides, at least about 80 nucleotides, or more.

[0304] The method of any one of the preceding claims, wherein the Pl or the P2 is a nucleotide length insufficient to produce amplicons of the target nucleic acid sequence without the presence of the third oligonucleotide.

[0305] The method of any one of the preceding claims, wherein the Pl and the P2 are present in molar excess relative to the target nucleic acid sequence.

[0306] In some embodiments, the Pl comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, or 99% identical to a region on a strand of a target nucleic acid sequence. In some embodiments, the P2 comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, or 99% identical to a region on another strand of the target nucleic acid sequence. In some embodiments, the third oligonucleotide comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, or 99% identical to a region of the target nucleic acid sequence.

[0307] In some embodiments, the Pl, the P2, the third oligonucleotide, the fourth oligonucleotide, or any combination thereof comprise a length of at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, atleastabout 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, at least about 100 nucleotides, or more. In some embodiments, the Pl ortheP2 is a nucleotide length insufficientto produce amplicons of the target region without the presence of third oligonucleotide. In some embodiments, the Pl and P2 are present in molar excess relative to the target nucleic acid sequence.

[0308] In some embodiments, the oligonucleotide comprises a 3 ' end blocking feature that blocks extension of a DNA polymerase. For example, the oligonucleotide can have a 3 ’ modification such as an inverted nucleotide, C3 -spacer, etc. which blocks 3’ extension by polymerase. In some embodiments, the 3' end blocking feature that blocks extension of a DNA polymerase comprises an inverted nucleotide, 2',3'-dideoxy-3'-aminonucleoside 5 '-triphosphates, or a C3 -spacer. In some embodiments, the blocking feature comprises a 3’ phosphorylation, a 3’ dideoxynucleotide (ddNTP), a 3’ amino acid, or a 3’ BHQ. Theoretically, any modification that makes the 3 ’ OH of the last nucleotide in a primer unavailable f or a DNA polymerase to start adding nucleotides can be a blocking feature.

[0309] In some embodiments, the oligonucleotides comprise label for detection of an amplification product, as described herein. In some embodiments, the Pl, P2, third oligonucleotide, the fourth oligonucleotide, or any combinationthereof comprises a label (such as a probe). In some embodiments, the oligonucleotide comprises a 3' end label, such as a label described herein. In some embodiments, the label comprises a xanthene core or a derivative thereof. In some embodiments, the label comprises a FAM. In some embodiments, the oligonucleotide comprises a 3 ' end fluorescein label, such as 6-FAM or fluorescein isothiocyanate (FITC).

[0310] In some embodiments, the oligonucleotides are target nucleic acid sequences asdescribed herein.

[0311] In some embodiments, the oligonucleotides described herein are primers. In some embodiments, the Pl is a primer. In some embodiments, theP2 is a primer. In some embodiments, the systems described herein comprise a third primer (P3). In some embodiments, the Pl, the P2, and the P3 are in a set of primers. In some embodiments, the Pl, the P2, and the P3, are in a set of primers. In some embodiments, the Pl, the P2, and the third oligonucleotide can be in a set of oligonucleotides. In some embodiments, the Pl , the P2, and the third oligonucleotide can be added to the target nucleic acid sequence as a set of oligonucleotides.

[0312] In some embodiments, the first oligonucleotide (Pl) and the second oligonucleotide (P2) are primers. In some embodiments, the third oligonucleotide can be an annexing oligonucleotide (AO). In some embodiments, an annexing probe (AP) can refer to a labeled annexing oligonucleotide (AO). In some embodiments, the third oligonucleotide can be an inreaction probe (IrP). In some embodiments, the fourth oligonucleotide can b e an in-reaction probe (IrP). In some embodiments, the in-reaction probe comprises any label as described herein. In some embodiments, the in-reaction probe comprises a detectable label. In some embodiments, the systems disclosed herein can comprise a fifth oligonucleotide, a sixth oligonucleotide, a seventh oligonucleotide, an eighth oligonucleotide, a ninth oligonucleotide, a tenth oligonucleotide, or more oligonucleotides. In some embodiments, the fifth oligonucleotide, the sixth oligonucleotide, the seventh oligonucleotide, the eighth oligonucleotide, the ninth oligonucleotide, or the tenth oligonucleotide can be a primer, an annexing oligonucleotide, or an in-reaction probe.

[0313] Some embodiments of systems described herein use a primer. In some embodiments of primer systems described herein, are present in a favorable ratio for amplification of target nucleotide sequence. In some embodiments, the method comprises introducing to a sample 2) a solution comprising enzymes, dNTPs, and a buffer comprising a buffering agent, salts, and crowding reagents. In some embodiments, the method comprises incubating the sample with the primers and the solution at a reaction temperature for a length of time. In some embodiments, the primer sequence and the in-reaction probe sequence do not overlap. In some embodiments, the primer sequence and in-reaction probe sequence overlap. In some embodiments, one of the primers is less than or equal to 15 nucleotides (nts) in length. In some embodiments, a primer as described herein comprises less than or equal to 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about ?, about 6, about 5, about 4, about 3, about 2, or less nucleotides (nts) in length. In some embodiments, such a primer comprising 15 or less nucleotides in length is a short primer. In some embodiments, such a short primer comprises a length insufficient to produce amplicons of the target sequence without the presence of an in-reaction probe (IrP) as described herein.

[0314] In an aspect described herein are systems of primers comprising: a first oligonucleotide (Pl), wherein the Pl is a primer comprising a sequence at least about 90% identical to a region on the plus strand of the target nucleic acid sequence, (i) a second oligonucleotide (P2), wherein the P2 is a primer comprising a sequence at least about 90% identical to a region on the minus strand of the target nucleic acid sequence, (ii) a third primer (P3), wherein the P3 comprises a sequence at least about 90% identical to a region on the plus or minus strand of the target nucleic acid sequence; and (iii) an annexing oligonucleotide (AO) comprising: a nucleotide sequence at least about 90% identical to a region on the plus or minus strand of the target nucleic acid sequence, wherein the AO nucleotide sequence is: flanked by Pl sequence, P2 sequence, and P3 sequence or at least partially overlaps with Pl sequence, P2 sequence, or P3 sequence, a blocking feature that blocks extension of a DNA polymerase; wherein the system is for producing target nucleic acid sequence comprises: i) target single-stranded (ss) amplicons, ii) two target double-stranded (ds) amplicons, oriii) target ss amplicons and two target ds amplicons. In some embodiments, sequence of one of the primers and the AO sequence do not overlap. In some embodiments, the sequence of one of the primers and the AO sequence at least partially overlap. In some embodiments, one of the primers is 15-20 nucleotides in length. In some embodiments, a 3’ end of one of the primers is linked to the 5’ end of the in -reaction probe. In some embodiments, the system prevents nonspecific binding. In some embodiments, the system decreases non-specific annealing of the primers. In some embodiments, one of the primers is at least 20 nucleotides in length. In some embodiments, one of the primers is at least 25 nucleotides in length. In some embodiments, one of the primers is at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100 or more nucleotides in length. In some embodiments, one of the primers is 10 to 40 nucleotides in length. In some embodiments, an annexing oligonucleotide (AO) as described herein is 20 to 75 nucleotides in length. In some embodiments, an AO as described herein is about 30 to about 75 nucleotides in length. In some embodiments, an AO as described herein is at least about25, atleast about 30, atleast about 35, at least about 40, at least about 45, at least about 50, atleast about 55, at least about 60, atleast about 65, at least about 70, atleast about 75, ormore nucleotides in length.

[0315] Systems described herein can utilize primers designed to replicate and amplify a target nucleic acid sequence. Primers may be designed for annealing to target location. In some embodiments, oligonucleotide primers comprise a stretch of nucleotide sequence that is at least about 90% identical to a region on a strand of the target nucleic acid sequence. Primers bind by sequence-specific annealing to complementary sequence at a target site. In some instances, it isdesirable to design primers with a short length (e.g., less than 20 nt) to limit annealing to off-target nucleic acid sequences. In some instances, short primers may not possess a long enough sequence region of complementarity to anneal efficiently to a complementary sequence at a target site (even in presence of a DNA processing enzyme). In some instances, presence of an annexing oligonucleotide within the reaction mix aids in on-target annealing of one of the primers. Without wishing to limit the present disclosure to any theory or mechanism, a combination of a first oligonucleotide or a second oligonucleotide as described herein and an in -reaction probe (IrP) as described herein may increase specificity and sensitivity of an amplification of a target region as compared to amplification without the IrP. In some embodiments, the first oligonucleotide or second oligonucleotide is a primer. In some embodiments, the primer is a short primer. Accordingly, in some embodiments, an IrP increases specificity and / or sensitivity of a binding of a primer as described herein to a target region, and increases specificity and / or sensitivity of amplification of the target region. In some embodiments, oligonucleotide primers comprise a stretch of nucleotide sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a region on a strand of the target nucleic acid sequence. In some embodiments, oligonucleotide primers comprise a stretch of nucleotide sequence that is identical to a region on a strand of the target nucleic acid sequence. In some embodiments, the stretch of nucleotide sequence in the primer that binds to a complementary target site is at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides in length. In some embodiments, the complementary target site is at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides in length. In some embodiments, primers are designed to anneal to a particular strand of a template sequence. In some embodiments, primers are designed to anneal to a strand designated the plus strand (in which case the annealed primer would reside on the minus strand). In some embodiments, primers are designed to anneal to a strand designated the minus strand (in which case the annealed primer would reside on the plus strand). In some embodiments, the system of amplification is configured for amplification in an absence of a denaturing of the target nucleic acid.

[0316] In some embodiments, one of the primers is from about 6 to about 24 nucleotides in length. In some embodiments, such a primer is a shorter primer. In other embodiments, one of the primers is about 10 to 24 nucleotides, or about 10 to 22 nucleotides, or about 10 to 20 nucleotides, or about 10 to 18 nucleotides, or about 10 to 16 nucleotides, or about 10 to 14 nucleotides, or about 10 to 12 nucleotides, or about 12 to 24 nucleotides, or about 12 to 22 nucleotides, or about 12 to 20 nucleotides, or about 12 to 18 nucleotides, or about 12 to 16 nucleotides, or about 12 to 14nucleotides, or about 14 to 24 nucleotides, or about 14 to 22 nucleotides, or about 14 to 20 nucleotides, or about 14 to 18 nucleotides, or about 14 to 16 nucleotides, or about 16 to 24 nucleotides, or about 16 to 22 nucleotides, or about 16 to 20 nucleotides, or about 16 to 18 nucleotides, or about 18 to 24 nucleotides, or about 18 to 22 nucleotides, or about 18 to 20 nucleotides, or about 20 to 24 nucleotides, or about 20 to 22 nucleotides, or about 22 to 24 nucleotides. In some embodiments, one of the primers is 10-21 nucleotides in length.

[0317] In some embodiments, one of the primers is at least 20 nucleotides (nts) in length without an adapter region. In some embodiments, one of the primers is 20 to 75 nucleotides in length with an adaptor region. In other embodiments, one of the primers is about 20 to 65 nucleotides, or about 20 to 40 nucleotides, or about 20 to 35 nucleotides, or about 20 to 30 nucleotides, or about 20 to 25 nucleotides, or about 25 to 35 nucleotides, or about 25 to 30 nucleotides, in length with an adaptor region.

[0318] In some embodiments, the adapter region of one of the primers is 5 to 40 nucleotides in length. In other embodiments, the adapter region of one of the primers is about 5 to 35 nucleotides, or about 5 to 30 nucleotides, or about 10 to 25 nucleotides, or about 10 to 20 nucleotides, or about10 to 15 nucleotides, or about 15 to 35 nucleotides, or about 15 to 30 nucleotides, or about 15 to 25 nucleotides, or about 15 to 20 nucleotides, or about 20 to 35 nucleotides, or about 20 to 30 nucleotides, or about 20 to 25 nucleotides, or about 25 to 35 nucleotides, or about 25 to 30 nucleotides, or about 30 to 35 nucleotides.

[0319] In some embodiments, the target binding region of one of the primers is 15 to 40 nucleotides in length. In other embodiments, the target binding region of one of the primers is about20 to 40 nucleotides, or about25 to 35 nucleotides, or about 25 to 30 nucleotides, or about 30 to 40 nucleotides, or about 30 to 35 nucleotides, or about 35 to 40 nucleotides.

[0320] In some embodiments, the adaptor region and the target binding region of a primer are spaced a distance apart, e.g., aprimer comprises a sequence (e.g., a spacer) between the two regions that are at least 1 nucleotide apart, e.g., 1 nucleotide apart, 2, 3, 4, 5, 6, 7, 8, 9, 10, more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, etc.

[0321] In some embodiments, one of the medium or longer length primers is at least 16 nucleotides (nts) in length without an adapter region. In some embodiments, one of the medium or longer length primers is 30 to 75 nucleotides in length with an adaptor region. In other embodiments, one of the medium or longer length primer is about 18 to 35 nts, or about 20 to 30 nts.

[0322] In some embodiments, the adapter region of a long primer is 5 to 40 nucleotides in length. In other embodiments, the adapter region of a long primer is about 10 to 35 nts, or about10 to 30 nts, or about 10 to 25 nts, or about 10 to 20 nts, or about 10 to 15 nts, or about 15 to 35 nts, or about 15 to 30 nts, or about 15 to 25 nts, or about 15 to 20 nts, or about 20 to 35 nts, or about 20 to 30 nts, or about 20 to 25 nts, or about 25 to 35 nts, or about 25 to 30 nts, or about 30 to 35 nts.

[0323] In some embodiments, thetargetbindingregion of a long primer is 15 to 40 nucleotides in length. In other embodiments, the target binding region of a long primer is about 20 to 40 nts, or about 20 to 35 nts, or about 20 to 30 nts, or about 30 to 40 nts, or about 30 to 35 nts, or about 35 to 40 nts.

[0324] In some embodiments, the adaptorregion and the targetbindingregion of alongprimer are spaced a distance apart, e.g., Pl comprises a sequence (e.g., a spacer) between the two regions that are at least 1 nucleotide apart, e.g., 1 nucleotide apart, 2, 3, 4, 5, 6, 7, 8, 9, 10, more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, etc.

[0325] In some embodiments, systems as described herein may comprise a longer primer and a shorter primer. In some embodiments, the longer primer is 20 to 40 nucleotides in length. In other embodiments, the longer primer is about 24 to 40 nucleotides, or about 25 to 35 nucleotides, or about25 to 30 nucleotides in length. In some embodiments, the shorter primer is 6 to 20 nucleotides in length. In other embodiments, the shorter primer is about 10 to 20 nucleotides, or about 10 to 15 nucleotides in length.

[0326] In an aspect described herein are systems comprising: a) a first oligonucleotide (Pl) having a sequence comprising a nucleotide sequence complementary to a portion of a forward strand 5’ of a target region; b) a second oligonucleotide (P2) having a sequence that is complementary to at least a portion of a reverse strand 3 ’ of the target region; wherein the set of nucleotides for Pl is at least partially 5’ to the set of nucleotides forP2; and c) an in-reaction probe (IrP) having a sequence comprising: i) a sequence complementary to atleast a portion of target nucleotide sequencedownstream of the binding sites of both Pl and a third primer (P3), and upstream of the binding site of P2; and a blocking feature that blocks extension of a DNA polymerase; wherein the system is for producing target ds amplicons and target ss amplicons having a sequence comprising at least the portion of the target sequence region. In some embodiments, Pl is a first primer. In some embodiments, P2 is a second primer.

[0327] Without wishing to limit the present disclosure to any theory or mechanism, the combination of a shorter primer and the in-reaction probe or oligonucleotide may increasespecificity and sensitivity compared to an asymmetric semi-nested isothermal nucleotide amplification without the combination of the shorter primer and the in-reaction probe oligonucleotide (see for instance Example 24 and FIG. 96A- FIG. 97B). In some embodiments, the shorter primer is from 6 to 24 nucleotides in length. In other embodiments, the shorter primer is about 10 to 24 nts, or about 10 to 22 nts, or about 10 to 20 nts, or about 10 to 18 nts, or about 10 to 16 nts, or about 10 to 14 nts, or about 10 to 12 nts, or about 12 to 24 nts, or about 12 to 22 nts, or about 12 to 20 nts, or about 12 to 18 nts, or about 12 to 16 nts, or about 12 to 14 nts, or about 14 to 24 nts, or about 14 to 22 nts, or about 14 to 20 nts, or about 14 to 18 nts, or about 14 to 16 nts, or about 16 to 24 nts, or about 16 to 22 nts, or about 16 to 20 nts, or about 16 to 18 nts, or about 18 to 24 nts, or about 18 to 22 nts, or about 18 to 20 nts, or about 20 to 24 nts, or about 20 to 22 nts, or about 22 to 24 nts. In some embodiments, P2 is 10-21 nucleotides in length.

[0328] In some emb odiments, one of the longer length primers i s 21 to 40 nucleotides in length. In other embodiments, one of the longer length primers is about 24 to 40 nts, or about 24 to 35 nts, or about24 to 30 nts in length. In some embodiments, one of the shorter length primers is 6 to 20 nucleotides in length. In other embodiments, one of the shorter length primers is about 6 to 15 nts in length, 10 to 18 nts, or about 10 to 15 nts in length.

[0329] In some specific embodiments, one of the primers is linked to the 5 ’ end of an annexing oligonucleotide (AO). In some embodiments, the primer sequence and the AO sequence do not overlap. In other embodiments, the primer sequence and the AO sequence overlap. The present disclosure may further feature a system of primers for inhibiting non -specific binding. The system may comprise a first primer (Pl) having a sequence comprising a 5’ adapter region and a target binding region downstream of the adapter region, a second primer (P2) having a sequence that is a set of nucleotides complementary to at least a portion of the adapter region, and an annexing oligonucleotide (AO). In some embodiments, Pl binds to a first complementary binding region (CSBR1) which is on a strand opposite the target region. In some embodiments, the AO has a sequence comprising a non -complementary tail at its 5’ end, a binding region having a sequence complementary to a portion of the adapter region and at least a portion of the TSBR1 of the target binding strand, and a blocker on a 3’ end of the invasion oligonucleotide. In some embodiments, the tail is non-complementary to the adapter region and TSBR1 of the target binding strand.

[0330] The present disclosure may further feature an oligonucleotide for inhibiting the nonspecific binding of a target nucleotide region. The oligonucleotide has a sequence comprising a non-complementary tail at its 5 ’ end, a binding region downstream of the non-complementary tail, and a blocker at its 3’ end. The non-complementary tail is non-complementary to the adapter region and nucleotides downstream of the adapter region.

[0331] In some embodiments, the in-reaction probe (IrP) or the annexing oligonucleotide (AO), comprise a stretch of nucleotide sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a region on a strand of the target nucleic acid sequence. In some embodiments, the IrP orthe AO, comprise a stretch of nucleotide sequencethatis identical to a region on a strand of the target nucleic acid sequence. In some embodiments, the stretch of nucleotide sequence in the IrP or AO that binds to a complementary target site is at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 5 5 contiguous nucleotides in length. In some embodiments, the complementary target site is at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 contiguous nucleotides in length. In some embodiments, the IrP or AO is designed with sequence aligned to a particular strand of a template sequence. In some embodiments, the IrP or AO is designed with sequence aligned to a strand designated the plus strand. In some embodiments, the IrP or AO is designed with sequence aligned to a strand designated the minus strand. In some embodiments, the IrP or AO can partially overlap with one of the primers. In some embodiments, this partial overlap can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. In some embodiments, the IrP or AO can fully overlap with one of the primers. In some embodiments, this full overlap can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. In some embodiments, the IrP or AO is flanked by Pl and P2, wherein at least part of the nucleotide sequence of the IrP or AO is located in between regions of Pl and P2 sequence of the target site. In some embodiments, the IrP or AO is flanked by Pl, P2, and P3, wherein at least part of the nucleotide sequence of the IrP or AO is located in between regions of Pl and P2 sequence of the target site.

[0332] In some embodiments, the in-reaction probe (IrP) comprises sequence complementary to at least a portion of the target amplicon region. In some embodiments, the IrP is an annexing oligonucleotide or annexing probe. In some embodiments, the IrP oligonucleotide is 20 to 75 nucleotides in length. In other embodiments, the IrP oligonucleotide is about 20 to 75 nucleotides, or about 20 to 70 nucleotides, or about 20 to 65 nucleotides, or about 20 to 60 nucleotides, or about 20 to 55 nucleotides, or about 25 to 50 nucleotides, or about 30 to 45 nucleotides, or about 30 to 40 nucleotides, or about 30 to 35 nucleotides, or about 35 to 75 nucleotides, or about 35 to 70 nucleotides, or about 35 to 65 nucleotides, or about 35 to 60 nucleotides, or about 35 to 55 nucleotides, or about 35 to 50 nucleotides, or about 35 to 45 nucleotides, or about 35 to 40nucleotides, or about 40 to 75 nucleotides, or about 40 to 70 nucleotides, or about 40 to 65 nucleotides, or about 40 to 60 nucleotides, or about 40 to 55 nucleotides, or about 40 to 50 nucleotides, or about 40 to 45 nucleotides, or about 45 to 75 nucleotides, or about 45 to 70 nucleotides, or about 45 to 65 nucleotides, or about 45 to 60 nucleotides, or about 45 to 55 nucleotides, or about 45 to 50 nucleotides in length. In some embodiments, the IrP comprises nucleotide sequence that is non -complementary to at least a portion of the target region nucleotide sequence. In some embodiments, the IrP comprises a blocking feature. In some embodiments, the in-reaction probe is long enough (greater than or equal to 20 nucleotides) to be an efficient substrate for the second enzyme having the DNA processing function and wherein the in-reaction probe becomes an annexing probe (AP) capable of invading the target dsDNA amplicon sequence and helping anneal P2 to target region when P2 is less than or equal to 15 nucleotides in length. In some embodiments, the in-reaction probe serves as an annexing oligo (AO).

[0333] In some embodiments, the in-reaction probe (IrP) comprises sequence complementary to at least a portion of the target amplicon region. In some embodiments, the IrP is an annexing oligonucleotide. In some embodiments, the IrP oligonucleotide is 20 to 75 nucleotides in length. In other embodiments, the IrP oligonucleotide is about 20 to 75 nts, or about 20 to 70 nts, or about 20 to 65 nts, or about 20 to 60 nts, or about 20 to 55 nts, or about 20 to 50 nts, or about 20 to 45 nts, or about 20 to 40 nts, or about 25 to 40 nts, or about 30 to 40 nts, about 30 to 35 nts, or about35 to 75 nts, or about 35 to 70 nts, or about 35 to 65 nts, or about 35 to 60 nts, or about 35 to 55 nts, or about 35 to 50 nts, or about 35 to 45 nts, or about 35 to 40 nts, or about 40 to 75 nts, or about 40 to 70 nts, or about 40 to 65 nts, or about 40 to 60 nts, or about 40 to 55 nts, or about 40 to 50 nts, or about 40 to 45 nts, or about45 to 75 nts, or about 45 to 70 nts, or about45 to 65 nts, or about 45 to 60 nts, or about 45 to 55 nts, or about 45 to 50 nts, In some embodiments, the IrP comprises nucleotide sequence that is non -complementary to at least a portion of the target region nucleotide sequence. In some embodiments, the IrP comprises a blocking feature. In some embodiments, the in-reaction probe is long enough (greater than or equal to 20 nts) to be an efficient substrate for the second enzyme having the DNA processing function and wherein the inreaction becomes an annexing probe (AP) capable of invading the target dsDNA amplicon sequence and helping anneal a primer to target region when the primer is less than or equal to 15 nucleotides in length. In some embodiments, the in-reaction probe serves as an annexing oligo (AO).

[0334] In some embodiments, one or more amplicons (ss amplicons, ds amplicons, and / or ss and ds amplicons) can be detected following a length of reaction time. In some embodiments, the IrP is bound to one or more amplicons, allowing for detection without an additional reaction stepthat modifies the one or more amplicons. In some embodiments, the in -reaction probe bound to target labelled ds amplicons is detected via a lateral flow assay (LFA). In some embodiments, the in-reaction probe bound to target labelled ss amplicons is detected via a lateral flow assay (LFA). In some embodiments, a 5’ end ofone ofthe primers is linked to the 5’ or 3 ’ end of the in -reaction probe. In some embodiments of methods described herein, the method comprises detecting the target ds amplicons. In some embodiments of methods described herein, the method comprises detecting the target ss amplicons. In some embodiments, the detecting comprises analyzing reaction products using a molecular lateral flow assay (LFA). In some embodiments, the detecting comprises analyzing reaction products using an intercalating dye or fluorophore. In some embodiments, the detecting comprises analyzing reaction products using a nucleic acid hybridization technique. In some embodiments, the detecting comprises analyzing reaction products using a fluorescence detection technique. In some embodiments, the detecting comprises analyzing reaction products using a colorimetric technique. In some embodiments, the detecting comprises analyzing reaction products using nucleic acid sequencing. In some embodiments, the detecting comprises analyzing reaction products usingDNA fragment size discrimination. In some embodiments, the detecting comprising determining a quantitative measure of an amplified reaction product comprising the target ds amplicons. In some embodiments, the detecting comprising determining a quantitative measure of an amplified reaction product comprising the target ss amplicons.

[0335] In some embodiments, a first oligonucleotide (Pl), a second oligonucleotide (P2) and an in-reaction probe (IrP) are provided to a target region of a nucleic acid sequence. In some embodiments, the Pl or the P2 does not overlap with the IrP. In some embodiments, the P2 sequence and the IrP oligonucleotide sequence do not overlap. In some embodiments, the P2 sequence and the IrP oligonucleotide sequence overlap. In some of the embodiments, P2 is about 10 to 24 nts, or about 10 to 22 nts, or about 10 to 20 nts, or about 10 to 18 nts, or about 10 to 16 nts, or about 10 to 14 nts, or about 10 to 12 nts, or about 12 to 24 nts, or about 12 to 22 nts, or about 12 to 20 nts, or about 12 to 18 nts, or about 12 to 16 nts, or about 12 to 14 nts, or about 14 to 24 nts, or about 14 to 22 nts, or about 14 to 20 nts, or about 14 to 18 nts, or about 14 to 16 nts, or about 16 to 24 nts, or about 16 to 22 nts, or about 16 to 20 nts, or about 16 to 18 nts, or about 18 to 24 nts, or about 18 to 22 nts, or about 18 to 20 nts, or about 20 to 24 nts, or about 20 to 22 nts, or about 22 to 24 nts. In some embodiments, P2 is 15-20 nucleotides in length. In some embodiments, P2 is from 10 to 24 nucleotides in length. In some embodiments, P2 is 10 -21 nucleotides in length. In some embodiments, Pl is at least 20 nucleotides (nts) in length without an adapter region. In some embodiments, Pl is 50 to 75 nucleotides in length with an adaptorregion. In other embodiments, Pl is about 50 to 75 nts, or about 50 to 70 nts, or about 50 to 65 nts, or about 50 to 60 nts, or about 50 to 55 nts, or about 55 to 75 nts, or about 55 to 70 nts, or about 55 to 65 nts, or about 55 to 60 nts, or about 60 to 75 nts, or about 60 to 70 nts, or about 60 to 65 nts, or about 65 to 75 nts, or about 65 to 70 nts, or about 70 to 75 nts in length with an adaptor region. In some embodiments, the adapter region of Pl is 10 to 40 nucleotides in length. In other embodiments, the adapter region of Pl is about 10 to 35 nts, or about 10 to 30 nts, or about 10 to 25 nts, or about 10 to 20 nts, or about 10 to 15 nts, or about 15 to 35 nts, or about 15 to 30 nts, or about 15 to 25 nts, or about 15 to 20 nts, or about 20 to 35 nts, or about 20 to 30 nts, or about 20 to 25 nts, or about 25 to 35 nts, or about 25 to 30 nts, or about 30 to 35 nts. In some embodiments, the target binding region of Pl is 25 to 40 nucleotides in length. In other embodiments, the target binding region of Pl is about 25 to 40 nts, or about 25 to 35 nts, or about 25 to 30 nts, or about 30 to 40 nts, or about 30 to 35 nts, or about 35 to 40 nts. In some embodiments, the adaptor region and the target binding region of Pl are spaced a distance apart, e.g., Pl comprises a sequence (e.g, a spacer) between the two regions that are at least 1 nucleotide apart, e.g., 1 nucleotide apart, 2, 3, 4, 5, 6, 7, 8, 9, 10, more than 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19 20, 21, 22, 23, 24, 25, etc. In some embodiments, Pl and P2 are primers. In some embodiments, a third primer is provided to the target region of the nucleic acid sequence. In some embodiments, the third primer is 25 to 40 nucleotides in length. In other embodiments, the third primer is about 25 to 40 nts, or about 25 to 35 nts, or about 25 to 30 nts in length. In some embodiments, the third primer is 10 to 20 nucleotides in length. In other embodiments, the third primer is about 10 to 20 nts, or about 10 to 15 nts in length. In some embodiments, the IrP oligonucleotide is 30 to 75 nucleotides in length. In other embodiments, the IrP oligonucleotide is about 30 to 75 nts, or about 3 Oto 70 nts, or about 30 to 65 nts, or about 30 to 60 nts, or about 30 to 55 nts, or about 30 to 50 nts, or about 30 to 45 nts, or about 30 to 40 nts, or about 30 to 35 nts, or about 35 to 75 nts, or about 35 to 70 nts, or about 35 to 65 nts, or about 35 to 60 nts, or about 35 to 55 nts, or about 35 to 50 nts, or about 35 to 45 nts, or about 35 to 40 nts, or about 40 to 75 nts, or about 40 to 70 nts, or about 40 to 65 nts, or about 40 to 60 nts, or about 40 to 55 nts, or about 40 to 50 nts, or about 40 to 45 nts, or about 45 to 75 nts, or about 45 to 70 nts, or about 45 to 65 nts, or about 45 to 60 nts, or about 45 to 55 nts, or about 45 to 50 nts, or about 50 to 75 nts, or about 50 to 70 nts, or about 50 to 65 nts, or about 50 to 60 nts, or about 50 to 55 nts, or about 55 to 75 nts, or about 55 to 70 nts, or about 55 to 65 nts, or about 55 to 60 nts, or about 60 to 75 nts, or about 60 to 70 nts, or about 60 to 65 nts, or about 65 to 75 nts, or about 65 to 70 nts, or about 70 to 75 nts in length. In some embodiments, wherein a 3 ’ end of P2 is linked to the 5’ end of the IrP oligonucleotide. In some embodiments, the IrP oligonucleotide improves amplification efficiency. In some embodiments, the IrP oligonucleotideimproves amplification specificity. In some embodiments, the IrP oligonucleotide is required for efficient amplification.

[0336] In some embodiments, one or more the primers or IrP has a non -complementary 5’ adaptor region. In some embodiments, one or more of the primers or IrP has a non-complementary 5 ’ adaptor region and has an additional primer that can bind to the adaptor region only. In some embodiments, Pl andP2 areprimers. In some embodiments, Pl andP2 are added at a concentration to the reaction admixture in molar excess relative to the target nucleic acid sequence. In some embodiments, primers Pl, P2, and a third primer are added at a concentration to the reaction admixture in molar excess relative to the target nucleic acid sequence.

[0337] One of the unique and inventive technical features of the present disclosure is the use of a first forward primer (FP1), a second forward primer (FP2), wherein FP2 is downstream from FP1, and a reverse primer (RP) or the use of a first reverse primer (RP1), a second reverse primer (RP2) wherein RP2 is upstream from RP1, and a forward primer (FP1) in addition to the use of a specific ratio of the forward primers to reverse primer for each primer set. Additionally, the primer design process is similar to that of PCR, instead of complicated methods used for other INAs, such as LAMP, RCA, SIBA. The semi-nested approach also increases the specificity of the amplification over a non-nested approach, while the specific ratio of the primers increases the amplification rate of the single strand target region compared to traditional asymmetric amplification strategies. In some embodiments, the IrP acts as an invasion oligonucleotide. In some embodiments, a 3’ end of P2 is linked to the 5’ end of the IrP oligonucleotide . In some embodiments, the IrP oligonucleotide improves amplification efficiency. In some embodiments, the IrP oligonucleotide improves amplification specificity.Labels

[0338] The present disclosure provides labels, such as labels used in the detection of nucleic acid molecules. In some embodiments, the oligonucleotide comprises a label. In some embodiments, the label is located on a 5 ' end of the oligonucleotide, a 3 ' end of the oligonucleotide, or anywhere in between. In some embodiments, the label allows extension of a DNA polymerase. In some embodiments, the label comprises a fluorescein or derivative thereof. In some embodiments, the label comprises a xanthene core or derivative thereof. In some embodiments, the label comprises a fluorophore, or a redox molecule, or biotin, or digoxigenin. In some embodiments, the label comprises a fluorophore or biotin or digoxigenin. In some embodiments, the label comprises a redox molecule.

[0339] In some embodiments, the Pl , the P2, or the third oligonucleotide comprises a label. In some embodiments, the third oligonucleotide comprises the label. In some embodiments, the labelis located on a 5' end of the third oligonucleotide, a3'end of the third oligonucleotide, or anywhere in between the 5’ end and the 3’ end. In some embodiments, the third oligonucleotide comprises a label at its 5’ end. In some embodiments, the label comprises a fluorescein or derivative thereof, a fluorophore, a chromophore, a redox molecule, a fluorescence quencher, a biotin, a digoxigenin, or any combination thereof. In some embodiments, the label comprises a xanthene core or a derivative thereof. In some embodiments, the label comprises a carboxyfluorescein (FAM). In some embodiments, the label comprises a fluorescein isothiocyanate (FITC). In some embodiments, the label comprises a biotin. In some embodiments, the label comprises a digoxigenin. In some embodiments, the Pl or the P2 comprise a label. In some embodiments, the label is located on a 5' end, a 3' end, or anywhere in between the 5’ end and the 3’ end of the Pl or the P2. In some embodiments, the label comprises a fluorescein or derivative thereof, a fluorophore, a chromophore, a redox molecule, a fluorescence quencher, a biotin, a digoxigenin, or any combination thereof. In some embodiments, the label comprises a biotin. In some embodiments, the label comprises a digoxigenin. In some embodiments, the label comprises a carboxyfluorescein (FAM). In some embodiments, the label comprises a fluorescein isothiocyanate (FITC). In some embodiments, the fourth oligonucleotide comprises a label. In some embodiments, the label comprises a fluorescein or derivative thereof, a fluorophore, a chromophore, a redox molecule, a fluorescence quencher, a biotin, a digoxigenin, or any combination thereof.

[0340] In some embodiments, the third oligonucleotide is an annexing oligonucleotide (AO). In some embodiments, the AO comprises a label and is termed an annexing probe (AP). In some embodiments, the AO comprises a label allowing the labeled AO to function as an in-reaction probe (IrP). In some embodiments, the AO comprises a label allowing the labeled AO to function as an in-reaction probe (IrP). In some embodiments, one of the primers is 6-21 nucleotides in length. In some embodiments, the AO is labeled. In some embodiments, the AO is labeled with a fluorophore, redox molecule, or biotin. In some embodiments, the in -reaction probe comprises a label. In some embodiments, the label comprises a fluorophore or a redox molecule or biotin or digoxigenin. In some embodiments, the label comprises a fluorophore. In some embodiments, the label comprises a redox molecule. In some embodiments, at least one primer comprises a label either on the 5 ’ end or internally in the sequence so as not to prevent extension by polymerase but leading to amplification of labelled ss and ds amplicons. In some embodiments, Pl is a primer. In some embodiments, P2 is a primer. In some embodiments, Pl , P2 and a third primer are provided to the target nucleic acid sequence. In some embodiments, the label on the Pl , the P2, and / or the third primer comprises a fluorophore or a redox molecule or biotin or digoxigenin. In some embodiments, the label on the Pl, the P2, and / or the third primer comprises biotin. In someembodiments, the primers may be labeled with a fluorophore (e.g., 6 -FAM) such that when a probe is labeled with an appropriate quencher (e.g., BHQ 1 ), the decrease in fluorescence can be measured to detect amplification in a qualitative or quantitative manner. In other embodiments, the primers may be labeled with a fluorophore (e.g., Alexa-488), such that if a probe is labeled with an appropriate fluorophore (e.g., Alexa-555), then an increase in emission of probe tag (e.g., Alexa- 555) on excitation of primer tag (e.g., Alexa-488), e.g., a FRET assay, can be used to detect amplification in a qualitative or quantitative manner. In further embodiments, the primers may be labeled with a tag (e.g., biotin) such that a probe labeled with FITC can be used to conduct lateral flow assay with the strips containing biotin -ligand and gold nanoparticles labeled with anti -FITC antibodies and can be used to detect amplification in a qualitative or quantitative manner.

[0341] In some embodiments, the IrP oligonucleotide comprises a label. In some embodiments, the label comprises a fluorophore or biotin. In some embodiments, the IrP oligonucleotide is required for efficient amplification. In some embodiments, the IrP oligonucleotide comprises a label. In some embodiments, the label comprises a fluorophore or biotin. In some embodiments, the label comprises a redox molecule.

[0342] A label used in the systems, devices, methods, and kits as described herein can be any known oligonucleotide label. The following are non -limiting examples of labels that can be used in the systems, devices, methods, and kits as described herein. In some embodiments, a labeled oligonucleotide (e.g., labeled annexing oligonucleotide) decreases non-specific amplification relative to an unlabeled oligonucleotide. In some embodiments, the label comprises a xanthene core ora derivative thereof. In some embodiments, an oligonucleotide labeled with a xanthene core label (e.g., xanthene core labeled annexing oligonucleotide) decreases non-specific amplification relative to an oligonucleotide labeled with a non -xanthene core label. In some embodiments, the label comprises carb oxy fluorescein (FAM).Enzyme Mix

[0343] The present disclosure provides systems as disclosedherein comprising an enzymemix. In some embodiments, the enzyme mix comprises a DNA polymerase or functional fragment thereof, a second enzyme having a DNA processing function, one or more enzymes (e.g., Reverse Transcriptase, Creatine Kinase), one or more nucleotides in an amount sufficient to support amplification of the target nucleic acid sequence, one or more salts (e.g. Magnesium Acetate, Potassium Acetate, Phosphocreatine) in an amount sufficient to support amplification of the target nucleic acid sequence, a single-strand binding protein, a crowding reagent, or any combination thereof.

[0344] In some embodiments, the system comprises a crowding agent. As used herein, a “crowding agent” refers to inert, non-charged polymers of certain sizes, that occupy space but do not interact with target proteins. Non-limiting examples of crowding agents that may be used in the present disclosure include, but are not limited to, polyvinylpyrr...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of amplifying a target nucleic acid sequence at an ambient temperature, the method comprising: a) binding a first oligonucleotide (Pl) primer to a strand of the target nucleic acid sequence; b) binding a second oligonucleotide (P2) primer to another strand of the target nucleic acid sequence; c) binding a third oligonucleotideto the target nucleic acid sequence, wherein the third oligonucleotide comprises a 3' end blocking feature that blocks extension of the third oligonucleotide by a polymerase, and wherein the third oligonucleotide does not facilitate a localizing and a binding of the Pl or the P2 to the target nucleic acid sequence; d) providing a polymerase and a DNA processing enzyme to the target nucleic acid sequence; and e) extending a 3 ' end of the Pl and a 3 ' end of the P2, thereby amplifying the target nucleic acid sequence at the ambient temperature.

2. A method of amplifying a target nucleic acid sequence at an ambient temperature, the method comprising: a) binding a first oligonucleotide primer (Pl) to a strand of the target nucleic acid sequence; b) binding a second oligonucleotide primer (P2) to another strand of the target nucleic acid sequence; c) binding a third oligonucleotideto the target nucleic acid sequence, wherein the third oligonucleotide comprises a 3' end blocking feature that blocks extension of the third oligonucleotide by a polymerase, and wherein a 5’ end of the third oligonucleotide and a 5' end of the Pl bind to the strand of the target nucleic acid sequence at a distance of about 3 nucleotides to about 25 nucleotides apart; d) providing a polymerase and a DNA processing enzyme to the target nucleic acid sequence; and e) extending a 3 ' end of the Pl and a 3 ' end of the P2, thereby amplifying the target nucleic acid sequence at the ambient temperature.

3. The method of claim 1 or 2, wherein the Pl localizesandbindstothe strandofthetargetnucleic acid sequence independently of the third oligonucleotide.

4. The method of any one of the preceding claims, wherein the P2 localizes and binds to the another strand of the target nucleic acid sequence independently of the third oligonucleotide.

5. The method of any one of the preceding claims, wherein a 5 ’ end of the third oligonucleotide binds to the strand of the target nucleic acid sequence.

6. The method of claim 1, wherein the 5’ end of the third oligonucleotide and a 5' end of the Pl bind to the strand of the target nucleic acid sequence at a distance from about 3 nucleotides to about 25 nucleotides apart.

7. The method of claim 2 or 6, wherein the 5 ' end of the third oligonucleotide and the 5 ' end of thePl bindtothe strand ofthe target nucleic acid sequence at a distance ofab out 15 nucleotides apart.

8. The method of claim 2 or 6, wherein the 5 ' end of the third oligonucleotide and the 5 ' end of thePl bindtothe strand ofthe target nucleic acid sequence at a distance of about 10 nucleotides apart.

9. The method of claim 2 or 6, wherein the 5 ' end of the third oligonucleotide and the 5 ' end of the Pl bind to the strand of the target nucleic acid sequence at a distance of about 8 nucleotides apart.

10. The method of any one ofthe preceding claims, wherein the 5' end of the third oligonucleotide and the 3 ’ end of Pl bind to the strand of the target nucleic acid sequence with 1 base pair (bp) of overlap.11 . The method of any one of the preceding claims, wherein the 5' end of the third oligonucleotide and the 3 ’ end of Pl are adjacent and bind to the strand of the target nucleic acid sequence.

12. The method of any one of the preceding claims, wherein the 5' end of the third oligonucleotide and the 3 ’ end of Pl bind to the strand of the target nucleic acid sequence with a gap in contiguous sequence of the target nucleic acid sequence of between about 1 -9 bp.

13. The method of any one of the preceding claims, wherein the 3 ' end blocking feature that blocks extension of the third oligonucleotide by a DNA polymerase comprises an inverted nucleotide or a C3 -spacer.

14. The method of any one ofthe preceding claims, wherein the Pl comprises a sequence at least about 80% identical to a region on the strand of the target nucleic acid sequence .

15. The method of any one of the preceding claims, wherein the P2 comprises a sequence at least about 80% identical to a region on the another strand of the target nucleic acid sequence .

16. The method of any one of the preceding claims, wherein the third oligonucleotide comprises a sequence at least about 80% identical to a region of the target nucleic acid sequence.

17. The method of any one of the preceding claims, further comprising providing the Pl, the P2, and the third oligonucleotide in a set of oligonucleotides prior to the binding of the Pl, the P2, and the third oligonucleotide to the target nucleic acid sequence.

18. The method of any one of the preceding claims, wherein the DNA processing enzyme is comprised in an enzyme mix.

19. The method of claim 18, wherein the enzyme mix comprises a DNA polymerase or functional fragmentthereof, the DNAprocessing enzyme, oneor more nucleotides in an amount sufficient to support amplification of the target nucleic acid sequence, a single -strand binding protein, a crowding reagent, a magnesium salt or any combination thereof.

20. The method of claim 19, wherein the DNA processing enzyme facilitates the binding of the Pl, P2, or the third oligonucleotide to the target region.21 . The method of claim 19, wherein the DNA processing enzyme facilitates the binding of the third oligonucleotide to the target nucleic acid sequence.

22. The method of any one of the preceding claims, wherein the method of amplification results in an amplified target nucleic acid sequence.

23. The method of claim 22, wherein the method of amplification results in target single-stranded (ss) amplicons.

24. The method of claim 22, wherein the method of amplification results in target double-stranded (ds) amplicons.

25. The method of claim 22, wherein the method of amplification results in target ds amplicons and target ss amplicons.

26. The method of any one of the preceding claims, wherein the Pl, the P2, or the third oligonucleotide comprises a label.

27. The method of claim 26, wherein the third oligonucleotide comprises the label.

28. The method of claim 27, wherein the label is located on a 5 ' end of the third oligonucleotide, a 3 ' end of the third oligonucleotide, or anywhere in between the 5’ end and the 3 ’ end.

29. The method of claim 27, wherein the third oligonucleotide comprises a label at its 5’ end.

30. The method of claim 29, wherein the label comprises a fluorescein or derivative thereof, a fluorophore, a chromophore, a redox molecule, a fluorescence quencher, a biotin, a digoxigenin, or any combination thereof.

31. The method of any one of claims 26-30, wherein the label comprises a xanthene core or a derivative thereof.

32. The method of claim 30, wherein the label comprises a carboxyfluorescein (FAM).

33. The method of claim 26, wherein the Pl or the P2 comprise a label.

34. The method of claim 33, wherein the label is located on a 5 ' end, a 3 ' end, or anywhere in between the 5’ end and the 3’ end of the Pl or the P2.

35. The method of any one of claims 33-34, wherein the label comprises a fluorescein or derivative thereof, a fluorophore, a chromophore, a redox molecule, a fluorescence quencher, a biotin, a digoxigenin, or any combination thereof.

36. The method of claim 35, wherein the label comprises a biotin or digoxigenin.

37. The method of any one of claims 33-36, wherein the label allows for extension of an end of the Pl or the P2 by a DNA polymerase.

38. The method of any one of the preceding claims, wherein the third oligonucleotide can form a binding complex with the nucleic acid molecule.

39. The method of any one of the preceding claims, wherein the third oligonucleotide promotes the localizing and binding of the Pl to the strand of the target nucleic acid sequence.

40. The method of claim 39, wherein the third oligonucleotide facilitates a localizing and a binding of P2 to the target nucleic acid sequence.41 . The method of claim 39, wherein the third oligonucleotide promotes annealing specificity of Pl or P2 or both to the target nucleic acid sequence.

42. The method of claim 39 or 40, wherein the third oligonucleotide promotes efficiency of the binding of Pl or P2 or both to the target nucleic acid sequence.

43. The method of any one of the preceding claims, wherein the third oligonucleotide binds between the Pl and the P2 bound to the target nucleic acid sequence.

44. The method of any one of the preceding claims, wherein the third oligonucleotide binds to a region at least partially overlapping with the Pl bound to the target nucleic acid sequence.

45. The method of any one of the preceding claims, wherein the Pl binds to a different region of the nucleic acid molecule than the third oligonucleotide.

46. The method of any one of the preceding claims, wherein the P2 binds to a different region of the target nucleic acid sequence than the third oligonucleotide.

47. The method of any one of the preceding claims, wherein the third oligonucleotide binds to a region at least partially overlapping with the P2 bound to the target nucleic acid sequence.

48. The method of any one of the preceding claims, further comprising providing a fourth oligonucleotide, wherein the fourth oligonucleotide does not promote the localizing and binding of the Pl to the strand of the target nucleic acid sequence.

49. The method of claim 48, wherein the fourth oligonucleotide binds to the target nucleic acid sequence between the Pl and the P2 that are bound to the target nucleic acid sequence.

50. The method of claim 48, wherein the fourth oligonucleotide binds to the target nucleic acid sequence upstream or downstream of both the Pl and the P2 bound to the target nucleic acid sequence.51 . The method of any one of claims 48-50, wherein the DNA processing enzyme facilitates the binding of the fourth oligonucleotide to the target nucleic acid sequence.

52. The method of any one of claims 48-51, wherein the Pl binds to a different region of the target nucleic acid sequence than the fourth oligonucleotide.

53. The method of any one of claims 48-52, wherein the P2 binds to a different region of the target nucleic acid sequence than the fourth oligonucleotide.

54. The method of any one of claims 48-53, wherein the fourth oligonucleotide comprises a nucleotide sequence at least about 80% identical to a region on the strand or the another strand of the target nucleic acid sequence.

55. The method of any one of claims 48-54, wherein the fourth oligonucleotide comprises a label.

56. The method of claim 55, wherein the label is located on a 5 ' end, a 3 ' end, or anywhere in between the 5’ end and the 3’ end of the fourth oligonucleotide.

57. The method of claim 55 or 56, wherein the label comprises a fluorescein or derivative thereof, a fluorophore, a chromophore, a redox molecule, a fluorescence quencher, a biotin, a digoxigenin, or any combination thereof.

58. The method of any one of the preceding claims, wherein the method further comprises providing to the nucleic acid molecule a crowding reagent.

59. The method of claim 58, wherein the crowding reagent comprises polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), Ficoll, Dextran, or any combination thereof.

60. The method of claim 59, wherein the crowding reagent comprises PEG.61 . The method of any one of claims 1-60, wherein the polymerase comprises a DNA polymerase or functional fragment thereof.

62. The method of claim 61, wherein the DNA polymerase or functional fragment thereof is a strand-displacing DNA polymerase.

63. The method of claim 61 or 62, wherein the DNA polymerase comprises Bacillus subtilis DNA polymerase I (Bsu), Sau, Bst, or Klenow Fragment.

64. The method of claim 63, wherein the DNA polymerase comprises Bacillus subtilis DNA polymerase I (Bsu).

65. The method of any one of the preceding claims, wherein the DNA processing enzyme comprises a recombinase or functional fragment thereof, a helicase or functional fragmentthereof, a topoisomerase or functional fragment thereof, single-stranded binding protein or functional fragment thereof, or any combination thereof.

66. The method of any one of the preceding claims, wherein the DNA processing enzyme comprises a recombinase protein.

67. The method of any one of the preceding claims, wherein the DNA processing enzyme comprises UvsX DNA recombinase (UvsX).

68. The method of any one of the preceding claims, wherein the DNA processing enzyme comprises bacterial recombinase A (RecA).

69. The method of any one of the preceding claims, wherein the DNA processing enzyme comprises T4 GP32, E. coli SSB, HELB helicase, RTEL1 helicase, Pifl helicase, Topi topoisomerase, or topoisomerase Illa.

70. The method of any one of the preceding claims, wherein the DNAprocessing enzyme facilitates binding or annealing of one or more of the Pl , the P2, the third oligonucleotide, and the fourth oligonucleotide to the target nucleic acid sequence.

71. The method of any one of the preceding claims, wherein the DNA processing enzyme comprises a plurality of DNA processing enzymes.

72. The method of any one of the preceding claims, wherein the DNA processing enzyme comprises a first DNA processing enzyme, a second DNApro cessing enzyme, and a third DNA processing enzyme.

73. The method of claim 72, where the first DNAprocessing enzyme is a single-stranded binding protein.

74. The method of claim 73, wherein the single-stranded binding protein comprises T4 GP32.

75. The method of any one of the preceding claims, wherein the method further comprises providing to the target nucleic acid sequence a reverse transcriptase (RT) or a functional fragment thereof to reverse transcribe an RNA template to a cDNA molecule, wherein the cDNA template comprises the target nucleic acid sequence.

76. The method of claim 75, wherein the RT uses one or more of the Pl, the P2 and the fourth oligonucleotide as primers for a first strand cDNA synthesis.

77. The method of claim 75 or 76, wherein the RT uses a set of random primers provided in a reaction mix as primers for a first strand cDNA synthesis.

78. The method any one of claims 75-77, wherein the RT is M-MuLV RT, AMV RT, SuperScript II RT, SuperScript III RT, SuperScript IV RT, or any other RT capable of reverse transcribing in ambient temperatures.

79. The method of any one of the preceding claims, wherein the method further comprises providing to the target nucleic acid sequence an RnaseH enzyme or a functional fragment thereof to promote reverse transcription.

80. The method of any one of the preceding claims, wherein the Pl, the P2, the third oligonucleotide, the fourth oligonucleotide or any combination thereof has a non- complementary 5' adaptor region.

81. The method of claim 80, wherein the Pl, the P2, the third oligonucleotide, the fourth oligonucleotide or any combination thereof has a non -complementary 5' adaptor region.

82. The method of any one of the preceding claims, wherein the Pl, the P2, the third oligonucleotide, or the fourth oligonucleotide comprises a length of at least about 6 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 60 nucleotides, at least about 80 nucleotides, or more.

83. The method of any one of the preceding claims, wherein the Pl or the P2 is a nucleotide length insufficient to produce amplicons of the target nucleic acid sequence without the presence of the third oligonucleotide.

84. The method of any one of the preceding claims, wherein thePl and theP2 are present in molar excess relative to the target nucleic acid molecule.

85. The method of any one of the preceding claims, wherein the method further comprises an incubation step for a length of time sufficient to replicate and amplify the target nucleic acid sequence.

86. The method of claim 85, wherein an incubation temperature is within 15 -45 °C for the length of time.

87. The method of claim 86, wherein the incubation temperature is within 20-37°C for the length of time.

88. The method of any one of claims 86 or 87, wherein the incubation temperature is maintained within 22-25 °C for the length of time.

89. The method of any one of claims 86-88, wherein the incubation temperature is room temperature for the length of time.

90. The method of any one of claims 86-89, wherein the incubation temperature is maintained under isothermal conditions without previously denaturing the nucleic acid molecule.

91. The method of claim 90, wherein maintaining isothermal conditions does not involve incubation in a temperature-regulating or temperature-maintaining device.

92. The method of claim 90 or 91, wherein maintaining isothermal conditions does not involve incubation in an incubator.

93. The method of any one of the proceeding claims, wherein amplification of the target nucleic acid region is conducted in an absence of a thermocycler.

94. The method of any one of claims 85-93, wherein the incubation remains within 10% of the starting temperature until sufficient amplification of the target nucleic acid sequence to be detected is achieved.

95. The method of any one of claims 85-94, wherein the length of time is between about 1-60 minutes.

96. The method of any one of claims 85-95, wherein the length of time is between about 10-40 minutes.

97. The method of any one of claims 85-96, wherein the length of time is between 20-30 minutes.

98. The method of any one of claims 85-96, wherein the length of time is about 20 minutes.

99. The method of any one of claims 85-96, wherein the length of time is about 30 minutes.

100. The method of any one of the proceeding claims, wherein the method further comprises detecting an amplified target nucleic acid sequence.

101. The method of claim 100, wherein a presence of a third oligonucleotide comprising a label increases production of the target ds amplicons.

102. The method of claim 100, wherein a presence of a third oligonucleotide comprising a label increases production of the target ss amplicons.

103. The method of any one of claims 100- 102, wherein the target ss and ds amplicons or the two target ds amplicons are produced at least at a linear rate of amplification.

104. The method of any one of claims 100-103, wherein the target ds amplicons or the two target ds amplicons are produced at an exponential rate of amplification.

105. The method of any one of claims 100-104, wherein the target ss amplicons are produced at least at a linear rate of amplification.

106. The method of any one of claims 100-105, wherein the target ss amplicons are produced at an exponential rate of amplification.

107. The method any one of claims 100-106, wherein the method further comprises detecting target ss amplicons, target ds amplicons, or target ss amplicons and target ds amplicons via a third oligonucleotide comprising a label.

108. The method of any one of claims 100-107, wherein a third oligonucleotide comprising a label can detect two target amplicons, and wherein the detection of the two target ampliconsproduces increased sensitivity of target nucleic acid sequence detection compared to detecting single target amplicons.

109. The method of any one of claims 100-108, wherein a 5' end of atleast one of the Pl orthe P2 is linked to the 5 ' or 3 ' end of the fourth oligonucleotide.

110. The method of any one of claims 100-109, comprising detecting the target ds amplicons.

111. The method of any one of claims 100-110, comprising detecting the target ss amplicons.

112. The method of any one of claims 100-111, wherein the detecting comprises analyzing reaction products using a molecular lateral flow assay (LFA).

113. The method of any one of claims 100-111, wherein the detecting comprises analyzing reaction products using an intercalating dye or fluorophore.

114. The method of claim 113, comprising use of FRET in the detecting.

115. The method of any one of claims 48-114, wherein the Pl or P2 or fourth oligonucleotide comprises a fluorophore label or fragment thereof for FRET detection of a labeled third oligonucleotide.

116. The method of any one of claims 48-114, wherein the Pl, the P2, or the fourth oligonucleotide comprises a quencher molecule label for fluorescence quenching detection of a labeled third oligonucleotide.

117. The method of any one of claims 100-116, wherein the detecting comprises analyzing reaction products using a nucleic acid hybridization technique.

118. The method of any one of claims 100-116, wherein the detecting comprises analyzing reaction products using a fluorescence detection technique.

119. The method of any one of claims 100-116, wherein the detecting comprises analyzing reaction products using a colorimetric technique.

120. The method of any one of claims 100-116, wherein the detecting comprises analyzing reaction products using nucleic acid sequencing.

121. The method of any one of claims 100-116, wherein the detecting comprises analyzing reaction products using DNA fragment size discrimination.

122. The method of any one of claims 100-116, wherein an oligonucleotide probeis added to ss amplicons or ds amplicons produced from the reaction to aid in the detecting.

123. The method of any one of claims 100-116, wherein a separate oligonucleotide probe comprising a quencher is added to ss amplicons or ds amplicons produced from the reaction to aid in the detecting.

124. The method of any one of claims 100-123, wherein the detecting comprises determining a quantitative measure of the target template in a sample.

125. The method of any one of claims 100-124, wherein the detecting comprises determining a quantitative measure of the target template present in a sample by using nucleic acid standards of known quantity.

126. The method of any one of claims 100-125, wherein the target ds amplicons are amplified and detected in real time.

127. The method of any one of claims 100-126, wherein the target ss amplicons and target ds amplicons are amplified and detected in real time.

128. The method of claim 126 or 127, wherein target ds amplicons are amplified and detected in less than about 60 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 12 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, or 3 minutes.

129. The method of claim 126 or 127, wherein target ss amplicons are amplified and detected in less than about 60 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 12 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, or 3 minutes.

130. The method of any one of claims 100-129, further comprising detection of two different regions of the target nucleic acid sequence.

131. The method of any one of claims 100-130, wherein two or more distinct pathogenic variants are detected within a single amplification reaction.

132. The method of any one of claims 100-131, wherein two or more distinct disease mutations are detected within a single amplification reaction.

133. The method of any one of claims 100-132, wherein a single nucleotide polymorphism (SNP) within the target nucleic acid sequence is detected.

134. The method of any one of the preceding claims, wherein a nucleic acid molecule is extracted from a sample.

135. The method of claim 134, wherein the nucleic acid molecule extracted from the sample comprises DNA or RNA.

136. The method of claim 135, wherein the nucleic acid molecule extracted from the sample comprises the extracted nucleic acid molecule comprises dsDNA, ssDNA, circulating DNA, cell free DNA, cDNA, plasmid DNA, mRNA, miRNA, tRNA, viral RNA, siRNA, sgRNA, or any combination thereof.

137. The method of claim 136, wherein the nucleic acid molecule extracted from the sample comprises DNA.

138. The method of claim 137, wherein the nucleic acid molecule extracted from the sample comprises double-stranded DNA.

139. The method of claim 137, wherein the nucleic acid molecule extracted from the sample comprises single-stranded DNA.

140. The method of claim 136, wherein the nucleic acid molecule extracted from the sample comprises RNA.

141. The method of any one of the preceding claims, wherein a template to the target nucleic acid sequence is one or more copies of a target genome, and wherein the target nucleic acid sequence is amplified at about room temperature to a level of a detectable range.

142. The method of any one of the preceding claims, wherein the method comprises amplification and detection of presence of a target nucleic acid in the extracted sample.

143. The method of any one of claims 134-142, wherein the sample is obtained from a biological fluid, a tissue sample, a mammalian sample, an animal sample, a eukaryotic sample, a bacterial sample, a viral sample, a plant sample, a bacterial cell, a eukaryotic cell, an animal tissue, whole blood, saliva, urine, a nasal swab, a buccal swab, a tongue swab, a cervical fluid, a vaginal fluid, an animal cell, a muscle tissue, a spleen tissue, a skin tissue, or a liver tissue.

144. The method of any one of claims 134-142, wherein the sample comprises nucleic acid extracted from a subject.

145. The method of claim 144, wherein the nucleic acid extracted from the subject comprises nucleic acid derived from a pathogen.

146. The method of claim 144 or 145, wherein the pathogen is a virus.

147. The method of claim 145, wherein the virus is listed in Table 1.

148. The method of claim 144 or 145, wherein the pathogen is a bacterium.

149. The method of claim 148, wherein the bacterium is listed in Table 2.

150. The method of claim 144 or 145, wherein the pathogen is a fungus.

151. The method of claim 150, wherein the fungus is listed in Table 3.

152. The method of claim 144 or 145, wherein the pathogen is an animal pathogen.

153. The method of claim 152, wherein the animal pathogen is listed in Table 4.

154. The method of any one of claims 134-142, wherein the sample is obtained from an animal.

155. The method of claim 154, wherein the animal is a livestock species.

156. The method of claim 155, wherein the livestock species is a type of cattle, horse, goat, sheep, pig, swine, rabbit, donkey, mule, bison, llama, cat, dog, deer, camel, yak, turkey, alpaca, elk, chicken, duck, emu, ostrich, pheasant, goose, fish, shrimp, crustacean, mosquito, fly, or bee.

157. The method of claim 154, wherein the animal is a human.

158. The method of any one of claims 134-142, wherein the sample is obtained from plant.

159. The method of any one of the preceding claims, wherein the target nucleic acid sequence is a selected region of a genomic DNA sequence from a pathogen.

160. The method of any one of the preceding claims, wherein the target nucleic acid sequence is a selected region of an RNA sequence from a pathogen.

161. The method of claim 159 or 160, wherein the target nucleic acid sequence is from a pathogen listed in Table 1, Table 2, Table 3, Table 4, or Table 5.

162. The method of any one of claims 159-161, wherein the target nucleic acid sequence is amplified in the presence of host genomic DNA.

163. A system of amplifying a target nucleic acid sequence at an ambient temperature, the system comprising: a) a first oligonucleotide (Pl) primer; b) a second oligonucleotide (P2) primer; c) a third oligonucleotide comprising a 3 ' end blocking feature that blocks extension of the third oligonucleotide by a polymerase, wherein the third oligonucleotide does not facilitate a localizing and a binding of the Pl or the P2 to the target nucleic acid sequence; d) a polymerase; and e) a DNA processing enzyme.

164. A system of amplifying a target nucleic acid sequence at an ambient temperature, the system comprising: a) a first oligonucleotide (Pl) primer; b) a second oligonucleotide (P2) primer; c) a third oligonucleotide comprising a 3 ' end blocking feature that blocks extension of the third oligonucleotide by a polymerase, wherein a 5' end of the third oligonucleotide and a 5 ' end of the Pl bind to the target nucleic acid sequence at a distance from about 3 nucleotides to about 25 nucleotides apart; d) a polymerase; and e) a DNA processing enzyme.

165. The system of claim 163 or 164, wherein the Pl localizes and binds to the strand of the target nucleic acid sequence independently of the third oligonucleotide.

166. The system of claim 163 or 164, wherein the P2 localizes and binds to the another strand of the target nucleic acid sequence independently of the third oligonucleotide.

167. The system of any one of claims 163-166, wherein a 5 ’ end of the P3 binds to the strand of the target nucleic acid sequence.

168. The system of claim 163 , wherein the 5 ’ end of the third oligonucleotide and a 5 ' end of the Pl bind to the strand of the target nucleic acid sequence at a distance from about 3 nucleotides to about 25 nucleotides apart.

169. The system of claim 164 or 168, wherein the 5' end of the third oligonucleotide and the 5' end of the Pl bind to the strand of the target nucleic acid sequence at a distance of about 15 nucleotides apart.

170. The system of claim 164 or 168, wherein the 5' end of the third oligonucleotide and the 5' end of the Pl bind to the strand of the target nucleic acid sequence at a distance of about 10 nucleotides apart.

171. The system of claim 164 or 168, wherein the 5' end of the third oligonucleotide and the 5' end of the Pl bind to the strand of the target nucleic acid sequence at a distance of about 8 nucleotides apart.

172. The system of any one of the claims 163-171, wherein the 3 ' end blocking feature that blocks extension of the third oligonucleotide by a DNA polymerase comprises an inverted nucleotide or a C3 -spacer.

173. The system of any one of claims 163-172, wherein the Pl comprises a sequence at least about 80% identical to a region on the strand of the target nucleic acid sequence .

174. The system of any one of claims 163-173, wherein the P2 comprises a sequence at least about 80% identical to a region on the another strand of the target nucleic acid sequence .

175. The system of any one of claims 163-174, wherein the third oligonucleotide comprises a sequence at least about 80% identical to a region of the target nucleic acid sequence.

176. The system of any one of claims 163-175, further comprising providing the Pl, the P2, and the third oligonucleotide in a set of oligonucleotides prior to the binding of the Pl, the P2, and the third oligonucleotide to the target nucleic acid sequence.

177. The system of any one of claims 163-176, wherein an enzyme mix comprises the DNA processing enzyme.

178. The system of claim 177, wherein the enzyme mix comprises the polymerase or functional fragmentthereof, the DNAprocessing enzyme, oneor more nucleotides in an amount sufficient to support amplification of the target nucleic acid sequence, a single-strand binding protein, a crowding reagent, a magnesium salt or any combination thereof.

179. The system of claim 178, wherein the DNA processing enzyme facilitates the binding of the third oligonucleotide to the target nucleic acid sequence.

180. The system of any one of claims 163-179, wherein the system of amplificationis configured to produce an amplified target nucleic acid sequence.

181. The system of claim 180, wherein the system of amplification is configured to produce target single-stranded (ss) amplicons.

182. The system of claim 180, wherein the system of amplification is configured to produce target double-stranded (ds) amplicons.

183. The system of claim 180, wherein the system of amplification is configured to produce target ds amplicons and target ss amplicons.

184. The system of any one of claims 163-183, wherein the Pl, the P2, or the third oligonucleotide comprises a label.

185. The system of claim 184, wherein the third oligonucleotide comprises the label.

186. The system of claim 185, wherein the label is located on a 5 ' end of the third oligonucleotide, a 3 ' end of the third oligonucleotide, or anywhere in between the 5 ’ end and the 3’ end.

187. The system of claim 186, wherein the third oligonucleotide comprises a label at its 5’ end.

188. The system of claim 187, wherein the label comprises a fluorescein or derivative thereof, a fluorophore, a chromophore, a redox molecule, a fluorescence quencher, a biotin, a digoxigenin, or any combination thereof.

189. The system of claim 188, wherein the label comprises a xanthene core or a derivative thereof.

190. The system of claim 189, wherein the label comprises a carboxyfluorescein (FAM).

191. The system of any one of claims 163-190, wherein the Pl or the P2 comprise a label.

192. The system of claim 191, wherein the label is located on a 5 ' end, a 3 ' end, or anywhere in between the 5’ end and the 3’ end of the Pl or the P2.

193. The system of claim 191 or 192, wherein the label comprises a fluorescein or derivative thereof, a fluorophore, a chromophore, a redox molecule, a fluorescence quencher, a biotin, a digoxigenin, or any combination thereof.

194. The system of any one of claims 191-193, wherein the label comprises a xanthene core or a derivative thereof.

195. The system of claim 194, wherein the label comprises a carboxyfluorescein (FAM).

196. The system of any one of claims 163 -195, wherein the system further comprises a crowding reagent.

197. The system of any one of claims 163-196, wherein the polymerase comprises a DNA polymerase or functional fragment thereof.

198. The system of any one of claims 163-197, wherein the DNA processing enzyme comprises a recombinase or functional fragment thereof, a helicase or functional fragment thereof, a topoisomerase or functional fragment thereof, single-stranded binding protein or functional fragment thereof, or any combination thereof.

199. The system of claim 198, wherein the DNA processing enzyme comprises UvsX DNA recombinase (UvsX), bacterial recombinase A (RecA), T4 GP32, E. coli SSB, HELB helicase, RTEL1 helicase, Pifl helicase, Topi topoisomerase, topoisomerase Illa, or any combination thereof.

200. The system of any one of claims 163-199, wherein the system is configured to detect an amplified target nucleic acid sequence.

201. The system of any one of claims 163-200, wherein the system comprises a detection assay.

202. The system of any one of claims 163-200, wherein the detection assay comprises a lateral flow assay strip, a colorimetric strip, an electrochemical sensor, a colorimetric sensor, or a fluorometric sensor.

203. The system of any one of claims 163-200, wherein the system comprises: a) a first channel comprising a first opening, wherein the first channel is configured to couple thereto a biological sample; b) a second channel comprising (i) a cellulose substrate coupled thereto and (ii) a second opening, wherein the second opening is configured to couple to the first opening, and wherein upon the coupling of the second opening to the first opening, the biological sample is transferred from the first channel to the second channel; and c) a third channel comprising (i) an elution solution and (ii) a third opening, wherein the third opening is configured to couple to the second opening, and wherein upon the coupling of the second opening to the third opening, the elution solution is transferred from the third channel to the second channel.

204. The system of claim 203, wherein the first channel, the second channel, and the third channel comprise a beaker, a bottle, a burette, a container, a cylinder, a vial, a tube, a pipette, a syringe, and any other combination thereof.

205. The system of claim 203, wherein the channel is configured for extraction of the target nucleic acid sequence.

206. The system of any one of claims 203-205, wherein the biological sample is a biological fluid, a tissue sample, a mammalian sample, an animal sample, a eukaryotic sample, a bacterial sample, a viral sample, or a plant sample.

207. The system of claim 206, wherein the biological sample is a bacterial cell, a eukaryotic cell, an animal tissue, whole blood, saliva, urine, a nasal swab, a buccal swab, a tongue swab, a cervical fluid, a vaginal fluid, an animal cell, a muscle tissue, a spleen tissue, a skin tissue, or a liver tissue.

208. The system of claim 207, wherein the biological sample is E. coli, N. gonorrhoeas, RSV, WSSV, EBV, or human genomic DNA.

209. The system of any one of claims 203-208, wherein the first channel further comprises a lysis solution.

210. The system of any one of claims 203-209, wherein the system comprises a processed biological sample.

211. The system of claim 210, wherein the processed biological sample comprises an extracted nucleic acid molecule.

212. The system of claim 211, wherein the nucleic acid molecule extracted from the sample comprises the extracted nucleic acid molecule comprises dsDNA, ssDNA, circulating DNA, cell free DNA, cDNA, plasmid DNA, mRNA, miRNA, tRNA, viral RNA, siRNA, sgRNA, or any combination thereof.

213. The system of claim 211, wherein the extracted nucleic acid molecule comprises a DNA or an RNA.

214. The system of any one of claims 163-213, wherein the amplification of the nucleic acid molecule determines the presence or absence of the target nucleic acid sequence.

215. A system comprising: a) the system of any one of claims 163-214; and b) a detection system for detecting the target nucleic acid.

216. The system of claim 215, wherein the second channel comprises an isolation chamber.

217. The system of claim 215 or 216, wherein the device further comprises an amplification chamber.

218. The system of any one of claims 215-217, wherein the device further comprises a detection buffer chamber.

219. The system of claim 218, wherein the i) isolation chamber and the amplification chamber, ii) the amplification chamber and the detection buffer chamber, and iii) the detection buffer chamber are fluidly connected via a plurality of channels.

220. The system of claim 218 or 219, further comprising an aliquot valve disposed between the isolation chamber, the amplification chamber, and the detection buffer chamber.

221. The system of any one of claims 216-220, further comprising a coupler fluidly connectedto the isolation chamber, wherein the coupler comprises an opening to the isolation chamber and is configured to engage at least a cap or a vial.

222. The system of any one of claims 216-221, further comprising a coupler fluidly connected to the isolation chamber, wherein the coupler comprises an openingto the isolation chamber and is configured to engage at least a means for preventing access to the isolation chamber through the coupler.

223. The system of claim 222, wherein the means for preventing access to the isolation chamber through the coupler comprises a cap, a seal, a plug, or a vial.

224. The system of any one of claims 216-223, further comprising a first chamber fluidly connected to a first end of the isolation chamber.

225. The system of claim 224, wherein the first chamber further comprises a first coupler, wherein the first coupler comprises an openingto the first chamber and is configured to engage at least a cap or a first vial.

226. The system of claim 225, wherein the first coupler comprises an opening to the first chamber and is configured to engage at least a means for preventing access to the first chamber through the first coupler.

227. The system of claim 226, wherein the means for preventing access to the first chamber through the first coupler comprises a cap, a seal, a plug, or a vial.

228. The system of claim 227, wherein the vial comprises a first vial.

229. The system of any one of claims 216-228, further comprising an elution chamber fluidly connected to a first end of the isolation chamber.

230. The system of claim 229, wherein the elution chamber further comprises a second coupler, wherein the second coupler comprises an openingto the elution chamber and is configured to engage at least a cap or a second vial.

231. The system of claim 230, wherein the second coupler comprises an openingto the elution chamber and is configured to engage at least a means for preventing access to the elution chamber through the second coupler.

232. The system of claim 231, wherein the means for preventing accessto the elution chamber through the second coupler comprises a cap, a seal, a plug, or a vial.

233. The system of claim 232, wherein the vial comprises a second vial.

234. The system of any one of claims 216-233, wherein a sample is either i) added directly to the isolation chamber or the first chamber or ii) is added to the isolation chamber or the first chamber via the use of the first vial.

235. The system of claim 234, wherein the sample comprises a biological sample or anenvironmental sample.

236. The system of claim 234 or claim 235, wherein the sample further comprisesa lysis buffer.

237. The system of any one of claims 216-236, further comprising one or more actuators configured to move a fluid through each of the aforementioned chambers.

238. The system of any one of claims 216-237, wherein the means of detection comprises a detection unit.

239. The system of claim 238, wherein the detection unit comprises a lateral flow assay strip, electrochemical sensor, or colorimetric sensor.

240. A kit comprising the system of any one of claims 163-239 and instructions for use.

241. The kit of claim 240, wherein the instructions designate a protocol for use as a field kit.

242. The kit of claim 241, wherein the instructions designate a detection of a viral infection.

243. The kit of claim 241, wherein the instructions designate a detection of abacterial infection.

244. The kit of claim 241 , wherein the instructions designate a detection of a specific antibioticresistant bacterial strain.

245. The kit of claim 241, wherein the instructions designate a detection of a DNA virus.

246. The kit of claim 241, wherein the instructions designate a detection of an RNA virus.

247. The kit of claim 241, wherein the instructions designate a detection ofWSSV, EBV, H1N1, H5N 1 , or any combination thereof.

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