Isothermal amplification of pathogens

Abstract

Disclosed herein are methods, compositions, and kits for use in detecting a target nucleic acid sequence in a sample. The method may include using a lysis buffer containing a lysis agent and / or a reducing agent to process the sample and detect the presence of the target nucleic acid sequence. In some embodiments, the method includes contacting a reagent composition containing an amplification agent and one or more protective agents capable of sequestering the lysis agent (e.g., cyclodextrin compounds) with the processed sample, e.g., under isothermal conditions, to generate an amplification reaction mixture, and detection.

Description

[Technical field]

[0001] Related Applications This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 163,399, filed March 19, 2021, and U.S. Provisional Application No. 63 / 307,099, filed February 5, 2022. The entire contents of these applications are hereby expressly incorporated by reference in their entireties. Sequence Listing Reference This application has been filed with a Sequence Listing in electronic format. The Sequence Listing is provided in a file entitled Sequence_Listing_68EB-317309-WO, created on March 17, 2022, and is 16.0 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. The present disclosure relates generally to methods and compositions for amplifying (eg, isothermal amplification) nucleic acids. [Background technology]

[0002] Nucleic acid-based diagnostics can be useful for the rapid detection of infections, diseases, and / or genetic mutations. For example, identification of bacterial or viral nucleic acids in a sample can be useful for diagnosing certain types of infections. Other examples include identification of single nucleotide polymorphisms for disease control or forensics, and identification of genetic mutations indicative of genetically modified foods. Nucleic acid-based diagnostic assays often require the amplification of specific portions of nucleic acids in a sample. A common technique for nucleic acid amplification is the polymerase chain reaction (PCR). This technique typically requires temperature cycling (i.e., thermal cycling) to proceed through the steps of denaturation (e.g., separation of strands of a double-stranded DNA (dsDNA) complex), annealing of oligonucleotide primers (short strands of complementary DNA sequences), and extension of the primers along their complementary targets by a polymerase. Such thermal cycling can be a time-consuming process that generally requires specialized machinery. Thus, there is a need for faster nucleic acid amplification methods that can be performed without thermal cycling. Such methods can be useful, for example, for on-site testing and point-of-care diagnostics. Additionally, a need exists for compositions and methods of nucleic acid detection in which the lysis agents used to lyse biological entities (e.g., virus particles, bacteria) are prevented from inactivating amplification reagents (e.g., polymerases) and in which the deleterious activity of nucleases (e.g., reduced deoxyribonuclease, reduced ribonuclease) is inhibited at all stages. Summary of the Invention

[0003] Disclosed herein is a method for detecting a target nucleic acid sequence in a sample. In some embodiments, the method includes (a) contacting a sample containing biological entities with a lysis buffer to generate a processed sample, the lysis buffer including one or more lysis agents capable of lysing the biological entities to release sample nucleic acids contained therein, the sample nucleic acids being suspected to contain a target nucleic acid sequence. The method may include (b) contacting a reagent composition (e.g., a dry composition) with the processed sample to generate an amplification reaction mixture, the reagent composition including one or more protectants and one or more amplification reagents. The method may include (c) amplifying the target nucleic acid sequence in the amplification reaction mixture, thereby generating a nucleic acid amplification product. The method may include (d) detecting the nucleic acid amplification product, the detection being performed less than about 20 minutes from the time the reagent composition is contacted with the processed sample. The lysis buffer and / or the reagent composition may include one or more reducing agents. In some embodiments, the pH of the lysis buffer is about 1.0 to about 10.0 (e.g., about 2.2). In some embodiments, the lysis buffer includes one or more of magnesium sulfate, ammonium sulfate, EDTA, and EGTA. In some embodiments, the sample nucleic acid includes a sample ribonucleic acid and / or a sample deoxyribonucleic acid. The sample ribonucleic acid may include cellular RNA, mRNA, microRNA, bacterial RNA, viral RNA, or any combination thereof. The one or more amplification reagents may include a reverse transcriptase and / or an enzyme having hyperthermophile polymerase activity. In some embodiments, the enzyme having hyperthermophile polymerase activity has reverse transcriptase activity. In some embodiments, contacting the dry composition with the sample to be processed includes dissolving the dry composition in the sample to be processed. In some embodiments, the reagent composition includes one or more of a reverse transcriptase, an enzyme having hyperthermophile polymerase activity, a first primer, a second primer, and a reverse transcription primer. The amplification can be carried out under isothermal amplification conditions. The detection of the nucleic acid amplification product can include the use of real-time detection methods. The one or more reducing agents can include one or more of 2-mercaptoethanol, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), dithioerythritol (DTE), reduced glutathione, cysteamine, tri-n-butylphosphine (TBP), dithioerythriol, tris(3-hydroxypropyl)phosphine (THPP), 2-mercaptoethylamine-HCl, dithiobutylamine (DTBA), cysteine, cysteine-thioglycolate, salts of sulfurous acid, thioglycolic acid, and hydroxyethyl disulfide (HED).

[0004] The reagent composition may be lyophilized and / or heat dried. The reagent composition may include one or more additives (e.g., amino acids, polymers, sugars, or sugar alcohols). The sugar or sugar alcohol may include sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof. The polymer may include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptides, collagen peptides, or any combination thereof.

[0005] The molar ratio of the one or more protective agents to the one or more amplification reagents may be about 10:1 to about 1:10 (e.g., about 2:1). The molar ratio of the one or more protective agents to the one or more lysis reagents may be about 10:1 to about 1:10 (e.g., about 2:1). The one or more additives may include Tween 20, Triton X-100, Tween 80, a non-ionic detergent (e.g., a non-ionic surfactant), or any combination thereof. In some embodiments, the one or more additives aid in lyophilization of the reaction composition and / or dissolution of the dried pellet. The one or more additives may include a non-ionic detergent at a concentration of about 0.01% in the reagent composition (e.g., a dried composition such as a dried pellet). In some embodiments, the presence of one or more additives (e.g., Tween 80 + Triton x-100, non-ionic detergents) in the dried pellets at low concentrations (e.g., 0.01%) improves APA reaction reproducibility. In some embodiments, even in the absence of non-ionic detergents in the lyophilized pellets, neutralization of ionic detergents by one or more protective agents (e.g., HP-beta CD) alone is sufficient. In some embodiments, the one or more protective agents include a cyclodextrin compound of formula (I), or a salt, ester, solvate, or hydrate thereof.

[0006] [ka] (In the formula, Each R is independently H, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which may be substituted; or -C(O)OR B , -OC(O)R B , -C(O)R B or -C(O)NR A R B and; Each R1 is independently H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, halogen, hydroxy, amino, -CN, -CF3, -N3, -NO2, -OR B , -SR B , -SOR B , -SO2R B , -N(R B )S(O2)-R B , -N(R B )S(O2)NR A R B , -NR A R B , -C(O)OR B , -OC(O)R B , -C(O)R B , -C(O)NR A R B , or -N(R B )C(O)R B each of which may be substituted; Each R A is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; Each R B is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each m is independently 0, 1, 2, 3, 4, or 5.

[0007] In some embodiments, each R is independently H, optionally substituted alkyl, -C(O)OR B , -OC(O)R B , -C(O)R B or -C(O)NR A R B In some embodiments, n is 1, 2, or 3. In some embodiments, each R is independently H, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, or octyl; each is straight or branched. In some embodiments, the cyclodextrin compound is 2-hydroxypropyl-α-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin (2HPβCD), hydroxypropyl-β-cyclodextrin (HPβCD), methyl-β-cyclodextrin (MβCD), 2-hydroxypropyl-γ-cyclodextrin, α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, or a salt, ester, solvate, or hydrate thereof. In some embodiments, 2-hydroxypropyl-α-cyclodextrin is more effective against smaller inhibitors than HPβCD.

[0008] The one or more lysis reagents may comprise about 0.001% (w / v) to about 1.0% (w / v) of the sample to be processed, for example, about 0.2% (w / v) of the sample to be processed. In some embodiments, the one or more lysis agents include a detergent. In some embodiments, the detergent includes one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. The anionic surfactant may be any one or more of the anionic surfactants disclosed herein. In some embodiments, the anionic surfactant includes NH4 as a counterion. + , K + , Na + , or Li +The cationic surfactant may be any one or more of the cationic surfactants disclosed herein. In some embodiments, the cationic surfactant comprises I as a counterion. - , Br - , or Cl - Includes.

[0009] The sample nucleic acid may comprise a nucleic acid that comprises a target nucleic acid sequence, hi some embodiments, the target nucleic acid sequence comprises a first strand and a second strand that are complementary to each other. In some embodiments, amplifying a target nucleic acid sequence comprises amplifying a target nucleic acid sequence comprising a first strand and a second strand that are complementary to one another under isothermal amplification conditions, the amplification comprising contacting a nucleic acid comprising the target nucleic acid sequence with: i) a first primer and a second primer, where the first primer is capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence and the second primer is capable of hybridizing to a sequence of the second strand of the target nucleic acid sequence; and ii) an enzyme having hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product, where the nucleic acid amplification product comprises (1) a sequence of the first primer and its reverse complement, (2) a sequence of the second primer and its reverse complement, and (3) a spacer sequence flanked by the (1) sequence of the first primer and its reverse complement and the (2) sequence of the second primer and its reverse complement, the spacer sequence being between 1 and 10 bases in length. In some embodiments, the amplification does not include the use of an enzyme other than an enzyme having hyperthermophile polymerase activity, and the amplification does not include thermal and / or enzymatic denaturation of the nucleic acid. In some embodiments, the method does not include contacting the nucleic acid with a single-stranded DNA binding protein before or during step (c). In some embodiments, the nucleic acid is double-stranded DNA. In some embodiments, the nucleic acid is a product of a reverse transcription reaction. In some embodiments, the nucleic acid is a product of a reverse transcription reaction generated from a sample ribonucleic acid. In some embodiments, step (c) includes generating the nucleic acid by a reverse transcription reaction. In some embodiments, the method does not include thermal or enzymatic denaturation of the sample nucleic acid.

[0010] In some embodiments, the sample nucleic acid comprises a sample ribonucleic acid, and the method comprises contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to generate cDNA. In some embodiments, amplifying the target nucleic acid sequence comprises: (c1) contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to generate cDNA; (c2) contacting the cDNA with an enzyme having a hyperthermophile polymerase activity to generate double-stranded DNA (dsDNA), the dsDNA comprising the target nucleic acid sequence, the target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other; and (c3) amplifying the target nucleic acid sequence under isothermal amplification conditions. In some embodiments, the amplification comprises contacting the dsDNA with (i) a first primer and a second primer, the first primer capable of hybridizing to a sequence of a first strand of a target nucleic acid sequence and the second primer capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence; and (ii) an enzyme having hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product, the nucleic acid amplification product comprising (1) a sequence of the first primer and its reverse complement, (2) a sequence of the second primer and its reverse complement, and (3) a spacer sequence flanked by the sequence of (1) the first primer and its reverse complement and the sequence of (2) the second primer and its reverse complement, the spacer sequence being 1-10 bases in length. In some embodiments, the method does not include the use of an enzyme other than a reverse transcriptase and / or an enzyme having hyperthermophile polymerase activity.

[0011] In some embodiments, step (d) further comprises determining the amount of dsDNA and / or nucleic acid comprising the target nucleic acid sequence in the sample. In some embodiments, the enzyme with hyperthermophile polymerase activity has an amino acid sequence that is at least about 90% identical or at least about 95% identical to the amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof. In some embodiments, the enzyme with hyperthermophile polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the enzyme with hyperthermophile polymerase activity has low or no exonuclease activity. Amplification of the target nucleic acid sequence can be performed at a constant temperature of about 55 degrees Celsius ("°C") to about 75°C, for example about 65°C. The first primer, the second primer, or both may be about 8-17 bases in length. In some embodiments, the first primer, the second primer, or both include one or more of DNA bases, modified DNA bases, or combinations thereof. The nucleic acid amplification product may be about 20-40 bases in length. In some embodiments, the spacer sequence includes a portion of the target nucleic acid sequence. In some embodiments, the spacer sequence is 1-10 bases in length.

[0012] In some embodiments, the method comprises contacting the nucleic acid amplification product with a signal generating oligonucleotide capable of hybridizing to the amplification product, the signal generating oligonucleotide comprising a fluorophore, a quencher, or both. In some embodiments, detecting the nucleic acid amplification product comprises detecting a fluorescent signal. In some embodiments, the fluorescent signal is from a molecular beacon. In some embodiments, the method is performed in a single reaction vessel. In some embodiments, the sample ribonucleic acid is contacted simultaneously with the reverse transcriptase and the enzyme having hyperthermophile polymerase activity. In some embodiments, the sample ribonucleic acid is contacted simultaneously with the reverse transcriptase, the enzyme having hyperthermophile polymerase activity, and the first and second primers. In some embodiments, the sample ribonucleic acid is contacted simultaneously with the reverse transcriptase, the enzyme having hyperthermophile polymerase activity, the first primer, the second primer, and the reverse transcription primer.

[0013] In some embodiments, the one or more lysis agents are capable of denaturing the one or more amplification reagents, and the one or more protection agents are capable of preventing the one or more lysis agents from denaturing the one or more amplification reagents. In some embodiments, the one or more protection agents are capable of sequestering the one or more lysis agents, thereby preventing the one or more lysis agents from denaturing the one or more amplification reagents. In some embodiments, more than about 90% of the one or more lysis agents are sequestered by the one or more protection agents. In some embodiments, less than about 10% of the one or more amplification reagents are denatured by the one or more lysis agents.

[0014] In some embodiments, the sample comprises multiple nucleases (e.g., deoxyribonucleases, ribonucleases). In some embodiments, the one or more lysis agents can denature the nucleases (e.g., deoxyribonucleases, ribonucleases) to generate modified nucleases (e.g., modified deoxyribonucleases, modified ribonucleases), the one or more reducing agents can reduce the nucleases to generate reduced nucleases (e.g., reduced deoxyribonucleases, reduced ribonucleases), and the one or more reducing agents and the one or more lysis agents can generate reduced modified nucleases (e.g., reduced modified deoxyribonucleases, reduced modified ribonucleases). In some embodiments, the denatured ribonuclease and / or reduced denatured ribonuclease can be renatured over time and / or upon reduced physical interaction with one or more lysis agents, thereby generating renatured ribonuclease. In some embodiments, the denatured deoxyribonuclease and / or reduced denatured deoxyribonuclease can be renatured over time and / or upon reduced physical interaction with one or more lysis agents, thereby generating renatured deoxyribonuclease. In some embodiments, sequestration of one or more lysis agents reduces physical interaction between one or more lysis agents and the denatured deoxyribonuclease, reduced denoxyribonuclease, denatured ribonuclease, and / or reduced denatured ribonuclease. In some embodiments, the denatured DNase, reduced DNase, reduced DNase, denatured ribonuclease, reduced ribonuclease, and / or reduced denatured ribonuclease are enzymatically inactive, and the renatured ribonuclease and / or renatured DNase are enzymatically active. In some embodiments, the reduced renatured ribonuclease renatures at least about 1.1 times slower than the renatured ribonuclease. In some embodiments, the reduced renatured DNase renatures at least about 1.1 times slower than the renatured DNase.In some embodiments, about 1.1 times more sample ribonucleic acid is used by the reverse transcriptase as a template to generate cDNA compared to a comparable method in which the lysis buffer does not contain one or more reducing agents. In some embodiments, about 1.1 times more sample deoxyribonucleic acid is used by the enzyme having hyperthermophile polymerase activity as a template to generate nucleic acid amplification products compared to a comparable method in which the lysis buffer does not contain one or more reducing agents. In some embodiments, the amplification reaction mixture contains at least 1.1 times less nuclease (e.g., deoxyribonuclease, ribonuclease) activity 10 minutes after step (b) and / or step (c) compared to a comparable method in which the lysis buffer does not contain one or more reducing agents.

[0015] The biological entity may include one or more of a prokaryotic cell, a eukaryotic cell, a virus particle, an exosome, a protoplast, and a microvesicle. In some embodiments, the biological entity includes a virus, a bacterium, a fungus, a protozoa, a part thereof, or any combination thereof. In some embodiments, the target nucleic acid sequence is a viral, bacterial, fungal, or protozoan nucleic acid sequence. In some embodiments, the sample nucleic acid is derived from a virus, a bacterium, a fungus, or a protozoa. The virus may include one or more of SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex, herpes virus 6, herpes virus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella zoster virus, JC virus, parvovirus B19, influenza A, influenza B, influenza C, rotavirus, human adenovirus, rubella virus, human enterovirus, genital human papillomavirus (HPV), and hantavirus.In some embodiments, the bacteria is selected from the group consisting of Mycobacteria tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella dumophila, and the like. dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumonia, S. agalactiae, and Listeria monocytogenes. In some embodiments, the fungus comprises one or more of Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, and Trichophyton rubrum.In some embodiments, the protozoa include one or more of Trypanosoma cruzi, Leishmania sp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., and Eimeria sp. In some embodiments, the amplifying step comprises multiplex amplification of two or more target nucleic acid sequences and the detecting step comprises multiplex detection of two or more nucleic acid amplification products derived from said two or more target nucleic acid sequences. In some embodiments, the two or more target nucleic acid sequences are specific to two or more different organisms. In some embodiments, the two or more different organisms include one or more of Chlamydia trachomatis, Neisseria gonorrhoeae, SARS-CoV-2, influenza A, influenza B, and / or influenza C.

[0016] In some embodiments, the amplification includes and / or does not include one or more of the following: loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal mediated amplification of RNA technology (SMART), self-sustained sequence replication (3SR), genome exponential amplification reaction (GEAR), and isothermal multiple displacement amplification (IMDA). In some embodiments, the amplification does not include LAMP. In some embodiments, the method does not include one or more of the following: (i) diluting the sample to be processed; (ii) diluting the amplification reaction mixture; (iii) heat denaturing the sample to be processed; (iv) sonicating the sample to be processed; (v) sonicating the amplification reaction mixture; (vi) adding RNase inhibitors and / or DNase inhibitors to the sample to be processed; (vii) adding RNase inhibitors and / or DNase inhibitors to the amplification reaction mixture; (viii) purifying the sample; (ix) purifying the sample nucleic acid; (x) purifying the nucleic acid amplification product; (xi) removing one or more lysis agents from the sample to be processed or the amplification reaction mixture; (xii) heat and / or enzymatic denaturation of the sample nucleic acid before and / or during amplification; and (xiii) adding RNase H to the sample to be processed or the amplification reaction mixture. In some embodiments, the sample is held at an amplification temperature (e.g., 67° C.). In some embodiments, the sample is held at a temperature between room temperature and the reaction temperature for 1-2 minutes prior to the amplification reaction to facilitate reverse transcription of the RNA sample.

[0017] Disclosed herein is a kit for detecting a target nucleic acid sequence in a sample. In some embodiments, the kit comprises: (a) a lysis buffer comprising one or more lysis agents, the one or more lysis agents being capable of lysing biological entities to release sample nucleic acids contained therein, the sample nucleic acids being suspected to contain a target nucleic acid sequence; and (b) a reagent composition (e.g., a dry composition) comprising one or more protection agents and one or more amplification reagents, the one or more amplification reagents comprising one or more components for amplifying a target nucleic acid sequence under isothermal amplification conditions. In some embodiments, the components comprise: (i) a first primer and a second primer, the first primer being capable of hybridizing to a sequence of a first strand of a target nucleic acid sequence, and the second primer being capable of hybridizing to a sequence of a second strand of a target nucleic acid sequence; and (ii) an enzyme having a hyperthermophile polymerase activity capable of generating a nucleic acid amplification product. In some embodiments, the lysis buffer and / or reagent composition comprises one or more reducing agents, which may include one or more of 2-mercaptoethanol, DTT, TCEP, DTE, reduced glutathione, cysteamine, TBP, dithioerythriol, THPP, 2-mercaptoethylamine-HCl, DTBA, cysteine, cysteine-thioglycolate, salts of sulfite, thioglycolic acid, and HED.

[0018] The kit may include at least one component that provides real-time detection activity of the nucleic acid amplification product. In some embodiments, the real-time detection activity is provided by a molecular beacon. In some embodiments, the reagent composition (e.g., dry composition) includes a reverse transcriptase and / or a reverse transcription primer. The enzyme having hyperthermophile polymerase activity may have an amino acid sequence that is at least about 90% identical or at least about 95% identical to the amino acid sequence of SEQ ID NO: 1 or a functional fragment thereof. In some embodiments, the enzyme having hyperthermophile polymerase activity is a polymerase that includes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid amplification product is about 20-40 bases in length, and the nucleic acid amplification product includes (1) a first primer sequence and its reverse complement, (2) a second primer sequence and its reverse complement, and (3) a spacer sequence flanked by (1) the first primer sequence and its reverse complement and (2) the second primer sequence and its reverse complement, the spacer sequence being 1-10 bases in length.

[0019] The molar ratio of the one or more protecting agents to the one or more amplification reagents may be about 10:1 to about 1:10, for example, about 2:1. In some embodiments, the one or more additives include Tween 20, Triton X-100, Tween 80, a non-ionic detergent (e.g., a non-ionic surfactant), or any combination thereof. In some embodiments, the one or more protecting agents include a cyclodextrin compound of formula (I). In some embodiments, the one or more protecting agents can sequester the one or more lysis agents, thereby preventing the one or more amplification reagents from being denatured by the one or more lysis agents. In some embodiments, the one or more lysis reagents comprise about 0.001% (w / v) to about 1.0% (w / v) of the sample to be processed, for example, about 0.2% (w / v) of the sample to be processed. In some embodiments, the one or more lysis agents include a detergent. The detergent may include one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. [Brief description of the drawings]

[0020] [Figure 1A-1B] FIG. 1 shows a non-limiting, exemplary schematic diagram of an isothermal amplification reaction provided herein. [Figure 2A-2B] Figure 2 illustrates data on the effect of the anionic detergents SDS and HP-βCD on Neisseria gonorrhea DNA amplification. Assays without protectant (Figure 2A) or with 4% HP-βCD (Figure 2B) are shown. [Figure 3A-3B] FIG. 1 illustrates data regarding the effect of the cationic detergent CTAB on Chlamydia trachomatis DNA amplification. [Figure 4] FIG. 1 illustrates data regarding lysis of Chlamydia trachomatis. [Figure 5A-5B] FIG. 1 illustrates data regarding the effect of hydroxypropyl-β-cyclodextrin (HPβCD) protection on influenza B virus detection. [Figure 6] FIG. 13 illustrates data relating to an addition order assay. [Figure 7] FIG. 13 graphically depicts data regarding the effect of DTT on the irreversibility of RNase A inactivation in the presence of CTAB at various temperatures. [Figure 8] FIG. 13 graphically depicts data regarding the effect of DTT and HP-βCD on RNase A inactivation. [Figure 9] FIG. 1 illustrates data regarding requirements for irreversible RNase inactivation. [Figure 10A] FIG. 13 illustrates data regarding protection of RNA from nasal swab ribonucleases. [Figure 10B] FIG. 1 illustrates data regarding assay calibration to assess degree of protection. [Figure 11A-11B] FIG. 11A illustrates data regarding RNase A activity at 58° without (FIG. 11A) and with (FIG. 11B) βCD. [Figure 12A-12B]FIG. 13 graphically depicts data regarding RNase A inactivation in an influenza B assay with DTT in ELB (elution / lysis buffer). [Figure 13] FIG. 1 illustrates data regarding the rapid irreversibility of nasal ribonuclease inactivation. [Figure 14A-14B] FIG. 1 illustrates data on direct lysis for isothermal influenza B virus detection in the presence and absence of clinical matrix. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, similar symbols typically identify similar components unless the context dictates otherwise. The exemplary embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments can be used and other changes can be made without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein and form part of the disclosure herein. All patents, published patent applications, other publications, and GenBank sequences and other databases referenced herein are incorporated by reference in their entirety with respect to the relevant art.

[0022] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of this disclosure, the following terms are defined below.

[0023] Disclosed herein includes a method for detecting a target nucleic acid sequence in a sample. In some embodiments, the method includes (a) contacting a sample containing biological entities with a lysis buffer to generate a processed sample, the lysis buffer including one or more lysis agents capable of lysing the biological entities to release sample nucleic acids contained therein, the sample nucleic acids suspected to contain the target nucleic acid sequence. The method may include (b) contacting a reagent composition (e.g., a dry composition) with the processed sample to generate an amplification reaction mixture, the reagent composition including one or more protectants and one or more amplification reagents. The method may include (c) amplifying the target nucleic acid sequence in the amplification reaction mixture, thereby generating a nucleic acid amplification product. The method may include (d) detecting the nucleic acid amplification product, the detection being performed less than about 20 minutes, about 15 minutes, about 10 minutes, about 5 minutes, or about 2.5 minutes from the time the reagent composition is contacted with the processed sample.

[0024] Disclosed herein is a kit for detecting a target nucleic acid sequence in a sample. In some embodiments, the kit comprises: (a) a lysis buffer comprising one or more lysis agents, the one or more lysis agents being capable of lysing biological entities to release sample nucleic acids contained therein, the sample nucleic acids being suspected to contain a target nucleic acid sequence; and (b) a reagent composition (e.g., a dry composition) comprising one or more protection agents and one or more amplification reagents, the one or more amplification reagents comprising one or more components for amplifying a target nucleic acid sequence under isothermal amplification conditions. In some embodiments, the components comprise: (i) a first primer and a second primer, the first primer being capable of hybridizing to a sequence of a first strand of a target nucleic acid sequence, and the second primer being capable of hybridizing to a sequence of a second strand of a target nucleic acid sequence; and (ii) an enzyme having a hyperthermophile polymerase activity capable of generating a nucleic acid amplification product. The lysis buffer and / or reagent composition may include one or more reducing agents, including, but not limited to, one or more of 2-mercaptoethanol, DTT, TCEP, DTE, reduced glutathione, cysteamine, TBP, dithioerythriol, THPP, 2-mercaptoethylamine-HCl, DTBA, cysteine, cysteine-thioglycolate, salts of sulfite, thioglycolic acid, and HED.

[0025] Provided herein are methods and compositions for amplifying nucleic acids. Conventional nucleic acid amplification methods typically require a thermal cycling process, a protein (e.g., an enzyme) (e.g., a helicase, a recombinase) that promotes nucleic acid denaturation, strand unwinding, strand separation, and / or strand exchange, and / or an endonuclease agent (e.g., a restriction enzyme, a nicking enzyme), and often require a reaction time of at least 20 to 30 minutes. The nucleic acid amplification method provided herein can be performed without thermal cycling, without thermal and / or enzymatic denaturation of the sample nucleic acid, without the addition of a protein (e.g., an enzyme) that promotes strand unwinding, strand separation, and / or strand exchange, without the use of an endonuclease agent, and within a reaction time of about 10 to 15 minutes.

[0026] Nucleic Acid Amplification Methods Provided herein are methods and compositions for amplifying (e.g., isothermal amplification) nucleic acids from a sample, for example, in an ionic detergent-based lysis solution. In some embodiments, the methods include lysis of biological entities (e.g., pathogens) in a sample, followed by immediate isothermal amplification, and real-time detection of the released RNA target sequences. In some embodiments, such methods have the advantage of exhibiting inactivation of nucleases (e.g., deoxyribonucleases, ribonucleases) and prevention of DNA and / or RNA degradation in clinical and physiological samples. Also provided herein are methods and compositions for directly lysing viral and bacterial pathogens in clinical samples prior to isothermal nucleic acid amplification and real-time detection, without any purification or separation steps. Disclosed herein, in some embodiments, is a two-step method consisting of a first step of chemically lysing viral particles or bacterial cells in a clinical sample containing nucleases (e.g., deoxyribonucleases, ribonucleases) using a lysis agent that inactivates the nucleases, followed by a second step in which the chemical agent can prevent reactivation of the nucleases under subsequent reaction conditions that favor enzyme activity. In some embodiments, the two-step method provided herein does not include sample purification and / or nucleic acid isolation / purification. Also provided herein is a one-step method that includes simultaneous chemical lysis and nuclease (e.g., deoxyribonucleases, ribonucleases) inactivation when a lysis agent and a reducing agent are both present in the lysis buffer. In some embodiments, the method provided herein is a two-step method.

[0027] Provided herein include methods and compositions for direct pathogen lysis in clinical samples that allow isothermal amplification and real-time detection of nucleic acids using archaeal polymerases. Rapid point-of-care (POC) diagnostics that do not require sample purification can be developed. Virus particles, bacterial cells, or other pathogens still need to be lysed so that their DNA and RNA can be released from the cells and made available for amplification reactions. Traditional chemical lysis methods (e.g., using strong bases, ionic detergents, and chaotropic agents) are not compatible with enzyme function because they would also inactivate DNA polymerase or other enzymes. Therefore, an effective chemical lysis method that is compatible with enzyme function and does not require any nucleic acid purification step to remove the lysis reagent would be of significant value.

[0028] In some embodiments, a clinical sample containing a pathogen is added to a lysis buffer containing a detergent (e.g., a strong ionic detergent) to dissolve biological entities (e.g., target pathogens) and release nucleic acids. The lysis solution can be directly transferred to a reaction containing lyophilized amplification reagents and a protectant against ionic detergents that effectively neutralize the chemical detergents used in the lysis buffer, allowing the reaction to proceed without intermediate purification or separation steps. The methods and compositions provided herein advantageously allow for direct and rapid target amplification reactions on patient samples (as used herein, the term "direct" can mean no purification of nucleic acids or dilution of the sample after delivery to an ionic detergent-based lysis solution designed to break down the protective coating of target organisms). This solution can then be used to dissolve the dried reaction reagents. Since soluble ionic detergents are incompatible with amplification chemistry, dry reagents to achieve that chemistry may contain cyclodextrins to sequester the ionic detergents and prevent what would otherwise be considered detrimental interactions with the enzymes in the reaction chemistry. Since even very brief exposure to the ionic detergents is believed to be sufficient to inactivate the enzymes, the reagents and drying conditions provided herein may be such that the cyclodextrin dissolves more quickly than the enzymes and / or acts quickly enough to protect the enzymes when the lysis solution is delivered to the dry reagent. For nucleic acid amplification methods such as PCR and RT-PCR assays, the theoretical detection limit is one genome copy per reaction, but the presence of inhibitors in clinical samples can limit the function of the enzymes for nucleic acid amplification. Therefore, for molecular detection of pathogens in clinical samples, currently available methods often include purification or separation steps for nucleic acid (DNA or RNA) amplification. Ionic detergents are commonly used for bacterial cell lysis and RNA or DNA extraction.Ionic detergents cannot be used for pathogen lysis without a separation or purification step, because they have a strong denaturing and inhibitory effect on amplification methods such as PCR in which Taq polymerase is used. In contrast to other methods, the compositions and methods of the present disclosure provide direct lysis of pathogens in clinical samples using ionic detergents, allowing isothermal amplification and real-time detection of the released nucleic acids without a purification or separation step, thereby reducing assay time and complexity and contamination risk.

[0029] The compositions and methods disclosed herein can overcome a series of obstacles encountered in developing a system for rapid point-of-care isothermal targeted amplification / detection of RNA viruses such as influenza or coronavirus in patient respiratory samples. As the most effective virus lysis method, a strong ionic detergent such as sodium dodecyl sulfate (SDS) was selected for proof-of-principle experiments. SDS can inhibit a variety of harmful enzymes that are normally present in samples. In direct tests, the same lysis agent that can inhibit harmful enzymes may also inhibit the enzymes necessary to achieve the amplification / detection reaction. To overcome this obstacle, SDS was sequestered by including a cyclodextrin compound in the lyophilized reaction mixture that contained the enzymes for the subsequent amplification of the released ribonucleic acid. However, this sequestration was then found to also allow for rapid reactivation of harmful ribonuclease enzyme activity originating from the sample. To overcome this reactivation obstacle, a reducing agent can be included during the lysis step. Thus, harmful ribonuclease activity can be rapidly and irreversibly inactivated, resulting in intact ribonucleic acid ready for amplification without the need for separation or purification. More precisely, respiratory samples from patients containing RNA viruses may necessarily contain ubiquitous ribonucleases. During the intended procedure of rapid molecular archaeal polymerase amplification (APA) testing of such viral RNA, there may be two steps where the target RNA may be susceptible to destruction by such ribonucleases. The first lysis step occurs after delivery of the sample to a lysis buffer containing a lysis agent designed to release the viral RNA from its protective coating to produce a lysate. Thus, the released RNA will no longer be protected from ribonuclease activity. The second reaction step can be initiated by directly dissolving the dry APA reagent in this lysate, with the susceptibility of the RNA to the sample ribonucleases remaining ongoing. The risk of RNA destruction may persist until the included enzyme reverse transcriptase completes the DNA copy of the RNA target segment as a precursor to the amplification and detection of the DNA copy by the APA process or any isothermal process.Ionic detergents such as SDS and / or cetyltrimethylammonium bromide (CTAB) can be used as lysis agents in the methods and compositions provided herein, allowing efficient lysis and also inhibiting ribonucleases by denaturation, thus protecting the target RNA during the lysis step. However, in subsequent direct and rapid molecular testing reactions initiated by dissolving the dry reaction mix directly into the lysis sample without separation or nucleic acid purification, SDS (and / or CTAB) may need to be removed quickly, as it will also denature the enzymes reverse transcriptase and polymerase required for the reaction. However, removal of some agents used to denature ribonucleases may allow reactivation of ribonucleases, i.e., recovery of their enzymatic activity. As disclosed herein, inclusion of cyclodextrin in the dry reaction mix can allow rapid removal of SDS (and / or CTAB). In fact, this rapid removal may also accelerate the refolding of some denatured proteins. As disclosed herein, it has been demonstrated that in the presence of cyclodextrin, reactivation of ribonuclease is very rapid, leaving the target RNA unprotected. Addition of a reducing agent during ribonuclease denaturation with some denaturants can delay reactivation after the relatively slow removal of the denaturant and reducing agent, usually over several hours. As described herein, addition of a reducing agent during the dissolution step in combination with SDS (and / or CTAB) made ribonuclease inactivation immediate and sufficiently irreversible to allow enzyme-mediated molecular detection, even though ribonuclease refolding is likely accelerated due to the rapid removal of SDS (and / or CTAB) by cyclodextrin after a short dissolution step. The reducing agent may be compatible with enzyme detection, so in some embodiments it does not need to be removed.

[0030] The methods and compositions provided herein offer a variety of unexpected advantages over currently available nucleic acid detection methods and compositions, such as those described in U.S. Pat. Nos. 5,705,345, 5,422,241, 6,777,210, and 6,204,375; U.S. Patent Application Publication Nos. 20170152546 and 20140322761; Anfinsen, Christian B., et al. (Proc Natl Acad Sci USA 47.9 (1961):1309), Anand (IJC 2013), and Yamaguchi, Hiroshi (Biomolecules 4.1 (2014): 235-251).

[0031] For example, currently available methods use cyclodextrin to neutralize ionic detergent-containing lysis solutions to allow subsequent amplification in wet reactions (without dry reagents). These available methods require the addition of cyclodextrin to detergent lysates, an extra step that is incompatible with point-of-care molecular diagnostics as disclosed herein. In some embodiments of the methods provided herein, the lysate is added to dry reagents containing CDs under conditions that allow the CDs to dissolve prior to the enzyme. In contrast, currently available methods use dilution in the amplification reaction mixture after neutralization of the detergent with 0.5% SDS to bring the SDS to a final 0.05% (i.e., dilution in the amplification reaction, which is incompatible with point-of-care). As provided herein, neutralization and amplification can occur in the same solution with 0.2% SDS. Currently available methods also include immobilizing nucleic acids on a solid support, such as FTE paper, and embedding the lysis reagent in the solid matrix. However, even if sample types can be expanded to human blood, which is easy to lyse, such methods do not provide efficient lysis or amplification of difficult-to-lyse pathogens. Thus, these currently available methods are limited in their use with clinical samples and are significantly different from those disclosed herein. Furthermore, currently available methods cannot achieve equivalent performance without SDS or SDS+cyclodextrin for RNA targets with reverse transcriptase.

[0032] Currently available compositions and methods (e.g., for ribonuclease inactivation) have been shown to be inadequate in the context of the applications contemplated herein. For example, currently available compositions and methods are effective only after inactivation, and not during inactivation (e.g., in this context, during the reaction and lysis steps, respectively). Furthermore, currently available methods require at least 4 minutes at 60°C for irreversibility, whereas the compositions and methods provided herein can allow virtually instantaneous irreversible inactivation at 68°C, allowing for a more rapid lysis step, which is essential for point-of-care applications. For example, Myhrvold et al. (Science. 2018 April 27;360(6387):444-448) disclose inactivation of RNases by prior art methods that are multi-step, cumbersome, and unacceptably slow, but do not disclose the compositions (e.g., lysis buffers, protectants) and methods provided herein. In addition, currently available methods use uncalibrated methods of unknown sensitivity to assess residual ribonuclease activity, whereas as provided herein, the assessment of ribonuclease activity after inactivation uses the intended molecular diagnostic test as a validity test. Furthermore, currently available methods can only achieve irreversible ribonuclease inactivation at up to 200 ng / ml, whereas the compositions and methods provided herein have shown utility at 10 μg / ml to accommodate potentially larger amounts in patient samples. Furthermore, currently available methods attempt to demonstrate efficacy after ribonuclease inactivation, but not during inactivation. Additionally, currently available methods are designed for analytical applications with additional extraction, separation, or purification steps, including intentionally mixing and incubating pure RNA and ribonuclease prior to the addition of denaturants, and treating with reagents that will purify undigested RNA and ribonuclease by precipitation, but the effect on recovery of ribonuclease activity is unknown, making this process incompatible with rapid point-of-care applications.Unlike currently available methods that show delayed reactivation of ribonucleases after extended denaturation in the presence of denaturants urea and reducing agents followed by relatively slow removal of both, conditions are provided herein that achieve a similar, but rapid and immediate effect on adequate prevention of ribonuclease reactivation when rapid renaturation is achieved by rapid removal of the denaturing ionic detergent alone. Furthermore, the current hypothesis that cyclodextrins affect the refolding of various proteins but not necessarily their reactivation has not been applied to ribonucleases. Furthermore, it is currently unclear whether the time scales involved in renaturation and reactivation of ribonucleases (e.g., RNaseA) following cyclodextrin treatment follow the same unfolding and refolding pathways as the model proteins studied. Furthermore, currently available methods contemplate the inclusion of ribonuclease inhibitors (rather than the inclusion of reducing agents during inactivation) so that the process can proceed to the next enzymatic step after removal of the ionic detergent.

[0033] Currently available methods are intended to effectively reverse transcribe RNA in samples containing ribonucleases without immediate nucleic acid purification after ribonuclease inactivation with reducing agents, but none have been designed to allow for the inclusion of harsh chemical virus lysis reagents such as the anionic detergent SDS or the cationic detergent CTAB, required for the rapid molecular diagnostics described herein. Unexpectedly, as shown herein, ribonuclease inactivation with denaturants and reducing agents occurs essentially instantaneously, allowing immediate reverse transcription as quickly as required for the applications contemplated herein.

[0034] Unexpectedly, as described herein, ribonuclease irreversibility operation under conditions required for rapid molecular testing required an ionic detergent in addition to a reducing agent if followed by selective rapid removal of the detergent. Bender et al (J Mol Diagn. 2020 Aug;22(8):1030-1040) discloses inactivation of endogenous ribonucleases using methods involving long incubations, proteinase K, and / or high incubation temperatures, conditions that are advantageously avoided by the methods and compositions provided herein. The methods and compositions provided herein overcome various problems in currently available methods to achieve practical and straightforward rapid molecular RNA testing in patient samples. Specifically, the lysis agent provided herein may be an ionic detergent such as SDS or CTAB, which also inactivates nucleases (e.g., deoxyribonucleases, ribonucleases) during the lysis step. The inclusion of a second component, a reducing agent such as dithiothreitol (DTT) or cysteine, ensures that removal of the detergent with cyclodextrin during the reaction step prevents ribonuclease reactivation, at least for a sufficient time until a DNA copy of the viral RNA is produced. As disclosed herein, irreversible operation of ribonuclease inactivation can be achieved virtually immediately during lysis at 68°C. Using only ionic detergents without reducing agents prevented ribonuclease activity during lysis (in the presence of detergent), but after treatment with cyclodextrin to remove the detergent, ribonuclease activity was fully and rapidly restored, preventing viral RNA detection. As disclosed herein, without ionic detergents but using only reducing agents, ribonuclease activity rendered RNA undetectable, presumably due to ribonuclease activity both during lysis and during the reaction. Thus, as described herein, successful detection under these conditions required simultaneous treatment with an ionic detergent and a reducing agent.

[0035] In some embodiments, the disclosed methods and compositions allow for direct pathogen lysis in clinical samples, isothermal amplification and real-time detection of nucleic acids without the need to separate or purify samples for point-of-care molecular diagnostics. In some embodiments, a lysis buffer is provided that includes a strong ionic detergent that can be used to lyse pathogens in clinical samples. The amplification reagent may include a protectant for the lysis reagent, may be dried (e.g., lyophilized, heat-dried), and can be used in a point-of-care setting to amplify the released nucleic acids.

[0036] The compositions and methods provided herein enable point-of-care molecular diagnosis of RNA / RNA viruses in clinical samples that can rapidly and irreversibly inactivate nucleases (e.g., deoxyribonucleases, ribonucleases) without the need for separation, purification, or extraction to amplify deoxyribonucleic acid and / or ribonucleic acid. The methods and compositions provided herein are compatible with a variety of lysis buffers and lysis agents, including, but not limited to, anionic detergents, cationic detergents including alkyl sulfates, alkyl sulfonates, bile salts, and alkyl trimethylammonium surfactants with various alkyl chain lengths.

[0037] Ionic detergents are powerful chemicals for cell lysis. Ionic detergents such as SDS solubilize phospholipid and protein components of cell membranes, resulting in cell lysis and release of the cell contents. SDS can have an inhibitory effect at concentrations higher than 0.01% (w / v) in PCR with Taq polymerase and amplification with archaeal polymerases. Cyclodextrins (CDs) are cyclic oligosaccharides that resemble truncated cones with a hydrophobic internal cavity and a hydrophilic external surface. Natural cyclodextrins include α-, β-, and γ-CD. The solubility of these CDs can be limited, but chemically modified CD derivatives such as hydroxypropyl β-CD can improve solubility in aqueous media by up to 50%. β-CD forms strong inclusion complexes (stronger than α-CD and γ-CD) with ionic detergents such as sodium dodecyl sulfate (SDS), mainly with a 1:1 stoichiometry. Ionic detergents can be used in lysis buffers for cell lysis / sample preparation. As described herein, cyclodextrins can be incorporated into dried (e.g., lyophilized, heat-dried) pellets containing enzymes and other assay components as a protectant against the deleterious effects of ionic detergents. After the lysis step, the sample solution can be mixed with dried (e.g., lyophilized, heat-dried) pellets containing cyclodextrin to promote CD and SDS inclusion complex formation.

[0038] Rapid and direct molecular testing of RNA viruses in respiratory samples requires an initial lysis step to release the viral RNA from its protective coating composed of carbohydrates, fats, and proteins. The lysis must sufficiently disrupt, e.g., denature, the protein structure in an irreversible manner so that the RNA remains intact and is available for the subsequent enzyme-mediated amplification step. Direct molecular testing means that this same harsh lysis solution must then be compatible with the enzyme-mediated amplification step, since it dissolves the reaction reagents / enzymes. Since enzymes are proteins, the protein-denaturing elements that achieved lysis must be removed, and in this situation, it must be done quickly. To make the lysis solution compatible with the amplification step, in some embodiments, a dry amplification reagent is provided that includes a cyclodextrin compound that sequesters the ionic detergent before it denatures the amplification enzyme as soon as the dry reagent is dissolved in the lysis solution. In some embodiments, the dissolution of the dry reagent is designed to obtain this dissolution order. In some embodiments, the dissolution of the dry reagent is designed to occur in this order of events. Respiratory samples containing RNA viruses also contain ribonuclease enzymes that may interfere. Depending on the method used to achieve lysis, the release of viral RNA may make it susceptible to destruction by such ribonucleases. The advantage of lysis with ionic detergents is that the protein ribonuclease is also denatured and thus inactivated during the lysis step. However, ribonucleases, compared to most other proteins, exhibit a very unusual property in that denaturation may be reversible, e.g., renaturation (with recovery of enzyme activity) occurs upon removal of the denaturing agent. Thus, after dissolving the dry reagent, removal of the ionic detergent by the cyclodextrin provided herein may reactivate the sample ribonuclease, which may prevent RNA reverse transcription. It has not previously been shown that sequestration of ionic detergents after treatment accelerates renaturation of ribonucleases.Addition of a reducing agent during denaturation of ribonuclease with a denaturing agent, such as urea, rather than a detergent, can delay renaturation when both removals are relatively slow. This is believed to be because the reducing agent prevents the important role of causing modification of sulfhydryl amino acid residues of ribonuclease, leading to refolding of ribonuclease and recovery of enzymatic activity. As described herein, the reducing agent prevents or sufficiently delays the subsequent reactivation of ribonuclease, which would interfere with RNA detection upon sequestration of ionic detergent denaturing agent by cyclodextrin. The present disclosure provides the first demonstration that the treatment combination of the present disclosure, such as reducing agent with ionic detergent, is effective and that ribonuclease inactivation is sufficiently rapid and sustained after selective and rapid removal of ionic detergent by cyclodextrin. All these properties may be essential for rapid and direct molecular testing of RNA viruses in respiratory samples.

[0039] The methods and compositions provided herein can be applied to other amplification methods that do not involve purification or separation, such as PCR, RT-PCR, or other isothermal amplification methods. The methods and compositions provided herein can also be applied to genome sequencing methods or any nucleic acid (DNA or RNA) amplification or detection methods that require a sample preparation step. The methods and compositions provided herein can also find use in genotyping, diagnostics, and forensics. The methods and compositions of the present disclosure are not limited to isothermal amplification methods, but rather can be applied to other amplification / detection methods, such as RT-PCR, WGS sequencing, and RNA purification / extraction without separation.

[0040] As described herein, it was unexpectedly found that the kinetics of hydroxypropyl β-CD was sufficiently rapid at a molar ratio of hydroxypropyl β-CD and SDS of 2:1. Inactivation of ribonucleases by denaturation in the presence of reducing agents and reactivation during or after removal of the inactivating agent may occur. However, the kinetics of reactivation upon rapid detergent removal by cyclodextrin after denaturation by ionic detergents in the presence of reducing agents has not been shown before. It was unexpectedly found, as described herein, that the kinetics of reactivation under these conditions in the absence of reducing agents was sufficiently rapid to prevent the subsequent production of DNA copies of RNA mediated by reverse transcriptase. This was true despite the fact that only very short RNA segments of about 30 nucleotides were reverse transcribed in a matter of seconds. Although not previously shown in the art, as described herein, it has been unexpectedly found that the inclusion of a reducing agent prior to cyclodextrin treatment delays reactivation of ribonuclease sufficiently to allow completion of reverse transcription and subsequent amplification and detection of DNA copies. Advantageously, the substantially irreversible inactivation of ribonuclease thus observed during the practice of the methods, kits, and compositions disclosed herein was virtually instantaneous, unlike currently available methods based on heat, which currently available methods such as those described above under a variety of different conditions were not sufficiently irreversible under the conditions described herein to be effective for rapid diagnostic applications.

[0041] Disclosed herein is a method for detecting a target nucleic acid sequence in a sample. In some embodiments, the method includes (a) contacting a sample containing biological entities with a lysis buffer to generate a processed sample, the lysis buffer including one or more lysis agents capable of dissolving the biological entities to release sample nucleic acids contained therein, the sample nucleic acids being suspected to contain a target nucleic acid sequence. The method may include (b) contacting a reagent composition (e.g., a dry composition) with the processed sample to generate an amplification reaction mixture, the reagent composition including one or more protective agents and one or more amplification reagents. The method may include (c) amplifying the target nucleic acid sequence in the amplification reaction mixture, thereby generating a nucleic acid amplification product. The method may include (d) detecting the nucleic acid amplification product, wherein the detection is performed less than about 20 minutes (e.g., about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minutes, or any value or range between any two of such values) from the time the reagent composition is contacted with the process sample. In some embodiments, steps (b) and (c) are performed simultaneously (e.g., amplification begins once contact of the dry composition and the process sample occurs). In some embodiments, the reagent composition comprises two or more reagent compositions (comprising the same or different components). In some embodiments, the lysis buffer comprises two or more lysis buffers (comprising the same or different components).

[0042] The sample nucleic acid may include sample ribonucleic acid and / or sample deoxyribonucleic acid. The sample ribonucleic acid may include cellular RNA, mRNA, microRNA, bacterial RNA, viral RNA, or any combination thereof. The one or more amplification reagents may include a reverse transcriptase and / or an enzyme having hyperthermophile polymerase activity. In some embodiments, the enzyme having hyperthermophile polymerase activity has reverse transcriptase activity. Contacting the reagent composition (e.g., a dry composition) with the process sample may include dissolving the reagent composition in the process sample. The reagent composition may include one or more of a reverse transcriptase, an enzyme having hyperthermophile polymerase activity, a first primer, a second primer, and a reverse transcription primer. The amplification may be performed under isothermal amplification conditions. Detecting the nucleic acid amplification products may include using a real-time detection method. The one or more lysis reagents may comprise about 0.001% (w / v) to about 1.0% (w / v) of the sample to be processed, for example, about 0.2% (w / v) of the sample to be processed. The sample nucleic acid may include a nucleic acid that includes a target nucleic acid sequence. The target nucleic acid sequence may include a first strand and a second strand that are complementary to each other.

[0043] Amplifying a target nucleic acid sequence can include amplifying a target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other under isothermal amplification conditions, the amplification comprising contacting a nucleic acid comprising the target nucleic acid sequence with: i) a first primer and a second primer, where the first primer is capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence and the second primer is capable of hybridizing to a sequence of the second strand of the target nucleic acid sequence; and ii) an enzyme having hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product, the nucleic acid amplification product comprising: (1) a sequence of the first primer and its reverse complement, (2) a sequence of the second primer and its reverse complement, and (3) a spacer sequence flanked by the (1) sequence of the first primer and its reverse complement and the (2) sequence of the second primer and its reverse complement, the spacer sequence being 1-10 bases in length. In some embodiments, the amplification does not include using an enzyme other than an enzyme having hyperthermophile polymerase activity, and the amplification does not include denaturing the nucleic acid. In some embodiments, the method does not include contacting the nucleic acid with a single-stranded DNA binding protein before or during step (c). In some embodiments, the method does not include thermal or enzymatic denaturation of the sample nucleic acid.

[0044] The nucleic acid may be double-stranded DNA. The nucleic acid may be a product of a reverse transcription reaction. The nucleic acid may be a product of a reverse transcription reaction generated from a sample ribonucleic acid. Step (c) may include generating the nucleic acid by a reverse transcription reaction. The sample nucleic acid may include a sample ribonucleic acid. The method may include contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to generate cDNA. Amplifying the target nucleic acid sequence includes the steps of: (c1) contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to generate cDNA; (c2) contacting the cDNA with an enzyme having hyperthermophile polymerase activity to generate dsDNA, the dsDNA comprising the target nucleic acid sequence, the target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other; and (c3) amplifying the target nucleic acid sequence under isothermal amplification conditions, the amplification including: amplifying the dsDNA with (i) a first primer and a second primer, the first primer capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence and the second primer capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence. and (ii) an enzyme having hyperthermophilic polymerase activity, thereby generating a nucleic acid amplification product, the nucleic acid amplification product comprising (1) the sequence of the first primer and its reverse complement, (2) the sequence of the second primer and its reverse complement, and (3) a spacer sequence flanked by (1) the sequence of the first primer and its reverse complement and (2) the sequence of the second primer and its reverse complement, the spacer sequence being 1 to 10 bases in length.

[0045] In some embodiments, the method does not include the use of an enzyme other than a reverse transcriptase and / or an enzyme having hyperthermophile polymerase activity. Step (d) may further comprise determining the amount of dsDNA and / or nucleic acid comprising the target nucleic acid sequence in the sample. In some embodiments, the enzyme having hyperthermophile polymerase activity has an amino acid sequence that is at least about 90% or at least about 95% identical to the amino acid sequence of SEQ ID NO: 1 or a functional fragment thereof. The enzyme having hyperthermophile polymerase activity may be a polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the enzyme having hyperthermophile polymerase activity has low or no exonuclease activity. Amplification of the target nucleic acid sequence may be carried out at a constant temperature of about 55°C to about 75°C, for example about 65°C. The first primer, the second primer, or both may be about 8 to 17 bases long. The first primer, the second primer, or both may contain one or more of DNA bases, modified DNA bases, or combinations thereof. The nucleic acid amplification product may be about 20 to 40 bases long. The spacer sequence may contain a portion of the target nucleic acid sequence. The spacer sequence may be 1 to 10 bases long.

[0046] In some embodiments, the method includes contacting the nucleic acid amplification product with a signal generating oligonucleotide capable of hybridizing to the amplification product. The signal generating oligonucleotide may include a fluorophore, a quencher, or both. Detecting the nucleic acid amplification product may include detecting a fluorescent signal. The fluorescent signal may be from a molecular beacon. The method may be performed in a single reaction vessel. The sample ribonucleic acid may be contacted simultaneously with the reverse transcriptase and the enzyme having hyperthermophile polymerase activity. The sample ribonucleic acid may be contacted simultaneously with the reverse transcriptase, the enzyme having hyperthermophile polymerase activity, and the first and second primers. In some embodiments, the sample ribonucleic acid is contacted simultaneously with the reverse transcriptase, the enzyme having hyperthermophile polymerase activity, the first primer, the second primer, and the reverse transcription primer. Reverse transcription of the sample ribonucleic acid may occur by addition of a reverse transcription primer. In some embodiments, the reverse transcription primer is an oligo(dT) primer, a random hexanucleotide primer, or a target specific oligonucleotide primer. In some embodiments, the oligo(dT) primer is 12-18 nucleotides in length and binds to an endogenous poly(A)+ tail at the 3' end of the mRNA. The random hexanucleotide primer can bind to various complementary sites on the sample ribonucleic acid. The target-specific oligonucleotide primer typically selectively primes the sample ribonucleic acid of interest. The first primer and / or the second primer may be a reverse transcription primer.

[0047] The amplification step may comprise multiplex amplification of two or more target nucleic acid sequences. The detection step may comprise multiplex detection of two or more nucleic acid amplification products derived from said two or more target nucleic acid sequences. The two or more target nucleic acid sequences may be specific for two or more different organisms, including, but not limited to, one or more of Chlamydia trachomatis, Neisseria gonorrhoeae, SARS-CoV-2, influenza A, influenza B, and / or influenza C. In some embodiments, the amplification comprises one or more of the following amplification methods: APA, LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR, and IMDA. In some embodiments, the amplification does not comprise one or more of the following: LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR, and IMDA. In some embodiments, the amplification does not comprise LAMP.

[0048] In some embodiments, the method does not include one or more of, or does not include, any of the following: (i) diluting the sample to be processed; (ii) diluting the amplification reaction mixture; (iii) heat denaturing the sample to be processed; (iv) sonicating the sample to be processed; (v) sonicating the amplification reaction mixture; (vi) adding nuclease inhibitors to the sample to be processed; (vii) adding nuclease inhibitors to the amplification reaction mixture; (viii) purifying the sample; (ix) purifying the sample nucleic acid; (x) purifying the nucleic acid amplification product; (xi) removing one or more lysis agents from the sample to be processed or the amplification reaction mixture; (xii) heat and / or enzymatic denaturation of the sample nucleic acid before and / or during amplification; and (xiii) adding RNase H to the sample to be processed or the amplification reaction mixture. In some embodiments, the sample is held at an amplification temperature (e.g., 67° C.). Step (a), step (b), step (c), and / or step (d) may be performed for about 20, 15, 10, 5, 2.5, or 1 minute. In some embodiments, step (a), step (b), step (c), and / or step (d) comprises sonication, osmotic shock, chemical treatment, heating, or any combination thereof.

[0049] The one or more lysis agents may be capable of denaturing the one or more amplification reagents. The one or more protection agents may be capable of preventing the one or more lysis agents from denaturing the one or more amplification reagents. The one or more protection agents may be capable of sequestering (e.g., complexing) the one or more lysis agents, thereby preventing the one or more amplification reagents from denaturing by the one or more lysis agents. In some embodiments, at least about 5% (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or a number or range between any two of such values) of the one or more lysis agents in the amplification reaction mixture is sequestered by the one or more protection agents. In some embodiments, less than about 40%, 30%, 20%, 10%, 5%, 3%, 1%, 0.1%, 0%, or less than any value or range between these values, of one or more amplification reagents is denatured by one or more lysis agents.

[0050] The sample may include multiple nucleases (e.g., deoxyribonucleases, ribonucleases). The one or more lysis agents may be capable of denaturing the nuclease to generate a denatured nuclease (e.g., a denatured deoxyribonuclease, a denatured ribonuclease). The one or more reducing agents may be capable of reducing the ribonuclease to generate a reduced nuclease (e.g., a reduced deoxyribonuclease, a reduced ribonuclease). The one or more reducing agents and the one or more lysis agents may be capable of generating a reduced denatured nuclease (e.g., a reduced denoxyribonuclease, a reduced denatured ribonuclease). The denatured ribonuclease and / or the reduced denatured ribonuclease may be capable of renaturing over time and / or upon reduced physical interaction with the one or more lysis agents, thereby generating a renatured ribonuclease. In some embodiments, the denatured deoxyribonuclease and / or reduced denatured deoxyribonuclease may be able to renature over time and / or reduced physical interaction with one or more lysis agents, thereby producing renatured deoxyribonuclease. In some embodiments, sequestration of one or more lysis agents reduces physical interaction between one or more lysis agents and the denatured deoxyribonuclease, reduced denatured deoxyribonuclease, denatured ribonuclease, and / or reduced denatured ribonuclease. The denatured deoxyribonuclease, reduced deoxyribonuclease, reduced denatured deoxyribonuclease, denatured ribonuclease, reduced ribonuclease, and / or reduced denatured ribonuclease may be enzymatically inactive. The renatured nuclease may be enzymatically active. Renaturation may include refolding. "Refolding," as used herein, shall be given its ordinary meaning and shall describe any process, reaction, or method of converting a disulfide bond-containing polypeptide from an improperly folded or unfolded state to a native or correctly folded conformation with respect to the disulfide bonds.

[0051] The reduced modified nuclease (e.g., reduced modified deoxyribonuclease, reduced modified ribonuclease) can be renatured at least about 1.1 times (e.g., 1.1 times, 1.3 times, 1.5 times, 1.7 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 1000 times, 10000 times, or any value or range between any of these values) slower than the modified nuclease (e.g., modified deoxyribonuclease, modified ribonuclease). Compared to a comparable method in which the lysis buffer does not contain one or more reducing agents, at least about 1.1 times (e.g., 1.1 times, 1.3 times, 1.5 times, 1.7 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 1000 times, 10000 times, or any value or range between any of these values) more sample ribonucleic acid and / or sample deoxyribonucleic acid can be used as a template by an enzyme having reverse transcriptase and / or hyperthermophile polymerase activity to generate cDNA and / or nucleic acid amplification products, respectively.

[0052] The amplification reaction mixture may contain at least 1.1-fold (e.g., 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 1000-fold, 10000-fold, or any value or range between any of these values) less nuclease (e.g., deoxyribonuclease, ribonuclease) activity after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and / or 20 minutes of step (b) and / or step (c) of the methods provided herein, compared to a comparable method in which the lysis buffer does not contain one or more reducing agents. In some embodiments, at least about 5% (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or a number or range between any two of such values) of the nucleases in the sample, processed sample, and / or amplification reaction mixture are enzymatically inactive.

[0053] In some embodiments, cDNA and / or nucleic acid amplification products are generated from at least about 5% (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or any value or range between any two of these values) of the sample ribonucleic acid and / or sample deoxyribonucleic acid, respectively. In some embodiments, less than about 30% (e.g., 30%, 20%, 10%, 5%, 3%, 1%, 0.1%, or any value or range between any of these values) of the sample ribonucleic acid and / or sample deoxyribonucleic acid is degraded by nucleases before being used as a template in the reverse transcription reaction and / or amplification reaction, respectively. The term "isothermal amplification reaction" shall be given its ordinary meaning and shall include reactions in which the temperature does not change significantly during the reaction. In some embodiments, the temperature of the isothermal amplification reaction does not deviate by more than 10°C during the main enzymatic reaction step in which amplification occurs, e.g., by no more than 5°C, or no more than 2°C. Depending on the method of isothermal amplification of nucleic acids, different enzymes may be used for amplification. Isothermal amplification compositions and methods are described in the PCT application published as WO2017176404, the contents of which are incorporated herein by reference in their entirety.

[0054] In some embodiments, the methods and compositions described herein include a storage-stable lysis buffer. In some embodiments, the lysis buffer is resistant to the formation of precipitates over a period of time under storage conditions (e.g., a storage-stable lysis buffer). Compositions, kits, and methods in which the lysis buffer is resistant to precipitation are described in U.S. Provisional Patent Application No. 63 / 307,092, filed February 5, 2022, entitled "NON-OPAQUE LYTIC BUFFER COMPOSITION FORMULATIONS," the contents of which are incorporated herein by reference in their entirety. Some embodiments of the methods and compositions provided herein do not include agents and / or conditions that denature nucleic acids (e.g., promote strand separation and / or promote unwinding) other than acid and / or low pH conditions. Compositions, kits, and methods for nucleic acid detection in which nucleic acid strands are dissociated under low pH conditions (e.g., by contact with an acidic lysis buffer) to facilitate subsequent rapid amplification and detection are described in U.S. Provisional Patent Application No. 63 / 307,085, filed February 5, 2022, and entitled "METHOD FOR SEPARATING GENOMIC DNA FOR SEPARATING GENOMIC DNA FOR AMPLIFICATION OF SHORT NUCLEIC ACID TARGETS," the contents of which are incorporated herein by reference in their entirety.

[0055] Provided herein are methods for amplifying nucleic acids. In some embodiments, the methods include contacting a sample nucleic acid under isothermal amplification conditions with a) at least one oligonucleotide comprising a polynucleotide complementary to a target sequence in the sample nucleic acid, and b) at least one component providing a hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product. In some embodiments, the methods include contacting a sample nucleic acid under isothermal amplification conditions with a) non-enzymatic components comprising at least one oligonucleotide comprising a polynucleotide complementary to a target sequence in the sample nucleic acid, and b) enzymatic components consisting of a hyperthermophile polymerase or a polymerase comprising an amino acid sequence at least about 90% identical to a hyperthermophile polymerase, thereby generating a nucleic acid amplification product. In some embodiments, the methods include contacting a sample nucleic acid under isothermal amplification conditions with a) non-enzymatic components comprising at least one oligonucleotide comprising a polynucleotide complementary to a target sequence in the sample nucleic acid, and b) enzymatic activities consisting of i) a hyperthermophile polymerase activity and ii) optionally a reverse transcriptase activity, thereby generating a nucleic acid amplification product.

[0056] Also provided herein is a method for determining the presence, absence, or amount of a target sequence in a sample nucleic acid, comprising the steps of: a) amplifying a target sequence in the sample nucleic acid, the target sequence comprising a first strand and a second strand, the first strand and the second strand being complementary to one another, the amplification comprising amplifying the sample nucleic acid under helicase- and / or recombinase-free isothermal amplification conditions with: i) a first oligonucleotide and a second oligonucleotide, the first oligonucleotide comprising or consisting of a first polynucleotide that is sequentially complementary to a sequence of the first strand, and the second oligonucleotide comprising or consisting of a second polynucleotide that is sequentially complementary to a sequence of the second strand; and ii) at least one component providing a hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product. and b) contacting the nucleic acid amplification product with at least one component comprising or consisting of: 1) a first nucleotide sequence that is contiguous with or substantially identical to a first polynucleotide of a first oligonucleotide, 2) a second nucleotide sequence that is contiguous with or substantially identical to a second polynucleotide of a second oligonucleotide, and 3) a spacer sequence comprising 1 to 10 bases flanked by the first nucleotide sequence and the second nucleotide sequence; and b) detecting the nucleic acid amplification product, wherein the detection of the nucleic acid amplification product includes the use of a real-time detection method and is performed in 10 minutes or less from the time of contacting the sample nucleic acid with (a)(i) and (a)(ii), thereby determining the presence, absence, or amount of the target sequence in the sample nucleic acid.

[0057] Nucleic Acids, Subjects, Samples, and Nucleic Acid Processing Provided herein are methods and compositions for amplifying nucleic acids. The terms "nucleic acid" and "nucleic acid molecule" can be used interchangeably herein. The terms refer to nucleic acids of any composition, such as DNA (e.g., complementary DNA (cDNA) and genomic DNA (gDNA)), RNA (e.g., message RNA (mRNA), short inhibitory RNA (siRNA), ribosomal RNA (rRNA), tRNA, microRNA, and / or DNA or RNA analogs (e.g., including base analogs, sugar analogs, and / or non-natural backbones), RNA / DNA hybrids, and polyamide nucleic acids (PNAs), all of which may be in single-stranded or double-stranded form and, unless otherwise limited, may include known analogs of natural nucleotides that can function in a manner similar to naturally occurring nucleotides. Nucleic acids may be present in plasmids, phages, autonomously replicating sequences (ARS), centromeres, artificial chromosomes, chromosomes, or in vivo. It may be or be derived from other nucleic acids that can be replicated or replicated in vitro or in a host cell, cell, cell nucleus, mitochondria, or cell cytoplasm. Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses not only the sequence explicitly indicated, but also its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed-base and / or deoxyinosine residue. The term nucleic acid can be used interchangeably with locus, gene, cDNA, and mRNA encoded by a gene.The term may also include nucleotide analogs, single-stranded polynucleotides ("sense" or "antisense", "plus" or "minus" strands, "forward" or "reverse" reading frames, "forward" or "reverse" strands) and RNA or DNA equivalents, derivatives, variants, and analogs synthesized from double-stranded polynucleotides. The term "gene" refers to a segment of DNA involved in producing a polypeptide chain, and generally includes the regions preceding and following the coding region (leader and trailer) involved in the transcription / translation of the gene product and the regulation of transcription / translation, as well as the intervening sequences (introns) between individual coding segments (exons). Nucleotides or bases generally refer to the purine and pyrimidine molecular units of nucleic acids (e.g., adenine (A), thymine (T), guanine (G), and cytosine (C)). In the case of RNA, the base thymine is replaced by uracil. The length or size of a nucleic acid may be expressed as the number of bases.

[0058] In some embodiments of the methods provided herein, one or more nucleic acid targets are amplified. The target nucleic acid may be referred to as a target sequence, a target polynucleotide, and / or a target polynucleotide sequence, and may include double-stranded and single-stranded nucleic acid molecules. The target nucleic acid may be, for example, DNA or RNA. When the target nucleic acid is an RNA molecule, the molecule may be, for example, double-stranded, single-stranded, or the RNA molecule may include a target sequence that is single-stranded. When the target nucleic acid is double-stranded, the target nucleic acid generally includes a first strand and a second strand. The first strand and the second strand may be referred to as a forward strand and a reverse strand, and are generally complementary to each other. When the target nucleic acid is single-stranded, the complementary strand may be generated, for example, by polymerization and / or reverse transcription, making the target nucleic acid double-stranded and having a first / forward strand and a second / reverse strand.

[0059] A target sequence may refer to either the sense or antisense strand of a nucleic acid sequence, or to a sequence present in a target nucleic acid, an amplified copy of the original target sequence, or an amplification product. A target sequence may be a subsequence within a larger polynucleotide. For example, a target sequence may be a short sequence (e.g., 20-50 bases) within a nucleic acid fragment, chromosome, or plasmid that is targeted for amplification. In some embodiments, a target sequence may refer to a sequence within a target nucleic acid that is complementary to an oligonucleotide (e.g., a primer) used to amplify the nucleic acid. Thus, a target sequence may refer to the entire sequence that is targeted for amplification, or may refer to a subsequence within a target nucleic acid to which an oligonucleotide binds. An amplification product may be a larger molecule that includes the target sequence as well as at least one other sequence, or other nucleotide. An amplification product may be approximately the same length as the target sequence, e.g., exactly the same length as the target sequence. An amplification product may comprise or consist of the target sequence. The target sequence length and / or guanosine cytosine (GC) concentration (percent) may depend, in part, on the temperature at which the amplification reaction is carried out, which may depend, in part, on the stability of the polymerase used in the reaction. Trial assays may be performed to determine the appropriate target sequence length and GC concentration for a set of reaction conditions. For example, if the polymerase is stable up to 60° C.-65° C., the target sequence may be, for example, 19-50 nucleotides long, or, for example, about 40-50, 20-45, 20-40, or 20-30 nucleotides long. The GC concentration under these conditions may be, for example, less than 60%, less than 55%, less than 50%, or less than 45%.

[0060] Target nucleic acids may include, for example, genomic nucleic acids, plasmid nucleic acids, mitochondrial nucleic acids, cellular nucleic acids, extracellular nucleic acids, bacterial nucleic acids, and viral nucleic acids. In some embodiments, target nucleic acids may include genomic DNA, chromosomal DNA, plasmid DNA, mitochondrial DNA, genes, any type of cellular RNA, messenger RNA, bacterial RNA, viral RNA, or synthetic oligonucleotides. Genomic nucleic acids may include any nucleic acid derived from any genome, for example, animal genomes, plant genomes, insect genomes, viral genomes, and bacterial genomes (e.g., genomes present in spores). In some embodiments, genomic target nucleic acids are present within a particular genomic locus or multiple genomic loci. A genomic locus may include any or a combination of open reading frame DNA, non-transcribed DNA, intronic sequences, exonic sequences, promoter sequences, enhancer sequences, flanking sequences, or any sequences considered to be associated with a given genomic locus.

[0061] The target sequence may contain one or more repetitive elements (e.g., multiple repeats, inverted repeats, palindromic sequences, tandem repeats, microsatellites, and minisatellites, etc.). In some embodiments, the target sequence is present in the sample nucleic acid (e.g., in a nucleic acid fragment, in a chromosome, in a genome, in a plasmid) as a repetitive element (e.g., multiple repeats, inverted repeats, palindromic sequences, tandem repeats, microsatellite repeats, and minisatellite repeats, etc.). For example, the target sequence may occur multiple times as a repetitive element, and one, some, or all occurrences of the target sequence in the repetitive element can be amplified (e.g., using a single pair of primers) using the methods described herein. In some embodiments, the target sequence is present in the sample nucleic acid (e.g., in a nucleic acid fragment, in a chromosome, in a genome, in a plasmid) as a duplicate and / or paralog. The target nucleic acid may include microRNA. MicroRNA, miRNA, or small temporal RNA (stRNA) are short (e.g., about 21-23 nucleotides long), single-stranded RNA sequences involved in gene regulation. MicroRNAs can interfere with the translation of messenger RNA and are partially complementary to messenger RNA. The target nucleic acid may include microRNA precursors, such as primary transcripts (pri-miRNA) and pre-miRNA stem-loop structure RNAs that are further processed into miRNA. The target nucleic acid may include small interfering RNA (siRNA), which are short (e.g., about 20-25 nucleotides long), at least partially double-stranded RNA molecules involved in RNA interference (e.g., downregulation of viral replication or gene expression).

[0062] The nucleic acids used in the methods described herein can be obtained from any suitable biological specimen or sample, for example, isolated from a sample obtained from a subject. The subject can be any living or non-living organism, including, but not limited to, humans, non-human animals, plants, bacteria, fungi, viruses, or protists. Any human or non-human animal can be selected, including, but not limited to, mammals, reptiles, birds, amphibians, fish, ungulates, ruminants, bovines (e.g., cows), equines (e.g., horses), caprines and ovines (e.g., sheep, goats), porcines (e.g., pigs), camelids (e.g., camels, llamas, alpacas), monkeys, apes (e.g., gorillas, chimpanzees), ursidae (e.g., bears), poultry, dogs, cats, mice, rats, fish, dolphins, whales, and sharks. The subject can be male or female, and the subject can be of any age (eg, embryo, fetus, infant, child, adult).

[0063] A sample or test sample may be any specimen isolated or obtained from a subject or a portion thereof. Non-limiting examples of specimens include fluids or tissues from a subject, including but not limited to blood or blood products (e.g., serum or plasma, etc.), umbilical cord blood, bone marrow, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal, ear, arthroscopic), serum, plasma, urine, aspirate, biopsy sample, intestinal puncture sample, cells (e.g., blood cells) or portions thereof (e.g., mitochondria, nuclei, or extracts, etc.), female reproductive tract lavage fluid, urine, feces, sputum, saliva, nasal mucosa, prostatic fluid, lavage, semen, lymph, bile, tears, sweat, breast milk, mammary fluid, hard tissue (e.g., liver, spleen, kidney, lung, or ovary), etc., or combinations thereof. The term blood, as conventionally defined, includes whole blood, blood products, or any fraction of blood such as serum, plasma, or buffy coat. Plasma refers to the fraction of whole blood obtained from centrifugation of blood that has been treated with an anticoagulant. Serum refers to the aqueous portion of the fluid remaining after a blood sample has clotted. Fluid or tissue samples are often collected according to standard protocols typically followed by hospitals or clinics. In the case of blood, an appropriate amount of peripheral blood (e.g., 3-40 milliliters) is often collected and can be stored according to standard procedures before or after preparation.

[0064] The sample may include nucleic acid from spores, viruses, cells, prokaryotes or eukaryotes, and / or samples containing any free nucleic acid. For example, the methods described herein can be used to detect nucleic acid outside of spores (e.g., without the need for lysis). The sample can be isolated from any material suspected of containing the target sequence, such as from a subject as described above. In some embodiments, the target sequence is present in air, plants, soil, or other material suspected of containing biological organisms. Nucleic acids can be derived (e.g., isolated, extracted, purified) from one or more sources by methods known in the art. Any suitable method for isolating, extracting, and / or purifying nucleic acids from biological samples can be used, including DNA preparation methods in the art and various commercially available reagents or kits such as Qiagen's QIAamp Circulating Nucleic Acid Kit, QiaAmp DNA Mini Kit or QiaAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany), GenomicPrep™ Blood DNA Isolation Kit (Promega, Madison, Wis.), and GFX™ Genomic Blood DNA Purification Kit (Amersham, Piscataway, N.J.), or combinations thereof. U.S. Patent No. 7,888,006 provides a DNA purification method, but does not disclose the compositions (e.g., lysis buffer, protection agent) and methods provided herein.

[0065] In some embodiments, a cell lysis procedure is performed. Cell lysis may be performed before starting the amplification reaction described herein (e.g., to release DNA and / or RNA from the cells for amplification). Cell lysis procedures and reagents are known in the art and can be performed by chemical methods (e.g., detergents, hypotonic solutions, and enzymatic procedures, or combinations thereof), physical methods (e.g., French press and sonication, or the like), or electrolytic lysis methods. For example, chemical methods generally involve disrupting cells using a lysis agent and extracting nucleic acids from the cells, followed by treatment with chaotropic salts. In some embodiments, cell lysis involves the use of detergents (e.g., ionic, nonionic, anionic, zwitterionic). In some embodiments, cell lysis involves the use of ionic detergents (e.g., sodium dodecyl sulfate (SDS), sodium lauryl sulfate (SLS), deoxycholate, cholate, sarkosyl). Physical methods such as freeze / thaw followed by grinding and the use of a cell press may also be useful. High salt lysis procedures can also be used. For example, alkaline lysis procedures can be used. The latter procedure traditionally incorporates the use of a phenol-chloroform solution, and an alternative phenol-chloroform-free procedure can be used that includes three solutions. In the latter procedure, for example, one solution can include 15 mM Tris, pH 8.0, 10 mM EDTA, and 100 μg / ml RNase A, a second solution can include 0.2 N NaOH and 1% SDS, and a third solution can include 3 M KOAc, pH 5.5. In some embodiments, a cell lysis buffer is used in conjunction with the methods and components described herein.

[0066] Nucleic acids for performing the methods described herein can be provided without processing a sample containing nucleic acids. For example, nucleic acids for performing the amplification methods described herein can be provided without prior nucleic acid purification. In some embodiments, the target sequence is amplified directly from the sample (e.g., without any nucleic acid extraction, isolation, purification, and / or partial purification steps). In some embodiments, nucleic acids for performing the methods described herein after processing a sample containing nucleic acids are provided. For example, the nucleic acids can be extracted, isolated, purified, or partially purified from the sample. The term "isolated" generally refers to a nucleic acid that has been removed from its original environment (e.g., the natural environment if naturally occurring, or a host cell if exogenously expressed) and thus has been altered from its original environment by human intervention (e.g., "by the hand of man"). The term "isolated nucleic acid" can refer to a nucleic acid that has been removed from a subject (e.g., a human subject). Isolated nucleic acids can be provided that have less non-nucleic acid components (e.g., proteins, lipids, carbohydrates) than the amount of components present in the source sample. A composition that includes an isolated nucleic acid may contain no more than about 50%-99% of non-nucleic acid components. A composition containing an isolated nucleic acid may contain no more than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% of non-nucleic acid components. The term "purified" generally refers to a nucleic acid that contains less non-nucleic acid components (e.g., proteins, lipids, carbohydrates) than the amount of non-nucleic acid components present before the nucleic acid is subjected to a purification procedure. A composition containing a purified nucleic acid may contain no more than about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% of other non-nucleic acid components.

[0067] Nucleic acids can be provided for carrying out the methods described herein without modifying the nucleic acid, e.g., by denaturation, digestion, nicking, unwinding, incorporating and / or ligating heterologous sequences, adding epigenetic modifications, adding labels (e.g., 32P, 33 P, 125 I, or 35 The label may include a radioactive label such as S; an enzyme label such as alkaline phosphatase; a fluorescent label such as fluorescein isothiocyanate (FITC); or other labels such as biotin, avidin, digoxigenin, antigens, haptens, and fluorescent dyes. Thus, in some embodiments, unmodified nucleic acids are amplified.

[0068] The methods disclosed herein for detecting a target nucleic acid sequence (single-stranded or dsDNA and / or RNA) in a sample can detect a target nucleic acid sequence (e.g., DNA or RNA) with high sensitivity. In some embodiments, the methods can be used to detect a target DNA / RNA present in a sample containing multiple RNAs / DNAs (including a target RNA / DNA and multiple non-target RNAs / DNAs), where the target RNA / DNA is 10, 20, 25, 50, 100, 500, 10 3 , 5×10 3 , 10 4 , 5×10 4 , 10 5 , 5×10 5 , 10 6 , or 10 7 There are one or more copies per non-target DNA / RNA. As used herein, the terms "RNA / DNA" and "RNAs / DNAs" shall be given their ordinary meanings and shall refer to DNA, or RNA, or a combination of DNA and RNA.

[0069] The detection threshold of a method for detecting target RNA / DNA in a sample can be, for example, a concentration of 10 nM or lower. The term "detection threshold" shall be given its ordinary meaning and shall describe the minimum amount of target RNA / DNA that must be present in a sample for detection to occur. As an illustrative example, if the detection threshold is 10 nM, a signal can be detected when the target RNA / DNA is present in the sample at a concentration of 10 nM or higher. In some embodiments, the method of the present disclosure can detect a signal at a concentration of 5 nM or lower, 1 nM or lower, 0.5 nM or lower, 0.1 nM or lower, 0.05 nM or lower, 0.01 nM or lower, 0.005 nM or lower, 0.001 nM or lower, 0.0005 nM or lower, 0.0001 nM or lower, 0.00005 nM or lower, 0.00001 ... or lower, 10 pM or lower, 1 pM or lower, 500 fM or lower, 250 fM or lower, 100 fM or lower, 50 fM or lower, 500 aM (atomolar) or lower, 250 aM or lower, 100 aM or lower, 50 aM or lower, 10 aM or lower, or 1 aM or lower. In some embodiments, a composition or method of the present disclosure exhibits attomolar (aM), femtomolar (fM), picomolar (pM), and / or nanomolar (nM) detection sensitivity.

[0070] A sample may contain sample nucleic acids (e.g., multiple types of sample nucleic acids). The term "multiple types" is used herein to mean two or more. Thus, in some embodiments, a sample contains two or more (e.g., three or more, five or more, ten or more, twenty or more, fifty or more, one hundred or more, five hundred or more, one thousand or more, or five thousand or more) types of sample nucleic acids (e.g., DNA / RNA). The method of the present disclosure can be used as a highly sensitive method for detecting target nucleic acids present in a sample (e.g., in a complex mixture of nucleic acids such as DNA / RNA). In some embodiments, a sample contains 5, 10, 20, 25, 50, 100, 500, 1 ... 3 Seeds, 5x10 3 seeds, 10 4 Seeds, 5x10 4 seeds, 10 5 Seeds, 5x10 5 seeds, 10 6 Seeds or 10 7 The sample may contain DNA / RNA from more than 10, 50, or more species that differ in sequence from one another. In some embodiments, the sample contains DNA / RNA from a cell (e.g., a eukaryotic cell, a mammalian cell, or a human cell) or a cell lysate (e.g., a eukaryotic cell lysate, a mammalian cell lysate, a human cell lysate, a prokaryotic cell lysate, a plant cell lysate, etc.).

[0071] The term "sample" as used herein shall be given its ordinary meaning and shall include any sample containing RNA and / or DNA (e.g., to determine whether target DNA and / or target RNA is present in an RNA and / or DNA population). A sample may be from any source, for example, a sample may be a synthetic combination of purified DNA and / or RNA. A sample may be a cell lysate, a DNA / RNA enriched cell lysate, or DNA / RNA isolated and / or purified from a cell lysate. A sample may be from a patient (e.g., for diagnostic purposes). A sample may be from permeabilized cells, crosslinked cells, tissue sections, or combinations thereof. A sample may be from tissue prepared by crosslinking followed by delipidation and adjustment to a uniform refractive index. A sample may include a target nucleic acid (e.g., target DNA / RNA) and multiple species of non-target DNA / RNA. In some embodiments, the target DNA / RNA is 10, 20, 25, 50, 100, 500, 10 3 , 5×10 3 , 10 4 , 5×10 4 , 10 5 , 5×10 5 , 10 6 , or 10 7 Present in the sample at 1 copy per non-target DNA / RNA.

[0072] Samples related to patients include blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny, as well as samples that have been manipulated in some way after procurement (such as treatment with a reagent); washed; or enriched for a particular cell population (e.g., cancer cells) or a particular type of molecule (e.g., RNA). Samples may include biological samples including, but are not limited to, clinical samples such as blood, plasma, serum, aspirates, cerebrospinal fluid (CSF), tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, and bone marrow. Biological samples may also include biological fluids derived therefrom (e.g., cancerous cells, infected cells, etc.), such as samples containing RNA obtained from such cells (e.g., cell lysates or other cell extracts containing RNA).

[0073] The source of the sample may be a diseased (or suspected) cell, fluid, tissue, or organ, or may be a normal (non-diseased) cell, fluid, tissue, or organ. In some embodiments, the source of the sample is a cell, tissue, or organ infected (or suspected) with a pathogen. For example, the source of the sample may be an individual, which may or may not be infected, and the sample may be any biological sample collected from the individual (e.g., blood, saliva, biopsy, plasma, serum, bronchoalveolar lavage fluid, sputum, fecal sample, cerebrospinal fluid, fine needle aspirate, swab sample (e.g., buccal swab, cervical swab, nasal swab), interstitial fluid, synovial fluid, nasal secretion, tears, buffy coat, mucosal sample, epithelial cell sample (e.g., epithelial cell scraping), etc.). The sample may be a cell-free liquid sample or a liquid sample containing cells. Pathogens may be viruses, fungi, helminths, protozoans, malarial parasites, Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites. "Helminths" include roundworms, heartworms, and plant-eating nematodes (Nematoda), trematodes (Tematoda), thorny head worms, and cestodes (Taenia). Protozoan infections include infections with Giardia spp., Trichomonas spp., African trypanosomiasis, amebic dysentery, babesiosis, balantidiosis, Chagas disease, coccidiosis, malaria, and toxoplasmosis. Examples of pathogens, such as parasitic / protozoan pathogens, include, but are not limited to, Plasmodium falciparum, Plasmodium vivax, Trypanosoma cruzi, and Toxoplasma gondii. Fungal pathogens include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans, and the like.albicans). Pathogenic viruses include, but are not limited to, immunodeficiency viruses (e.g., HIV), influenza viruses, dengue fever, West Nile virus, herpes viruses, yellow fever viruses, hepatitis C viruses, hepatitis A viruses, hepatitis B viruses, and papilloma viruses. Pathogenic viruses include, but are not limited to, papovaviruses (e.g., HPV, polyomaviruses); hepadnaviruses; herpes viruses (e.g., HSV (e.g., HSV I, HSV II), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, pityriasis rosea, and the like. Rosea), Kaposi's sarcoma-associated herpesvirus); adenoviruses (e.g., atadenovirus, aviadenovirus, ichtadenovirus, mastadenovirus, siadenovirus); poxviruses (e.g., smallpox, vaccinia virus, cowpox virus, monkeypox virus, orf virus, pseudocowpox, bovine papular stomatitis virus; variola virus, yaba monkey tumor virus; molluscum contagiosum virus (MCV)); parvoviruses (e.g., adeno-associated virus (AAV), parvovirus B19, human bocavirus, bufavirus, human parv4 G1); Geminiviridae; Nanoviridae; and Phycodnaviridae. Non-limiting examples of pathogens include Mycobacterium tuberculosis and Streptococcus agalactiae, Methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum, Haemophilus influenzae type B, and the like.B), Treponema pallidum, Lyme disease spirochete, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, Rabies virus, Human serum bulbo-like virus, Respiratory syncytial virus, Measles virus, Adenovirus, Human T-cell leukemia virus, Murine leukemia virus, Mumps virus, Vesicular stomatitis virus, Sindbis virus, Lymphocytic choriomeningitis virus, Wart virus, Bluetongue virus, Sendai virus, Feline leukemia virus, Reovirus, Poliovirus, Simian virus 40, Mouse mammary tumor virus, Dengue virus, Rubella virus, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeria tenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis arthritidis, M.hyorhinis, M.orale, M.arginini, Acholeplasma laidlowiilaidlawii, M. salivarium, and M. pneumoniae.

[0074] amplification Provided herein are methods for amplifying nucleic acids. In some embodiments, the nucleic acids are amplified using a suitable amplification process. Nucleic acid amplification typically involves enzymatic synthesis of nucleic acid amplicons (copies) that contain sequences complementary to the nucleotide sequence being amplified. In some embodiments, the amplification method is carried out in a single vessel, a single chamber, and / or a single volume (i.e., a continuous volume). In some embodiments, the amplification method and the detection method (e.g., the detection methods described herein) are carried out in a single vessel, a single chamber, and / or a single volume (i.e., a continuous volume).

[0075] The terms "amplify", "amplification", "amplification reaction", or "amplifying" refer to any in vitro process for multiplying copies of a target nucleic acid. Amplification can also refer to an "exponential" increase in the target nucleic acid. "Amplifying" can also refer to a linear increase in the number of target nucleic acids, but is distinct from a one-time, single primer extension step. In some embodiments, a limited amplification reaction, also known as preamplification, can be performed. Preamplification is a method in which a limited amount of amplification occurs because a small number of cycles, e.g., 10 cycles, are performed. Preamplification allows some amplification, but stops the amplification before the exponential phase, typically producing about 500 copies of the desired nucleotide sequence. Preamplification can be used to limit the inaccuracies associated with reactant exhaustion in a particular amplification reaction and can also reduce amplification bias due to target nucleotide sequence or species abundance. In some embodiments, a one-time primer extension can be performed as a pre-step to linear or exponential amplification.

[0076] A general description of the amplification process is provided herein. For example, when a primer (e.g., an oligonucleotide described herein) and a target nucleic acid are contacted, the complementary sequences anneal or hybridize to each other. The primer can anneal to the target nucleic acid at or near (e.g., adjacent, abutting, etc.) the sequence of interest. A primer annealed to a target may be referred to as a primer-target hybrid, hybridized primer-target, or primer-target duplex. The term "near" or "adjacent" when referring to a nucleotide sequence of interest refers to the distance (e.g., number of bases) or region between the end of the primer and one or more nucleotides (e.g., a nucleotide sequence) of the target. Generally, adjacent is within a range of about 1 nucleotide to about 50 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nucleotides) from the nucleotide or nucleotide sequence of interest. In some embodiments, a set of primers (e.g., a pair of primers, a forward primer and a reverse primer, a first oligonucleotide and a second oligonucleotide) anneals within about 1-20 nucleotides of a nucleotide or nucleotide sequence of interest to generate an amplification product. In some embodiments, the primers anneal within the nucleotide or nucleotide sequence of interest. After annealing, each primer is extended along the target (i.e., the template strand) by a polymerase to generate a complementary strand. For example, several cycles of primer annealing and extension can be performed until a detectable amount of amplification product is generated. In some embodiments, when the target nucleic acid is RNA, a DNA copy (cDNA) of the target RNA is synthesized by reverse transcription before or during the amplification step.

[0077] Components of an amplification reaction (e.g., one or more amplification reagents) can include, for example, one or more primers (e.g., individual primers, primer pairs, primer sets, oligonucleotides, and multiple primer sets for multiplex amplification), a nucleic acid target (e.g., a target nucleic acid derived from a sample), one or more polymerases, nucleotides (e.g., dNTPs, and the like), and a suitable buffer (e.g., a buffer containing a detergent, a reducing agent, monovalent ions, and divalent ions). The amplification reaction may further include one or more of a reverse transcriptase, a reverse transcription primer, and one or more detection agents. Nucleic acid amplification can be performed in the presence of natural nucleotides, such as dideoxyribonucleoside triphosphates (dNTPs) and / or derivatized nucleotides. Natural nucleotides generally refer to adenylic acid, guanylic acid, cytidylic acid, thymidylic acid, or uridylic acid. Derivatized nucleotides generally are nucleotides other than natural nucleotides. Ribonucleoside triphosphates are referred to as NTPs or rNTPs, where N can be A, G, C, U. Deoxynucleoside triphosphate substrates are referred to as dNTPs, where N can be A, G, C, T, or U. Monomeric nucleotide subunits may be represented herein as A, G, C, T, or U, without specific reference to DNA or RNA. In some embodiments, non-naturally occurring nucleotides or nucleotide analogs can be used, such as analogs that include a detectable label (e.g., a fluorescent or colorimetric label). For example, nucleic acid amplification can be performed using labeled dNTPs, such as dNTPs, where N can be A, G, C, T, or U. 32 P, 33 P, 125 I, or 35 It can be performed in the presence of a radioactive label such as S; an enzyme label such as alkaline phosphatase; a fluorescent label such as fluorescein isothiocyanate (FITC); or other labels such as biotin, avidin, digoxigenin, antigens, haptens, or fluorescent dyes. In some embodiments, nucleic acid amplification can be performed in the presence of modified dNTPs, e.g., heat-activated dNTPs (e.g., CleanAmp™ dNTPs from TriLink).

[0078] The one or more amplification reagents may include non-enzymatic and enzymatic components. Non-enzymatic components may include, for example, primers, nucleotides, buffers, salts, reducing agents, detergents, and ions. In some embodiments, the non-enzymatic components do not include proteins (e.g., nucleic acid binding proteins), enzymes, or proteins with enzymatic activity, such as polymerases, reverse transcriptases, helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, and recombinases. In some embodiments, the enzymatic components consist of polymerases or consist of polymerases and reverse transcriptases. Thus, such enzymatic components would exclude other proteins (e.g., nucleic acid binding proteins and / or proteins with enzymatic activity), such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, and recombinases.

[0079] In some embodiments, the amplification conditions include an enzymatic activity (e.g., an enzymatic activity provided by a polymerase, or an enzymatic activity provided by a polymerase and a reverse transcriptase). In some embodiments, the enzymatic activity does not include an enzymatic activity provided by an enzyme other than a polymerase and / or a reverse transcriptase, such as, for example, a helicase, a topoisomerase, a ligase, an exonuclease, an endonuclease, a restriction enzyme, a nicking enzyme, and a recombinase. The polymerase activity and the reverse transcriptase activity may be provided by separate enzymes or separate enzyme types (e.g., a polymerase and a reverse transcriptase), or may be provided by a single enzyme or enzyme type (e.g., a polymerase). The amplification of nucleic acids may include non-thermal cycling type PCR. In some embodiments, the amplification of nucleic acids includes isothermal amplification processes, such as isothermal polymerase chain reaction (iPCR). Isothermal amplification is generally an amplification process carried out at a constant temperature. Terms such as isothermal conditions, isothermally, and constant temperature generally refer to reaction conditions in which the reaction temperature is kept essentially constant during the course of the amplification reaction. Isothermal amplification conditions generally do not include a thermal cycling (i.e., cycling between upper and lower temperature limits) component to the amplification process. When amplifying under isothermal conditions, the reaction can be kept at an essentially constant temperature, meaning that the temperature does not have to be maintained at exactly one temperature. For example, isothermal amplification processes may experience small variations in temperature (e.g., ±1-5°C, etc.) due to environmental or equipment-based variables, etc. In many cases, the entire reaction volume is kept at an essentially constant temperature, and isothermal reactions herein generally do not include amplification conditions that rely on temperature cycling based on temperature gradients and / or convection generated within the reaction vessel.

[0080] The isothermal amplification reaction herein can be carried out at an essentially constant temperature. In some embodiments, the isothermal amplification reaction herein is carried out at a temperature of about 55°C to about 75°C, for example, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75°C or about these values, or a value or range between any two of these values. In some embodiments, the temperature element (e.g., heat source) is maintained at an essentially constant temperature, for example, about 75°C or less, about 70 degrees Celsius or less, about 65°C or less, or about 60°C or less.

[0081] The amplification process herein can be carried out for a certain period of time, for example, until a detectable nucleic acid amplification product is produced. The nucleic acid amplification product can be detected by any suitable detection process and / or detection process described herein. The amplification process can be carried out within about 20 minutes or less, or within about 10 minutes or less. For example, the amplification process can be carried out within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, or within any value or range between any two of such values. In some embodiments, the nucleic acid target can be amplified without exposure to agents or conditions that denature the nucleic acid. In some embodiments, the nucleic acid target can be amplified without exposure to agents or conditions that promote strand separation during the amplification step (and / or other steps). In some embodiments, the nucleic acid target can be amplified without exposure to agents or conditions that promote unwinding during the amplification step (and / or other steps). Agents or conditions that denature the nucleic acid and / or promote strand separation and / or promote unwinding can include, for example, thermal conditions (e.g., high temperature), pH conditions (e.g., high or low pH), chemical agents, and proteins (e.g., enzymatic agents), etc.

[0082] In some embodiments, the methods disclosed herein do not include heat denaturation (e.g., heating a solution containing nucleic acid to an elevated temperature, such as, for example, 75°C, 80°C, 90°C, or 95°C, or higher) or protein-based (e.g., enzymatic) denaturation of nucleic acid. Protein-based (e.g., enzymatic) denaturation may include contacting the nucleic acid with one or more of a helicase, a topoisomerase, a ligase, an exonuclease, an endonuclease, a restriction enzyme, a nicking enzyme, a recombinase, an RNA replicase, and a nucleic acid binding protein (e.g., a single-stranded binding protein). In some embodiments, the compositions provided herein do not include a helicase, a topoisomerase, a ligase, an exonuclease, an endonuclease, a restriction enzyme, a nicking enzyme, a recombinase, an RNA replicase, and / or a nucleic acid binding protein (e.g., a single-stranded binding protein). In some embodiments, the compositions and methods provided herein do not include intercalating agents, alkylating agents, and / or chemicals such as formamide, glycerol, urea, dimethylsulfoxide (DMSO), or N,N,N-trimethylglycine (betaine). In some embodiments, the methods of the disclosure do not include contacting the nucleic acid with a denaturing agent (e.g., formamide). In some embodiments, the amplification step does not include agents and / or conditions that denature the nucleic acid (e.g., promote strand separation and / or promote unwinding). In some embodiments, the amplification step (e.g., step (c)) does not include agents and / or conditions that denature the nucleic acid (e.g., promote strand separation and / or promote unwinding) other than a polymerase (e.g., a hyperthermophile polymerase). In some embodiments, the methods and compositions provided herein do not include agents and / or conditions that denature the nucleic acid (e.g., promote strand separation and / or promote unwinding) other than a polymerase (e.g., a hyperthermophile polymerase) and / or low pH conditions (e.g., contact with acid).

[0083] Nucleic acid targets can be amplified without exposure to agents or conditions that promote strand separation and / or unwinding, such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, recombinases, RNA replicases, nucleic acid binding proteins (e.g., single-stranded binding proteins), or any combination thereof. For example, nucleic acid targets can be amplified without exposure to helicases, including, but not limited to, DNA helicases and RNA helicases. Amplification conditions that do not include the use of a helicase are helicase-free amplification conditions.

[0084] The nucleic acid target can be amplified without exposure to a recombinase, including but not limited to Cre recombinase, Hin recombinase, Tre recombinase, FLP recombinase, RecA, RAD51, RadA, T4 uvsX. In some embodiments, the nucleic acid target is amplified without exposure to a recombinase accessory protein, such as a recombinase loading factor (e.g., T4 uvsY). The nucleic acid target can be amplified without exposure to a nucleic acid binding protein (e.g., single-stranded binding protein or single-stranded DNA binding protein (SSB)), such as T4 gp32. In some embodiments, the nucleic acid target is amplified without exposure to a topoisomerase. The nucleic acid target can be amplified with or without exposure to agents or conditions that destabilize the nucleic acid. As used herein, the term "destabilization" shall be given its ordinary meaning and shall refer to the disruption of the overall organization and geometric orientation (e.g., double helix structure) of a nucleic acid molecule by one or more of tilt, rotation, twist, slip, and flip effects (e.g., as described in Lenglet et al., (2010) Journal of Nucleic Acids Volume 2010, Article ID 290935, 17 pages). Destabilization generally does not refer to melting or separation (e.g., denaturation) of nucleic acid strands. Nucleic acid destabilization can be achieved, for example, by exposure to agents such as intercalating or alkylating agents, and / or chemicals such as formamide, urea, dimethylsulfoxide (DMSO), or N,N,N-trimethylglycine (betaine). In some embodiments, the methods provided herein include the use of one or more destabilizing agents. In some embodiments, the methods provided herein exclude the use of destabilizing agents. In some embodiments, the nucleic acid target is amplified without exposure to a ligase and / or an RNA replicase.

[0085] In some embodiments, the nucleic acid target can be amplified without cleavage or digestion. For example, the nucleic acid target can be amplified without prior exposure to one or more cleavage agents, and intact nucleic acid is amplified. In some embodiments, the nucleic acid target is amplified without exposure to one or more cleavage agents during amplification. In some embodiments, the nucleic acid target is amplified without exposure to one or more cleavage agents after amplification. Amplification conditions that do not include the use of a cleavage agent may be referred to as cleavage agent-free amplification conditions herein. The term "cleavage agent" generally refers to an agent, sometimes a chemical or an enzyme, that can cleave a nucleic acid at one or more specific or non-specific sites. A specific cleavage agent often specifically cleaves a specific site according to a specific nucleotide sequence. Cleavage agents can include endonucleases (such as, for example, restriction enzymes and nicking enzymes); exonucleases (DNAses, RNAses (e.g., RNAse H), 5'-3' exonucleases (e.g., exonuclease II), 3'-5' exonucleases (e.g., exonuclease I), and poly(A)-specific 3'-5' exonucleases); and chemical cleavage agents.

[0086] Nucleic acid targets can be amplified without the use of restriction enzymes and / or nicking enzymes. In some embodiments, nucleic acids are amplified without prior exposure to restriction enzymes and / or nicking enzymes. In some embodiments, nucleic acids are amplified without exposure to restriction enzymes and / or nicking enzymes during amplification. In some embodiments, nucleic acids are amplified without exposure to restriction enzymes and / or nicking enzymes after amplification. Nucleic acid targets can be amplified without exonuclease treatment. Exonucleases include, for example, DNAse, RNAse (e.g., RNAseH), 5'-3' exonucleases (e.g., exonuclease II), 3'-5' exonucleases (e.g., exonuclease I), and poly(A)-specific 3'-5' exonucleases. In some embodiments, nucleic acids are amplified without exonuclease treatment before, during, and / or after amplification. Amplification conditions that do not include the use of exonucleases are exonuclease-free amplification conditions. In some embodiments, the nucleic acid is amplified without DNAse and / or RNAse treatment, hi some embodiments, the nucleic acid is amplified without RNAseH treatment.

[0087] Amplified nucleic acids may be referred to herein as nucleic acid amplification products or amplicons. In some embodiments, amplification products include naturally occurring nucleotides, non-naturally occurring nucleotides, nucleotide analogs, and the like, as well as combinations of the foregoing. Amplification products typically have a nucleotide sequence that is identical or substantially identical to the sequence of the sample nucleic acid (e.g., the target sequence) or its complement. A "substantially identical" nucleotide sequence in an amplification product will generally have a high degree of sequence identity (e.g., about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity) to the nucleotide sequence being amplified or its complement, with variations being the result of poor polymerase fidelity or other variables.

[0088] In some embodiments, the nucleic acid amplification product comprises a polynucleotide that is contiguously complementary or substantially identical to a target sequence in the sample nucleic acid. Contiguously complementary generally refers to a nucleotide sequence of a first strand, for example, in which each base is paired in sequence (e.g., reading from 5' to 3') with a correspondingly ordered base of a second strand, and there are no gaps, additional sequences, or unpaired bases within the sequence considered to be contiguously complementary. In other words, contiguously complementary generally refers to every contiguous base of the nucleotide sequence of the first strand being complementary to the corresponding contiguous base of the nucleotide sequence of the second strand. For example, a first strand having the sequence 5'-ATGCATGCATGC-3' (SEQ ID NO: 3) would be considered to be contiguously complementary to a second strand having the sequence 5'-GCATGCATGCAT-3' (SEQ ID NO: 4), and every contiguous base of the first strand is complementary to every corresponding contiguous base of the second strand. However, a first strand having the sequence 5'-ATGCATAAAAAAGCATGC-3' (SEQ ID NO:5) would not be considered contiguously complementary to a second strand having the sequence 5'-GCATGCATGCAT-3' (SEQ ID NO:4) because the sequence of six adenines (six A's) in the middle of the first strand would not pair with the bases of the second strand. A contiguously complementary sequence is optionally about 5 to about 25 contiguous bases in length, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or a range between any two of these values. In some embodiments, the nucleic acid amplification product comprises a polynucleotide that is contiguously complementary or substantially identical to a target sequence in the sample nucleic acid. Thus, in some embodiments, the nucleic acid amplification product does not include any additional sequences (e.g., at the 5' and / or 3' ends or within the product) that are not contiguous complementary or substantially identical to the target sequence, such as additional sequences incorporated into the amplification product by tail primers or ligation, and / or additional sequences that provide cleavage agent recognition sites (e.g., nicking enzyme recognition sites). Generally, the amplification product will not include products in the form of tandem repeats unless the target sequence includes tandem repeats.

[0089] The nucleic acid amplification product may comprise sequences complementary or substantially identical to one or more primers used in the amplification reaction, hi some embodiments, the nucleic acid amplification product comprises a first nucleotide sequence that is contiguous complementary or identical to a first primer sequence, and a second nucleotide sequence that is contiguous complementary or identical to a second primer sequence.

[0090] The nucleic acid amplification product may include a spacer sequence. As described herein, a spacer sequence in an amplification product is a sequence (one or more bases) that is consecutively complementary or substantially identical to a portion of a target sequence in a sample nucleic acid, and is flanked by sequences in the amplification product that are complementary or substantially identical to one or more primers used in the amplification reaction. A spacer sequence flanked by sequences in an amplification product is generally located between a first sequence (complementary or substantially identical to a first primer) and a second sequence (complementary or substantially identical to a second primer). Thus, an amplification product typically includes a first sequence, followed by a spacer sequence, followed by a second sequence. The spacer sequence is generally neither complementary nor substantially identical to the sequence of the primer. The spacer sequence may be or include about 1 to 10 bases, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases. In some embodiments, the nucleic acid amplification product consists or consists essentially of a first nucleotide sequence that is contiguous complementary or identical to the first primer sequence, a second nucleotide sequence that is contiguous complementary or identical to the second primer sequence, and a spacer sequence. In some embodiments, the nucleic acid amplification product does not include any additional sequence that is not contiguous complementary or identical to the first and second primer sequences (e.g., at the 5' and / or 3' ends or within the product) and is not part of a spacer sequence, e.g., a tail or loop primer, ligation, or other additional sequence incorporated into the amplification product by other mechanisms. In some embodiments, the nucleic acid amplification product generally does not include any additional sequence that is not contiguous complementary or identical to the first and second primer sequences (e.g., at the 5' and / or 3' ends or within the product) and is not part of a spacer sequence, e.g., a tail or loop primer, ligation, or other additional sequence incorporated into the amplification product by other mechanisms.However, in such embodiments, the nucleic acid amplification product may contain some mismatched (i.e., non-complementary) bases or one extra base (e.g., at the 5' and / or 3' end, or within the product) introduced into the product, for example, by errors or promiscuity in the amplification process.

[0091] The nucleic acid amplification products may be up to 50 bases long, including 10, 15, 20, 25, 30, 35, 40, 45, 50, or any number or range of base lengths between any two of these values. In some embodiments, the nucleic acid amplification products of a given target sequence have the same length or substantially the same length (e.g., within 1-10 bases). Thus, the nucleic acid amplification products of a given target sequence can produce a single signal (e.g., a band in an electrophoretic gel) and generally do not produce multiple signals indicative of multiple lengths (e.g., ladders or smears in an electrophoretic gel). In a multiplex reaction, the nucleic acid amplification products of different target sequences may have different lengths.

[0092] The methods and components described herein can be used for multiplex amplification, which generally refers to the amplification of more than one nucleic acid of interest (e.g., the amplification of more than one target sequence). For example, multiplex amplification can refer to amplifying multiple sequences from the same sample, or amplifying one of several sequences in a sample. Multiplex amplification can also refer to amplifying one or more sequences present in multiple samples simultaneously or stepwise. For example, multiplex amplification can be used to amplify at least two target sequences that are amplifiable (e.g., the amplification reaction includes suitable primers and enzymes to amplify at least two target sequences). In some embodiments, the amplification reaction is arranged to detect at least two target sequences, but only one of the target sequences is present in the sample being tested, so that both sequences are amplifiable, but only one sequence is amplified. In some embodiments, when two target sequences are present, the amplification reaction results in the amplification of both target sequences. A multiplex amplification reaction including suitable primers and enzymes can result in the amplification of one, some, or all of the target sequences. In some embodiments, the amplification reaction is set up to detect two sequences using one pair of primers, one sequence being a target sequence and one sequence being a control sequence (e.g., a synthetic sequence that is amplifiable with the same primers as the target sequence but has different spacer bases or sequences than the target). In some embodiments, the amplification reaction is set up to detect multiple sets of sequences using corresponding primer pairs, each set including a target sequence and a control sequence.

[0093] Primer Nucleic acid amplification is generally carried out in the presence of one or more primers. A primer is generally characterized as an oligonucleotide that comprises a nucleotide sequence capable of hybridizing or annealing to a target nucleic acid at or near (e.g., adjacent to) a specific region of interest (i.e., a target sequence). A primer can, for example, allow for the specific determination of the nucleotide sequence of a target nucleic acid or the detection of a target nucleic acid or a feature thereof (e.g., the presence or absence of a sequence). A primer can be naturally occurring or synthetic. The term specific or specific generally refers to the binding or hybridization of one molecule, such as a primer to a target polynucleotide, with another molecule. That is, the term specific or specific refers to the recognition, contact, and formation of a stable complex between two molecules, compared to the substantially lesser recognition, contact, or complex formation of either of those two molecules with the other molecule. The term annealing or hybridization generally refers to the formation of a stable complex between two molecules. The terms primer, oligo, or oligonucleotide can be used interchangeably herein when referring to a primer.

[0094] Primers can be designed and synthesized using any suitable process and may be of any length suitable for hybridizing to a target sequence and performing the amplification process described herein. Primers are often designed according to the sequence of the target nucleic acid. In some embodiments, primers may be about 5 to about 30 bases long, e.g., 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, or 30 bases long. Primers may be composed of naturally occurring and / or non-naturally occurring nucleotides (e.g., modified nucleotides, labeled nucleotides) or mixtures thereof. Modifications and modified bases include, for example, phosphorylation (e.g., 3' phosphorylation, 5' phosphorylation); attachment chemistry or linker modification (e.g., Acrydite™, adenylation, azide (NHS ester), digoxigenin (NHS ester), cholesteryl-TEG, I-Linker™, amino modifiers (e.g., amino modifier C6, amino modifier C12, amino modifier C6dT, Uni-Link™ amino modifiers), alkynes (e.g., 5' hexynyl, 5-octadiynyl dU), biotinylation (e.g., biotin, biotin (azide), biotin dT, biotin-TEG, dual biotin, PC biotin, desthiobiotin-TEG), thiol modification (e.g., thiol modifier C3S-S, dithiol, thiol modifier C6S-S)); fluorophores (e.g., Freedom™ dyes, Alexa Fluor® dyes, LI-COR IRDyes®, ATTO™ dyes, rhodamine dyes, WellRED dyes, 6-FAM (azide), Texas Red®-X (NHS ester), Lightcycler® 640 (NHS ester), Dy750 (NHS ester); Iowa Black® dark quencher modifications (e.g., Iowa Black® FQ, Iowa Black® RQ); dark quencher modifications (e.g., Black Hole Quencher®-1, Black Hole Quencher®-2, Dabcyl);Spacers (C3 Spacer, PC Spacer, Hexanediol, Spacer 9, Spacer 18, 1',2'-dideoxyribose (dSpacer); modified bases (e.g., 2-aminopurine, 2,6-diaminopurine (2-amino-dA), 5-bromo-dU, deoxyuridine, inverted dT, inverted dideoxy-T, dideoxy-C, 5-methyl-dC, deoxyinosine, SuperT®, SuperG®, locked nucleic acid (LNA), 5-nitroindole, 2'-O-methyl RNA bases, hydroxymethyl dC, UNA unlocked nucleic acids (e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dC, Iso-dG, Fluoro-C, Fluoro-U, Fluoro-A, Fluoro-G); phosphorothioate (PS) bond modifications (e.g., phosphorothioated DNA bases, phosphorothioated RNA bases, phosphorothioated 2'O-methyl bases, phosphorothioated LNA bases);and click chemistry modifications. In some embodiments, the modifications and modified bases include uracil bases, ribonucleotide bases, O-methyl RNA bases, PS linkages, 3' phosphate groups, spacer bases (such as C3 spacer or other spacer bases). For example, the primers may include one or more O-methyl RNA bases (e.g., 2'-O-methyl RNA bases). 2'-O-methyl RNA is a post-transcriptional modification of RNA commonly found in tRNA and other small RNA molecules. Primers containing 2'-O-methyl RNA bases can be directly synthesized. This modification can, for example, increase the Tm of the RNA:RNA duplex and provide stability in the presence of single-stranded ribonucleases and DNases. 2'-O-methyl RNA bases can be included in the primers to, for example, increase stability and binding affinity with the target sequence. In some embodiments, the primers may include one or more phosphorothioate (PS) linkages (e.g., PS linkage modifications). PS linkages replace non-bridging oxygens in the phosphate backbone of the primer with sulfur atoms. This modification typically renders the internucleotide bond resistant to nuclease degradation. PS bonds can be introduced at the 5' or 3' end of the primer, between about the last 3-5 nucleotides, for example, to inhibit exonuclease degradation. In some embodiments, PS bonds included throughout the primer can help reduce attack by endonucleases. The primer can include, for example, a 3' phosphate group. 3' phosphorylation can inhibit degradation by certain 3'-exonucleases, and in certain cases can be used to block extension by DNA polymerase. In some embodiments, the primer includes one or more spacer bases (e.g., one or more C3 spacers). C3 spacer phosphoramidites can be incorporated internally or at the 5' end of the primer. Multiple C3 spacers can be added to either end of the primer, for example, to introduce long chain hydrophilic spacer arms for attachment of fluorophores or other pendant groups.;

[0095] A primer may comprise DNA bases, RNA bases, or both, where one or more of the DNA and RNA bases are modified or unmodified. For example, a primer may be a mixture of DNA bases and RNA bases. A primer may comprise DNA bases (e.g., modified DNA bases and / or unmodified DNA bases). In some embodiments, a primer comprises unmodified DNA bases. In some embodiments, a primer comprises modified DNA bases. A primer may comprise RNA bases (e.g., modified RNA bases and / or unmodified RNA bases). In some embodiments, a primer comprises unmodified RNA bases. In some embodiments, a primer comprises modified RNA bases. In some embodiments, a primer does not comprise an RNA base. In some embodiments, a primer does not comprise a DNA base. In some embodiments, a primer does not comprise a cleavage agent recognition site (e.g., does not comprise a nicking enzyme recognition site). In some embodiments, a primer does not comprise a tail (e.g., does not comprise a tail comprising a nicking enzyme recognition site).

[0096] In some embodiments, all or a portion of the primer sequence may be complementary or substantially complementary to the target nucleic acid. Substantially complementary in the context of sequences generally refers to nucleotide sequences that will hybridize to each other. The stringency of the hybridization conditions can be varied to allow for various amounts of sequence mismatch. The target sequence and the primer sequence may be at least 75% complementary to each other, including, for example, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to each other. A primer that is substantially complementary to a target nucleic acid sequence is typically also substantially identical to the complement of the target nucleic acid sequence (i.e., the sequence of the antisense strand of the target nucleic acid). The primer and the antisense strand of the target nucleic acid may be at least 75% identical in sequence, for example, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to each other.

[0097] In some embodiments, the primers comprise a pair of primers. A pair of primers may comprise a forward primer and a reverse primer (e.g., primers that bind to the sense and antisense strands of the target nucleic acid). In some embodiments, the primers consist of a pair of primers (i.e., a forward primer and a reverse primer). Thus, in some embodiments, the amplification of the target sequence is performed using a pair of primers, and the amplification of the target sequence does not include additional primers or oligonucleotides (e.g., the amplification reaction components do not include additional primer pairs for a given target sequence, nested primers, bumper primers, oligonucleotides other than primers, and probes, etc.). In some embodiments, the primers consist of a pair of primers. In some embodiments, the amplification reaction may include additional primer pairs for amplifying different target sequences, such as in multiplex amplification. In some embodiments, the primers consist of a pair of primers, but in some embodiments, the amplification reaction may include additional primers, oligonucleotides, or probes for detection processes that are not considered part of the amplification. In some embodiments, the primers are used in sets. An amplification primer set may comprise a pair of forward and reverse primers for a given target sequence. In the case of multiplex amplification, the primers that amplify a first target sequence are considered to be a primer set and the primers that amplify a second target sequence are considered to be a different primer set.

[0098] The amplification reaction components may include or consist of a first primer (first oligonucleotide) that is complementary to a target sequence of a first strand (e.g., sense strand, forward strand) of a sample nucleic acid, and a second primer (second oligonucleotide) that is complementary to a target sequence of a second strand (e.g., antisense strand, reverse strand) of the sample nucleic acid. In some embodiments, the first primer (first oligonucleotide) comprises a first polynucleotide that is sequentially complementary to a target sequence of a first strand of a sample nucleic acid, and the second primer (second oligonucleotide) comprises a second polynucleotide that is sequentially complementary to a target sequence of a second strand of a sample nucleic acid. Continuously complementary with respect to primer-target generally refers to a nucleotide sequence of a primer in which each base pairs sequentially with a corresponding sequential base in the target sequence, with no gaps, additional sequences, or unpaired bases in the sequence considered to be continuously complementary. In some embodiments, the primer does not include any additional sequence (e.g., at the 5' and / or 3' end or within the primer) that is not contiguous with the target sequence, such as additional sequence present in a tail primer or loop primer, and / or additional sequence that provides a cleavage agent recognition site (e.g., a nicking enzyme recognition site). In some embodiments, the amplification reaction components do not include primers that include additional sequence (i.e., sequence other than sequence that is contiguous with the target sequence), such as tail primers, loop primers, primers capable of forming step-loop structures, hairpin structures, and / or additional sequence that provides a cleavage agent recognition site (e.g., a nicking enzyme recognition site).

[0099] In some embodiments, a primer may include modifications such as one or more inosines, abasic sites, locked nucleic acids, minor groove binders, duplex stabilizers (e.g., acridine, spermidine), Tm modifiers, or any modifier that alters the binding characteristics of the primer. In some embodiments, a primer may include a detectable molecule or entity (e.g., a fluorophore, a radioisotope, a colorimetric reagent, a particle, an enzyme, and the like).

[0100] Polymerase Amplification reaction components (e.g., one or more amplification reagents) may include one or more polymerases. A polymerase is a protein capable of catalyzing the specific incorporation of nucleotides that extend the 3' hydroxyl end of a primer molecule, such as an amplification primer described herein, to a nucleic acid target sequence (e.g., to which the primer anneals). Non-limiting examples of polymerases include thermophilic or hyperthermophilic polymerases that can exhibit activity at high reaction temperatures (e.g., greater than 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100°C). Hyperthermophilic polymerases are sometimes referred to as hyperthermophilic polymerases. Polymerases may or may not have strand displacement capabilities. In some embodiments, the polymerase is capable of incorporating from about 1 to about 50 nucleotides in a single synthesis, e.g., about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides, or a number or range between any two of these values ​​in a single synthesis.

[0101] The amplification reaction components may include one or more DNA polymerases selected from the following: 9°N DNA polymerase; 9°Nm™ DNA polymerase; Therminator™ DNA polymerase; Therminator™ II DNA polymerase; Therminator™ III DNA polymerase; Therminator™ Gamma DNA polymerase; Bst DNA polymerase; Bst DNA polymerase (large fragment); Phi29 DNA polymerase, DNA polymerase I (E. coli), DNA polymerase I, large (Klenow) fragment; Klenow fragment (3'-5' exo-); T4 DNA polymerase; T7 DNA polymerase; Deep VentR™ (exo-) DNA polymerase; Deep VentR™ DNA polymerase; DyNAzyme™ EXT DNA; DyNAzyme™ II Hot Start DNA polymerase; Phusion™ high fidelity DNA polymerase; VentR™ DNA polymerase; VentR™ (exo-) DNA polymerase; RepliPHI™ Phi29 DNA polymerase; rBst DNA polymerase, large fragment (IsoTherm™ DNA polymerase); MasterAmp™ AmpliTherm™ DNA polymerase; Tag DNA polymerase; Tth DNA polymerase; Tfl DNA polymerase; Tgo DNA polymerase; SP6 DNA polymerase; Tbr DNA polymerase; DNA polymerase beta; and ThermoPhi DNA polymerase.

[0102] In some embodiments, the amplification reaction components include one or more hyperthermophilic DNA polymerases (e.g., hyperthermophilic DNA polymerases that are thermostable at high temperatures). The half-life of the hyperthermophilic DNA polymerase may be about 5-10 hours at 95°C and about 1-3 hours at 100°C. For example, the amplification reaction components may include one or more hyperthermophilic DNA polymerases from Archaea (e.g., a hyperthermophilic DNA polymerase from Thermococcus or a hyperthermophilic DNA polymerase from Thermococcaceaen archaean). In some embodiments, the amplification reaction components include one or more hyperthermophilic DNA polymerases from the genus Pyrococcus, Methanococcaceae, Methanococcus, or Thermus. In some embodiments, the amplification reaction components include one or more hyperthermophilic DNA polymerases from Thermus thermophiles.

[0103] In some embodiments, the amplification reaction components include a hyperthermophile DNA polymerase or a functional fragment thereof. A functional fragment generally retains one or more functions of a full-length polymerase, such as the ability to polymerize DNA (e.g., in an amplification reaction). In some cases, the functional fragment performs a function (e.g., polymerizing DNA in an amplification reaction) at a level of at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the functional level of the full-length polymerase. The level of polymerase activity can be assessed using a detectable nucleic acid amplification method, such as, for example, a method described herein. In some embodiments, the amplification reaction components include a hyperthermophile DNA polymerase that includes the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or a functional fragment of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, an amplification reaction component (e.g., one or more amplification reagents) comprises a polymerase that comprises an amino acid sequence that is at least about 90% identical to a Hyperthermophile polymerase or a functional fragment thereof, hi some embodiments, an amplification reaction component comprises a polymerase that comprises an amino acid sequence that is at least about 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or a functional fragment thereof.

[0104] The polymerase may have reverse transcriptase ability. In such an embodiment, the amplification reaction may, for example, amplify an RNA target in a single step without the use of a separate reverse transcriptase. Non-limiting examples of polymerases with reverse transcriptase ability include Bst (large fragment), 9°N DNA polymerase, 9°Nm™ DNA polymerase, Therminator™, and Therminator™ II. The amplification reaction components may include one or more separate reverse transcriptases. In some embodiments, more than one polymerase is included in the amplification reaction. For example, the amplification reaction may include a polymerase with reverse transcriptase activity and a second polymerase without reverse transcriptase activity. In some embodiments, one or more polymerases with exonuclease activity are used during amplification. In some embodiments, one or more polymerases with no or low exonuclease activity are used during amplification. In some embodiments, the polymerase with no or low exonuclease activity comprises one or more modifications (e.g., amino acid substitutions) that reduce or eliminate the exonuclease activity of the polymerase. For example, a modified polymerase with low exonuclease activity may have 10% or less exonuclease activity compared to an unmodified polymerase, for example, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% exonuclease activity compared to an unmodified polymerase. In some embodiments, the polymerase has no or low 5'-3' exonuclease activity and / or no or low 3'-5' exonuclease activity. In some embodiments, the polymerase has no or low single-strand dependent exonuclease activity and / or no or low double-strand dependent exonuclease activity. Non-limiting examples of modifications that can reduce or eliminate the exonuclease activity of a polymerase include one or more amino acid substitutions at or corresponding to positions 141 and / or 143 and / or 458 of SEQ ID NO:1 (e.g., D141A, E143A, E143D, and A485L).

[0105] Detection and Quantification The methods described herein may include detecting and / or quantifying the nucleic acid amplification products. The amplification products may be detected and / or quantified, for example, by any suitable detection and / or quantification method described herein. Non-limiting examples of detection and / or quantification methods include molecular beacons (e.g., real-time, end-point), lateral flow, fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), surface capture, 5'-3' exonuclease hydrolysis probes (e.g., TAQMAN), intercalating / binding dyes, absorbance (e.g., colorimetric, turbidity), electrophoresis (e.g., gel electrophoresis, capillary electrophoresis), mass spectrometry, nucleic acid sequencing, digital amplification, primer extension (e.g., iPLEX™), Affymetrix's molecular inversion probe (MIP) technology, restriction fragment length polymorphism (RFLP analysis), allele-specific oligonucleotide (ASO) analysis, molecular quantification (MIP ... Chilled specific PCR (MSPCR), pyrosequencing analysis, acycloprime analysis, reverse dot blot, GeneChip microarray, dynamic allele specific hybridization (DASH), peptide nucleic acid (PNA) and locked nucleic acid (LNA) probes, AlphaScreen, SNPstream, gene bit analysis (GBA), multiplex minisequencing, SNaPshot, GOOD assay, microarray miniseq, array primer extension (APEX), microarray primer extension, Tag array, coded microspheres, template-directed integration (TDI, incorporation), colorimetric oligonucleotide ligation assay (OLA), sequence-coded OLA, microarray ligation, ligase chain reaction, padlock probes, invader assay, hybridization using at least one probe, hybridization using at least one fluorescently labeled probe, cloning and sequencing, use of hybridization probes and quantitative real-time polymerase chain reaction (QRT-PCR), nanopore sequencing, chips, and combinations thereof.In some embodiments, detection of nucleic acid amplification products includes the use of real-time detection methods (i.e., products are detected and / or continuously monitored during the amplification process). In some embodiments, detection of nucleic acid amplification products includes the use of end-point detection methods (i.e., products are detected after the amplification process is completed or stopped). Nucleic acid detection methods can also employ the use of labeled nucleotides that are directly incorporated into the target sequence or that are incorporated into a probe that contains a complementary sequence to the target. Such labels may be radioactive and / or fluorescent in nature and can be resolved in any of the manners discussed herein. In some embodiments, quantification of nucleic acid amplification products can be achieved using one or more detection methods described below. In some embodiments, detection methods can be used in conjunction with measuring signal intensity and / or creating (or referencing) a standard curve and / or look-up table for quantification of nucleic acid amplification products.

[0106] The detection of nucleic acid amplification products may include the use of molecular beacon technology. The term molecular beacon generally refers to a detectable molecule, whose detectable property is detectable under certain conditions, thereby allowing the molecule to function as a specific informative signal. Non-limiting examples of detectable properties include optical properties (e.g., fluorescence), electrical properties, magnetic properties, chemical properties, and time or speed of passage through an aperture of known size. Molecular beacons for detecting nucleic acid molecules may be, for example, hairpin-shaped oligonucleotides containing a fluorophore at one end and a quenching dye at the opposite end. The loop of the hairpin may contain a probe sequence complementary to the target sequence, and the stem is formed by annealing complementary arm sequences located on either side of the probe sequence. The fluorophore and quenching molecule may be covalently linked to the opposite ends of each arm. Under conditions that prevent the oligonucleotide from hybridizing to its complementary target, or when the molecular beacon is free in solution, the fluorescent molecule and the quenching molecule are in close proximity to each other, preventing FRET. When a molecular beacon encounters a target molecule (e.g., a nucleic acid amplification product), hybridization can occur, converting the loop structure into a stable, more rigid conformation, causing the fluorophore and quencher molecules to separate, resulting in fluorescence. Because the probe is specific, the generation of fluorescence is generally only due to the synthesis of the intended amplification product. In some cases, the molecular beacon probe sequence hybridizes to a sequence in the amplification product that is identical or complementary to a sequence in the target nucleic acid. In some cases, the molecular beacon probe sequence hybridizes to a sequence in the amplification product that is not identical or complementary to a sequence in the target nucleic acid (e.g., hybridizes to a tail amplification primer or a sequence added to the amplification product by ligation). Molecular beacons are highly specific and can distinguish single nucleotide polymorphisms. Molecular beacons can also be synthesized with different color fluorophores and different target sequences, allowing for the simultaneous detection of several products in the same reaction (e.g., in a multiplex reaction).In the case of a quantitative amplification process, molecular beacons can specifically bind to the amplified target after each cycle of amplification, and since unhybridized molecular beacons are dark in color, it is not necessary to isolate the probe-target hybrid to quantitatively determine the amount of amplified product. The signal obtained is proportional to the amount of amplified product. Detection using molecular beacons can be performed in real time or as an end-point detection method.

[0107] The detection of the nucleic acid amplification product may include the use of lateral flow. The use of lateral flow typically includes the use of lateral flow devices, including, but not limited to, dipstick assays and thin-layer chromatography plates with various suitable coatings. In the flow path, various binding reagents for the sample, binding partners for the sample or conjugates containing binding partners, and a signal generating system are immobilized.

[0108] Detection of nucleic acid amplification products may involve the use of FRET, an energy transfer mechanism between two chromophores: a donor molecule and an acceptor molecule. Briefly, a donor fluorophore molecule is excited at a specific excitation wavelength. Subsequent emission of the donor molecule as it returns to its ground state can transfer the excitation energy to the acceptor molecule through long-range dipole-dipole interactions. The emission intensity of the acceptor molecule can be monitored, which is a function of the distance between the donor and acceptor, the overlap of the donor emission spectrum with the acceptor absorption spectrum, and the orientation of the donor emission dipole moment and the acceptor absorption dipole moment. FRET can be useful, for example, to quantify the molecular dynamics of DNA-DNA interactions, as described for molecular beacons. To monitor the production of a specific product, a probe can be labeled with a donor molecule at one end and an acceptor molecule at the other end. Upon probe-target hybridization, the distance or orientation between the donor and acceptor changes and a FRET change is observed.

[0109] Detection of nucleic acid amplification products may involve the use of fluorescence polarization (FP). FP techniques are based on the principle that when a fluorescently labeled compound is excited with linearly polarized light, it will emit fluorescence with a degree of polarization inversely proportional to its rate of rotation. Thus, when a molecule, such as a fluorescently labeled tracer-nucleic acid conjugate, is excited with linearly polarized light, the emission remains highly polarized because the rotation of the fluorophore is constrained between the time the light is absorbed and the time it is emitted. When a free tracer compound (i.e., not bound to a nucleic acid) is excited with linearly polarized light, its rotation is much faster than that of the corresponding tracer-nucleic acid conjugate, and the molecular orientation becomes more random, resulting in depolarized emitted light. Thus, fluorescence polarization provides a quantitative means to measure the amount of tracer-nucleic acid conjugate produced in an amplification reaction.

[0110] Detection of nucleic acid amplification products may include the use of surface capture, achieved, for example, by immobilizing specific oligonucleotides on a surface to create a biosensor that is both highly sensitive and selective. Examples of surfaces that can be used to attach probes include gold and carbon. Detection of nucleic acid amplification products may include the use of 5'-3' exonuclease hydrolysis probes (e.g., TAQMAN). For example, TAQMAN probes are hydrolysis probes that can increase the specificity of quantitative amplification methods (e.g., quantitative PCR). The principle of TAQMAN probes relies on 1) the 5'-3' exonuclease activity of Taq polymerase, which cleaves a dual-labeled probe during hybridization to a complementary target sequence, and 2) fluorophore-based detection. The resulting fluorescent signal allows for quantitative measurement of the accumulation of amplification products during the exponential phase of amplification, and TAQMAN probes can significantly increase the specificity of detection.

[0111] Detection of nucleic acid amplification products may include the use of intercalating and / or binding dyes, including dyes that specifically stain nucleic acids (e.g., intercalating dyes exhibit enhanced fluorescence when bound to DNA or RNA). Dyes may include DNA or RNA intercalating fluorophores, including, but not limited to, SYTO® 82, acridine orange, ethidium bromide, Hoechst dyes, PicoGreen®, propidium iodide, SYBR® I (asymmetric cyanine dye), SYBR® II, TOTO (thiazole orange dimer), and YOYO (oxazole yellow dimer). Detection of nucleic acid amplification products may include the use of absorbance methods (e.g., colorimetry, turbidity). In some embodiments, detection and / or quantification of nucleic acids may be achieved by directly converting absorbance (e.g., UV absorbance measurement at 260 nm) to concentration. Direct measurements of nucleic acids can be converted to concentrations using the Beer-Lambert law, which relates absorbance to concentration using the path length and extinction coefficient of the measurement. Detection of nucleic acid amplification products may include the use of electrophoresis (e.g., gel electrophoresis, capillary electrophoresis) and / or the use of mass spectrometry. Mass spectrometry is an analytical technique that can be used to determine the structure and amount of nucleic acids and can be used to provide rapid analysis of complex mixtures. After amplification, the sample can be ionized and the resulting ions can be separated according to their mass-to-charge ratio in an electric and / or magnetic field, and the mass-to-charge ratio of the ions is measured by a detector. Mass spectrometry methods include, for example, MALDI, MALDI-TOF, and electrospray. Such methods can be combined with gas chromatography (GC / MS) and liquid chromatography (LC / MS). Mass spectrometry methods (e.g., matrix-assisted laser desorption / ionization mass spectrometry (MALDI MS)) can achieve high throughput due to fast signal acquisition from solid surfaces and automated analysis.

[0112] The detection of nucleic acid amplification products may include the use of nucleic acid sequencing. The entire or partial sequence of the amplification product may be determined, and the determined nucleotide sequence may be referred to as a read. For example, linear amplification products may be directly analyzed without further amplification (e.g., by using single molecule sequencing methods). In some embodiments, linear amplification products are subjected to further amplification and then analyzed (e.g., using sequencing by ligation or pyrosequencing methods). Non-limiting examples of sequencing methods include single-end sequencing, paired-end sequencing, reversible terminator-based sequencing, sequencing by ligation, pyrosequencing, sequencing by synthesis, single molecule sequencing, multiplex sequencing, solid-phase single nucleotide sequencing, and nanopore sequencing. The detection of nucleic acid amplification products may include the use of digital amplification (e.g., digital PCR). Systems for digital amplification and analysis of nucleic acids are available (e.g., Fluidigm® Corporation).

[0113] Lysis buffer Solubilizer As disclosed herein, the dissolution agent may include a detergent. The detergent may include one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. Anionic surfactants have NH4 as a counterion. + , K + , Na + , or Li + The cationic surfactant may contain, as a counter ion, I - , Br - , or Cl - may also include

[0114] The anionic surfactant may be selected from potassium laurate, triethanolamine stearate, ammonium lauryl sulfate, lithium dodecyl sulfate, sodium lauryl sulfate, sodium alkyl sulfate (C8-16), sodium dodecyl sulfate, alkyl polyoxyethylene sulfate, sodium alginate, dioctyl sodium sulfosuccinate, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidic acid and its salts, glyceryl esters, sodium carboxymethylcellulose, bile acids and its salts, cholic acid, deoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid, alkyl sulfonates, aryl sulfonates, alkyl phosphates, alkyl sulfonates, stearic acid and its salts, calcium stearate, phosphates, sodium carboxymethylcellulose, dioctyl sulfosuccinate, dialkyl esters of sodium sulfosuccinate, phospholipids, and calcium carboxymethylcellulose.

[0115] The cationic surfactants can be selected from the following: quaternary ammonium compounds, benzalkonium chloride, cetyltrimethylammonium bromide, chitonic acid, lauryldimethylbenzylammonium chloride, acylcarnitine hydrochloride, alkylpyridinium halides, cetylpyridinium chloride, cationic lipids, polymethylmethacrylate trimethylammonium bromide, sulfonium compounds, polyvinylpyrrolidone-2-dimethylaminoethyl methacrylate dimethyl sulfate, hexadecyltrimethylammonium bromide, phosphonium compounds, quaternary ammonium compounds, benzyl-di(2-chloroethyl)ethylammonium bromide, coconut trimethylammonium chloride, coconut trimethylammonium bromide, coconut methyldihydroxyethylammonium chloride, coconut methyldihydroxyethylammonium bromide, decyltriethylammonium chloride, decyldimethylhydroxyethylammonium chloride, decyldimethylhydroxyethylammonium chloride bromide, C 12-15-Dimethylhydroxyethylammonium chloride, C 12-15 -Dimethylhydroxyethylammonium chloride bromide, coconut dimethylhydroxyethylammonium chloride, coconut dimethylhydroxyethylammonium bromide, myristyltrimethylammonium methylsulfate, lauryldimethylbenzylammonium chloride, lauryldimethylbenzylammonium bromide, lauryldimethyl(ethenoxy)4 ammonium chloride, lauryldimethyl(ethenoxy)4 ammonium bromide, N-alkyl(C 12-18 ) Dimethylbenzylammonium chloride, N-alkyl(C 14-18 ) Dimethyl-benzyl ammonium chloride, N-tetradecyl dimethylbenzyl ammonium chloride monohydrate, dimethyldidecyl ammonium chloride, N-alkyl(C 12-14 ) dimethyl 1-naphthylmethyl ammonium chloride, trimethyl ammonium halide alkyl-trimethyl ammonium salt, dialkyl-dimethyl ammonium salt, lauryl trimethyl ammonium chloride, ethoxylated alkyl amide alkyl dialkyl ammonium salt, ethoxylated trialkyl ammonium salt, dialkyl benzene dialkyl ammonium chloride, N-didecyl dimethyl ammonium chloride, N-tetradecyl dimethyl benzyl ammonium chloride monohydrate, N-alkyl (C 12-14 ) Dimethyl 1-naphthylmethyl ammonium chloride, dodecyl dimethyl benzyl ammonium chloride, dialkyl benzene alkyl ammonium chloride, lauryl trimethyl ammonium chloride, alkyl benzyl methyl ammonium chloride, alkyl benzyl dimethyl ammonium bromide, C 8-16 Trimethylammonium bromide, C 8-16 Trimethylammonium chloride, C 15 Trimethylammonium bromide, C 17Trimethylammonium bromide, dodecylbenzyltriethylammonium chloride, polydiallyldimethylammonium chloride, dimethylammonium chloride, alkyldimethylammonium halides, tricetylmethylammonium chloride, decyltrimethylammonium bromide, dodecyltriethylammonium bromide, tetradecyltrimethylammonium bromide, methyltrioctylammonium chloride, POLYQUAT 10, tetrabutylammonium bromide, benzyltrimethylammonium bromide, choline esters, benzalkonium chloride, stearalkonium chloride, cetylpyridinium bromide, cetylpyridinium chloride, halide salts of quaternized polyoxyethylalkylamines, MIRAPOL Alkaquat, alkylpyridinium salts, amines, amine salts, imidoazolinium salts, protonated quaternary acrylamides, methylated quaternary polymers, cationic guar gum, benzalkonium chloride, dodecyltrimethylammonium bromide, triethanolamine, and poloxamine.

[0116] The nonionic surfactants may be selected from the following: polyoxyethylene fatty alcohol ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, sorbitan esters, glyceryl esters, glycerol monostearate, polyethylene glycol, polypropylene glycol, polypropylene glycol esters, cetyl alcohol, cetostearyl alcohol, stearyl alcohol, aryl alkyl polyether alcohols, polyoxyethylene polyoxypropylene copolymers, poloxamers, poloxamines, methylcellulose, hydroxycellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, noncrystalline cellulose, polysaccharides, starch, starch derivatives, hydroxyethyl starch, polyvinyl alcohol, triethanolamine stearate, amine oxides, dextran, glycerol, gum acacia, cholesterol, tragacanth, and polyvinylpyrrolidone. Nonionic surfactants include alkyl sulfates, alkyl sulfonates, fatty acid soaps, hydroxy fatty acids, hydroperoxy fatty acids, polyhydroxy fatty acids, salts of epoxy fatty acids, salts of mono- and polycarboxylic acids, prostanoic acids and prostaglandins, leukotrienes and lipoxins, alkyl phosphates, alkyl phosphonates, sodium dialkyl sulfosuccinates, n-alkyl ethoxylated sulfates, bile salts cholates and deoxycholates, perfluorocarboxylic acids, fluoroacliphatic phosphonates, or fluoroaliphatic sulfates.

[0117] The lysis agents provided herein may be capable of acting as denaturing agents. As used herein, "denaturing agent" or "denaturant" shall be given its ordinary meaning and include any compound or substance that will cause reversible unfolding of proteins. The strength of a denaturing agent or denaturant will be determined by both the properties and the concentration of the particular denaturing agent or denaturant. Suitable denaturing agents or denaturants include chaotropes, detergents, organic solvents, water-miscible solvents, phospholipids, or combinations of two or more such agents. Suitable chaotropes include, but are not limited to, urea, guanidine, and sodium thiocyanate. Useful detergents include strong detergents such as sodium dodecyl sulfate or polyoxyethylene ethers (e.g., Tween or Triton detergents), sarkosyl, mild non-ionic detergents (e.g., digitonin), mild cationic detergents (e.g., N->2,3-(dioleoxy)-propyl-N,N,N-trimethylammonium), mild ionic detergents (e.g., sodium cholate or sodium deoxycholate), or zwitterionic detergents, including, but not limited to, sulfobetaine (Zwittergent), 3-(3-chloramidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and 3-(3-chloramidopropyl)dimethylammonio-2-hydroxy-1-propane sulfonate (CHAPSO). Organic water-miscible solvents such as acetonitrile, lower alkanols (especially C2-C4 alkanols such as ethanol or isopropanol), or lower alkanediols (especially C2-C4 alkanediols such as ethylene-glycol) can be used as denaturants. The phospholipids may be naturally occurring phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol, or synthetic phospholipid derivatives or variants such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

[0118] Suitable surfactant levels may be from about 0.1% to about 25%, from about 0.25% to about 10%, or from about 0.5% to about 5% by weight of the total composition. In some embodiments, the surfactant is an anionic surfactant, an amphoteric surfactant, a nonionic surfactant, a zwitterionic surfactant, a cationic surfactant, and mixtures thereof. In some embodiments, it may be advantageous to use anionic, amphoteric, nonionic, and zwitterionic surfactants (and mixtures thereof). Anionic surfactants useful herein include the water-soluble salts of alkyl sulfates and alkyl ether sulfates having from 10 to 18 carbon atoms in the alkyl radical, and the water-soluble salts of sulfonated monoglycerides of fatty acids having from 10 to 18 carbon atoms. Sodium lauryl sulfate and sodium coconut monoglyceride sulfonate are examples of this type of anionic surfactant.

[0119] Suitable cationic surfactants can be broadly defined as derivatives of aliphatic quaternary ammonium compounds having one long alkyl chain containing about 8 to 18 carbon atoms, such as lauryltrimethylammonium chloride, cetylpyridinium chloride, benzalkonium chloride, cetyltrimethylammonium bromide, diisobutylphenoxyethyl-dimethylbenzylammonium chloride, coconut alkyltrimethyl-ammonium nitrite, cetylpyridinium fluoride, etc. Certain cationic surfactants can also act as germicides in the compositions disclosed herein.

[0120] Suitable nonionic surfactants that can be used in the compositions, methods, and kits of the present disclosure can be broadly defined as compounds that are produced by condensation of an alkylene oxide group (hydrophilic in nature) with an organic hydrophobic compound, which may be aliphatic and / or aromatic in nature. Examples of suitable nonionic surfactants include: poloxamers; sorbitan derivatives, such as sorbitan diisostearate; ethylene oxide condensates of hydrogenated castor oil, such as PEG-30 hydrogenated castor oil; ethylene oxide condensates of aliphatic alcohols or alkylphenols; products derived from the condensation of ethylene oxide with the reaction product of propylene oxide and ethylenediamine; long-chain tertiary amine oxides; long-chain tertiary phosphine oxides; long-chain dialkyl sulfoxides; and mixtures of such materials. Such materials are useful for stabilizing air bubbles without contributing to excessive viscosity increase of consumer product compositions.

[0121] Zwitterionic surfactants can be broadly described as derivatives of aliphatic quaternary ammonium, phosphonium, and sulfonium compounds, where the aliphatic radical may be straight or branched chain, one of the aliphatic substituents contains from about 8 to 18 carbon atoms, and one contains an anionic water-solubilizing group, such as carboxy, sulfonate, sulfate, phosphate, or phosphonate. Examples of anionic short chain surfactants include alkyl sulfates, alkyl sulfonates, alkyl benzene sulfonates, saturated or unsaturated fatty acids, and their salts.The moiety containing a polar head group in cationic surfactants can include, for example, quaternary ammonium, pyridinium, sulfonium, and / or phosphonium groups.For example, the polar head group can include trimethylammonium.Exemplary cationic short chain surfactants include alkyl trimethylammonium halides, alkyl trimethylammonium tosylates, and N-alkyl pyridinium halides.

[0122] Alkyl sulfates can include sodium octyl sulfate, sodium decyl sulfate, sodium dodecyl sulfate, and sodium tetra-decyl sulfate. Alkyl sulfonates can include sodium octyl sulfonate, sodium decyl sulfonate, and sodium dodecyl sulfonate. Alkyl benzene sulfonates can include sodium octyl benzene sulfonate, sodium decyl benzene sulfonate, and sodium dodecyl benzene sulfonate. Fatty acid salts can include sodium octanoate, sodium decanoate, sodium dodecanoate, and the sodium salt of oleic acid. The alkyl trimethyl ammonium halide can include octyl trimethyl ammonium bromide, decyl trimethyl ammonium bromide, dodecyl trimethyl ammonium bromide, myristyl trimethyl ammonium bromide, and cetyl trimethyl ammonium bromide.The alkyl trimethyl ammonium tosylate can include octyl trimethyl ammonium tosylate, decyl trimethyl ammonium tosylate, dodecyl trimethyl ammonium tosylate, myristyl trimethyl ammonium tosylate, and cetyl trimethyl ammonium tosylate.For example, the N-alkyl pyridinium halide can include decyl pyridinium chloride, dodecyl pyridinium chloride, cetyl pyridinium chloride, decyl pyridinium bromide, dodecyl pyridinium bromide, cetyl pyridinium bromide, decyl pyridinium iodide, dodecyl pyridinium iodide, and cetyl pyridinium iodide.

[0123] The cationic surfactant may comprise at least one compound selected from the following: dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, cetyltrimethylammonium bromide, cetyldimethylethylammonium bromide, (C1-C30 alkyl)-trimethylammonium bromide, (C1-C30 alkyl)amines, (C1-C30 alkyl)imidazolines, ethoxylated amines, quaternary compounds, quaternary esters, (C1-C30 alkyl)amine oxides, lauramine oxide, dicetyldimonium chloride, cetri ... ammonium halides, primary polyethoxylated fatty amine salts, secondary polyethoxylated fatty amine salts, tertiary polyethoxylated fatty amine salts, quaternary ammonium salts, tetra(C1-C30 alkyl)ammonium halides, (C1-C30 alkyl)amido-(C1-C30-alkyl)ammonium halides, tri(C1-C30 alkyl)benzylammonium halides, tri(C1-C30 alkyl)hydroxy-(C1-C30 alkyl)ammonium halides, (C1-C30 alkyl)pyridinium chloride, (C1-C30 alkyl)pyridinium bromide, and amine oxides.

[0124] The anionic surfactant may comprise at least one compound selected from the following: sodium dodecyl sulfate, (C6-C30 alkyl)benzenesulfonates, C6-C30 alpha olefin sulfonates, paraffin sulfonates, (C6-C30 alkyl)ester sulfonates, (C6-C30 alkyl)sulfates, (C6-C30 alkyl alkoxy)sulfates, (C6-C30 alkyl)sulfonates, (C6-C30 alkyl alkoxy)carboxylates, (C6-C30 alkyl alkoxylated)sulfates, mono(C1-C30 alkyl)(ether)phosphates, di(C6-C30 alkyl)(ether)phosphates, ( ... C6-C30 alkyl) sarcosinate, sulfosuccinate, sodium bis(2-ethylhexyl) sulfosuccinate, ethoxylated 4-nonylphenyl ether glycolate, (C1-C30 alkyl) isethionate, taurate, ammonium lauryl sulfate, ammonium laureth sulfate, triethylamine lauryl sulfate, triethylamine laureth sulfate, triethanolamine lauryl sulfate, triethanolamine laureth sulfate, monoethanolamine lauryl sulfate, monoethanolamine laureth sulfate, diethanolamine lauryl sulfate, diethanolamine laureth sulfate, lauric monoglyceride sodium sulfatesulfate), sodium lauryl sulfate, sodium laureth sulfate, potassium lauryl sulfate, potassium laureth sulfate, sodium lauryl phosphate, sodium tridecyl phosphate, sodium behenyl phosphate, sodium laureth-2 phosphate, sodium ceteth-3 phosphate, sodium trideceth-4 phosphate, sodium dilauryl phosphate, sodium ditridecyl phosphate, sodium ditrideceth-6 phosphate, sodium lauroyl sarcosinate, lauroyl sarcosine, cocoyl sarcosine, ammonium cocoyl sulfate, sodium cocoyl sulfate, sodium trideceth sulfate, sodium tridecyl sulfate, ammonium tridecyl sulfate, ammonium tridecyl sulfate, sodium cocoyl isethionate, disodium laureth sulfosuccinate, sodium oleoyl methyl taurate, sodium laureth carboxylate, sodium trideceth carboxylate, sodium lauryl sulfate, potassium cocoyl sulfate, potassium lauryl sulfate, monoethanolamine cocoyl sulfate, sodium tridecyl benzenesulfonate, sodium dodecyl benzenesulfonate, and sodium dodecyl sulfate.

[0125] The nonionic surfactant may comprise at least one compound selected from the following: C6-C18 alkyl alcohols, (C6-C18 alkyl)phenols, (C6-C18 alkyl)ethoxylates, (C6-C18 alkyl)phenols (C1-C3 alkoxylates), block oxy(C1-C3 alkylene) condensates of C6-C18 alkylphenols, oxy(C1-C3 alkylene) condensates of alkanols, oxyethylene / oxypropylene block copolymers, amine oxides, phosphine oxides, alkylamine oxides having 8 to 50 carbon atoms, mono- or di(C8-C30) alkyl alkanolamides, (C6-C30 alkyl) polysaccharides, sorbitan fatty acid esters, polyoxyethylene ... esters, polyoxyethylene sorbitol esters, polyoxyethylene nonylphenyl ether, polyoxyethylene acid, polyoxyethylene alcohols, coco monoethanolamide, coco diethanolamide, coco diglycoside, (C8-C30 alkyl) polyglycosides, cocamidopropyl, lauramine oxide, polyoxyethylene (20) sorbitan monolaurate, ethoxylated linear C8-C30 alcohols, cetearyl alcohol, lanolin alcohol, stearic acid, glyceryl stearate, polyethylene glycol 100 stearate, 4-(1,1,3,3-tetramethylbutyl)phenyl polyethylene glycol, polyoxyethylene (10) cetyl ether, eicosaethylene glycol octadecyl ether, and HO(CH2CH2O). 20 (CH2CH(CH3)O) 70 (CH2CH2O) 20 H.

[0126] Reducing Agent The lysis buffer and / or reagent composition (e.g., dry composition) may include one or more reducing agents. A "reducing agent" may be a compound or a group of compounds. As used herein, a "reducing agent" is also known as a "reductant," "reducer," or "reducing equivalent," and may refer to an element or compound that donates electrons to another species. In particular, a reducing agent is generally a compound that breaks disulfide bonds by reduction, thereby overcoming tertiary protein folding structures and quaternary protein structures (oligomeric subunits) that are stabilized by disulfide bonds. Examples of suitable reducing agents include, but are not limited to, 2-mercaptoethanol, DTT, TCEP, DTE, reduced glutathione, cysteamine, TBP, dithioerythriol, THPP, 2-mercaptoethylamine-HCl, DTBA, cysteine, cysteine-thioglycolate, salts of sulfite, thioglycolic acid, and HED. In some embodiments of the methods, compositions, and kits provided herein, the lysis buffer and / or reagent composition (e.g., a dry composition) does not include one or more reducing agents.

[0127] Reagent Composition The reagent compositions (e.g., dry compositions) described herein can be provided in a "dry form" or in a form not suspended in a liquid medium. A "dry form" of a composition can include a dry powder, a lyophilized composition, a spray-dried composition, or a precipitated composition. A "dry form" composition can include one or more lyoprotectants such as sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, and mannitol; amino acids, such as arginine and histidine; lyotropic salts, such as magnesium sulfate; polyols, such as propylene glycol, glycerol, poly(ethylene glycol), or polypropylene glycol; and combinations thereof. Additional exemplary lyoprotectants include gelatin, dextrin, modified starch, and carboxymethylcellulose. As used herein, the terms "lyophilization," "lyophilized," and "freeze-drying" refer to a process in which the material to be dried is first frozen and then the ice or freezing solvent is removed by sublimation in a vacuum environment. "Lyophile" refers to a material that has been freeze-dried.

[0128] The reagent composition (e.g., dry composition) may be frozen, lyophilized, or spray dried. The reagent composition may be heat dried. The reagent composition may include one or more additives (e.g., amino acids, polymers, sugars, or sugar alcohols). The sugar or sugar alcohol may include sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof. The polymer may include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptides, collagen peptides, or any combination thereof. The lyophilized reagent may include poly rA, EGTA, EDTA, Tween 80, and / or Tween 20. The frozen or lyophilized or spray-dried or heat-dried composition, or the aqueous composition for preparing the frozen or lyophilized or spray-dried composition, may include one or more of the following: (i) non-aqueous solvents such as ethylene glycol, glycerol, dimethylsulfoxide, and dimethylformamide. (ii) surfactants such as Tween 80, Brij 35, Brij 30, Lubrol-px, Triton X-10; Pluronic F127 (polyoxyethylene-polyoxypropylene copolymer), also known as poloxamer, poloxamine, and sodium dodecyl sulfate. (iii) disaccharides such as trehalose, sucrose, lactose, and maltose. (iv) polymers (which may have different MW) such as polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropylmethylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, and albumin. (v) Amino acids such as glycine, proline, 4-hydroxyproline, L-serine, glutamic acid, alanine, lysine, sarcosine, and gamma-aminobutyric acid. As disclosed herein, a reagent composition (eg, a dry composition) may include one or more protectants and one or more amplification reagents.

[0129] Protective Agent Provided herein are methods and compositions that include one or more protective agents and / or one or more additives. The one or more additives may include Tween 20, Triton X-100, Tween 80, non-ionic detergents (e.g., non-ionic surfactants), or any combination thereof. The one or more protective agents may include a cyclodextrin compound (e.g., a compound of formula I). ​​Cyclodextrins (CDs) can be used for complexation with solubilizing agents (e.g., SDS). Cyclodextrins (CDs) can be cyclic oligosaccharides that resemble a truncated cone with a hydrophobic internal cavity and a hydrophilic external surface. The most commonly used natural cyclodextrins include those with 6, 7, and 8 glucose units, referred to as α, β, and γ-CD. Natural CDs can be soluble. Chemically modified CDs, such as hydroxypropyl derivatives, increase solubility in aqueous media by up to 50%. CAVASOL® is a trade name for cyclodextrin derivatives manufactured by WACKER, and includes a variety of α-, β-, and γ-CD derivatives. β-CD can form a strong inclusion complex (stronger than α-CD and β-CD) with sodium dodecyl sulfate (SDS) mainly in a 1:1 stoichiometry. The binding constant between β-CD and SDS is 2100 M -1 ~2500M -1 The range may be:

[0130] The protectants (e.g., cyclodextrin compounds) described herein can be used with a lysis buffer containing a lysis agent (e.g., SDS) for complex formation, such as a lysis buffer for Chlamydia trachomatis and / or Neisseria gonorrhea detection. SDS is one of the most commonly used reagents for Chlamydia cell lysis. SDS can solubilize phospholipid and protein components of the cell membrane, resulting in cell lysis and release of the cell contents. SDS can also bind to proteins quite specifically, thereby denaturing the proteins at high concentration ranges. SDS can exhibit inhibitory effects at concentrations higher than 0.01% (weight / volume) for PCR with Taq polymerase. Provided herein are compositions in methods where CD-SDS complex formation occurs in real-time assays for Chlamydia trachomatis and / or Neisseria gonorrhea detection. SDS can be used in lysis buffers for cell lysis / sample preparation. CD can be incorporated into a dry (e.g., lyophilized, heat-dried) pellet containing the enzyme and other assay components. After the lysis step, the sample solution can be mixed with dried (e.g., freeze-dried, heat-dried) pellets containing cyclodextrin to promote CD and SDS inclusion complex formation. The methods and compositions provided herein can include a chlamydia cell lysis buffer containing SDS (e.g., RIPA buffer: 50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40; 1% SDS buffer: 50 mM Tris (pH 7.5), 150 mM NaCl, 1% SDS). The one or more protecting agents can include (2-hydroxypropyl)-β-cyclodextrin (catalog number 332593-5G), which can include an average Mw of about 1,380 and 0.6 molar substitution. The one or more protecting agents may include (2-hydroxypropyl)-β-cyclodextrin (catalog number 389145-5G), which may include an average Mw of about 1,540 and a 1.0 molar substituted dextrin.The one or more protecting agents may include (2-hydroxypropyl)-γ-cyclodextrin (catalog number H125-5G-I), which may have a water solubility of 450 mg / mL. The one or more protecting agents may include α-cyclodextrin (catalog number C4642-5G), which may have a water solubility of 50 mg / mL.

[0131] The molar ratio of one or more protecting agents to one or more amplification reagents (e.g., amplification reaction components) in the amplification reaction mixture can be from about 10:1 to about 1:10, e.g., about 2:1. This ratio can be about, at least, or at most 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1 , 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 9 The ratio may be 5:1, 96:1, 97:1, 98:1, 99:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 10000:1, or a number or range between any two of these values.

[0132] The one or more protecting agents may include a cyclodextrin compound, such as a cyclodextrin compound of formula I, or a salt, ester, solvate, or hydrate thereof. [ka] (In the formula, Each R is independently H, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which may be optionally substituted; or -C(O)OR B , -OC(O)R B , -C(O)R B or -C(O)NR A R B and; Each R1 is independently H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, halogen, hydroxy, amino, -CN, -CF3, -N3, -NO2, -OR B , -SR B , -SOR B , -SO2R B , -N(R B )S(O2)-R B , -N(R B )S(O2)NR A R B , -NR A R B , -C(O)OR B , -OC(O)R B , -C(O)R B , -C(O)NR A R B , or -N(R B )C(O)R B each of which may be substituted; Each R A is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; Each R Bis independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each m is independently 0, 1, 2, 3, 4, or 5.

[0133] In some embodiments, each R is independently H, optionally substituted alkyl, -C(O)OR B , -OC(O)R B , -C(O)R B or -C(O)NR A R B In some embodiments, n is 1, 2, or 3. Each R may independently be H, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, or octyl; each may be straight or branched chain. The cyclodextrin compound may be 2-hydroxypropyl-α-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin (2HPβCD), hydroxypropyl-β-cyclodextrin (HPβCD), methyl-β-cyclodextrin (MβCD), 2-hydroxypropyl-γ-cyclodextrin, α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, or a salt, ester, solvate, or hydrate thereof.

[0134] The number of carbon atoms in the hydrocarbyl substituent is indicated by the prefix "C x -C y " where x is the minimum number of carbon atoms in the substituent and y is the maximum number. x Chain means a hydrocarbyl chain containing x carbon atoms. The prefix "halo" indicates that the substituent to which the prefix is ​​attached is substituted with one or more independently selected halogen radicals. For example, "C1-C6 haloalkyl" means a C1-C6 alkyl substituent in which at least one hydrogen radical is replaced with a halogen radical. When a linking element of a depicted structure is "absent" or a "bond", the element to the left of the depicted structure is directly connected to the element to the right of the depicted structure. For example, if a chemical structure is X-(L) n -Y, where L is absent or n is 0, then the chemical structure is XY.

[0135] The term "alkyl" as used herein refers to a saturated straight or branched chain hydrocarbon radical. For example, a "C1-C8 alkyl" contains from 1 to 8 carbon atoms. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, ieri-butyl, neopentyl, n-hexyl, heptyl, and octyl radicals. The term "alkenyl" as used herein refers to a straight or branched chain hydrocarbon radical containing one or more double bonds. For example, "C2-C8 alkenyl" contains from 2 to 8 carbon atoms. Alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl, and octenyl. The term "alkynyl" as used herein refers to a straight or branched chain hydrocarbon radical containing one or more triple bonds. For example, "C2-C8 alkynyl" contains from 2 to 8 carbon atoms. Representative alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, heptynyl, and octynyl.

[0136] The term "cycloalkyl" refers to a monovalent group derived from a monocyclic or polycyclic saturated carbocyclic ring compound. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptyl, and bicyclo[2.2.2]octyl. The terms "carbocycle" or "carbocyclic" or "carbocyclyl" refer to a saturated (e.g., "cycloalkyl") ring system, partially saturated (e.g., "cycloalkenyl" or "cycloalkynyl") ring system, or fully unsaturated (e.g., "aryl") ring system containing zero heteroatom ring atoms. A carbocyclyl may be, but is not limited to, a single ring, or two or more fused, or bridged, or spirocyclic rings. A carbocyclyl may be, for example, a ring system having 3 to 10 ring members (i.e., C3-C 10 C3-C such as cycloalkyl 10 Substituted carbocyclyls may have either cis or trans geometry. Representative examples of carbocyclyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclopentadienyl, cyclohexadienyl, adamantyl, decahydro-naphthalenyl, octahydro-indenyl, cyclohexenyl, phenyl, naphthyl, fluorenyl, indanyl, 1,2,3,4-tetrahydro-naphthyl, indenyl, isoindenyl, bicyclodecanyl, anthracenyl, phenanthrene, benzonaphthenyl (also known as "phenalenyl"), decalinyl, and norpinanyl. A carbocyclyl group may be attached to the parent molecular moiety through any substitutable carbon atom of the group.

[0137] The term "aryl" refers to an aromatic carbocyclyl containing 6 to 14 carbon ring atoms. Non-limiting examples of aryl include phenyl, naphthalenyl, anthracenyl, indenyl, and the like. An aryl group may be attached to the parent molecular moiety through any substitutable carbon atom of the group. The term "heteroaryl" typically refers to an aromatic heterocyclyl containing 5 to 18 ring atoms, with at least one ring atom being a heteroatom. Heteroaryls may be single ring or may have two or more fused rings. Non-limiting examples of 5-membered heteroaryls include imidazolyl; furanyl; thiophenyl (or thienyl or thiofuranyl); pyrazolyl; oxazolyl; isoxazolyl; thiazolyl; 1,2,3-, 1,2,4-, 1,2,5-, and 1,3,4-oxadiazolyl; and isothiazolyl. Non-limiting examples of 6-membered heteroaryls include pyridinyl; pyrazinyl; pyrimidinyl; pyridazinyl; and 1,3,5-, 1,2,4-, and 1,2,3-triazinyl. Non-limiting examples of 6 / 5 membered fused ring heteroaryls include benzothiofuranyl, isobenzothiofuranyl, benzisoxazolyl, benzoxazolyl, purinyl, and anthranilyl. Non-limiting examples of 6 / 6 membered fused ring heteroaryls include quinolinyl, isoquinolinyl, and benzoxazinyl (including cinnolinyl and quinazolinyl).

[0138] The term "heterocycloalkyl" refers to a non-aromatic 3-, 4-, 5-, 6-, or 7-membered ring, or bicyclic or tricyclic fused ring system in which at least one of the ring atoms is a heteroatom, (i) each 5-membered ring has 0-1 double bonds, each 6-membered ring has 0-2 double bonds, (ii) the nitrogen and sulfur heteroatoms are optionally oxidized, (iii) the nitrogen heteroatom is optionally quaternized, and (iv) any of the above rings are optionally fused to a benzene ring. Representative heterocycloalkyl groups include, but are not limited to, [1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

[0139] The terms "heterocyclic" or "heterocycle" or "heterocyclyl" refer to a saturated (e.g., "heterocycloalkyl") ring system, partially unsaturated (e.g., "heterocycloalkenyl" or "heterocycloalkynyl") ring system, or fully unsaturated (e.g., "heteroaryl") ring system in which at least one of the ring atoms is a heteroatom (i.e., nitrogen, oxygen, or sulfur), and the remaining ring atoms are independently selected from carbon, nitrogen, oxygen, and sulfur. A heterocyclyl group may be connected to the parent molecular moiety through any substitutable carbon or nitrogen atom in the group as long as a stable molecule is obtained. A heterocyclyl may be, without limitation, a monocyclic ring. Non-limiting examples of single-ring heterocyclyls include furanyl, dihydrofuranyl, pyrrolyl, isopyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, isoimidazolyl, imidazolinyl, imidazolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, triazolyl, tetrazolyl, dithiolyl, oxathiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, thiazolinyl, isothiazolinyl, thiazolin ... Heterocyclyl may include, but is not limited to, zolidinyl, isothiazolidinyl, thiodiazolyl, oxathiazolyl, oxadiazolyl, pyranyl, dihydropyranyl, pyridinyl, piperidinyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl, triazinyl, isoxazinyl, oxazolidinyl, isoxazolidinyl, oxathiazinyl, oxadiazinyl, morpholinyl, azepinyl, oxepinyl, thiepinyl, or diazepinyl. Heterocyclyl may also include, but is not limited to, two or more rings fused together, such as naphthyridinyl, thiazolepyrimidinyl, thienopyrimidinyl, pyrimidopyrimidinyl, or pyridopyrimidinyl. Heterocyclyl may contain one or more sulfur atoms as ring members, and in some cases, the sulfur atoms are oxidized to SO or SO2. The nitrogen heteroatom of a heterocyclyl may or may not be quaternized and may or may not be oxidized to an N-oxide. In addition, the nitrogen heteroatom may or may not be N-protected.

[0140] The terms "optionally substituted", "optionally substituted alkyl", "optionally substituted", "optionally substituted alkenyl", "optionally substituted alkynyl", "optionally substituted carbocyclic", "optionally substituted aryl", "optionally substituted heteroaryl", "optionally substituted heterocyclic", and any other optionally substituted group as used herein refer to a group that is substituted or unsubstituted by independently replacing one, two, three or more of its hydrogen atoms with exemplary substituents, including but not limited to: -alkyl, -alkenyl, -alkynyl, -aryl, -arylalkyl, -heteroaryl, -heteroarylalkyl, -heterocycloalkyl, -cycloalkyl, -carbocyclic, -heterocyclic, -F, -CI, -Br, -I, -OH, protected hydroxy, alkoxy, oxo, thiooxo, -NO2, -CN, CF3, N3, -NH2, protected amino, -NHalkyl, -NHalkenyl, -NHalkynyl, -NHcycloalkyl, -NH-Aryl, -NH-Heteroaryl, -NH-Heterocyclic, -Dialkylamino, -Diarylamino, -Diheteroarylamino, -O-Alkyl, -O-Alkenyl, -O-Alkynyl, -O-Cycloalkyl, -O-Aryl, -O-Heteroaryl, -O-Heterocyclic, -C(O)-Alkyl, -C(O)-Alkenyl, -C(O)-Alkynyl, -C(O)-Cycloalkyl, -C(O)-Aryl, -C(O)-Heteroaryl, -C(O)-Heterocycloalkyl, -CONH2, -CONH-Alkyl, -CONH-Alkenyl , -CONH-alkynyl, -CONH-cycloalkyl, -CONH-aryl, -CONH-heteroaryl, -CONH-heterocycloalkyl, -OCO2-alkyl, -OCO2-alkenyl, -OCO2-alkynyl, -OCO2-cycloalkyl, -OCO2-aryl, -OCO2-heteroaryl, -OCO2-heterocycloalkyl, -OCONH2, -OCONH-alkyl, -OCONH-alkenyl, -OCONH-alkynyl, -OCONH-cycloalkyl, -OCONH-aryl, -OCONH-heteroaryl,-OCONH-heterocycloalkyl, -NHC(O)-alkyl, -NHC(O)-alkenyl, -NHC(O)-alkynyl, -NHC(O)-cycloalkyl, -NHC(O)-aryl, -NHC(O)-heteroaryl, -NHC(O)-heterocycloalkyl, -NHCO2-alkyl, -NHCO2-alkenyl, -NHCO2-alkynyl, -NHCO2-cycloalkyl, -NHCO2-aryl, -NHCO2-heteroaryl, -NHCO2-heterocycloalkyl, -NHC(O)NH2, -NHC(O)NH-alkyl, - NHC(O)NH-alkenyl, -NHC(O)NH-alkenyl, -NHC(O)NH-cycloalkyl, -NHC(O)NH-aryl, -NHC(O)NH-heteroaryl, -NHC(O)NH-heterocycloalkyl, NHC(S)NH2, -NHC(S)NH-alkyl, -NHC(S)NH-alkenyl, -NHC(S)NH-alkynyl, -NHC(S)NH-cycloalkyl, -NHC(S)NH-aryl, -NHC(S)NH-heteroaryl, -NHC(S)NH-heterocycloalkyl, -NHC(NH)NH2, -NHC( NH)NH-alkyl, -NHC(NH)NH-alkenyl, -NHC(NH)NH-alkenyl, -NHC(NH)NH-cycloalkyl, -NHC(NH)NH-aryl, -NHC(NH)NH-heteroaryl, -NHC(NH)NH-heterocycloalkyl, -NHC(NH)-alkyl, -NHC(NH)-alkenyl, -NHC(NH)-alkenyl, -NHC(NH)-cycloalkyl, -NHC(NH)-aryl, -NHC(NH)-heteroaryl, -NHC(NH)-heterocycloalkyl, -C(NH)NH-alkyl, - C(NH)NH-alkenyl, -C(NH)NH-alkynyl, -C(NH)NH-cycloalkyl, -C(NH)NH-aryl, -C(NH)NH-heteroaryl, -C(NH)NH-heterocycloalkyl, -S(O)-alkyl, -S(O)-alkenyl, -S(O)-alkynyl, -S(O)-cycloalkyl, -S(O)-aryl, -S(O)-heteroaryl, -S(O)-heterocycloalkyl, -SONH2, -SONH-alkyl, -SONH-alkenyl, -SONH-alkynyl, -SONH-cycloalkyl,-SO2NH-aryl, -SO2NH-heteroaryl, -SO2NH-heterocycloalkyl, -NHSO2-alkyl, -NHSO2-alkenyl, -NHSO2-alkynyl, -NHSO2-cycloalkyl, -NHSO2-aryl, -NHSO2-heteroaryl, -NHSO2-heterocycloalkyl, -CH2NH2, -CH2SO2CH3, polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -methoxyethoxy, -SH, -S-alkyl, -S-alkenyl, -S-alkynyl, -S-cycloalkyl, -S-aryl, -S-heteroaryl, -S-heterocycloalkyl, or methylthiomethyl.,

[0141] It is understood that the aryl, heteroaryl, carbocycle, heterocycle, alkyl, and the like can be further substituted. The terms "halo" and "halogen," as used herein, refer to an atom selected from fluorine, chlorine, bromine, and iodine.

[0142] The term "leaving group" or "LG," as used herein, refers to any group that leaves during the course of a chemical reaction in which it participates, including, but not limited to, halogens, brosylate, mesylate, tosylate, triflate, p-nitrobenzoate, and phosphonate groups. The term "protected hydroxy", as used herein, refers to a hydroxy group protected with a hydroxy-protecting group as defined above, including, for example, a benzoyl, acetyl, trimethylsilyl, triethylsilyl, methoxymethyl group.

[0143] The term "hydroxy protecting group" as used herein refers to a reactive chemical moiety known in the art to protect a hydroxy group from undesired reactions during a synthetic procedure. Following said synthetic procedure, the hydroxy protecting groups described herein can be selectively removed. Non-limiting examples of hydroxy protecting groups include: benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, methoxycarbonyl, tert-butoxycarbonyl, isopropoxycarbonyl, diphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, 2-furfuryloxycarbonyl, allyloxycarbonyl, acetyl, formyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl ... Such protecting groups include phenyl, methyl, t-butyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, benzyl, para-methoxybenzyldiphenylmethyl, triphenylmethyl (trityl), tetrahydrofuryl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, para-toluenesulfonyl, trimethylsilyl, triethylsilyl, and triisopropylsilyl. In some embodiments, the hydroxy protecting group is acetyl (Ac or -C(O)CH3), benzoyl (Bz or -C(O)CH5), and trimethylsilyl (TMS or -Si(CH3)3).

[0144] The term "amino protecting group" as used herein refers to a reactive chemical moiety known in the art to protect an amino group from undesired reactions during a synthetic procedure. After said synthetic procedure, the amino protecting group described herein can be selectively removed. Examples of amino protecting groups include, but are not limited to, t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, and benzyloxycarbonyl. The term "protected amino", as used herein, refers to an amino group protected with an amino-protecting group as defined above. The term “alkylamino” refers to the structure —N(R a R b ), wherein R a and R b is independently H or alkyl.

[0145] Examples of acceptable salts include, but are not limited to, non-toxic acid addition salts or salts of amino groups formed with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, and perchloric acids, or with organic acids such as acetic, maleic, tartaric, citric, succinic, or malonic acids, or by using other methods used in the art such as ion exchange. Other acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy- Ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate salts, etc. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, or magnesium salts, etc. Additional acceptable salts include amine cations, carboxylates, sulfates, phosphates, nitrates, alkyls, sulfonates, and arylsulfonates having 1 to 6 carbon atoms, formed as appropriate using non-toxic ammonium, quaternary ammonium, and halide, hydroxide, and other counterions.

[0146] The term "ester" as used herein may refer to an ester of a compound formed by the processes disclosed herein, and may include those that hydrolyze in vivo and decompose easily in the human body to leave the parent compound or a salt thereof. Suitable ester groups include, for example, those derived from acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic, and alkanedioic acids, each alkyl or alkenyl moiety advantageously having 6 or fewer carbon atoms. Examples of specific esters include, but are not limited to, formates, acetates, propionates, butyrates, acrylates, and ethylsuccinates.

[0147] The compounds described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be specified in terms of absolute stereochemistry as (R)- or (S)-, or in the case of amino acids, as (D)- or (L)-. The present disclosure is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optical isomers can be prepared from their respective optically active precursors by the procedures described above or by separating the racemic mixtures. Separation can be carried out in the presence of a resolving agent, by chromatography, or by repeated crystallization, or by some combination of techniques known to those skilled in the art. When the compounds described herein contain olefinic double bonds or other geometrically asymmetric centers, unless otherwise specified, the compounds are intended to include both E and Z geometric isomers. Likewise, all tautomeric forms are intended to be included. The configuration of any carbon-carbon double bonds appearing herein has been chosen merely as a matter of convenience, and no particular configuration is intended to be designated unless otherwise indicated in the context. Thus, a carbon-carbon double bond depicted herein for convenience as trans may also be cis, trans, or a mixture of the two in any proportion.

[0148] kit Disclosed herein is a kit for detecting a target nucleic acid sequence in a sample. In some embodiments, the kit comprises: (a) a lysis buffer comprising one or more lysis agents, the one or more lysis agents being capable of lysing biological entities to release sample nucleic acids contained therein, the sample nucleic acids being suspected to contain a target nucleic acid sequence; and (b) a dry composition comprising one or more protection agents and one or more amplification reagents, the one or more amplification reagents comprising one or more components for amplifying a target nucleic acid sequence under isothermal amplification conditions. In some embodiments, the components comprise: (i) a first primer and a second primer, the first primer being capable of hybridizing to a sequence of a first strand of a target nucleic acid sequence, and the second primer being capable of hybridizing to a sequence of a second strand of a target nucleic acid sequence; and (ii) an enzyme having a hyperthermophile polymerase activity capable of generating a nucleic acid amplification product. In some embodiments, the lysis buffer and / or reagent composition comprises one or more reducing agents, which may include one or more of 2-mercaptoethanol, DTT, TCEP, DTE, reduced glutathione, cysteamine, TBP, dithioerythriol, THPP, 2-mercaptoethylamine-HCl, DTBA, cysteine, cysteine-thioglycolate, salts of sulfite, thioglycolic acid, and HED.

[0149] The kit may include at least one component that provides real-time detection activity of the nucleic acid amplification product. The real-time detection activity may be provided by a molecular beacon. The reagent composition (e.g., a dry composition) may include a reverse transcriptase and / or a reverse transcription primer. In some embodiments, the molar ratio of the one or more protective agents to the one or more amplification reagents is about 10:1 to about 1:10 (e.g., about 2:1). In some embodiments, the one or more additives include Tween 20, Triton X-100, Tween 80, a non-ionic detergent (e.g., a non-ionic surfactant), or any combination thereof. In some embodiments, the one or more protective agents include a cyclodextrin compound of formula (I). In some embodiments, the one or more lysis reagents comprise about 0.001% (w / v) to about 1.0% (w / v) (e.g., about 0.2% (w / v)) of the treated sample. In some embodiments, the one or more lysis agents include a detergent. The detergent may include one or more of a cationic surfactant, an anionic surfactant, a non-ionic surfactant, and an amphoteric surfactant. In some embodiments, it may be advantageous for the one or more protecting agents to be capable of sequestering the one or more lysis agents, thereby preventing denaturation of the one or more amplification reagents by the one or more lysis agents.

[0150] The kit may include one or more polymerases and one or more primers, and optionally one or more reverse transcriptases and / or reverse transcription primers, for example as described herein. If one target is being amplified, one pair of primers (forward and reverse) may be included in the kit. If multiple target sequences are being amplified, multiple primer pairs may be included in the kit. The kit may include a control polynucleotide, and if multiple target sequences are being amplified, multiple control polynucleotides may be included in the kit. An enzyme with hyperthermophile polymerase activity may have an amino acid sequence that is at least about 90% or 95% identical to the amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof. For example, an enzyme with hyperthermophile polymerase activity may include the amino acid sequence of SEQ ID NO: 1. The nucleic acid amplification product may be about 20 to 40 bases in length. The nucleic acid amplification product may include (1) a first primer sequence and its reverse complement, (2) a second primer sequence and its reverse complement, and (3) a spacer sequence flanked by (1) the first primer sequence and its reverse complement and (2) the second primer sequence and its reverse complement, the spacer sequence being 1 to 10 bases in length.

[0151] The biological entity may include one or more of a prokaryotic cell, a eukaryotic cell, a virus particle, an exosome, a protoplast, and a microvesicle. The biological entity may include a virus, a bacterium, a fungus, a protozoan, a part thereof, or any combination thereof. The target nucleic acid sequence may be a viral, bacterial, fungal, or protozoan nucleic acid sequence. The sample nucleic acid may be derived from a virus, a bacterium, a fungus, or a protozoan. The kit may also include one or more of the components in any number of separate vessels, chambers, containers, packets, tubes, vials, microtiter plates, and the like, or the components may be combined in various combinations in such containers. The components of the kit may, for example, be present in one or more containers. In some embodiments, all of the components are provided in one container. In some embodiments, the enzymes (e.g., polymerase and / or reverse transcriptase) may be provided in a separate container from the primers. The components may, for example, be lyophilized, heat-dried, freeze-dried, or present in a stable buffer. In some embodiments, the polymerase and / or reverse transcriptase are present in a single container in lyophilized or heat-dried form, and the primers are either lyophilized, heat-dried, freeze-dried, or present in a buffer in a different container. In some embodiments, the polymerase and / or reverse transcriptase and the primers are present in a single container in lyophilized or heat-dried form.

[0152] The kit may further include, for example, dNTPs used in the reaction, or modified nucleotides used in the reaction, vessels, cuvettes, or other containers, or vials of water or buffer for rehydrating lyophilized or heat-dried components. The buffer used may, for example, be suitable for both polymerase activity and primer annealing activity. The kits may also include instructions for practicing one or more of the methods described herein and / or instructions for one or more of the components described herein. The instructions and / or instructions may be in printed form or may be included in a kit insert. The kits may also include a written description of an internet location that provides such instructions or instructions. The kit may further include reagents used in detection methods, such as reagents used in FRET, lateral flow devices, dipsticks, fluorescent dyes, colloidal gold particles, latex particles, molecular beacons, or polystyrene beads. EXAMPLES

[0153] Certain aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way. Example 1 Effect of anionic detergents SDS and HP-βCD on Neisseria gonorrhoeae DNA amplification This example provides an SDS complex formation analysis of the methods and compositions provided herein. In particular, this example demonstrates the effect of the anionic detergents SDS and HP-βCD on Neisseria gonorrhoeae DNA amplification. Although >0.01% of the anionic detergent SDS inhibited APA amplification of DNA, the presence of the protectant hydroxypropyl β-CD (βCD) allowed amplification to proceed despite ≦0.2% SDS. Thus, the kinetics of SDS sequestration of hydroxypropyl β-CD was sufficiently rapid at a molar ratio of 2:1 (approximately 14 mM:approximately 7 mM) to suppress the 9°N complex formation by SDS.m (TM) DNA polymerase inactivation was prevented.

[0154] Standard APA reactions with or without 4% Sigma 389145 HPβCD containing "hot start" CleanAmp™ deoxynucleotides (CAdNTPs) and sequence-specific molecular beacon detection probes were performed using lyophilized ((dextran 40, supplemented with trehalose), FIG. 2B) or "wet" (no HPβCD, FIG. 2A) reagents containing Tris pH 8.4, KCl, MgSO4, and (NH4)2SO4 Triton X-100, 9°Nm™ DNA polymerase, CAdNTPs, primers, and probes. For lyophilized reactions, samples were prepared in 50 μl solution containing MgSO4 and (NH4)2SO4 with (graphic symbols) or without (dashed lines) 500 copies of N. gonorrhoeae genomic DNA. After dissolving the dried reagents in the samples, various amounts of SDS were added as indicated, and the solutions were vortexed. For wet reactions, 45 μl master mix containing all reaction components was added to 5 μl samples containing 10x SDS with or without 500 copies of N. gonorrhoeae genomic DNA (final concentrations as indicated). CAdNTPs were activated at 90°C for 2 min, followed by APA at 68°C for 10 min, and NG amplification products were detected by fluorescence collected every 15 s. Results showed that in the absence of βCD, SDS was inactivated by >0.01% SDS, but was tolerated to at least 0.2% in the presence of βCD (Figures 2A-2B).

[0155] Example 2 Effect of cationic detergent CTAB on Chlamydia trachomatis DNA amplification This example is similar to Example 1, but provides a demonstration of the effect of cationic detergent CTAB on C. trachomatis DNA amplification. APA reactions containing CAdNTPs and sequence-specific molecular beacon detection probes were performed as above with varying amounts of cationic detergent CTAB in lyophilized reactions (with HP-βCD, Figure 3B) or wet reactions (without HP-βCD, Figure 3A) without ("NTC") or with the addition of 500 copies of C. trachomatis whole cell DNA. CAdNTPs were activated by heating at 90°C for 2 min, followed by APA at 68°C with fluorescence collected every 15 s. Results showed that in the absence of HP-βCD, it was completely inactivated by >0.01% CTAB, but was fully tolerated up to at least 0.2% in the presence of HP-βCD.

[0156] Example 3 Lysis of Chlamydia trachomatis This example demonstrates the unexpected advantages of the methods and compositions provided herein over currently available nucleic acid detection methods and compositions, for example, currently available methods can be extended to sample types such as human blood, which is easy to lyse, but such methods cannot provide efficient lysis or amplification of difficult to lyse pathogens. In this example, an APA-based assay was used to examine elementary body lysis (approximately 40 / reaction). Elementary bodies (Acrometrix) were added to either chilled Chlamydia transport medium (SPD containing sucrose, phosphate buffer, and glutamic acid), TE (Tris-EDTA10-1), or lysis buffer pH 2.2 containing 0.4% SDS. Samples in TE or SDS were heated at 95°C for 5 min (hot lysis) or 78°C for 2 min (SDS lysis), respectively. 5 μl (containing 40 elementary bodies) were then added as above to a "wet" APA reaction mix (pH 7.6, to facilitate isothermal CAdNTP activation) containing 4% cyclodextrin HP-βCD for isothermal APA (68°C), and fluorescence from Chlamydia-specific molecular beacons was collected every 15 seconds. SDS lysis (dashed line) was as effective at releasing target DNA as heat lysis (solid line). In the absence of lysis reagent, elementary bodies, which are unique in that they contain a rigid cross-linked proteinaceous cell wall, were not detectable (squares) as were non-template controls (NTC dotted line) (Figure 4). Despite the presence of 0.4% ionic detergent to lyse the organisms, the continued presence of detergent in the APA did not affect 9°Nm™ DNA polymerase-mediated amplification because cyclodextrin sequestered SDS. The presence of detergent would otherwise be inhibitory.

[0157] Example 4 Effect of hydroxypropyl-β-cyclodextrin (HPβCD) protection on influenza B virus detection This example demonstrates the unexpected advantages of the methods and compositions provided herein over currently available nucleic acid detection methods and compositions. For example, unlike previous methods that do not disclose the sensitivity of the assay to determine the degree of protection, this example shows that the detection time of RNA target is comparable with + / - SDS, and determines the assay sensitivity. APA was performed with lyophilized reagents (including β-cyclodextrin, dNTPs, 35 units of reverse transcriptase, KCl, EGTA (or EDTA), Tx-100, Tween 80, poly rA, Tris pH 8.4, dextran 40k, 4% beta CD, trehalose, primers, probes, 9°Nm™).

[0158] As shown in Figure 5A, the dried reagent preheated to 58°C was lysed by pipetting 50 ml lysis solution containing 0.2% SDS (RBSR) or 0.1% Triton X-100 (RBTR), MgSO4, and (NH4)2SO4 preheated to 58°C for 2 min, with or without (NTC, dotted line) influenza B RNA equivalent to 500 IU (infectious units). After 2 min at 58°C (to facilitate reverse transcription), the temperature was increased to 68°C for APA and fluorescence detection. As shown in Figure 5B, the APA demonstrated an assay detection range of 0 (NTC) to 5,000 IU of influenza B (with SDS and virus), with the 500 IU used in Figure 5A being well within the assay dynamic range, suggesting that the assay sensitivity was sufficient and that assay performance was virtually fully restored by HP-β-cyclodextrin in the presence of SDS under these conditions.

[0159] Example 5 Order of addition assay This example demonstrates the unexpected advantages of the methods and compositions provided herein. For example, it was unexpectedly found that the kinetics of hydroxypropyl β-CD was sufficiently rapid at a molar ratio of hydroxypropyl β-CD and SDS of 2:1. Data for the order of addition assay are shown in Figure 6. Order of addition assay: Detection time depends on the addition of cyclodextrin before the brief interaction of the enzyme with SDS. SDS is added at 9°N m Since .TM. DNA polymerase was rapidly and irreversibly inactivated, if the enzyme were instead exposed simultaneously to SDS and β-CD, the kinetics of complex formation of the latter two substances must be very rapid compared to the rate of interaction between SDS and the enzyme. Figure 6 shows the following: Tris pH 7.6, KCl, (NH4)2SO4, MgSO4, Triton X-100, SYBRI, CAdNTP, 9°N mData are shown for detection of S. enterica DNA target (50,000 copies) by APA reaction in a wet "hot start" format mediated by CleanAmp™ deoxynucleotides (CAdNTPs) with CleanAmp™ DNA polymerase. 0.4% SDS (X), 4% hydroxypropyl β-CD (squares), SDS then immediately β-CD (circles), or β-CD then SDS (solid lines) were added and mixed. None was added to the control (ctrl dotted line). All samples contained DNA target. Isothermal APA was performed at 68°C and intercalating dye fluorescence was collected every 15 seconds to monitor all exponential DNA amplifications. The barrier to amplification from SDS (X, flat) was counteracted by prior addition of β-CD (solid line), reducing the plateau but restoring detection times. Even a brief exposure to SDS prior to β-CD addition (circles, flat lines) reduced the amplification time to 9°N. m (TM) DNA polymerase enzyme, suggesting that the kinetics of sequestration were rapid at this 2:1 molar ratio of β-CD:SDS. Negative controls for each condition lacking S. enterica DNA confirmed the specificity of the indicated amplification products (not shown).

[0160] Example 6 RNaseA inactivation assay Ionic detergents such as sodium dodecyl sulfate (SDS) and / or cetyltrimethylammonium bromide (CTAB) can be used as lysis agents in the methods and compositions provided herein, allowing efficient lysis and also inhibiting ribonucleases by denaturation, thus protecting the target RNA during the lysis step. However, in subsequent direct and rapid molecular testing reactions initiated by dissolving the dry reaction mix directly into the lysis sample without separation or nucleic acid purification, SDS (and / or CTAB) may need to be removed quickly, as it will also denature the enzymes reverse transcriptase and polymerase required for the reaction. However, removal of some agents used to denature ribonucleases may allow reactivation of ribonucleases, i.e., recovery of their enzymatic activity. As shown herein, inclusion of cyclodextrin in the dry reaction mix can allow rapid removal of SDS (and / or CTAB). In fact, this rapid removal may also accelerate the refolding of some denatured proteins. As demonstrated in this example, it was indeed shown that ribonuclease reactivation was very rapid in the presence of cyclodextrin, leaving the target RNA unprotected.

[0161] Figure 7 shows data on the effect of DTT on the irreversibility of RNase A inactivation in the presence of CTAB at various temperatures. mStandard APA reaction reagents lacking HP-βCD but containing 4% HP-βCD were prepared in lyophilized format. The lyophilized reagents also contained RNaseAlert® Assay Substrate, a synthetic quenched RNA probe that emits fluorescence in the presence of active RNaseA. The reagents were preincubated for 2 minutes and dissolved in a 50 ml solution containing 20 ng RNaseA (0.4 mg / ml) with (dotted line) or without (symbols) 10 mM DTT at the indicated temperature. This solution had also been preincubated at the indicated temperature in a Bio-Rad CFx thermocycler with a programmed temperature gradient. In all cases, the immediate reversibility of putative CTAB-mediated RNase activity inactivation (without DTT, symbol) following CTAB sequestration by 4% HP-βCD was irreversible when DTT was included; i.e., no recovery of activity was detected (flat line).

[0162] Example 7 RNaseA inactivation assay Currently available methods can only achieve irreversible ribonuclease inactivation at up to 200 ng / ml, but the compositions and methods provided herein have demonstrated utility at 10 μg / ml to accommodate potentially larger amounts in patient samples. Data on the effect of DTT and HP-βCD on RNase A inactivation are illustrated in FIG. 8. The RNase Alert assay was used to study the effect of DTT and 0.2% cationic detergent CTAB on RNase inactivation. RNase A was spiked into a solution containing 0.2% CTAB, which was then mixed with a lyophilized APA reagent pellet lacking the enzyme but containing RNase Alert substrate and protectant (4% HP-β CD) after all solutions were preincubated at 68° C., followed by collection of fluorescence data at 68° C. In the presence of 10 mM DTT (dotted and dashed lines; FIG. 8), RNase A was effectively irreversibly inactivated, and sequestering the detergent did not restore activity. However, when detergent alone was used, activity was rapidly restored after sequestration (solid line 10 μg / ml, circles 1 μg / ml).

[0163] Example 8 Requirements for irreversible RNase inactivation This example provides a demonstration that, unlike some prior art, the combined action of DTT and SDS, but not either alone, allowed the undiluted sample to be used to irreversibly inactivate RNase A in the crude sample eluate without further processing prior to dissolving the lyophilized APA reagent for APA (Figure 9). 500 ng of RNase A (i.e., 10 μg / ml) was added to 50 ml of APA running buffer with MgSO4 and (NH4)2SO4, containing the indicated additives (0.2% SDS, 10 mM DTT, 0.1% Triton X-100) without (dotted line) or with (graphic symbols) purified influenza B RNA equivalent to 2500 infectious units, and preheated to 58°C for 2 minutes (Figure 9). The heated solution was then used to perform a 9°N run of RNase A similar to that used in Example 4. mSimilar pre-heated lyophilized APA reagent containing HP™ DNA polymerase, reverse transcriptase, and 4% βCD was dissolved. After an additional 2 min at 58°C (to facilitate reverse transcription with this primer set), APA was monitored by collecting fluorescence every 15 s after adjusting the temperature to 68°C. In this format, SDS alone (squares) did not prevent significant recovery of RNaseA activity after SDS sequestration with 4% HP βCD (it has been shown elsewhere that there is no RNase activity in the presence of SDS during the initial 2 min 58°C incubation). However, the combined action of DTT and SDS (X) allowed subsequent APA performance equivalent to a positive control without RNaseA (not shown). In the case of Triton X-100, which does not affect RNaseA activity, no RNA was detected (diamonds), presumably indicating that RNaseA is active both before and after dissolution of the APA reagent and initiation of reverse transcription. DTT and Triton did not protect RNA, although they did not include ionic detergent (triangles). Again, this indicates that DTT pretreatment is insufficient to adequately protect RNA in this format. Assay calibration (not shown) was consistent with a >99% reduction in RNA detectability (Figure 9). Importantly, this example shows that prior art RNase inactivation (DTT without ionic detergent) is ineffective in protecting RNA as detected in this POC assay system using purified RNase A as the representative RNase. Furthermore, ionic detergent alone was ineffective. Currently available methods can only achieve irreversible ribonuclease inactivation up to 200ng / ml, whereas the compositions and methods provided herein have shown utility at 10μg / ml to accommodate potentially larger amounts in patient samples. Thus, as shown here, simultaneous treatment with ionic detergent and reducing agent is necessary for successful detection under these conditions.

[0164] Example 9 Protection of RNA from nasal swab ribonucleases This example provides a demonstration that the methods and compositions provided herein protected RNA from nasal swab ribonucleases. In FIG. 10A, nasal swab eluate (4% of swab contents, which is the amount likely to be routinely encountered during patient testing) was heated at 58° C. for 2 minutes in a 50 ml solution containing 0.2% SDS (X) or 0.2% SDS + 10 mM DTT (triangles). After adjusting to 4% βCD to sequester SDS, influenza B RNA was added and incubation at 58° C. was continued. To monitor the ribonuclease activity recovered during the second incubation (after SDS sequestration), 5 ml (containing 2500 infectious units worth of RNA) was tested in a standard APA (with standard RNase protection measures built in). Briefly, lyophilized reagents and RBS DTT were pre-incubated at 58° C., and then the reverse transcription APA proceeded as in Example 4. No detection was observed without DTT (x), but the addition of DTT restored detection (triangles). This suggests that RNase activity was restored without DTT compared to the control (no nasal eluate, squares). The NTC contained nasal eluate but no RNA. Assay calibration (FIG. 10B) showed that the restored RNase activity was sufficient to eliminate >95% of RNA detectability during a 2 minute incubation at 58° C. Thus, as shown here, simultaneous treatment with ionic detergents and reducing agents is necessary for successful detection under these conditions. Consistent with the data above, the compositions and methods of the present disclosure address a potential risk to influenza RNA detectability by demonstrating SDS+DTT-dependent irreversible ribonuclease inactivation in the human nasal cavity (e.g., as may be encountered during customer use).

[0165] Example 10 RNaseA activity assay This example demonstrates the unexpected advantages of the methods and compositions provided herein over currently available nucleic acid detection methods and compositions. 2 ng RNase A was incubated in 50 ml solutions containing 10 mM DTT + 0.2% SDS (+), 0.2% SDS (triangles), 10 mM DTT (squares), or neither (solid line) at 58°C for 2 minutes, and then the RNase Alert assay was used to study the recovery of any RNase activity upon sequestering SDS after dissolving lyophilized APA reagent without enzyme but containing RNase Alert substrate and protectant (4% βCD), continuing incubation at 58°C and fluorescence data collection. In the presence of RNase activity, the RNase Alert indicator substrate emits detectable fluorescence. As shown in FIG. 11B, only the combination of DTT and SDS(+) prevented the subsequent recovery of RNase, consistent with its irreversible inactivation, whereas neither DTT (squares) nor SDS (triangles) alone was sufficient to prevent the virtually complete recovery of enzyme activity, nearly equal to that of the control (solid line). FIG. 11A illustrates the recovery of RNaseA activity after the same pretreatment without the addition of βCD, in this case where activity was monitored in 5 ml of solution by transferring to the RNase alert "wet" reaction format. In this format, SDS would continue to inactivate RNaseA during the RNase alert assay, so that relevant conclusions can only be drawn from samples without SDS, but the results showed that pretreatment with DTT alone (squares) did not affect the recovery of RNaseA activity without subsequent addition of βCD. Thus, as shown here, simultaneous treatment with ionic detergent and reducing agent is necessary for successful irreversible inactivation under the conditions described herein. Furthermore, as demonstrated in this example, it was indeed shown that ribonuclease reactivation was very rapid in the presence of cyclodextrin, leaving the target RNA unprotected. Without being bound to any particular theory, this may be related to the effect of cyclodextrin and other APA additives on the refolding process.This example also provides further confirmation using an independent commercial RNase assay that prior art and currently available methods (e.g., using reducing agents but not denaturing agents) are inadequate under these conditions.

[0166] Example 11 Influenza B Assay This example demonstrates the unexpected advantages of the methods and compositions provided herein. Figures 12A-12B show data on RNase A inactivation in an influenza B assay similar to that of Example 4, using DTT in ELB (elution / lysis buffer). This example (Figure 12A) demonstrates the recovery of inhibition levels of RNAse A (10 μg / ml) activity after heating for 2 minutes at 58°C in ELB containing 0.2% SDS without (NTC dotted line) or with (solid and dashed lines) various amounts of influenza B virus. After incubation, lyophilized APA reagent with β-CD (4% final) co-incubated for 2 minutes at 58°C was dissolved in the heated solution and APA was monitored by collecting FAM fluorescence every 15 seconds. In Figure 12B, the same conditions were used, except that RNAse A was pre-incubated with 10 mM DTT in 0.2% SDS. This allowed for comparable performance to the no RNAse A control (not shown). The inclusion of DTT allows for uninterrupted operation of this POC influenza detection system in simulated samples containing very high levels of purified RNase A (10 mg / ml). Furthermore, currently available methods can only achieve irreversible ribonuclease inactivation up to 200 ng / ml, whereas the compositions and methods provided herein demonstrate utility at 10 μg / ml to accommodate potentially larger amounts in patient samples.

[0167] Example 12 Rapid irreversibility of nasal ribonuclease inactivation. This example demonstrates the unexpected advantages of the methods and compositions provided herein over currently available nucleic acid detection methods and compositions. Figure 13 illustrates data on the rapid irreversibility of nasal ribonuclease inactivation. Five microliters of concentrated nasal swab eluate in TE, an amount equivalent to 2% of a typical nasal swab, was mixed with 45 μl of either lysis buffer containing 0.2% SDS (RBS, solid line), RBS containing 40 mM cysteine ​​(squares), or water (dotted line), all preheated to 60°C. Five microliters were immediately diluted with 45 μl of 4% β-cyclodextrin to sequester detergent. Recovered RNase activity was measured by RNase alert assay. As an assay control, an equal volume of nasal swab eluate was added directly without pretreatment (X). The results were consistent with the idea that RNase inactivation mediated by the combined action of SDS and cysteine ​​as a reducing agent is an immediate, irreversible operation (squares). In the presence of SDS alone (solid line), RNase inactivation was virtually completely reversible after SDS sequestration by cyclodextrin, i.e., indistinguishable from the control (dotted line or X). Unexpectedly, the effectively irreversible inactivation of ribonucleases thus observed during the practice of the present invention was virtually instantaneous (monitored by directly testing ribonuclease activity), unlike heat-based prior art methods. This example also shows that the inactivation characteristics described for RNase A also apply to ribonucleases naturally present in nasal exudates.

[0168] Example 13 Direct lysis for isothermal influenza B virus detection in the presence and absence of clinical matrices This example provides a demonstration of equivalent detection with and without nasal swab samples using the methods disclosed herein. Live influenza B virus was lysed in 50 μl of solution containing 0.2% SDS at 68° C. for 2 min and then used to lyse lyophilized APA reagent containing HP-βCD for isothermal reverse transcription and APA as above with reverse transcriptase; 9dN+poly rA+BSA without (FIG. 14A) and with (FIG. 14B) at the same temperature. A volume of nasal swab eluate equivalent to 4% of the volume of a typical nasal swab sample (containing ribonuclease activity) from a presumptive negative individual was included in the lysis. Fluorescein-labeled molecular beacons were used for detection of amplified influenza B. There was little or no difference with or without nasal "matrix" ribonuclease (FIGS. 14A-14B).

[0169] In at least some of the foregoing embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment unless such substitution is technically infeasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter defined by the appended claims. With respect to the use of substantially any plural and / or singular terminology herein, one of ordinary skill in the art can translate from plural to singular and / or from singular to plural as appropriate to the context and / or application. For clarity, various singular / plural permutations may be expressly set forth herein. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Any reference to "or" herein is intended to include "and / or" unless otherwise indicated.

[0170] Those skilled in the art will understand that the terms used in this specification, generally, and in the appended claims in particular (e.g., the body of the appended claims), are generally intended to be "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including, but not limited to," etc.). Moreover, those skilled in the art will understand that if a specific number is intended in the introduced claim language, such intent will be expressly recited in the claim, and that in the absence of such recitation, no such intent exists. For example, to aid in understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce the claim language. However, the use of such phrases should not be interpreted as meaning that the introduction of a claim recitation with the indefinite article "a" or "an" limits any particular claim containing such an introduced claim recitation to an embodiment containing only one such recitation, even if the same claim includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"). The same applies to the use of definite articles used to introduce claim recitations. In addition, even if a specific number is explicitly recited in an introduced claim recitation, a person skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the basic recitation of "two recitations" means at least two recitations, or two or more recitations, in the absence of other modifiers).Furthermore, when a convention similar to "at least one of A, B, and C, etc." is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). When a convention similar to "at least one of A, B, or C, etc." is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Moreover, those skilled in the art will appreciate that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of those terms, either such terms, or both terms.

[0171] Additionally, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also described in terms of every individual member or subgroup of members of the Markush group. As will be appreciated by those of skill in the art, for any and all purposes, including in terms of providing a written description, all ranges disclosed herein encompass any and all possible subranges and combinations of those subranges. Any recited range can be readily recognized as being capable of dividing the same range into at least two, three, four, five, ten, etc., as fully described. As a non-limiting example, each range discussed herein can be readily divided into a lower third, a middle third, and an upper third, etc. Also, as will be appreciated by those of skill in the art, all terms such as "up to," "at least," "greater than," and "less than" refer to ranges that are inclusive of the numerical values ​​recited and that can be subsequently divided into subranges as discussed above. Finally, as will be appreciated by those of skill in the art, ranges include each individual member. Thus, for example, a group containing 1-3 items refers to a group containing 1, 2, or 3 items. Similarly, a group containing 1-5 items refers to a group containing 1, 2, 3, 4, or 5 items, etc.

[0172] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those of ordinary skill in the art. The various aspects and embodiments disclosed herein are intended to be illustrative and not limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. 1. A method for detecting a target nucleic acid sequence in a sample, comprising: (A) contacting a sample containing biological entities with a lysis buffer to generate a processed sample, wherein the lysis buffer comprises one or more lysis agents capable of lysing the biological entities to release sample nucleic acids contained therein, and wherein the sample nucleic acids are suspected of containing a target nucleic acid sequence; (B) contacting a reagent composition with the processed sample to produce an amplification reaction mixture, where the reagent composition comprises one or more protectants and one or more amplification reagents; (C) amplifying a target nucleic acid sequence in the amplification reaction mixture, thereby producing a nucleic acid amplification product; and (D) detecting said nucleic acid amplification products, wherein said detecting step is performed less than about 20 minutes from the time said reagent composition contacts the sample to be processed. The method includes: (a) the lysis buffer and / or reagent composition comprises one or more reducing agents, optionally comprising one or more of 2-mercaptoethanol, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), dithioerythritol (DTE), reduced glutathione, cysteamine, tri-n-butylphosphine (TBP), dithioerythriol, tris(3-hydroxypropyl)phosphine (THPP), 2-mercaptoethylamine-HCl, dithiobutylamine (DTBA), cysteine, cysteine-thioglycolate, salts of sulfurous acid, thioglycolic acid, and hydroxyethyl disulfide (HED); and / or (b) the lysis buffer comprises one or more of magnesium sulfate, ammonium sulfate, EDTA, and EGTA; and / or (c) the pH of the lysis buffer is from about 1.0 to about 10.0, optionally the pH of the lysis buffer is about 2.2; and / or (d) the sample nucleic acid comprises a sample ribonucleic acid and / or a sample deoxyribonucleic acid, optionally the sample nucleic acid comprises cellular RNA, mRNA, microRNA, bacterial RNA, viral RNA, or a combination thereof; and / or (e) the one or more amplification reagents comprise a reverse transcriptase and / or an enzyme with hyperthermophilic polymerase activity, optionally the enzyme with hyperthermophilic polymerase activity has reverse transcriptase activity; and / or (f) the reagent composition comprises one or more of a reverse transcriptase, an enzyme having hyperthermophile polymerase activity, a first primer, a second primer, and a reverse transcription primer, wherein: (i) optionally, the first primer, the second primer, and / or the reverse transcription primer are about 8-17 bases in length, and optionally, the first primer, the second primer, and / or the reverse transcription primer comprise one or more of DNA bases, modified DNA bases, or a combination thereof; (ii) optionally, the nucleic acid amplification product is about 20-40 bases in length; and / or (iii) optionally, the spacer sequence comprises a portion of the target nucleic acid sequence, and optionally, the spacer sequence is 1-10 bases in length; and / or (g) the amplifying step is carried out under isothermal amplification conditions; and / or (h) the step of detecting the nucleic acid amplification products comprises the use of a real-time detection method; and / or (i) the reagent composition is lyophilized, heat dried, and / or comprises one or more additives, optionally the one or more additives comprising: Tween 20, Triton X-100, Tween 80, non-ionic detergent, or combinations thereof; amino acid; a sugar or sugar alcohol, optionally wherein the sugar or sugar alcohol comprises sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof; and / or a polymer, optionally wherein the polymer comprises polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptides, collagen peptides, or any combination thereof; and / or (j) the molar ratio of the one or more protection agents to the one or more amplification reagents is from about 10:1 to about 1:10, optionally, the molar ratio is about 2:1; The method of claim 1.

3. The method according to any one of claims 1 to 2, wherein the one or more protecting agents comprise a cyclodextrin compound of formula (I) or a salt, ester, solvate, or hydrate thereof: 【Chemistry 1】 During the ceremony, Each R is independently H, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or -C(O)OR B , -OC(O)R B , -C(O)R B or -C(O)NR A R B and Each R 1 are independently H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, halogen, hydroxy, amino, -CN, -CF 3 , -N 3 , -NO 2 , -OR B , -SR B , -SOR B , -SO 2 R B , -N(R B ) S (O 2 )-R B , -N(R B ) S (O 2 ) N.R. A R B , -NR A R B , -C(O)OR B , -OC(O)R B , -C(O)R B , -C(O)NR A R B , or -N(R B ) C(O)R B each of which may be substituted; Each R A is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; Each R B is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each m is independently 0, 1, 2, 3, 4, or 5.

4. (a) each R is independently H, optionally substituted alkyl, or -C(O)OR B , -OC(O)R B , -C(O)R B or -C(O)NR A R B and / or (b) each R is independently H, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, or octyl, optionally each is straight or branched chain, and further optionally, n is 1, 2, or 3; and / or (c) the cyclodextrin compound is 2-hydroxypropyl-α-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin (2HPβCD), hydroxypropyl-β-cyclodextrin (HPβCD), methyl-β-cyclodextrin (MβCD), 2-hydroxypropyl-γ-cyclodextrin, α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, or a salt, ester, solvate, or hydrate thereof; The method according to claim 3.

5. The one or more lytic reagents include from about 0.001% (w / v) to about 1.0 (w / v) of the treated sample, optionally about 0.2% (w / v) of the treated sample; and / or a detergent, optionally comprising one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant; The method according to any one of claims 1 to 4, comprising: (a) the sample nucleic acid comprises a nucleic acid comprising a target nucleic acid sequence, optionally the target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other, wherein: (i) optionally, the nucleic acid is double-stranded DNA; and / or (ii) optionally, the nucleic acid is the product of a reverse transcription reaction, optionally, the nucleic acid is the product of a reverse transcription reaction generated from a sample ribonucleic acid, and further optionally, step (C) comprises generating the nucleic acid by a reverse transcription reaction; and / or (b) amplifying the target nucleic acid sequence, The method comprises amplifying a target nucleic acid sequence comprising a first strand and a second strand complementary to each other under isothermal amplification conditions, wherein the amplifying step comprises amplifying a nucleic acid comprising the target nucleic acid sequence by subjecting the nucleic acid to the following i) and ii): i) a first primer and a second primer, wherein the first primer is capable of hybridizing to a sequence of a first strand of the target nucleic acid sequence and the second primer is capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence; and ii) an enzyme having hyperthermophile polymerase activity, thereby producing a nucleic acid amplification product, wherein the nucleic acid amplification product is (1) the sequence of the first primer and its reverse complement; (2) the sequence of the second primer and its reverse complement, and (3) a spacer sequence flanked by (1) a sequence of a first primer and its reverse complement, and (2) a sequence of a second primer and its reverse complement, wherein the spacer sequence is 1-10 bases in length. and / or (c) the amplifying step does not include using an enzyme other than an enzyme having hyperthermophile polymerase activity, and / or the amplifying step does not include thermal and / or enzymatic denaturation of the nucleic acid, and optionally the method does not include contacting the nucleic acid with a single-stranded DNA binding protein prior to or during step (C); The method according to any one of claims 1 to 5. (a) the sample nucleic acid comprises a sample ribonucleic acid, and the method comprises contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to produce cDNA; and / or (b) amplifying the target nucleic acid sequence, (C1) contacting a sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to generate cDNA; (C2) contacting the cDNA with an enzyme having hyperthermophile polymerase activity to produce double-stranded DNA (dsDNA), wherein the dsDNA comprises a target nucleic acid sequence, the target nucleic acid sequence comprising a first strand and a second strand that are complementary to one another; (C3) amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein the amplifying comprises: amplifying the dsDNA by: (i) a first primer and a second primer, wherein the first primer is capable of hybridizing to a sequence of a first strand of the target nucleic acid sequence and the second primer is capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence; and (ii) an enzyme having hyperthermophile polymerase activity, thereby producing a nucleic acid amplification product, wherein the nucleic acid amplification product is (1) the sequence of the first primer and its reverse complement; (2) the sequence of the second primer and its reverse complement, and (3) a spacer sequence flanked by (1) a sequence of a first primer and its reverse complement, and (2) a sequence of a second primer and its reverse complement, wherein the spacer sequence is 1-10 bases in length. contacting the Including, Optionally, the method does not involve the use of any enzyme other than the reverse transcriptase and / or the enzyme having hyperthermophile polymerase activity. The method according to any one of claims 1 to 6. (a) step (D) further comprises determining the amount of dsDNA and / or nucleic acid comprising the target nucleic acid sequence in the sample; and / or (b) the enzyme with hyperthermophile polymerase activity has an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:1, or a functional fragment thereof, optionally, the enzyme with hyperthermophile polymerase activity has an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:1, further optionally, the enzyme with hyperthermophile polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO:1, optionally, the enzyme with hyperthermophile polymerase activity has reduced or no exonuclease activity; and / or (c) the step of amplifying the target nucleic acid sequence is carried out at a constant temperature of about 55°C to about 75°C, optionally, the step of amplifying the target nucleic acid sequence is carried out at a constant temperature of about 65°C; and / or (d) contacting the nucleic acid amplification product with a signal-generating oligonucleotide capable of hybridizing to the amplification product, wherein the signal-generating oligonucleotide comprises a fluorophore, a quencher, or both; and / or (e) the step of detecting the nucleic acid amplification product comprises detecting a fluorescent signal, optionally the fluorescent signal is from a molecular beacon; and / or (f) the one or more lysis agents are capable of denaturing the one or more amplification reagents and the one or more protection agents are capable of preventing denaturation of the one or more amplification reagents by the one or more lysis agents; and / or (g) the one or more protecting agents are capable of sequestering the one or more lysis agents, thereby preventing the one or more lysis agents from denaturing the one or more amplification reagents; and / or (h) greater than about 90% of the one or more lytic agents are sequestered by the one or more protective agents; and / or (i) less than about 10% of the one or more amplification reagents is denatured by the one or more lysis agents; The method according to any one of claims 1 to 7.

9. The method according to any one of claims 6 to 8, wherein the sample ribonucleic acid is contacted with the reverse transcriptase and the enzyme having hyperthermophile polymerase activity simultaneously, optionally, the sample ribonucleic acid is contacted with the reverse transcriptase, the enzyme having hyperthermophile polymerase activity, and the first and second primers simultaneously, and further optionally, the sample ribonucleic acid is contacted with the reverse transcriptase, the enzyme having hyperthermophile polymerase activity, the first primer, the second primer, and the reverse transcription primer simultaneously.

10. The sample comprises a plurality of nucleases, wherein the nucleases comprise ribonucleases and / or deoxyribonucleases, and the one or more lysing agents are capable of denaturing the ribonuclease to produce a modified ribonuclease, the one or more reducing agents are capable of reducing the ribonuclease to produce a reduced ribonuclease, the one or more reducing agents and the one or more lysing agents are capable of producing a reduced modified ribonuclease, and / or the one or more lysing agents are capable of denaturing deoxyribonuclease to generate a denatured deoxyribonuclease, the one or more reducing agents are capable of reducing deoxyribonuclease to generate a reduced deoxyribonuclease, and the one or more reducing agents and the one or more lysing agents are capable of generating a reduced denatured deoxyribonuclease; The method according to any one of claims 2 to 9.

11. (a) the denatured ribonuclease and / or reduced ribonuclease are capable of renaturing to produce renatured ribonuclease over time and / or upon reduced physical interaction with said one or more lysing agents, and optionally, sequestering said one or more lysing agents reduces physical interaction between said one or more lysing agents and the renatured ribonuclease and / or reduced ribonuclease; and / or (b) the denatured DNase and / or reduced DNase are capable of renaturing to produce renatured DNase over time and / or upon reduced physical interaction with said one or more lysing agents, and optionally sequestering said one or more lysing agents reduces physical interaction between said one or more lysing agents and the denatured DNase and / or reduced denatured DNase; and / or (c) the denatured deoxyribonuclease, reduced deoxyribonuclease, reduced modified deoxyribonuclease, modified ribonuclease, reduced ribonuclease, and / or reduced modified ribonuclease are enzymatically inactive, and the renatured ribonuclease and / or renatured deoxyribonuclease are enzymatically active; and / or (d) the reduced denatured ribonuclease renatures at least about 1.1 times slower than the denatured ribonuclease; and / or (e) the reduced denatured DNase renatures at least about 1.1 times slower than the denatured DNase; The method of claim 10. (a) about 1.1 times more sample ribonucleic acid is used by reverse transcriptase as a template to generate cDNA, as compared to a comparable method in which the lysis buffer does not contain one or more reducing agents; and / or (b) about 1.1 times more sample deoxyribonucleic acid is used by the enzyme having hyperthermophile polymerase activity as a template to generate nucleic acid amplification products, as compared to a comparable method in which the lysis buffer does not contain one or more reducing agents; and / or (c) the amplification reaction mixture comprises at least 1.1-fold less nuclease activity 10 minutes after step (B) and / or step (C) compared to a comparable method in which the lysis buffer does not contain one or more reducing agents, and optionally, the nuclease activity comprises deoxyribonuclease activity and / or ribonuclease activity; and / or (d) the biological entity is (i) one or more of a prokaryotic cell, a eukaryotic cell, a viral particle, an exosome, a protoplast, and a microvesicle; and / or (ii) a virus, a bacterium, a fungus, a protozoa, a part thereof, or any combination thereof where Optionally, the virus is SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex, herpes virus 6, herpes virus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella zoster virus, JC virus, parvovirus B19, influenza A, influenza B, influenza C, rotavirus, human adenovirus, rubella virus, human enterovirus, genital human papillomavirus (HPV), or hantavirus; Optionally, the bacterium is selected from the group consisting of Mycobacteria tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, and the like. pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumoniae, pneumonia, S. agalactiae, and Listeria monocytogenes; Optionally, the fungus comprises one or more of Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, and Trichophyton rubrum; and / or Optionally, the protozoa comprises one or more of Trypanosoma cruzi, Leishmania sp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., and Eimeria sp.; and / or (e) the target nucleic acid sequence is a viral, bacterial, fungal, or protozoan nucleic acid sequence, and optionally the sample nucleic acid is derived from a viral, bacterial, fungal, or protozoan, wherein: Optionally, the virus is SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex, herpes virus 6, herpes virus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella zoster virus, JC virus, parvovirus B19, influenza A, influenza B, influenza C, rotavirus, human adenovirus, rubella virus, human enterovirus, genital human papillomavirus (HPV), or hantavirus; Optionally, the bacterium is selected from the group consisting of Mycobacteria tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, and the like. pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumoniae, pneumonia, S. agalactiae, and Listeria monocytogenes; Optionally, the fungus comprises one or more of Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, and Trichophyton rubrum; and / or Optionally, the protozoa comprises one or more of Trypanosoma cruzi, Leishmania sp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., and Eimeria sp.; and / or (f) the amplifying step comprises multiplex amplification of two or more target nucleic acid sequences and the detecting step comprises multiplex detection of two or more nucleic acid amplification products derived from the two or more target nucleic acid sequences, optionally wherein the two or more target nucleic acid sequences are specific to two or more different organisms, optionally wherein the two or more different organisms comprise one or more of Chlamydia trachomatis, Neisseria gonorrhoeae, SARS-CoV-2, influenza A, influenza B, and / or influenza C; and / or (g) the amplifying step does not include one or more of the following: loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal-mediated RNA amplification technology (SMART), self-sustained sequence replication (3SR), genomic exponential amplification reaction (GEAR), and isothermal multiple displacement amplification (IMDA), optionally wherein the amplifying step does not include loop-mediated isothermal amplification (LAMP); and / or (h) the method does not include one or more of the following: (i) diluting the process sample; (ii) diluting the amplification reaction mixture; (iii) heat denaturing the process sample; (iv) sonicating the process sample; (v) sonicating the amplification reaction mixture; (vi) adding an RNase inhibitor and / or a DNase inhibitor to the process sample; (vii) adding an RNase inhibitor and / or a DNase inhibitor to the amplification reaction mixture; (viii) purifying the sample; (ix) purifying the sample nucleic acid; (x) purifying the nucleic acid amplification product; (xi) removing the one or more lysing agents from the process sample or the amplification reaction mixture; (xii) heat and / or enzymatically denaturing the sample nucleic acid before and / or during amplification; and (xiii) adding RNase H to the process sample or the amplification reaction mixture. The method according to any one of claims 1 to 11.

13. 1. A kit for detecting a target nucleic acid sequence in a sample, comprising: (a) a lysis buffer comprising one or more lysis agents capable of lysing biological entities to release sample nucleic acids contained therein, said sample nucleic acids being suspected of containing a target nucleic acid sequence; and (b) a reagent composition comprising one or more protection agents and one or more amplification reagents comprising one or more components for amplifying a target nucleic acid sequence under isothermal amplification conditions, wherein the one or more components for amplifying are selected from the following (i) and (ii): (i) a first primer and a second primer, wherein the first primer is capable of hybridizing to a sequence of a first strand of a target nucleic acid sequence and the second primer is capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence; and (ii) an enzyme having a hyperthermophilic polymerase activity capable of producing a nucleic acid amplification product; Includes.

14. (a) the lysis buffer and / or the reagent composition comprises one or more reducing agents, optionally comprising one or more of 2-mercaptoethanol, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), dithioerythritol (DTE), reduced glutathione, cysteamine, tri-n-butylphosphine (TBP), dithioerythriol, tris(3-hydroxypropyl)phosphine (THPP), 2-mercaptoethylamine-HCl, dithiobutylamine (DTBA), cysteine, cysteine-thioglycolate, salts of sulfurous acid, thioglycolic acid, and hydroxyethyl disulfide (HED); and / or (b) the kit further comprises at least one component providing real-time detection activity of nucleic acid amplification products, optionally wherein the real-time detection activity is provided by a molecular beacon; and / or (c) the reagent composition comprises a reverse transcriptase and / or a reverse transcription primer; and / or (d) the enzyme with hyperthermophile polymerase activity has an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:1 or a functional fragment thereof, optionally the enzyme with hyperthermophile polymerase activity has an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:1, and further optionally the enzyme with hyperthermophile polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO:1; and / or (e) the nucleic acid amplification product is about 20 to 40 bases in length, and the nucleic acid amplification product is (1) the sequence of the first primer and its reverse complement; (2) the sequence of the second primer and its reverse complement, and (3) a spacer sequence flanked by (1) the sequence of a first primer and its reverse complement, and (2) the sequence of a second primer and its reverse complement, wherein the spacer sequence is 1-10 bases in length. and / or (f) the biological entity comprises one or more of a prokaryotic cell, a eukaryotic cell, a virus particle, an exosome, a protoplast, and a microvesicle, optionally the biological entity comprises a virus, a bacterium, a fungus, a protozoa, a part thereof, or any combination thereof, wherein Optionally, the virus is SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex, herpes virus 6, herpes virus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella zoster virus, JC virus, parvovirus B19, influenza A, influenza B, influenza C, rotavirus, human adenovirus, rubella virus, human enterovirus, genital human papillomavirus (HPV), and hantavirus; Optionally, the bacterium is selected from the group consisting of Mycobacteria tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, and the like. pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumoniae, pneumonia, S. agalactiae, and Listeria monocytogenes; Optionally, the fungus comprises one or more of Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, and Trichophyton rubrum; and / or Optionally, the protozoa comprises one or more of Trypanosoma cruzi, Leishmania sp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., and Eimeria sp.; and / or (g) the target nucleic acid sequence is a viral, bacterial, fungal, or protozoan nucleic acid sequence, and optionally the sample nucleic acid is derived from a viral, bacterial, fungal, or protozoan nucleic acid sequence, Optionally, the virus is SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex, herpes virus 6, herpes virus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella zoster virus, JC virus, parvovirus B19, influenza A, influenza B, influenza C, rotavirus, human adenovirus, rubella virus, human enterovirus, genital human papillomavirus (HPV), and hantavirus; Optionally, the bacterium is selected from the group consisting of Mycobacteria tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, and the like. pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumoniae, pneumonia, S. agalactiae, and Listeria monocytogenes; Optionally, the fungus comprises one or more of Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, and Trichophyton rubrum; and / or Optionally, the protozoa comprises one or more of Trypanosoma cruzi, Leishmania sp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., and Eimeria sp.; and / or (h) the reagent composition is lyophilized and / or heat dried and comprises one or more additives, wherein the one or more additives are (i) Tween 20, Triton X-100, Tween 80, a non-ionic detergent, or a combination thereof; (ii) an amino acid; (iii) a sugar or sugar alcohol, optionally wherein the sugar or sugar alcohol comprises sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof; and / or (iv) a polymer, optionally wherein the polymer comprises polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptides, collagen peptides, or any combination thereof; and / or (j) the one or more lysis agents comprise a detergent, and the detergent comprises one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant; and / or (k) the one or more protecting agents are capable of sequestering the one or more lysing agents, thereby preventing denaturation of the one or more amplification reagents by the one or more lysing agents; The kit of claim 13.

15. The kit according to any one of claims 13 to 14, wherein the one or more protecting agents comprise a cyclodextrin compound of formula (I) or a salt, ester, solvate or hydrate thereof: 【Chemistry 2】 During the ceremony, Each R is independently H, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or -C(O)OR B , -OC(O)R B , -C(O)R B or -C(O)NR A R B and Each R 1 are independently H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, halogen, hydroxy, amino, -CN, -CF 3 , -N 3 , -NO 2 , -OR B , -SR B , -SOR B , -SO 2 R B , -N(R B ) S (O 2 )-R B , -N(R B ) S (O 2 ) N.R. A R B , -NR A R B , -C(O)OR B , -OC(O)R B , -C(O)R B , -C(O)NR A R B , or -N(R B ) C(O)R B each of which may be substituted; Each R A is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; Each R B is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each m is independently 0, 1, 2, 3, 4, or 5, where: (a) optionally, each R is independently H, optionally substituted alkyl, -C(O)OR B , -OC(O)R B , -C(O)R B , or -C(O)NR A R B , and optionally, n is 1, 2, or 3; and / or (b) optionally, each R may independently be H, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, or octyl, optionally each being straight or branched chain, and further optionally, n is 1, 2, or 3; and / or (c) Optionally, the cyclodextrin compound is 2-hydroxypropyl-α-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin (2HPβCD), hydroxypropyl-β-cyclodextrin (HPβCD), methyl-β-cyclodextrin (MβCD), 2-hydroxypropyl-γ-cyclodextrin, α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, or a salt, ester, solvate, or hydrate thereof.

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