Condensed loop-mediated isothermal amplification

Abstract

The present disclosure provides methods and compositions for nucleotide amplification.

Description

CONDENSED LOOP-MEDIATED ISOTHERMAL AMPLIFICATIONCross-Reference to Related Applications

[0001] The present application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 470,298, filed on June 1, 2023, and U.S. Provisional Patent Application No. 63 / 511,491, filed on June 30, 2023, the entirety of which are incorporated herein by reference.Background

[0002] Template-dependent nucleic acid synthesis methods using the Polymerase Chain Reaction (PCR) method have served as a major driving force for research in bioscience fields in recent years. The PCR method has made it possible to exponentially amplify nucleic acids composed of a nucleotide sequence complementary to a template by using a small amount of double-stranded nucleic acid as template. PCR is currently widely used as a tool for gene cloning and detection. Tn the PCR method, one set of primers comprising nucleotide sequences complementary to both ends of a target nucleotide sequence is used. One of the primers is designed to anneal to the elongation product generated by the other primer. In this manner, a synthesis reaction progresses in which annealing to a mutual elongation product and complementary strand synthesis are repeated, enabling exponential amplification to be achieved. In the PCR method, complex temperature control is essential. A special reaction apparatus must be used to accommodate this complex temperature control. A number of isothermal nucleic acid detection methods have been developed that do not require temperature cycling, such as Loop-mediated isothermal amplification (LAMP).

[0003] Templated synthesis, such as target nucleic acid synthesis and / or amplification is an important step in many applications (e.g., detection applications). Improvement of nucleic acid synthesis and / or amplification technologies, for example that may lead to increased speed, efficiency, and / or extent or quality of product generation are useful.Summary

[0004] The present disclosure provides insights and technologies that can achieve improvement of nucleotide amplicon production (e.g., nucleotide synthesis and / or nucleotide amplification).

[0005] In some embodiments, the present disclosure provides particular technologies (e.g., primers, compositions, kits, amplicons, and methods) for nucleotide amplicon production. Among other things, the present disclosure identifies the source of a problem with certain amplification methods (e.g., conventional LAMP) requiring a high number of target specific primers and consequently a corresponding region of the target nucleotide sequences to which the primers can bind. Conventional LAMP method employs six linear primers containing eight distinct domains (Fl, F2, F3, FLOOP, B l, B2, B3, and BLOOP), each of which must match the target sequence. This is necessary because the LAMP dumbbell amplicon is constructed via direct extension of the F3 and FIP (forward-inner) primers on the template, followed by extension of the B3 and BIP (backward- inner) primers on the template reverse-complement. As a result, LAMP primer design is highly constrained, requiring eight ~20-mer domains to be chosen within a ~300-base region of the template sequence, while minimizing cross-reactivity between the domains and avoiding undesirable secondary structures. This problem becomes even more constrained when detecting a highly variable target region and may even require redundant LAMP primers and / or multiplexed primer sets, compounding the complexity problem. Most highly mutated regions (e.g., highly mutated target nucleotide sequences) would not be efficiently amplified using conventional LAMP unless a highly multiplexed set of LAMP primers (e.g., ~50-100 primers for HIV) was used taking the target nucleotide sequence mutations into consideration.

[0006] Attempts to circumvent the template-specificity and complexity of conventional LAMP have included ligation-initiated LAMP process. Ligation step carries some inherent disadvantages. First, ligation and LAMP amplification cannot be easily combined into a one-pot reaction format due to temperature incompatibilities, which is inconvenient for e.g., diagnostic assays. Second, ligation requires additional time to perform, and generally increases the time-to-result by 20-30 minutes or more. Third, ligation is often sequence-dependent, constraining the set of template regions which can be targeted. Finally, a ligase enzyme is an additional point of failure in the reaction, and would require additionaloptimization of the buffer composition, lyophilization conditions, and other factors before the assay could be deployed into a point-of-need test.

[0007] The present disclosure provides solutions to these identified problems, including particularly useful technologies (e.g., primers, compositions, kits, amplicons, and methods) comprising at least one hybrid primer without ligation-initiated LAMP strategies.

[0008] Condensed loop-mediated isothermal amplification (cLAMP) is an improved method of isothermal nucleic acid amplification. The primary limitation, which the present disclosure addresses, is the constrained primer design process of conventional LAMP. In some embodiments, compositions and methods of the present disclosure use at least one hybrid primer. In some embodiments, a hybrid primer comprises a hairpin- loop structure and represents a large fragment of the intermediate hairpin- loop amplicon and the dumbbellshaped amplicon produced during cLAMP. Using one or more hybrid primers reduces the number of target-specific domains required for nucleotide amplicon production compared to other conventional nucleotide amplicon production methods (e.g., conventional LAMP). Among other things, the present disclosure surprisingly demonstrates that a relatively small target nucleotide sequence is required for amplifying a target nucleotide sequence. In cLAMP, as described in the present disclosure, fewer target-specific domains are required than in standard LAMP. Using a hybrid primer as initiation primer makes the initiation process more streamlined than conventional LAMP, since, in some embodiments, cLAMP begins with half of the dumbbell already formed. Additionally, in some embodiments cLAMP hairpin primers each require just one target- specific domain. In some embodiments, the remainder of the hairpin primer is composed of purely target-agnostic nucleotide sequences. This substantially reduces the number of target- specific domains required for amplification. The reduction in the number of target- specific domains is important for heavily mutated targets, where locating a long enough conserved region may be difficult or impossible. cLAMP uses fewer target- specific domains and hence a smaller conserved target nucleotide region is needed. If a highly variable target nucleotide sequence is used, then the low target-specific domain requirement means that cLAMP reactions will require fewer multiplexed or degenerate primers than LAMP.

[0009] cLAMP primer design is less constrained than conventional LAMP primer design. For efficient nucleotide synthesis and amplification, conventional LAMP requires eight domains to be contained within a -300 base region of the target nucleotide sequence.cLAMP, as described in the present disclosure, greatly reduces the number of adjacent domains which must be chosen on the target nucleotide sequence, increasing primer design flexibility.

[0010] Non-target-specific primer domains allow better control over the hairpin-loop amplicon and dumbbell-shaped amplicon as compared to LAMP, allowing more flexibility for amplicon detection (e.g., Cas-mediated readout). In some embodiments, this provides additional options for guide polynucleotides when combining cLAMP with Cas-mediated detection.

[0011] Additionally, the present disclosure provides solutions that do not include ligation-initiated LAMP strategies. In contrast to ligation-initiated LAMP, cLAMP initiates its amplicon formation purely through polymerase-mediated mechanisms. Thus, in some embodiments the cLAMP reaction as described herein can be performed in a one-pot assay format, which is more convenient for detection (e.g., diagnostic assays). Furthermore, cLAMP avoids the time-intensive ligation step (-20-30 minutes or more) and therefore reduces time-to-result. Finally, the compositions and methods of the present disclosure are an improvement relative to those known in the art since no additional reagent optimization is required for ligase lyophilization, buffer and temperature compatibility, etc.

[0012] In some embodiments, the present disclosure provides particular technologies (e.g., primers, compositions, kits, amplicons, and methods) for target nucleotide detection.

[0013] The present disclosure provides a composition comprising (i) a first hybrid primer whose nucleotide sequence comprises: a) a 3’ T1 domain complementary to a Tic domain of a target nucleotide sequence; b) a first 5’ hairpin nucleotide sequence comprising four domains from 3’ to the 5’ : 1) a B 1 domain; 2) a LBc domain; 3) a B2 domain; and 4) a Bic domain complementary to the B l domain, wherein at least one domain is not complementary to the target nucleotide sequence; and (ii) a backward inner primer (BIP) whose nucleotide sequence comprises a 3’ B2 domain and a 5’ Bic domain complementary to the B 1 domain.

[0014] In some embodiments, at least two domains from the first 5’ hairpin nucleotide sequence are not complementary to the target nucleotide sequence. In some embodiments, at least three domains from the first 5’ hairpin nucleotide sequence are not complementary to the target nucleotide sequence. In some embodiments, a first hairpinnucleotide acid sequence is not complementary to the target nucleotide sequence. In some embodiments, a BIP is not complementary to the target nucleotide sequence.

[0015] In some embodiments, a composition provided herein further comprises: a LB primer whose nucleic acid sequence comprises a LB domain that is complementary to the LBc domain. In some embodiments, a LB primer is not complementary to the target nucleotide sequence.

[0016] In some embodiments, a composition provided herein further comprises: a second hybrid primer whose nucleotide sequence comprises: a) a 3’ T2 domain complementary to a T2c domain of the target nucleotide sequence, wherein Tic and T2c are non-overlapping domains located on opposite or complementary strands of the target nucleotide sequence; b) a second 5’ hairpin nucleotide sequence comprising four domains from 3’ to the 5’ : 1) a Fl domain; 2) a LFc domain; 3) a F2 domain; and 4) a Flc domain complementary to the Fl domain, wherein at least one domain is not complementary to the target nucleotide sequence. In some embodiments, at least two domains from the second 5’ hairpin nucleotide sequence are not complementary to the target. In some embodiments, at least three domains from the second 5 ’ hairpin nucleotide sequence are not complementary to the target. In some embodiments, a second hairpin nucleotide acid sequence is not complementary to the target nucleotide sequence.

[0017] In some embodiments, a composition provided here further comprises a forward inner primer (FIP) whose nucleotide sequence comprises a 3 ’ F2 domain and a 5 ’ Flc domain complementary to the Fl domain. In some embodiments, a FIP is not complementary to the target nucleotide sequence. In some embodiments, a FIP is a FIPp.

[0018] In some embodiments, a composition provided herein further comprises an LFp primer whose nucleotide sequence is or comprises a LFp domain that is complementary to the LFc domain. In some embodiments, a LF primer is not complementary to the target nucleotide sequence.

[0019] In some embodiments, a composition provided herein further comprises a FlPt primer whose nucleotide sequence comprises a) a 3 ’ F2t domain complementary to a F2ct domain of the target nucleotide sequence, wherein F2ct and Tic are non-overlapping domains located on opposite or complementary strands of the target nucleotide sequence; and b) a 5’ Flct domain complementary to a Fit domain of the target nucleotide sequence. In some embodiments, a Flct domain is located 5’ to the F2ct domain on the same strand.

[0020] In some embodiments, a composition provided herein further comprises a F3 primer whose nucleotide sequence is complementary to a F3c domain of the target nucleotide sequence. In some embodiments, a F3c domain is located 3’ to the F2c domain on the same target strand.

[0021] In some embodiments, a composition provided herein further comprises an LFt primer whose nucleotide sequence is complementary to an LFct domain of the target nucleotide sequence. In some embodiments, a LFct domain is located between Fit and F2t on the same target strand.

[0022] In some embodiments, a composition provided herein further comprises a B3 primer whose nucleotide sequence is complementary to a B3c domain of the target nucleotide sequence. In some embodiments, a B3c domain is located 3’ to the Tic domain on the same strand.

[0023] In some embodiments, a primer nucleotide sequence is a ribonucleotide sequences. In some embodiments, a primer nucleotide sequence is a deoxyribonucleotide sequence. In some embodiments, a primer nucleotide sequence is a mixture of deoxyribonucleotides and polyribonucleotides. In some embodiments, a primer nucleotide sequence includes peptide nucleic acids or locked nucleic acids. In some embodiments, a primer nucleotide sequence includes peptide nucleic acids or locked nucleic acids. In some embodiments, a primer nucleotide sequence includes 2’ Fluoro modifications, 2’-O-methyl modifications, or a combination thereof. In some embodiments, a primer ribonucleotide sequence includes 2- Aminopurine. In some embodiments, a primer deoxyribonucleotide sequence includes a phosphodiester deoxyribonucleotide. In some embodiments, a primer deoxyribonucleotide sequence includes a phosphorothioated deoxyribonucleotide. In some embodiments, one or more of the primer domains are about 9 to about 30 nucleotides. In some embodiments, a primer domain complementary to the target nucleotide sequence is at least 80% complementary to its hybridization site in the target nucleotide sequence or complement thereof. In some embodiments, a domain complementary to the target nucleotide sequence comprises 1, 2, 3, or 4 mismatches. In some embodiments, one or more of the primers comprises a fluorophore. In some embodiments, one or more of the primers comprises a quencher.

[0024] The present disclosure provides a kit comprising a composition as provided herein and a DNA polymerase. In some embodiments, a DNA polymerase has strand displacement activity. In some embodiments, a DNA polymerase is a Bsu Polymerase, BsmPolymerase, Bst Polymerase, or a combination thereof. In some embodiments, a kit further comprises amplification reagents. In some embodiments, a kit further comprises a reverse transcriptase. In some embodiments, a kit further comprises a guide RNA. In some embodiments, a kit further comprises a Cas enzyme. In some embodiments, a Cas enzyme is a Casl2 or Casl3 enzyme. In some embodiments, a Cas enzyme has collateral cleavage activity. In some embodiments, a Cas enzyme is a thermostable Cas enzyme. In some embodiments, a Cas enzyme is thermostable within a range of about 20°C to about 65°C. In some embodiments, a kit further comprises a detectably labeled nucleic acid probe. In some embodiments, a kit further comprises a double stranded DNA binding dye. In some embodiments, a double stranded DNA binding dye is a SYBR Green I and II, DAPI, PicoGreen, Ethidium Bromide, Propidium Iodide, EvaGreen, or a combination. In some embodiments, a kit further comprises a single stranded DNA binding dye. In some embodiments, a kit further comprises Thermostable Inorganic Pyrophosphatase (TIPP).

[0025] The present disclosure provides a method for producing a hairpin-loop amplicon comprising a target nucleotide sequence or complement thereof, comprising the steps of: (A) contacting a target nucleotide sequence with a DNA polymerase or a reverse transcriptase, amplification reagents and a first hybrid primer whose nucleotide sequence comprises: a) a 3’ T1 domain complementary to a Tic domain of the target nucleotide sequence; b) a first 5’ hairpin nucleotide acid sequence comprising four domains from 3’ to the 5’ : 1) a Bl domain; 2) a LBc domain; 3) a B2 domain; and 4) a Bic domain complementary to the Bl domain; wherein at least one domain is not complementary to the target nucleotide; and (B) incubating the target nucleotide sequence with the DNA polymerase or the reverse transcriptase, amplification reagents and the first hybrid primer so that the hairpin- loop product is generated. In some embodiments, the step of incubating is performed in the presence of TIPP. In some embodiments, a target nucleotide sequence is in a sample. In some embodiments, a sample is a crude sample. In some embodiments, a sample is a biological sample or environmental sample. In some embodiments, a biological sample is obtained from a subject.

[0026] In some embodiments, a method provided herein further comprises a step of: isolating the target nucleotide sequence. In some embodiments, a biological sample is saliva, blood, plasma, buffy coat, serum, teeth, urine, nasal fluid, nasopharyngeal swab, oropharyngeal swab, nasal aspirate, sputum, bronchoalveolar lavage, buccal swab, vaginal swab, rectal swab, wound swab, skin swab, bone, muscle, tissue, CSF, semen, fecalmatter, hair follicle, skin sample, or a combination hereof. In some embodiments, a target nucleotide sequence is a viral, a bacterial, a fungal, a protozoan, or a parasitic sequence.

[0027] The present disclosure provides a method for producing a dumbbell- shaped amplicon, comprising the steps of: (A) contacting a target nucleotide sequence with a DNA polymerase having strand displacement activity, amplification reagents and a plurality of primers comprising: i) a first hybrid primer whose nucleotide sequence comprises: a) a 3’ T1 domain complementary to a Tic domain of the target nucleotide sequence; b) a 5’ hairpin nucleotide acid sequence comprising four domains from 3’ to the 5’: 1) a B l domain; 2) a LBc domain; 3) a B2 domain; and 4) a Bic domain complementary to the Bl domain; wherein at least one domain is not complementary to the target nucleotide; and ii) a second primer selected from a second hybrid primer whose nucleotide sequence comprises: a) a 3’ T2 domain complementary to a T2c domain of the target nucleotide sequence, wherein Tic and T2c are non-overlapping domains located on opposite or complementary strands of the target nucleotide sequence; b) a second 5’ hairpin nucleotide acid sequence comprising four domains from 3’ to the 5’: 1) a Fl domain; 2) a LFc domain; 3) a F2 domain; and 4) a Flc domain complementary to the Fl domain; wherein at least one domain is not complementary to the target nucleotide sequence; or a FlPt primer whose nucleotide sequence comprises: a) a 3’ F2t domain complementary to a F2ct domain of the target nucleotide sequence, wherein F2ct and Tic are non-overlapping domains located on opposite or complementary strands of the target nucleotide sequence; and b) a 5’ Flct domain complementary to a Fit domain of the target nucleotide sequence, (B) incubating the target nucleotide sequence with the DNA polymerase, amplification reagents and the primers so that the dumbbell- shaped target nucleotide structure is generated.

[0028] In some embodiments, a target nucleotide sequence is also contacted with a reverse transcriptase. In some embodiments, a step of incubating is performed in the presence of TIPP. In some embodiments, a target nucleotide sequence is in a sample. In some embodiments, a sample is a crude sample. In some embodiments, a sample is a biological sample or environmental sample. In some embodiments, a biological sample is obtained from a subject.

[0029] In some embodiments, a method provided herein further comprises a step of: isolating the target nucleotide sequence. In some embodiments, a biological sample is saliva, blood, plasma, buffy coat, serum, teeth, urine, nasal fluid, nasopharyngeal swab, oropharyngeal swab, nasal aspirate, sputum, bronchoalveolar lavage, buccal swab,vaginal swab, rectal swab, wound swab, skin swab, bone, muscle, tissue, CSF, semen, fecal matter, hair follicle, skin sample, or a combination hereof. In some embodiments, a target nucleotide sequence is a viral, a bacterial, a fungal, a protozoan, or a parasitic sequence. In some embodiments, a plurality of primers further comprises a F3 primer whose nucleotide sequence is complementary to an F3c domain of the target nucleotide sequence.

[0030] The present disclosure provides a method for synthesizing a deoxyribonucleotide sequence comprising the steps of: (A) contacting a target ribonucleotide sequence with a reverse transcriptase, amplification reagents, and a composition as provided herein; and (B) incubating a target ribonucleotide with the reverse transcriptase, amplification reagents and the primers so a deoxyribonucleotide sequence comprising the target nucleotide sequence or a complement thereof is generated.

[0031] The present disclosure provides a method for amplifying a target nucleotide sequence comprising the steps of: (A) contacting a target nucleotide sequence with a DNA polymerase having strand displacement activity, amplification reagents, and the composition as provided herein; and (B) incubating the target nucleotide with the DNA polymerase, amplification reagents and the primers so an amplified nucleotide sequence comprising the target nucleotide sequence is generated. In some embodiments, a method provided herein further comprises the steps of: (a) contacting a target ribonucleotide sequence with a reverse transcriptase, amplification reagents, and the composition provided herein; (b) incubating the target ribonucleotide with the reverse transcriptase, amplification reagents and the primers so an deoxyribonucleotide sequence comprising the target nucleotide sequence is generated; before performing step (A) and (B). In some embodiments, a step of incubating is performed in the presence of TIPP.

[0032] In some embodiments, a method for amplifying a target nucleotide sequence is conducted in a single vessel. In some embodiments, amplification is an isothermal amplification reaction. In some embodiments, amplification is performed at ambient temperature. In some embodiments, a target nucleotide sequence is in a sample. In some embodiments, a sample is a crude sample. In some embodiments, a sample is a biological sample or environmental sample.

[0033] In some embodiments, a method provided herein further comprises obtaining a sample from a subject. In some embodiments, a method further comprises a step of: isolating the target nucleotide sequence. In some embodiments, a sample is saliva, blood, plasma, buffy coat, serum, teeth, urine, nasal fluid, nasopharyngeal swab, oropharyngealswab, nasal aspirate, sputum, bronchoalveolar lavage, buccal swab, vaginal swab, rectal swab, wound swab, skin swab, bone, muscle, tissue, CSF, semen, fecal matter, hair follicle, skin sample, or a combination hereof.

[0034] The present disclosure provides a method for detecting a target nucleotide sequence comprising the steps of: (a) contacting the target nucleotide sequence with a DNA polymerase having strand displacement activity, amplification reagents, and the composition as provided herein or kit as provided herein; (b) incubating the target nucleotide, the compositions and amplification reagents so an amplified nucleic acid is generated; and (c) detecting the amplified nucleic acid. In some embodiments, a step of incubating is performed in the presence of TIPP. In some embodiments, a nucleotide sequence is in a sample. In some embodiments, a sample is a crude sample. In some embodiments, a sample is a biological sample or environmental sample. In some embodiments, biological sample is obtained from a subject. In some embodiments, a method further comprises a step of: isolating the target nucleotide sequence. In some embodiments, a sample is saliva, blood, plasma, buffy coat, serum, teeth, urine, nasal fluid, nasopharyngeal swab, oropharyngeal swab, nasal aspirate, sputum, bronchoalveolar lavage, buccal swab, vaginal swab, rectal swab, wound swab, skin swab, bone, muscle, tissue, CSF, semen, fecal matter, hair follicle, skin sample, or a combination hereof. In some embodiments, a step of detecting the amplified nucleotide is performed by incubating the amplified nucleotide with a double DNA stranded binding dye. In some embodiments, a double stranded DNA binding dye binds to the amplified nucleotide it transits from a first undetectable state to a second detectable state. In some embodiments, a double stranded DNA binding dye emits fluorescence in its second detectable state. In some embodiments, an amplified nucleotide is incubated with a guide polynucleotide capable of binding the target nucleotide sequence, a detectably labeled nucleic acid probe, and a Cas enzyme. In some embodiments, a Cas enzyme is a Casl3 enzyme. In some embodiments, a Cas enzyme is a Casl2 enzyme. In some embodiments, a Cas enzyme is a thermostable Cas enzyme. In some embodiments, a detectably labeled nucleic acid probe comprises a fluorescent group end and a quenching group. In some embodiments, a detectably labeled nucleic acid probe comprises a fluorescent group at the 5' end and a quenching group at the 3’ end. In some embodiments, a step of detecting is performed by detecting a change in florescence as an indication of amplification of the target nucleotide sequence. In some embodiments, a change in thefluorescence is an increase in the intensity of fluorescence emission of the detectably labeled nucleic acid probe.

[0035] The present disclosure provides a hairpin-loop amplicon comprising a nucleotide sequence comprising: a) a 3’ target nucleotide sequence; b) a 5’ hairpin nucleotide sequence comprising four domains from the 3 ’ to the 5 ’ : 1) a B 1 domain; 2) a LBc domain; 3) a B2 domain; and 4) a Bic domain complementary to the Bl domain; wherein at least one domain is not complementary to the target nucleotide.

[0036] The present disclosure provides a dumbbell- shaped amplicon comprising a nucleotide sequence comprising from its 3’ end to its 5’ end: a) a first hairpin nucleotide sequence comprising four domains: 1) a Bl domain; 2) a B2c domain; 3) a LB domain; and 4) a Bic domain complementary to the Bl domain; wherein at least one domain is not complementary to the target nucleotide; b) a target nucleotide sequence or complement thereof; and c) a second hairpin nucleotide sequence comprising four domains: 1) a Fl domain; 2) a LFc domain; 3) a F2 domain; and 4) a Flc domain complementary to the Fl domain.

[0037] In some embodiments, at least two domains are not complementary to the target nucleotide sequence. In some embodiments, at least three domains are not complementary to the target nucleotide sequence. In some embodiments, at least four domains are not complementary to the target nucleotide sequence. In some embodiments, two hairpin nucleotide acid sequences are not complementary to the target nucleotide sequence. In some embodiments, at least half of the product is not complementary to the target nucleotide sequence. In some embodiments, a target nucleotide sequence is at the most 100 nucleotides.Brief Description of the Drawings

[0038] Figure 1 shows exemplary components useful in amplification processes as described herein. Each nucleic acid domain is indicated with a unique label (“Fl”, “F2”, “F3”, “LF’, “Bl”, “B2”, “LB”, and “T”), complementary sequences to a domain are indicated with a lowercase ‘c’ (e.g., “Flc”). Dotted line: target-specific sequences; Solid line: target-agonistic sequence.

[0039] Figure 2 shows exemplary steps of an amplification process as described herein. Dotted line: target- specific sequences; Solid line: target- agonistic sequence.

[0040] Figure 3 shows exemplary components useful in amplification processes as described herein. Each domain is indicated with a unique label (“Fl”, “F2”, “F3”, “LF”, “Bl”, “B2”, “B3”, “LB”, and “T”), complementary sequences to a domain are indicated with a lowercase ‘c’ (e.g., “Flc”). Dotted line: target- specific sequences; Solid line: target- agonistic sequence.

[0041] Figure 4 shows exemplary steps of an amplification process as described herein. Dotted line: target- specific sequences; Solid line: target-agonistic sequence.

[0042] Figure 5 shows exemplary components useful in amplification processes as described herein. Each domain is indicated with a unique label (“Fl”, “F2”, “LF”, “B l”, “B2”, “B3”, “LB”, and “T”), complementary sequences to a domain are indicated with a lowercase ‘c’ (e.g., “Flc”). Dotted line: target-specific sequences; Solid line: target- agonistic sequence.

[0043] Figure 6 shows exemplary steps of an amplification process as described herein. Dotted line: target- specific sequences; Solid line: target-agonistic sequence.

[0044] Figure 7 shows exemplary components useful in amplification processes as described herein. Each domain is indicated with a unique label (“Fl”, “F2”, “F3”, “LF”, “Bl”, “B2”, “LB”, and “T”), complementary sequences to a domain are indicated with a lowercase ‘c’ (e.g., “Flc”). Dotted line: target-specific sequences; Solid line: target- agonistic sequence.

[0045] Figure 8 shows exemplary steps of an amplification process as described herein. Dotted line: target- specific sequences; Solid line: target- agonistic sequence.

[0046] Figure 9 shows exemplary components useful in amplification processes as described herein. Each domain is indicated with a unique label (“Fl”, “F2”, “F3”, “LF”, “Bl”, “B2”, “LB”, “drl”, “dr2”, “si”, and “T”), complementary sequences to a domain are indicated with a lowercase ‘c’ (e.g., “Flc”), and truncated sequences from a domain are labeled with an apostrophe (e.g., t’). In this example, sic and LFc are domains that can be used for detection. Dotted line: target-specific sequences; Solid line: target- agonistic sequence; Dashed line: direct repeat sequence (template- agonistic).

[0047] Figure 10 shows exemplary steps of an amplification process as described herein. Dotted line: target- specific sequences; Solid line: target-agonistic sequence; Dashed line: direct repeat sequence (template-agonistic).

[0048] Figure 11 shows exemplary components useful in amplification processes as described herein. Each domain is indicated with a unique label (“Fl”, “F2”, “F3”, “LF”, “Bl”, “B2”, “LB”, “dr”, “si”, and “T”), complementary sequences to a domain are indicated with a lowercase ‘c’ (e.g., “Flc”), and truncated sequences from a domain are labeled with an apostrophe (e.g., T’). Single nucleotide polymorphism (SNP) sites within each domain are indicated with a single asterisk. Mismatch(es) are indicated with two asterisks. In this example, sic is a domain that can be used for detection. Dotted line: target- specific sequences; Solid line: target-agonistic sequence; Dashed line: direct repeat sequence (template-agonistic).

[0049] Figure 12 shows exemplary steps of an amplification process as described herein. Single nucleotide polymorphism (SNP) sites within each domain are indicated with a single asterisk. Mismatch(es) are indicated with two asterisks. Dotted line: target-specific sequences; Solid line: target-agonistic sequence; Dashed line: direct repeat sequence (template-agonistic).

[0050] Figure 13 shows exemplary components useful in amplification processes as described herein. Each domain is indicated with a unique label (“Fl”, “F2”, “F3”, “LF”, “Bl”, “B2”, “LB”, “drl”, “dr2”, “si”, and “T”), complementary sequences to a domain are indicated with a lowercase ’c’ (e.g., “Flc”), and truncated sequences from a domain are labeled with an apostrophe (e.g., I”). Single nucleotide polymorphism (SNP) sites within each domain are indicated with a single asterisk. Mismatch(es) are indicated with two asterisks. In this example, sic and LFc are domains that can be used for detection. Dotted line: target-specific sequences; Solid line: target-agonistic sequence; Dashed line: direct repeat sequence (template- agonistic).

[0051] Figure 14 shows exemplary steps of an amplification process as described herein. Single nucleotide polymorphism (SNP) sites within each domain are indicated with a single asterisk. Mismatch(es) are indicated with two asterisks. Dotted line: target-specific sequences; Solid line: target-agonistic sequence; Dashed line: direct repeat sequence (template-agonistic) .

[0052] Figure 15 shows exemplary components useful in amplification processes as described herein. Each domain is indicated with a unique label (“Fl”, “F2”, “LF”, “Bl”, “B2”, “LB”, “Tl” and “T2”), complementary sequences to a domain are indicated with alowercase ‘c’ (e.g., “Flc”). Dotted line: target-specific sequences; Solid line: target- agonistic sequence.

[0053] Figure 16 shows exemplary steps of an amplification process as described herein. Dotted line: target- specific sequences; Solid line: target-agonistic sequence.

[0054] Figure 17 shows exemplary components useful in amplification processes as described herein. Each domain is indicated with a unique label (“Fl”, “F2”, “LF”, “Bl”, “B2”, “B3”, “LB”, and “T”), complementary sequences to a domain are indicated with a lowercase *c’ (e.g., “Flc”). Dotted line: target-specific sequences; Solid line: target- agonistic sequence.

[0055] Figure 18 shows exemplary steps of an amplification process as described herein. Dotted line: target- specific sequences; Solid line: target-agonistic sequence.

[0056] Figure 19 shows exemplary guide polynucleotides. Single nucleotide polymorphism (SNP) sites within each domain are indicated with a single asterisk. Mismatch(es) are indicated with two asterisks. Dotted line: target-specific sequences; Solid line: target-agonistic sequence; Dashed line: direct repeat sequence (template-agonistic).

[0057] Figure 20 shows exemplary components useful in amplification and detection processes as described herein. Each nucleic acid domain is indicated with a unique label (“Fl”, “F2”, “F3”, “LF”, “Bl”, “B2”, “LB”, ”dr”, “si”, and “T”), complementary sequences to a domain are indicated with a lowercase ‘c’ (e.g., “Flc”), and truncated sequences from a domain are labeled with an apostrophe (e.g., I”), sic is a domain that can be used for detection. Dotted line: target-specific sequences; Solid line: target-agonistic sequence; Dashed line: direct repeat sequence (template- agonistic).

[0058] Figure 21 shows exemplary steps of an amplification and detection process as described herein. Dotted line: target-specific sequences; Solid line: target-agonistic sequence.

[0059] Figure 22 shows exemplary components useful in amplification and detection processes as described herein. Each domain is indicated with a unique label (“Fl”, “F2”, “F3”, “LF”, “B l”, “B2”, “B3”, “LB”, ”dr”, “si”, and “T”), complementary sequences to a domain are indicated with a lowercase 'c’ (e.g., “Flc”) , and truncated sequences from a domain are labeled with an apostrophe (e.g., T’). sic is a domain that can be used fordetection. Dotted line: target-specific sequences; Solid line: target-agonistic sequence; Dashed line: direct repeat sequence (template-agonistic).

[0060] Figure 23 shows exemplary steps of an amplification and detection process as described herein. Dotted line: target-specific sequences; Solid line: target-agonistic sequence; Dashed line: direct repeat sequence (template-agonistic).

[0061] Figure 24 shows exemplary components useful in amplification processes as described herein. Each domain is indicated with a unique label (“Fl”, “F2”, “LF”, “Bl”, “B2”, “B3”, “LB”, “dr”, “si”, and “T”), complementary sequences to a domain are indicated with a lowercase ‘c’ (e.g., “Flc”), and truncated sequences from a domain are labeled with an apostrophe (e.g., I”), sic is a domain that can be used for detection. Dotted line: targetspecific sequences; Solid line: target-agonistic sequence; Dashed line: direct repeat sequence (template-agonistic) .

[0062] Figure 25 shows exemplary steps of an amplification and detection process as described herein. Dotted line: target-specific sequences; Solid line: target-agonistic sequence; Dashed line: direct repeat sequence (template- agonistic).

[0063] Figure 26 shows exemplary components useful in amplification and detection processes as described herein. Each domain is indicated with a unique label (“Fl”, “F2”, “F3”, “LF”, “B 1”, “B2”, “LB”, “dr”, and “T”), complementary sequences to a domain are indicated with a lowercase ‘c’ (e.g., “Flc”). In this example, LF is a domain that can be used for detection. Dotted line: target-specific sequences; Solid line: target-agonistic sequence; Dashed line: direct repeat sequence (template- agonistic).

[0064] Figure 27 shows exemplary steps of an amplification and detection process as described herein. Dotted line: target-specific sequences; Solid line: target-agonistic sequence; Dashed line: direct repeat sequence (template-agonistic).

[0065] Figure 28 shows exemplary components useful in amplification and detection processes as described herein. Each domain is indicated with a unique label (“Fl”, “F2”, “LF’, “Bl”, “B2”, “LB”, “dr”, “Tl” and “T2”), complementary sequences to a domain are indicated with a lowercase ‘c’ (e.g., “Flc”), and truncated sequences from a domain are labeled with an apostrophe (e.g., Tl’). sic is a domain that can be used for detection. Dotted line: target-specific sequences; Solid line: target-agonistic sequence; Dashed line: direct repeat sequence (template-agonistic).

[0066] Figure 29 shows exemplary steps of an amplification and detection process as described herein. Dotted line: target-specific sequences; Solid line: target-agonistic sequence; Dashed line: direct repeat sequence (template-agonistic).

[0067] Figure 30 shows exemplary components useful in amplification and detection processes as described herein. Each domain is indicated with a unique label (“Fl”, “F2”, “LF’, “Bl”, “B2”, “B3”, “LB”, “dr”, “si”, and “T”), complementary sequences to a domain are indicated with a lowercase ‘c’ (e.g., “Flc”), and truncated sequences from a domain are labeled with an apostrophe (e.g., Tc’). sic is a domain that can be used for detection. Dotted line: target-specific sequences; Solid line: target-agonistic sequence; Dashed line: direct repeat sequence (template-agonistic).

[0068] Figure 31 shows exemplary steps of an amplification and detection process as described herein. Dotted line: target-specific sequences; Solid line: target-agonistic sequence; Dashed line: direct repeat sequence (template- agonistic).

[0069] Figures 32 A-D. A. shows amplification using a method described herein with a hybrid primer as shown in D. B. shows amplification using a method described herein without a hybrid primer. C. shows conventional LAMP amplification. D. shows a hybrid primer having a target region complementary to influenza B segment 8 and an off-target region based on influenza A segment 1. Dotted line: Region targeting FluB segment 8; Solid line: Resion based on FluA sequence (Segment 1). cLAMP: condensed Loop-mediated Isothermal Amplification; LAMP: Loop-mediated Isothermal Amplification.

[0070] Figures 33A-D. A. shows amplification using a method described herein with a hybrid primer as shown in D. B. shows amplification using a method described herein without a hybrid primer. C. shows conventional LAMP amplification. D. shows a hybrid primer having a target region complementary to influenza B segment 5 and an off-target region based on influenza A segment 1. Dotted line: Region targeting FluB segment 5; Solid line: Resion based on FluA sequence (Segment 1). cLAMP: condensed Loop-mediated Isothermal Amplification; LAMP: Loop-mediated Isothermal Amplification.

[0071] Figures 34 A-D. A. shows amplification using a method described herein with a hybrid primer as shown in D. B. shows amplification using a method described herein without a hybrid primer. C. shows conventional LAMP amplification. D. shows a hybrid primer having a target region complementary to influenza B segment 5 and an off-targetregion based on influenza A segment 2. Dotted line: Region targeting FluB segment 5; Solid line: Resion based on FluA sequence (Segment 2). cLAMP: condensed Loop-mediated Isothermal Amplification; LAMP: Loop-mediated Isothermal Amplification.

[0072] Figure 35 shows lysis of respiratory viruses. A) Influenza A (FLUA) and B) SARS-CoV-2 (SCV2) using 95°C heat lysis or 20 mM NaOH versus no treatment in individual nasal swab matrices. Viral lysis efficiency was assayed by first performing a room temperature reverse transcriptase reactions, followed by the inactivation of the reverse transcriptase and standard taqman qPCR of the produced cDNA. A lower Cq value corresponds to greater viral RNA release. cLAMP: condensed Loop-mediated Isothermal Amplification; LAMP: Loop-mediated Isothermal Amplification; SCV2: SARS-CoV-2.

[0073] Figure 36 shows RNase inhibition achieved in nasal swab matrix using an RNase inhibitor or Sodium hydroxide (NaOH). RNase activity was measured using the RNase Alert reagent from IDT.

[0074] Figure 37 shows lysis of respiratory viruses (FluA and SARS-CoV-2) with high pH solutions. Various concentrations of KOH and NaOH were used to lyse viral particles. Viral lysis efficiency was assayed by first performing a room temperature reverse transcriptase reactions, followed by the inactivation of the reverse transcriptase and standard taqman qPCR of the produced cDNA. A lower Cq value corresponds to greater viral RNA release, and therefore better lysis of the viral particle

[0075] Figure 38 shows hydroxide-based chemical lysis of a non-enveloped virus. A stock of Human adenovirus was treated the indicated concentration of NaOH at room temperature prior to qPCR to detect released viral DNA. A faster Cq value indicates a higher concentration of released viral DNA, and therefore better lysis of the viral particle.

[0076] Figure 39 shows room temperature lysis of bacteria A) N. gonorrhoeae and B) C. trachomatis. Bacterial cells were treated with the indicated concentration of KOH or subjected to bead beating. After lysis, the cell suspensions were centrifuged to pellet intact cells, and a portion of the remaining supernatant was assayed by qPCR. Results are presented as the difference in Cq values between the treated cells and the untreated control, with a larger delta indicating a more effective lysis.

[0077] Figure 40 shows lysis of bacteria N. gonorrhoeae with 50 mM KOH with the addition of a detergent. Bacterial cells were treated with the indicated concentration ofdetergent in the presence of 50 mM KOH, or subjected to bead beating. After lysis, the cell suspensions were centrifuged to pellet intact cells, and a portion of the remaining supernatant was assayed by qPCR. Results are presented as the difference in Cq values between the treated cells and the untreated control, with a larger delta indicating a more effective lysis.

[0078] Figure 41 shows lysis efficiency of N. gonorrhoeae treated with 50 mM KOH, 13.5 mM HCL + / - 0.5% Plutonic 64 detergent at various incubation temperatures. Bacterial cells were treated with the indicated concentration of KOH, HC1, and / or detergent at the indicated temperature for 5 minutes, or subjected to a heat lysis of 95C for 5 minutes. After lysis, a portion of the remaining supernatant was assayed by qPCR, with the concentration of gDNA in the samples quantified by a standard curve of extracted gDNA. Results are presented as the percent efficiency of lysis, assuming 100% lysis for the heat lysis control. The signal from untreated control cells (“no lysis”) was subtracted from all samples.

[0079] Figure 42 shows lysis efficiency testing of N. gonorrhoeae treated with 50 mM KOH or HCL+ESH9 at various incubation temperatures. Bacterial cells were treated with the indicated concentration of KOH, HC1, and / or detergent at the indicated temperature for 5 minutes, or subjected to a heat lysis of 95C for 5 minutes. After lysis, a portion of the remaining supernatant was assayed by qPCR, with the concentration of gDNA in the samples quantified by a standard curve of extracted gDNA. Results are presented as the percent efficiency of lysis, assuming 100% lysis for the heat lysis control. The signal from untreated control cells (“no lysis”) was subtracted from all samples.

[0080] Figure 43 shows lysis efficiency testing of N. gonorrhoeae treated with 50 mM KOH, + NP40 at various incubation temperatures. Bacterial cells were treated with the indicated concentration of KOH, HC1, and / or detergent at the indicated temperature for 5 minutes, or subjected to a heat lysis of 95C for 5 minutes. After lysis, the cell suspensions were centrifuged to pellet intact cells, and a portion of the remaining supernatant was assayed by qPCR. Results are presented as the percent efficiency of lysis, assuming 100% lysis for the heat lysis control.

[0081] Figure 44 shows lysis efficiency testing of N. gonorrhoeae treated with various KOH concentrations, + / - 3% NP40 at various incubation temperatures. Bacterial cells were treated with the indicated concentration of KOH and / or detergent at the indicatedtemperature for 5 minutes, or subjected to a heat lysis of 95C for 5 minutes. After lysis, a portion of the remaining supernatant was assayed by qPCR, with the concentration of gDNA in the samples quantified by a standard curve of extracted gDNA. Results are presented as the percent efficiency of lysis, assuming 100% lysis for the heat lysis control. The signal from untreated control cells (“no lysis”) was subtracted from all samples.

[0082] Figure 45 shows lysis technologies as described herein versus Heat lysis methods (95°C) upstream of LAMP-Cas detection. N. gonorrhoeae were diluted in TE, treated as indicated, and a portion of the lysate was used as template for a LAMP-Cas reaction.

[0083] Figure 46 shows KOH lysis of N. gonorrhoeae in vaginal matrix using LAMP-Cas. N. gonorrhoeae were diluted in TE, treated as indicated, and a portion of the lysate was used as template for a LAMP-Cas reaction.Definitions

[0084] 3’-end and 5’-end‘. The term as used herein do not solely refer to the nucleotide or either terminus, it also covers reference to a region located at the terminus that includes the single end nucleotide. More specifically, 500 nucleotides, such as 100 nucleotides, such as 20 nucleotides from either terminus are included in the term 3 ’-end and 5 ’-end.

[0085] Agent: In general, the term “agent”, as used herein, is used to refer to an entity (e.g., for example, a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc., or complex, combination, mixture or system [e.g., cell, tissue, organism] thereof), or phenomenon (e.g., heat, electric current or field, magnetic force or field, etc.). In appropriate circumstances, as will be clear from context to those skilled in the art, the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof. Alternatively or additionally, as context will make clear, the term may be used to refer to a natural product in that it is found in and / or is obtained from nature. In some instances, again as will be clear from context, the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and / or produced through action of the hand of man and / or is not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may beprovided as collections or libraries, for example that may be screened to identify or characterize active agents within them. In some cases, the term “agent” may refer to a compound or entity that is or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound or entity that is not a polymer and / or is substantially free of any polymer and / or of one or more particular polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety.

[0086] Ambient temperature: As used herein, the term “ambient temperature” is the temperature of surroundings. In general, the term ambient temperature is to be understood as the temperature of any object or environment surrounding an item. Measuring an ambient temperature can be accomplished by using a thermometer or sensor. The ambient temperature of an item is dependent on the temperature of the surrounding of the item. The surroundings can have any temperature, such as a temperature below 95°C, such as below 90°C, such as below 85°C, such as below 80°C, such as below 75°C, such as below 70°C, such as below 65 °C, such as below 60°C, such as below 55 °C, such as below 50°C, such as below 45°C, such as below 40°C, such as below 35°C, such as below 30°C, such as below 25°C, such as below 24°C, such as below 23 °C, such as below 22°C, such as below 21 °C, such as below 20°C. Exemplary ambient temperature ranges include 5°C to 50°C, such as 10°C to 40°C, such as 15°C to 35°C, such as 20°C to 30°C, such as 20°C to 25 °C, such as 20°C to 22°C.

[0087] Amino acid: in its broadest sense, as used herein, refers to any compound and / or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally- occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a polypeptide, can contain astructural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.

[0088] Approximately or About: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0089] Associated: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of and / or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0090] Binding: It will be understood that the term “binding”, as used herein, typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts - including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and / or in a biological system or cell).

[0091] Biological Sample: As used herein, the term “biological sample” typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell culture) of interest, as described herein. In some embodiments, a source of interest is or comprises an organism, such as an animal or human. In some embodiments, a biological sample is or comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and / or excretions; and / or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, obtained cells are or include cells from an individual from whom the sample is obtained. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane.Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of certain components, etc.

[0092] Cellular lysate: As used herein, the term “cellular lysate” or “cell lysate” refers to a fluid containing contents of one or more disrupted cells (i.e., cells whose membrane has been disrupted). In some embodiments, a cellular lysate includes both hydrophilic and hydrophobic cellular components. In some embodiments, a cellular lysate includes predominantly hydrophilic components; in some embodiments, a cellular lysate includes predominantly hydrophobic components. In some embodiments, a cellular lysate is a lysate of one or more cells selected from the group consisting of plant cells, microbial (e.g., bacterial or fungal) cells, animal cells (e.g., mammalian cells), human cells, and combinations thereof. In some embodiments, a cellular lysate is a lysate of one or more abnormal cells, such as cancer cells. In some embodiments, a cellular lysate is a crude lysate in that little or no purification is performed after disruption of the cells; in some embodiments, such a lysate is referred to as a “primary” lysate. In some embodiments, one or more isolation or purification steps is performed on a primary lysate; however, the term “lysate” refers to a preparation that includes multiple cellular components and not to pure preparations of any individual component.

[0093] Composition: Those skilled in the art will appreciate that the term “composition”, as used herein, may be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition may be of any form - e.g., gas, gel, liquid, solid, etc.

[0094] Complementary: As used herein, the term “complementary” refers to the overall relatedness between polymeric molecules, e.g., between polynucleotides. In some embodiments, polynucleotides such as nucleotides sequences (e.g., primer nucleotide sequences or target nucleotide sequences) are considered to be “complementary” to one another if their sequences are at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical. In some embodiments, polynucleotides are considered to be “complementary” to one another if their sequences are at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% similar. In some embodiments, polynucleotides are considered to be “complementary” to one another if they are capable of hybridize to each other.

[0095] Comprising: A composition or method described herein as "comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the compositionor method. To avoid prolixity, it is also understood that any composition or method described as "comprising" (or which "comprises") one or more named elements or steps also describes the corresponding, more limited composition or method "consisting essentially of" (or which "consists essentially of") the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of" one or more named elements or steps also describes the corresponding, more limited, and closed-ended composition or method "consisting of" (or "consists of") the named elements or steps to the exclusion of any other unnamed element or step. In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step may be substituted for that element or step.

[0096] Corresponding to: As used herein, the term “corresponding to” may be used to designate the position / identity of a structural element in a compound or composition through comparison with an appropriate reference compound or composition. For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid "corresponding to" a residue at position 190, for example, need not actually be the 190thamino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify "corresponding" amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHLLS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and / or nucleic acids in accordance with the present disclosure.

[0097] Designed: As used herein, the term “designed” refers to an agent (i) whose structure is or was selected by the hand of man; (ii) that is produced by a process requiring the hand of man; and / or (iii) that is distinct from natural substances and other known agents.

[0098] Detectable entity. The term “detectable entity” as used herein refers to any element, molecule, functional group, compound, fragment or moiety that is detectable. In some embodiments, a detectable entity is provided or utilized alone. In some embodiments, a detectable entity is provided and / or utilized in association with (e.g., joined to) another agent. Examples of detectable entities include, but are not limited to: various ligands, radionuclides (e.g.,3H,14C,18F,19F,32P,35S,135I,125I,123I,MCu,187Re,11 ]In,90Y,99mTc,177LU,89Zr etc.), fluorescent dyes (for specific exemplary fluorescent dyes, see below), chemiluminescent agents (such as, for example, acridinum esters, stabilized dioxetanes, and the like), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductors nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.) nanoclusters, paramagnetic metal ions, enzymes (for specific examples of enzymes, see below), colorimetric labels (such as, for example, dyes, colloidal gold, and the like), biotin, dioxigenin, haptens, and proteins for which antisera or monoclonal antibodies are available.

[0099] Determine: Many methodologies described herein include a step of “determining”. Those of ordinary skill in the art, reading the present specification, will appreciate that such “determining” can utilize or be accomplished through use of any of a variety of techniques available to those skilled in the art, including for example specific techniques explicitly referred to herein. In some embodiments, determining involves manipulation of a physical sample. In some embodiments, determining involves consideration and / or manipulation of data or information, for example utilizing a computer or other processing unit adapted to perform a relevant analysis. In some embodiments, determining involves receiving relevant information and / or materials from a source. In some embodiments, determining involves comparing one or more features of a sample or entity to a comparable reference.

[0100] Expression: As used herein, “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and / or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0101] GeV. As used herein, the term “gel” refers to viscoelastic materials whose rheological properties distinguish them from solutions, solids, etc. In some embodiments, a composition is considered to be a gel if its storage modulus (G') is larger than its modulus (G"). In some embodiments, a composition is considered to be a gel if there are chemical or physical cross-linked networks in solution, which is distinguished from entangled molecules in viscous solution.

[0102] Homology: As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g., between polypeptide molecules. In some embodiments, polymeric molecules such as antibodies are considered to be “homologous” to one another if their sequences are at least 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 80%, 85%, 90%, 95%, or 99% similar.

[0103] Identity. As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percentidentity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0104] In vitro". The term “in vitro” as used herein refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

[0105] Isolated: as used herein, refers to a substance and / or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) designed, produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered "isolated" or even "pure" , after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients. To give but one example, in some embodiments, a biological polymer such as a polypeptide or polynucleotide that occurs in nature is considered to be "isolated" when, a) by virtue of its origin or source of derivation is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; c) is expressed by or is otherwise in association with components from a cell or other expression systemthat is not of the species that produces it in nature. Thus, for instance, in some embodiments, a polypeptide that is chemically synthesized or is synthesized in a cellular system different from that which produces it in nature is considered to be an "isolated” polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide to the extent that it has been separated from other components a) with which it is associated in nature; and / or b) with which it was associated when initially produced.

[0106] Nucleic acid'. As used herein, in its broadest sense, refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, "nucleic acid" refers to an individual nucleic acid residue (e.g., a nucleotide and / or nucleoside); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a "nucleic acid" is or comprises RNA; in some embodiments, a "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the systems and / or methods provided herein. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxy adenosine, deoxythymidine, deoxy guanosine, and deoxycytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5 -methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5 -bromouridine, C5- fluorouridine, C5 -iodouridine, C5 -propynyl-uridine, C5 -propynyl-cytidine, C5- methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases,and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.

[0107] Polypeptide: As used herein refers to any polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and / or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and / ordoes not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and / or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and / or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.

[0108] Protein: As used herein, the term “protein” refers to a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.)and / or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and / or characteristic portions thereof.

[0109] Reference: As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0110] Sample: As used herein, the term “sample” typically refers to an aliquot of material obtained or derived from a source of interest, as described herein. In some embodiments, a source of interest is a biological or environmental source. In some embodiments, a source of interest may be or comprise a cell or an organism, such as a microbe, a plant, or an animal (e.g., a human). In some embodiments, a source of interest is or comprises biological tissue or fluid. In some embodiments, a biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breastmilk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secreations, vitreous humour, vomit, and / or combinations or component(s) thereof. In some embodiments, a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and / or a transcellular fluid. In some embodiments, a biological fluid may be or comprise a plant exudate. In some embodiments, a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., brocheoalvealar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and / or purification of certain components, etc.

[0111] Specific: The term “specific”, when used herein with reference to an agent having an activity, is understood by those skilled in the art to mean that the agent discriminates between potential target entities or states. For example, an in some embodiments, an agent is said to bind “specifically” to its target if it binds preferentially with that target in the presence of one or more competing alternative targets. In many embodiments, specific interaction is dependent upon the presence of a particular structural feature of the target entity (e.g., an epitope, a cleft, a binding site). It is to be understood that specificity need not be absolute. In some embodiments, specificity may be evaluated relative to that of the binding agent for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to that of a reference specific binding agent. In some embodiments specificity is evaluated relative to that of a reference non-specific binding agent. In some embodiments, the agent or entity does not detectably bind to the competing alternative target under conditions of binding to its targetentity. In some embodiments, binding agent binds with higher on-rate, lower off-rate, increased affinity, decreased dissociation, and / or increased stability to its target entity as compared with the competing alternative target(s).

[0112] Specificity. As is known in the art, “specificity” is a measure of the ability of a particular ligand to distinguish its binding partner from other potential binding partners.

[0113] Subject: As used herein, the term “subject” refers to an organism, for example, a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, a dog). In some embodiments a human subject is an adult, adolescent, or pediatric subject. In some embodiments, a subject is suffering from a disease, disorder or condition, e.g., a disease, disorder or condition that can be treated as provided herein, e.g. , a cancer or a tumor listed herein. In some embodiments, a subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to and / or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing the disease, disorder or condition. In some embodiments, a subject displays one or more symptoms of a disease, disorder or condition. In some embodiments, a subject does not display a particular symptom (e.g,. clinical manifestation of disease) or characteristic of a disease, disorder, or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.Detailed Description of Certain Embodiments

[0114] Target nucleotide synthesis, nucleotide amplification and / or nucleotide detection are critical tools in biomedical research and clinical medicine, including for use in a variety of diagnostic technologies.Conventional LAMP

[0115] Conventional LAMP technologies copy and / or amplify a target nucleotide sequence under isothermal conditions using two or more sets of primers (e.g., a plurality) and a polymerase with high strand displacement activity. Further, LAMP target nucleotide synthesis reactions can be conducted in a single reaction vessel (“one-pot” embodiment). Typically, four different primers are used, a Forward Primer (F3), a Backward Primer (B3), a Forward Inner Primer (FIP), and / or a Backward Inner Primer (BIP). Both a FIP and / or BIP contact complementary sequences nested within complementary sequences that F3 and / or B3 contact. Optionally, an additional pair of primers (e.g., loop primers) can be used that hybridize to stem-loops (e.g., as discussed below, step 5), except for loops that are hybridized by an inner primer. Use of loop primers can increase LAMP product generated and / or decrease duration of a reaction required to achieve a detection limit. The present disclosure, among other things, provides improved and / or alterative LAMP technologies and / or systems for target nucleotide sequence synthesis, target nucleotide sequence amplification, target nucleotide sequence detecting nucleic acid in a sample, or any combination thereof. As those skilled in the art will be aware, a conventional LAMP reaction often involves steps such as primer annealing and initiation of nucleotide synthesis. For example, a representative LAMP reaction. A representative LAMP reaction in described in e.g., W02002024902A.Condensed LAMP (cLAMP)

[0116] In some embodiments, technologies disclosed herein (e.g., primers, compositions, kits, amplicons, and methods) are useful in condensed Loop-mediated Isothermal Amplification (cLAMP). In some embodiments, the present disclosure demonstrates that use of a hybrid primer reduced the number of target nucleotide specific domains required for nucleotide synthesis and / or nucleotide amplification (e.g., relative to other methods of nucleotide synthesis and / or nucleotide amplification or LAMP without using a hybrid primer according to the present disclosure).Compositions

[0117] In some embodiments, a composition according to the present disclosure comprises a hybrid primer. In some embodiments, a composition according to the present disclosure comprises at least one hybrid primer. In some embodiments, a composition according to the present disclosure comprises one or more hybrid primer(s) (e.g., a first hybrid primer, a second hybrid primer, etc). In some embodiments, the present disclosure provides compositions useful for target nucleotide synthesis and amplicon production (e.g., producing one or more cLAMP amplicons, such as a hairpin-loop amplicon, a dumbbellshaped amplicon, etc.). In some embodiments, the present disclosure provides compositions and methods useful for target nucleotide amplification. In some embodiments, the present disclosure provides compositions and methods useful for target nucleotide detection.Primers

[0118] In some embodiments, technologies described herein use one or more primers. In some embodiments, a primer consists or comprises of a nucleotide sequence. In some embodiments, a primer consists or comprises of a ribonucleotide sequence. In some embodiments, a primer consists or comprises of a deoxyribonucleotide sequence. In some embodiments, a primer consists or comprises of a combination of ribonucleotides and deoxyribonucleotides. In some embodiments, a primer comprises one or more modification as described herein. In some embodiments, a primer comprises one or more nucleotides comprising modification as described herein.

[0119] In some embodiments, a primer as described herein comprises a primer domain. In some embodiments, a primer comprises at least one primer domain. In some embodiments, a primer comprises one or more primer domains. In some embodiments, a primer domain is a domain present within a primer as described herein below. In some embodiments, a primer comprises a plurality of primer domains. In some embodiments, primer domains are directly linked (e.g., covalently). In some embodiments, primer domains are separated by one or more nucleotides. In some embodiments, primer domains are separated by 2 to 10 nucleotides, such as 2 to 5 nucleotides, such as 4 nucleotides. In some embodiments, primer domains are separated by one or more domains. In some embodiments, a primer domain is about 9 to about 30 nucleotides. In some embodiments, primer domains are separated by a linker. In some embodiments, a linker comprises thymineone or more thymine (T). In some embodiments, a linker is a TTTT linker. A linker may improve spacing and flexibility.

[0120] Tn some embodiments, a primer consists or comprises of one or more primer domains that are not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof. In some embodiments, a primer comprises a first primer domain that is complementary to second primer domain, wherein none of the primer domains are complementary to a target nucleotide sequence or complement thereof (e.g., within the same primer or between two different primers). In some embodiments, a primer comprises a first primer domain and a second primer domain, wherein the two domains are complementary.

[0121] In some embodiments, a primer comprises one or more primer domains that are complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof and one or more primer domains that are not complementary to a target nucleotide sequence or complement thereof. In some embodiments, a primer comprises a first primer domain that is complementary to a second primer domain. In some embodiments, a primer comprises two primer domains that are complementary to each other. In some embodiments, a primer comprises a primer domain that is complementary to a primer domain in another primer.

[0122] In some embodiments, a primer consists or comprises of one or more primer domains that are complementary to a target nucleotide sequence (e.g., complementary target domain) or complement thereof. In some embodiments, the entire primer is complementary to a target nucleotide sequence or complementary target domain(s), or complements thereof.

[0123] A primer domain that is complementary to a target nucleotide sequence (e.g., target domain) is capable of hybridizing to the target nucleotide sequence, i.e., hybridization site. A primer domain that is complementary to a primer nucleotide sequence (e.g., a primer domain) is capable of hybridizing to the primer nucleotide sequence, i.e., hybridization site. In some embodiments, degree of complementarity or identity between a primer domain and a hybridization site in the target domain or a hybridization site in the primer domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In some embodiments, a primer domain is 100% identical to a hybridization site in the target domain. In some embodiments, a primer domain is 100% identical to a hybridization site in another primer domain. In some embodiments, a primer domain that is complementary to a target domain or primer domain comprises 1, 2, 3,or 4 mismatches (e.g., about 1, 2, 3, or 4 mismatches within a primer domain having a nucleotide sequence of about 18 to 25 nucleotides). In some embodiments, a primer domain complementary to a target nucleotide sequence or complement thereof comprises 1, 2, 3, or 4 mismatches relative to its hybridization site in the target nucleotide sequence.Hybrid Primer

[0124] In some embodiments, composition and methods described herein comprise one or more hybrid primer(s) (e.g., a first hybrid primer, a second hybrid primer, etc.). In some embodiments, a primer as described herein is a hybrid primer. In some embodiments, a hybrid primer is a first hybrid primer. In some embodiments, a hybrid primer is a second hybrid primer.

[0125] In some embodiments, a hybrid primer comprises a nucleotide sequence. In some embodiments, a hybrid primer comprises a nucleotide sequence of about 20 nucleotides to about 250 nucleotides. In some embodiments, a hybrid primer comprises a nucleotide sequence of about 60 nucleotides to about 250 nucleotides.

[0126] In some embodiments, a hybrid primer comprises one or more primer domains that are complementary to a target nucleotide sequence. In some embodiments, a hybrid primer comprises one or more primer domains that are complementary to a target nucleotide sequence or complement thereof and one or more primer domains that are not complementary to a target nucleotide sequence. In some embodiments, at least a part of the hybrid primer nucleotide sequence is not complementary to a target nucleotide sequence. In some embodiments, at least 50% of the hybrid primer nucleotide sequence is not complementary to a target nucleotide sequence. In some embodiments, at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80% of the hybrid primer nucleotide sequence is not complementary to a target nucleotide sequence.

[0127] In some embodiments, a hybrid primer comprises one or more 3’ Target (T) domain(s) and one or more 5’ hairpin nucleotide sequence(s). In some embodiments, a T domain is a T1 domain. In some embodiments, a T domain is a T2 domain. In some embodiments, a T1 domain and a T2 domain are non-overlapping. In some embodiments, a T1 domain and a T2 domain hybridize to opposite or complementary strands of the targetnucleotide sequence. In some embodiments, a first hybrid primer comprises a 3’ T1 domain and a 5’ first hairpin nucleotide sequence. In some embodiments, a second hybrid primer comprises a 3’ T2 domain and a 5’ second hairpin nucleotide sequence.Hybrid primer T domains

[0128] In some embodiments, a 3’ Target (T) domain (e.g., a 3’ T1 domain, a 3’ T2 domain, or both) is complementary to a target nucleotide sequence. In some embodiments, a 3’ T domain is complementary to a Tc domain of the target nucleotide sequence. In some embodiments, a 3’ T1 domain is complementary to a Tic domain of the target nucleotide sequence. In some embodiments, a 3’ T2 domain is complementary to a T2c domain of the target nucleotide sequence.

[0129] In some embodiments, only a 3’ T domain of a hybrid primer is capable of hybridizing to a Tc domain of a target nucleotide sequence. In some embodiments, one or more primer domains are capable of hybridizing to a Tc domain of the target nucleotide sequence. In some embodiments, a 3’ T domain of a hybrid primer and an inner primer domain (e.g., Flc domain, a Bic, or both) are both capable of hybridizing to the same Tc domain of the target nucleotide sequence. An example hereof is shown in Example 4.

[0130] In some embodiments, a hybrid primer T domain is capable of hybridizing to a target nucleotide sequence or its complement. In some embodiments, a hybrid primer T domain hybridizes to a target nucleotide sequence. In some embodiments, a hybrid primer T domain hybridizes to a complement of a target nucleotide sequence. A hybrid primer (e.g., a first hybrid primer and / or a second hybrid primer) can be used in more than one orientation. In some embodiments, a hybrid primer is the complement of a positive strand of a target nucleotide sequence. In some embodiments, a hybrid primer is the complement of a negative strand of a target nucleotide sequence. In some embodiments, a first hybrid primer is used as a forward hybrid primer. In some embodiments, a first hybrid primer is used as a backward hybrid primer. In some embodiments, a second hybrid primer is used as a forward hybrid primer. In some embodiments, a second hybrid primer is used as a backward hybrid primer. Examples 1 and 3 show a hybrid primer used in both directions (i.e. by hybridizing to a target forward nucleotide sequence or hybridizing to a target backward sequence).

[0131] In some embodiments, a 3’ T domain (e.g., a 3’T1 domain and / or 3’T2 domain) is or comprises a nucleotide sequence of about 9 nucleotides to about 45 nucleotides. In some embodiments, a 3’ T domain is or comprises a nucleotide sequence of about 10 nucleotides to about 40 nucleotides, such as about 15 nucleotides to about 35 nucleotides. In some embodiments, a 3’ T1 domain is complementary to a Tic domain of a target nucleotide sequence. In some embodiments, a 3’ T2 domain is complementary to a T2c domain of a target nucleotide sequence. In some embodiments, a Tic domain and a T2c domain are non-overlapping domains located on opposite or complementary strands of the target nucleotide sequence.Hybrid primer 5’ hairpin nucleotide sequences

[0132] In some embodiments, a 5’ hairpin nucleotide sequence is a first 5’ hairpin nucleotide sequence. In some embodiments, a 5’ hairpin nucleotide sequence is a second 5’ hairpin nucleotide sequence.

[0133] In some embodiments, a 5’ hairpin nucleotide sequence comprises a plurality of primer domains. In some embodiments, a 5’ hairpin nucleotide sequence comprises one or more primer domains. In some embodiments, a 5’ hairpin nucleotide sequence comprises two or more primer domains. In some embodiments, a 5’ hairpin nucleotide sequence comprises three or more primer domains. In some embodiments, a 5’ hairpin nucleotide sequence comprises four or more primer domains. In some embodiments, 5’ hairpin nucleotide sequence domains are directly linked (e.g., covalently) to each other. In some embodiments, 5’ hairpin nucleotide sequence domains are separated by one or more nucleotides. In some embodiments, 5’ hairpin nucleotide sequence domains are separated by one or more domains.

[0134] In some embodiments, a 5’ hairpin nucleotide sequence comprises one or more primer domains that are not complementary to a target nucleotide sequence. In some embodiments, a 5’ hairpin nucleotide sequence comprises two or more primer domains that are not complementary to a target nucleotide sequence. In some embodiments, a 5 ’ hairpin nucleotide sequence comprises three or more primer domains that are not complementary to a target nucleotide sequence. In some embodiments, a 5’ hairpin nucleotide sequence comprises four or more primer domains that are not complementary to a target nucleotidesequence. In some embodiments, at least a part of a 5’ hairpin nucleotide sequence is not complementary to a target nucleotide sequence. In some embodiments, a 5’ hairpin nucleotide sequence is not complementary to a target nucleotide sequence.

[0135] In some embodiments, a 5’ hairpin nucleotide sequence comprises two primer domains that are complementary to each other. In some embodiments, a 5’ hairpin nucleotide sequence comprises two primer domains that are not complementary to each other.

[0136] In some embodiments, a first 5’ hairpin nucleotide sequence comprises four domains from 3’ to the 5’1) a Bl domain;2) a LBc domain;3) a B2 domain; and4) a Bic domain complementary to the Bl domain.

[0137] In some embodiments, a second 5’ hairpin nucleotide sequence comprises four domains from 3’ to the 5’1) a Fl domain;2) a LFc domain;3) a F2 domain; and4) a Flc domain complementary to the Fl domain.

[0138] In some embodiments, at least one domain of the first 5’ hairpin nucleotide sequence (e.g., a Bl domain; an LBc domain; a B2 domain; or a Bic domain) is not complementary to a target nucleotide sequence. In some embodiments, at least one domain of the second 5’ hairpin nucleotide sequence (e.g., a Fl domain; an LFc domain; a F2 domain; or an Flc domain) is not complementary to a target nucleotide sequence. In some embodiments, at least two domains of the first 5’ hairpin nucleotide sequence are not complementary to a target nucleotide sequence. In some embodiments, at least two domains of the second 5 ’ hairpin nucleotide sequence are not complementary to a target nucleotide sequence. In some embodiments, at least three domains of the first 5’ hairpin nucleotide sequence are not complementary to the target nucleotide sequence. In some embodiments, atleast three domains of the second 5’ hairpin nucleotide sequence are not complementary to the target nucleotide sequence. In some embodiments, all four domains of the first 5’ hairpin nucleotide sequence are not complementary to a target nucleotide sequence. In some embodiments, a first 5’ hairpin nucleotide sequence is not complementary to the target nucleotide sequence. In some embodiments, all four domains of the second 5’ hairpin nucleotide sequence are not complementary to a target nucleotide sequence. In some embodiments, a second 5’ hairpin nucleotide sequence is not complementary to a target nucleotide sequence.

[0139] In some embodiments, a 5’ hairpin nucleotide sequence (e.g., a first 5’ hairpin nucleotide sequence and / or second 5 ’ hairpin nucleotide sequence) adapts a folded form. In some embodiments, a folded form comprises a hybrid primer stem portion and hybrid primer loop.

[0140] In some embodiments, a 1) domain (e.g., Bl domain and / or Fl domain) hybridizes to a 4) domain (e.g., Bic domain and / or Flc domain) resulting in formation of a stem- loop structure at the 5’ end of the hybrid primer. In some embodiments, a 5’ hairpin nucleotide sequence comprises a hybrid primer stem (e.g., a first hybrid primer stem and / or a second primer hybrid stem) and a hybrid primer loop (e.g., a first hybrid primer loop and / or second hybrid primer loop). In some embodiments, a hybrid primer loop is single stranded. In some embodiments, a hybrid primer stem is double stranded. In some embodiments, a Bl domain and a Bic domain hybridize to form a first hybrid primer stem. In some embodiments, an LBc domain and a B2 domain form a first hybrid primer loop. In some embodiments, a Fl domain and an Flc domain hybridize to form a second hybrid primer stem. In some embodiments, an LFc domain and a F2 domain form a second hybrid primer loop.Bl domains

[0141] In some embodiments, a Bl domain comprises or consists of a nucleotide sequence. In some embodiments, a Bl domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof. In some embodiments, a Bl domain is complementary to another primer domain (e.g., hybrid primer domain). In some embodiments, a Bl domain is complementary to a Bic domain. In some embodiments, a Bl domain is characterized in that it hybridizes to a Bic domain. In some embodiments, a Bl domain and a B 1c domain hybridize to form a first hybrid primer stem. In someembodiments, a Bl domain is directly connected to a T1 domain or linked (e.g., covalently) by one or more nucleotides to a T1 domain. In some embodiments, a Bl domain is directly connected to an LBc domain or linked (e.g., covalently) by one or more nucleotides to an LBc domain.

[0142] In some embodiments, a Bl domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides. In some embodiments, a Bl domain is or comprises a nucleotide sequence of about 10 nucleotides to about 40 nucleotides, such as about 15 nucleotides to about 35 nucleotides.LBc domains

[0143] In some embodiments, an LBc domain comprises or consists of a nucleotide sequence. In some embodiments, an LBc domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof. In some embodiments, an LBc domain is a part of a hybrid primer loop (e.g., a first hybrid primer loop). In some embodiments, an LBc domain is not complementary to any of the first 5’ hairpin nucleotide sequence domains. In some embodiments, an LBc domain is characterized in that it does not hybridize to any of the first 5’ hairpin nucleotide sequence domains. In some embodiments, a hybrid primer loop comprises an LBc domain and a B2 domain. In some embodiments, a hybrid primer loop comprises an LBc domain, a B2 domain, and one or more primer domains. In some embodiments, an LBc domain and a B2 domain form a first hybrid primer loop (e.g., a loop without any pairing nucleotides). In some embodiments, an LBc domain is complementary to a LB domain of a LB primer (e.g., a loop primer). Thus, at least a part of the hybrid primer loop is capable of base paring with a complementary LB primer. In some embodiments, an LBc domain comprises a nucleotide sequence of about 9 to about 30 nucleotides that is complementary to a LB primer. In some embodiments, about 20% to about 50% of a hybrid primer loop (e.g., a first hybrid primer loop) is complementary to a LB primer. In some embodiments, at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50% of a hybrid primer loop is complementary to a LB primer. In some embodiments, at the most 80%, such as at the most 75%, such as at the most 70%, such as at the most 65%, such as at the most 60%, such as at the most 55%, such as at the most 50% of a hybrid primer loop is complementary to a LB primer. In some embodiments, an LBc domain is directly connected to a B l domain or linked (e.g., covalently) by one or more nucleotides to a Bl domain. Insome embodiments, an LBc domain is directly connected to a B2 domain or linked (e.g., covalently) by one or more nucleotides to a B2 domain.

[0144] In some embodiments, an LBc domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides. In some embodiments, an LBc domain is or comprises a nucleotide sequence of about 10 nucleotides to about 40 nucleotides, such as about 15 nucleotides to about 35 nucleotides.B2 domains

[0145] In some embodiments, a B2 domain comprises or consists of a nucleotide sequence. In some embodiments, B2 domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof. In some embodiments, a B2 domain is not complementary to any of the first 5 ’ hairpin nucleotide sequence domains. In some embodiments, a B2 domain is complementary to a B2c domain. In some embodiments, a B2 domain of a first hybrid primer is identical to a B2 domain of a backward inner primer (BIP).

[0146] In some embodiments, a B2 domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides. In some embodiments, a B2 domain is or comprises a nucleotide sequence of about 10 nucleotides to about 40 nucleotides, such as about 15 nucleotides to about 35 nucleotides.Bic domain

[0147] In some embodiments, a Bic domain comprises or consists of a nucleotide sequence. In some embodiments, a Bic domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof. In some embodiments, a Bic domain is complementary to a primer domain (e.g., hybrid primer domain). In some embodiments, a Bic domain is complementary to a Bl domain. In some embodiments, a Bic domain is characterized in that it hybridizes to a Bl domain. In some embodiments, a Bic domain and a Bl domain hybridize to form a first hybrid primer stem. In some embodiments, a B 1c domain is directly connected to a B2 domain or linked (e.g., covalently) by one or more nucleotides to a B2 domain.

[0148] In some embodiments, a Bic domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides. In some embodiments, a Bic domain is orcomprises a nucleotide sequence of about 10 nucleotides to about 40 nucleotides, such as about 15 nucleotides to about 35 nucleotides.

[0149] Tn some embodiments, a composition comprises(i) a first hybrid primer whose nucleotide sequence comprises: a) a 3’ T1 domain complementary to a Tic domain of the target nucleotide sequence; b) a first 5’ hairpin nucleotide sequence comprising four domains from 3’ to the 5’1) a Bl domain;2) a LBc domain;3) a B2 domain; and4) a Bic domain complementary to the Bl domain, wherein at least one domain is not complementary to the target nucleotide sequence.

[0150] In some embodiments, a 3’ T1 domain of a first hybrid primer is capable of annealing to a Tic domain of a target nucleotide or a complement thereof when contacted under conditions (e.g., salt, temperature, etc.) that permit annealing. Annealing of the 3’ T1 domain to the Tic domain of the target nucleotide sequence or complement thereof permits extension of the first hybrid primer by a polymerase enzyme (e.g., with stand displacement activity) thereby synthesizing a new strand.Fl domains

[0151] In some embodiments, a Fl domain comprises or consists of a nucleotide sequence. In some embodiments, a Fl domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof. In some embodiments, a Fl domain is complementary to another primer domain (e.g., hybrid primer domain). In some embodiments, a Fl domain is complementary to an Flc domain. In some embodiments, a Fl domain is characterized in that it hybridizes to an Flc domain. In some embodiments, a Fl domain and an Flc domain hybridize to form a second hybrid primer stem. In some embodiments, a Fl domain is directly connected to a T2 domain or linked (e.g., covalently) by one or more nucleotides to a T2 domain. In some embodiments, a Fl domain is directly connected to an LFc domain or linked by one or more nucleotides to an LFc domain.

[0152] In some embodiments, a Fl domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides. In some embodiments, a Fl domain is or comprises a nucleotide sequence of about 10 nucleotides to about 40 nucleotides, such as about 15 nucleotides to about 35 nucleotides.LFc domains

[0153] In some embodiments, an LFc domain comprises or consists of a nucleotide sequence. In some embodiments, an LFc domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof. In some embodiments, an LFc domain is a part of a hybrid primer loop (e.g., a second hybrid primer loop). In some embodiments, an LFc domain is not complementary to any of the second 5’ hairpin nucleotide sequence domains. In some embodiments, an LFc domain is characterized in that it does not hybridize to any of the second 5’ hairpin nucleotide sequence domains. In some embodiments, a hybrid primer loop comprises an LFc domain and a F2 domain. In some embodiments, a hybrid primer loop comprises an LFc domain, a F2 domain, and one or more primer domains. In some embodiments, an LFc domain and a F2 domain form a first hybrid primer loop (e.g., a loop without any pairing nucleotides). In some embodiments, an LFc domain is complementary to a LF domain of a LF primer (e.g., a loop primer). Thus, at least a part of the hybrid primer loop is capable of base paring with a complementary LF domain. In some embodiments, an LFc domain comprises a nucleotide sequence of about 9 to about 30 nucleotides that is complementary to a LF primer. In some embodiments, about 20% to about 50% of the hybrid primer loop (e.g., a second hybrid primer loop) is complementary to a LF primer. In some embodiments, at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50% of a hybrid primer loop is complementary to a LF primer. In some embodiments, at the most 80%, such as at the most 75%, such as at the most 70%, such as at the most 65%, such as at the most 60%, such as at the most 55%, such as at the most 50% of a hybrid primer loop is complementary to a LF primer. In some embodiments, an LFc domain is directly connected to a Fl domain or linked (e.g., covalently) by one or more nucleotides to a Fl domain. In some embodiments, an LFc domain is directly connected to a F2 domain or linked (e.g., covalently) by one or more nucleotides to a F2 domain.

[0154] In some embodiments, an LFc domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides. In some embodiments, a LBc domain is orcomprises a nucleotide sequence of about 10 nucleotides to about 40 nucleotides, such as about 15 nucleotides to about 35 nucleotides.F2 domains

[0155] In some embodiments, a F2 domain comprises or consists of a nucleotide sequence. In some embodiments, F2 domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof. In some embodiments, a F2 domain is not complementary to any of the first 5 ’ hairpin nucleotide sequence domains. In some embodiments, a F2 domain of a second hybrid primer is the same as an F2p domain of a forward inner primer (FIPp).

[0156] In some embodiments, a F2 domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides. In some embodiments, a F2 domain is or comprises a nucleotide sequence of about 10 nucleotides to about 40 nucleotides, such as about 15 nucleotides to about 35 nucleotides.Flc domains

[0157] In some embodiments, an Flc domain comprises or consists of a nucleotide sequence. In some embodiments, an Flc domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof. In some embodiments, an Flc domain is complementary to a primer domain (e.g., hybrid primer domain). In some embodiments, an Flc domain is complementary to a Fl domain. In some embodiments, an Flc domain is characterized in that it hybridizes to a Fl domain. In some embodiments, an Flc domain and a Fl domain hybridize to form a second hybrid primer stem. In some embodiments, an Flc domain is directly connected to an F2 domain or linked (e.g., covalently) by one or more nucleotides to an F2 domain.

[0158] In some embodiments, a Flc domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides. In some embodiments, a Flc domain is or comprises a nucleotide sequence of about 10 nucleotides to about 40 nucleotides, such as about 15 nucleotides to about 35 nucleotides.

[0159] In some embodiments, a composition comprises(i) a second hybrid primer whose nucleotide sequence comprises:a) a 3’ T2 domain complementary to a T2c domain of the target nucleotide sequence; b) a second 5 ’ hairpin nucleotide sequence comprising four domains from 3’ to the 5’1) a Fl domain;2) a LFc domain;3) a F2 domain; and4) a Flc domain complementary to the Fl domain, wherein at least one domain is not complementary to the target nucleotide sequence.

[0160] In some embodiments, a 3’ T2 domain of a second hybrid primer is capable of annealing to a T2c domain of a target nucleotide when contacted under conditions (e.g., salt, temperature, etc.) that permit annealing. Annealing of the 3’ T2 domain to the T2c domain of the target nucleotide sequence or complement thereof permits extension of the second hybrid primer by a polymerase enzyme (e.g., with stand displacement activity) hereby synthesizing a new strand.Inner primers

[0161] In some embodiments, compositions and methods of the present disclosure comprise an inner primer (e.g., a backward inner primer (BIP) and / or forward inner primer (FIP)). In some embodiments, a primer as described herein is an inner primer. In some embodiments, an inner primer is a BIP. In some embodiments, an inner primer is a FIP (e.g., a FlPt or a FIPp).

[0162] In some embodiments, an inner primer comprises a nucleotide sequence. In some embodiments, an inner primer comprises a nucleotide sequence of about 18 nucleotides to about 60 nucleotides. In some embodiments, an inner primer comprises a nucleotide sequence of about 20 nucleotides to about 50nucleotides.

[0163] In some embodiments, an inner primer comprises one or more primer domains that are not complementary to a target nucleotide sequence. In some embodiments, an inner primer comprises two or more primer domains that are not complementary to a target nucleotide sequence. In some embodiments, at least a part of the inner primernucleotide sequence is not complementary to a target nucleotide sequence. In some embodiments, at least 50% of the inner primer nucleotide sequence is not complementary to a target nucleotide sequence. In some embodiments, at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80% of the inner primer nucleotide sequence is not complementary to a target nucleotide sequence.

[0164] In some embodiments, an inner primer comprises one or more primer domains that are complementary to a target nucleotide sequence. In some embodiments, an inner primer comprises two or more primer domains that are complementary to a target nucleotide sequence.BIP

[0165] In some embodiments, a BIP comprises a nucleotide sequence. In some embodiments, a BIP comprises one or more primer domains. In some embodiments, a BIP comprises two or more primer domains. In some embodiments, one or more BIP primer domains are not complementary to a target nucleotide sequence (e.g., target domain) or complement thereof. In some embodiments, two or more BIP primer domains are not complementary to a target nucleotide sequence (e.g., target domain) or complement thereof.

[0166] In some embodiments, a BIP comprises a B2 domain and a Bic domain. The B2 domain may be located 3’ to the Bic domain. In some embodiments, a BIP comprises a 3’ B2 domain and a 5’ Bic domain. In some embodiments, a B2 domain and a Bic domain are separated by a linker. In some embodiments, a linker is a TTTT linker.B2 domains

[0167] In some embodiments, a B2 domain comprises or consists of a nucleotide sequence. In some embodiments, a B2 domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof. In some embodiments, a B2 domain is not complementary to any of the first 5 ’ hairpin nucleotide sequence domains. In some embodiments, a B2 domain is complementary to a B2c domain. In some embodiments, a B2c domain is formed as a result of synthesis of a DNA strand that is complementary to a B2 domain (e.g., from a primer annealing to the stand synthesized using the first hybrid primer). In some embodiments, a B2 domain of a BIP is the same as a B2 domain of a first hybrid primer.

[0168] In some embodiments, a B2 domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides. In some embodiments, a B2 is or comprises a nucleotide sequence of about 10 nucleotides to about 40 nucleotides, such as about 15 nucleotides to about 35 nucleotides.Bic domains

[0169] In some embodiments, a Bic domain comprises or consists of a nucleotide sequence. In some embodiments, Bic domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof. In some embodiments, a Bic domain is complementary to a primer domain. In some embodiments, a B 1c domain is complementary to a Bl domain (e.g., a first hybrid primer Bl domain). In some embodiments, a Bic domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides. In some embodiments, a B 1c is or comprises a nucleotide sequence of about 10 nucleotides to about 40 nucleotides, such as about 15 nucleotides to about 35 nucleotides.

[0170] In some embodiments, a BIP is capable of annealing to a DNA strand that is complementary to a first hybrid primer (e.g., an extension product of a FlPt, FIPp, LT, or a second hybrid primer). In some embodiments, a BIP is capable of annealing to a B2c domain within a first hybrid primer (e.g., an extension product of a FlPt, FIPp, LT, or second hybrid primer). In some embodiments, a BIP is capable of annealing to a Bl domain and a B2c domain within a first hybrid primer (e.g., an extension product of a FlPt, FIPp, LT, or second hybrid primer). Annealing of the BIP to a complement strand of a first hybrid primer permits extension of the BIP by a polymerase enzyme (e.g., with stand displacement activity) thereby synthesizing a new strand.

[0171] In some embodiments, a composition comprises a BIP whose nucleotide sequence comprises a 3’ B2 domain and a 5’ Bic domain complementary to the Bl domain.

[0172] In some embodiments, a composition comprises(i) a first hybrid primer whose nucleotide sequence comprises: a) a 3’ T1 domain complementary to a Tic domain of the target nucleotide sequence; b) a first 5’ hairpin nucleotide sequence comprising four domains from 3’ to the 5’1) a Bl domain;2) a LBc domain;3) a B2 domain; and4) a Bic domain complementary to the Bl domain, wherein at least one domain is not complementary to the target nucleotide sequence; and(ii) a backward inner primer (BIP) whose nucleotide sequence comprises a 3’ B2 domain and a 5’ Bic domain complementary to the Bl domainFIP

[0173] In some embodiments, a forward inner primer (FIP) comprises a nucleotide sequence. In some embodiments, a FIP comprises one or more primer domains. In some embodiments, a FIP comprises two or more primer domains. A FIP or any of its primer domains may be capable of hybridizing to a hybrid primer (e.g., a second hybrid primer) or a target nucleotide sequence. In some embodiments, a FIP capable of hybridizing to a hybrid primer is a FIPp. In some embodiments, a FIP capable of hybridizing to a target nucleotide sequence is a FlPt.

[0174] In some embodiments, a composition comprises a FIP primer whose nucleotide sequence comprises a 3’ F2 domain and a 5’ Flc domain complementary to the Fl domain.FIPp

[0175] In some embodiments, one or more FIPp primer domains are not complementary to a target nucleotide sequence (e.g., target domain) or complement thereof. In some embodiments, two or more FIPp primer domains are not complementary to a target nucleotide sequence (e.g., target domain) or complement thereof. In some embodiments, one or more FIP primer domains are complementary to a primer (e.g., a primer domain). In some embodiments, two or more FIP primer domains are complementary to a primer (e.g., a primer domain).

[0176] In some embodiments, a FIPp comprises an F2p domain and an Flcp domain. The F2p domain may be located 3’ to the Flcp domain. In some embodiments, a FIPpcomprises a 3’ F2p domain and a 5’ Flcp domain. In some embodiments, an F2p domain and an Flcp domain are separated by a linker. In some embodiments, a linker is a TTTT linker.F2p domains

[0177] In some embodiments, an F2p domain comprises or consists of a nucleotide sequence. In some embodiments, an F2p domain is not complementary to a target nucleotide sequence (e.g., a target domain). In some embodiments, an F2p domain is identical to a primer domain of a second 5’ hairpin nucleotide sequence. In some embodiments, an F2p domain is identical to a F2 domain. In some embodiments, an F2p domain is complementary to an F2c domain. In some embodiments, an F2c domain is formed as a result of synthesis of a DNA stand that is complementary to an F2 domain (e.g., from a primer annealing to the stand synthesized using the second hybrid primer).

[0178] In some embodiments, an F2p domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides. In some embodiments, an F2p is or comprises a nucleotide sequence of about 10 nucleotides to about 40 nucleotides, such as about 15 nucleotides to about 35 nucleotides.Flcp domains

[0179] In some embodiments, an Flcp domain comprises or consists of a nucleotide sequence. In some embodiments, an Flcp domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof. In some embodiments, an Flcp domain is complementary to a primer domain. In some embodiments, an Flcp domain is complementary to an Fl domain (e.g., a second hybrid primer Fl domain). In some embodiments, an Flcp domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides. In some embodiments, an Flcp is or comprises a nucleotide sequence of about 10 nucleotides to about 40 nucleotides, such as about 15 nucleotides to about 35 nucleotides.

[0180] In some embodiments, a FIPp is capable of annealing to a DNA strand that is complementary to a second hybrid primer (e.g., an extension product of a BIP, LB, or a first hybrid primer). In some embodiments, a FIPp is capable of annealing to a Fl domain and an F2c domain within a second hybrid primer (e.g., an extension production of a BIP, LB, or a first hybrid primer). Annealing of the FIPp to a complement strand of the second hybridprimer permits extension of the FIPp by a polymerase enzyme (e.g., with stand displacement activity) thereby synthesizing a new strand.

[0181] Tn some embodiments, a composition comprises a FTPp whose nucleotide sequence comprises a 3’ F2p domain and a 5’ Flcp domain complementary to a Fl domain.FlPt

[0182] In some embodiments, one or more FlPt primer domains are complementary to a target nucleotide sequence (e.g., target domain) or complement thereof. In some embodiments, two or more FlPt primer domains are complementary to a target nucleotide sequence (e.g., target domain) or complement thereof. In some embodiments, a FlPt primer consists or comprises of a ribonucleotide sequence. In some embodiments, a FlPt ribonucleotide primer can be used to convert an ssRNA into an ssDNA and / or dsDNA by reverse transcription. In some embodiments, a FlPt primer consists or comprises of a deoxyribonucleotide sequence. In some such embodiments, a FlPt deoxyribonucleotide primer can be used to synthesize a complementary DNA strand a target deoxyribonucleotide sequence by a polymerase.

[0183] In some embodiments, a FlPt comprises an F2t domain and an Flct domain. The F2t domain may be located 3’ to the Flct domain. In some embodiments, a FlPt comprises a 3’ F2t domain and a 5’ Flct domain. . In some embodiments, an F2t domain and an Flct domain are separated by a linker. In some embodiments, a linker is a TTTT linker.

[0184] In some embodiments, a FlPt-linked nucleotide strand comprises complementary regions at the 5’ end, resulting in formation of a stem-loop structure at said 5’ end. In some embodiments, a FlPt-linked nucleotide strand may be released as a result of DNA synthesized initiated by a F3 primer.F2t domains

[0185] In some embodiments, an F2t domain comprises or consists of a nucleotide sequence. In some embodiments, an F2t domain is complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof. In some embodiments, an F2t domain is complementary to an F2ct domain of a target sequence. In some embodiments, anF2ct domain is formed as a result of synthesis of a DNA strand that is complementary to an F2t domain of the target sequence.

[0186] In some embodiments, an F2p domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides. In some embodiments, an F2t is or comprises a nucleotide sequence of about 10 nucleotides to about 40 nucleotides, such as about 15 nucleotides to about 35 nucleotides.Flct domains

[0187] In some embodiments, an Flct domain comprises or consists of a nucleotide sequence. In some embodiments, an Flct domain is complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof. In some embodiments, an Flcp domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides. In some embodiments, an Flcp is or comprises a nucleotide sequence of about 10 nucleotides to about 40 nucleotides, such as about 15 nucleotides to about 35 nucleotides.

[0188] In some embodiments, a FlPt is capable of annealing to an extension product of a target nucleotide sequence (i.e., a strand that is complementary to a target nucleotide sequence) or complement thereof. Annealing of the FlPt to a complement strand of a target nucleotide sequence or complement thereof permits extension of the FlPt by a polymerase enzyme (e.g., with stand displacement activity) hereby synthesizing a new strand.

[0189] In some embodiments, a composition comprises a FlPt whose nucleotide sequence comprises a 3’ F2t domain and a 5’ Flct domain complementary to the Fit domain.

[0190] In some embodiments, a composition comprises a FlPt primer whose nucleotide sequence comprises: a) a 3 ’ F2t domain complementary to an F2ct domain of the target nucleotide sequence, wherein F2ct and Tic are non-overlapping domains located on opposite or complementary strands of the target nucleotide sequence; and b) a 5’ Flct domain complementary to a Fit domain of the target nucleotide sequence.

[0191] In some embodiments, an Flct domain is located 5’ to an F2ct domain of the same strand, i.e., target stand or complement thereof.Outer primers

[0192] In some embodiments, technologies of the present disclosure utilize an outer primer (e.g., a B3 primer, F3 primer, or both). In some embodiments, a primer as described herein is an outer primer. In some embodiments, an outer primer is a B3 primer. In some embodiments, an outer primer is a F3 primer.

[0193] As described herein, a hybrid primer is capable of annealing to a Tc domain of a target nucleotide sequence. In some embodiments, a B3 primer binds to a B 3c domain directly upstream of the Tc domain (Figure 4). Annealing of the hybrid primer to its complement target nucleotide sequence permits extension by a polymerase hereby forming a complement to the target polynucleotide sequence. Extension of the B3 primer by a polymerase having strand-displacement activity releases the hybrid primer extension product. The FIP and F3 primers can then bind directly to this extension product, without the need to invade into a duplex region. In some embodiments, it is advantage to include a F3 primer, a B3 primer, or both.

[0194] In some embodiments, a polymerase (e.g., with strand displacement activity) extends form an outer primer (e.g., a B3 primer or F3 primer), displacing and releasing a hybrid primer or inner (e.g., FIP or BIP) -linked complementary strand.B3 primers

[0195] In some embodiments, a B3 primer comprises a nucleotide sequence. In some embodiments, a B3 primer is or comprises a primer domain. In some embodiments, a B3 primer is or comprises a B3 domain. In some embodiments, a B3 domain is complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof. In some embodiments, a B3 domain is complementary to a B3c domain of a target nucleotide sequence. In some embodiments, a B3c domain is located 3’ to a Tic domain on the same strand. Thus a B3 primer may anneal to a region on a target nucleotide that is outside of that which a first hybrid primer annealed (e.g., Tic domain). A nucleic acid polymerase enzyme (e.g., with stand displacement activity) can extend from the B3 primer, displacing and releasing a first hybrid primer-linked complementary strand.

[0196] In some embodiments, a composition comprises a B3 primer whose nucleotide sequence is complementary to a B3c domain of a target nucleotide sequence.F3 primers

[0197] In some embodiments, an F3 primer comprises a nucleotide sequence. In some embodiments, an F3 primer is or comprises a primer domain. In some embodiments, an F3 primer is or comprises a F3 domain. In some embodiments, an F3 domain is complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof. In some embodiments, an F3 domain is complementary to an F3c domain of a target nucleotide sequence. In some embodiments, an F3c domain is located 3’ to a T2c domain on the same target strand. Thus a F3 primer may anneal to a region on a target nucleotide that is outside of that which a first hybrid primer annealed (e.g., T2c domain). A nucleic acid polymerase enzyme e.g., with stand displacement activity) can extend from the F3 primer, displacing and releasing a first hybrid primer-linked complementary strand.

[0198] In some embodiments, a composition comprises a F3 primer whose nucleotide sequence is complementary to an F3c domain of a target nucleotide sequence.Loop-primers

[0199] In some embodiments, technologies of the present disclosure utilize a loop primer (e.g., a LB primer and / or LF primer). In some embodiments, a cLAMP reaction can further utilize loop primers which comprise nucleotide sequences complementary to a single stranded loop region produced in a cLAMP reaction described herein. Typically, when used in a cLAMP method according to the present disclosure, loop-primers hybridize to an intermediate cLAMP amplicon (e.g., amplicons produced by methods and compositions described herein, such as a hairpin-loop amplicon, a dumbbell-shaped amplicon, or a combination) and provide an increased number of starting points for DNA synthesis. Increasing cLAMP product generated and / or decreasing the duration of time required for detecting a nucleic acid. Loop primers are particular useful in facilitating subsequent rounds of amplification through extension on the loops and annealing of the primers.

[0200] In some embodiments, a primer as described herein is a loop-primer. In some embodiments, a loop-primer is a LB primer. In some embodiments, a loop-primer is a LF primer.LB primers

[0201] In some embodiments, a LB primer comprises a nucleotide sequence. In some embodiments, a LB primer is or comprises a primer domain. In some embodiments, a LB primer is or comprises a LB domain. In some embodiments, a LB domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof. In some embodiments, a LB domain is complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof. In some embodiments, a LB primer comprises a nucleotide sequence complementary to the single stranded loop region of the first hybrid primer. In some embodiments, a LB domain is complementary to an LBc domain (e.g., an LBc domain of a first hybrid primer).

[0202] In some embodiments, a LB primer is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides. In some embodiments, a LB primer is or comprises a nucleotide sequence of about 10 nucleotides to about 40 nucleotides, such as about 15 nucleotides to about 35 nucleotides.

[0203] Annealing of the LB domain to the LBc domain of the second hybrid primer or amplicons produced by methods and compositions described herein permits extension of the LB domain by a polymerase enzyme (e.g., with stand displacement activity) hereby synthesizing a new strand.

[0204] In some embodiments, a composition comprises a LB primer whose nucleic acid sequence comprises a LB domain that is complementary to the LBc domain.LF primers

[0205] In some embodiments, a LF primer comprises a nucleotide sequence. In some embodiments, a LF primer is or comprises a primer domain. In some embodiments, a LF primer is or comprises a LF domain. In some embodiments, a LF domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof. In some embodiments, a LB domain capable of hybridizing to a hybrid primer is an LBp. In some embodiments, a LF domain is complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof. In some embodiments, a LF domain capable of hybridizing to a target nucleotide sequence or complement thereof is an LFt.

[0206] In some embodiments, a composition comprises a LF primer whose nucleotide sequence is or comprises a LF domain that is complementary to an LFc domain.LFp

[0207] In some embodiments, an LFp primer comprises a nucleotide sequence complementary to the single stranded loop region of the second hybrid primer. In some embodiments, an LFp domain is complementary to an LFc domain (e.g., an LFc domain of a second hybrid primer).

[0208] In some embodiments, an LFp primer is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides. In some embodiments, an LFp primer is or comprises a nucleotide sequence of about 10 nucleotides to about 40 nucleotides, such as about 15 nucleotides to about 35 nucleotides.

[0209] Annealing of the LFp domain to the LFc domain of the second hybrid primer permits extension of the LFp domain by a polymerase enzyme (e.g., with stand displacement activity) hereby synthesizing a new strand.

[0210] In some embodiments, a composition comprises an LFp primer whose nucleotide sequence is or comprises an LFp domain that is complementary to an LFc domain.LFt

[0211] In some embodiments, an LFt primer comprises a nucleotide sequence complementary to a target nucleotide sequence. In some embodiments, an LFt domain is complementary to an LFc domain (e.g., an LFc domain of a target sequence).

[0212] In some embodiments, an LFt primer is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides. In some embodiments, an LFt primer is or comprises a nucleotide sequence of about 10 nucleotides to about 40 nucleotides, such as about 15 nucleotides to about 35 nucleotides.

[0213] LFt is capable of annealing to an extension product of a FlPt (i.e., a strand that is complementary to a first hybrid primer extension product). Annealing of the LFt to anLFc domain permits extension of the LFt by a polymerase enzyme (e.g., with stand displacement activity) hereby synthesizing a new strand.

[0214] Tn some embodiments, a composition comprises an LFt primer whose nucleotide sequence is or comprises an LFt domain that is complementary to an LFct domain.Modifications

[0215] In some embodiments, a primer described herein comprises one or more modified nucleotides (e.g., modified ribonucleotides, modified deoxyribonucleotides, or a combination hereof).

[0216] In some embodiments, a modified nucleotide is a peptide nucleic acid (PNA). In some embodiments, a modified nucleotide is a locked nucleic acid (LNA). Peptide nucleic acids, locked nucleic acids, or a combination hereof may be used to increase primer Tm and / or specificity. Primers comprising peptide nucleotides, locked nucleotides, or a combination may be particularly useful in methods of detecting a target nucleotide sequence having one or more SNP sites in order to increase specificity.

[0217] In some embodiments, a modified nucleotide is a 2’-Fluoro-nucleic acid or a 2’-O-methyl-nucleic acid. Primers comprising 2’-Fluoro-nucleic acid modifications, 2’-O- methyl-nucleic acid modifications or a combination have increased nuclease resistance compared to non-modified primers, as well as increased Tm of the 2’-Fluoro-nucleic acid modifications and / or 2’-O-methyl-nucleotide modified domain(s).

[0218] In some embodiments, a modified deoxyribonucleotide is a phosphorothioated deoxyribonucleotide. In some embodiments, a modified deoxyribonucleotide is a phosphodiester deoxyribonucleotide. In some embodiments, a modified deoxy ribonucleotide as described herein destabilizes helices. In some embodiments, a nucleic acid comprising a modified deoxyribonucleotide melts at lower temperatures relative to a control without modified deoxyribonucleotide. In some embodiments, a nucleic acid comprising a modified deoxyribonucleotide can be amplified at lower temperatures relative to a control without modified deoxyribonucleotide.

[0219] In some embodiments, a primer comprises a modified nucleotide in place of at least one guanine or adenine. In some embodiments, a modified nucleotide is a 2- Aminopurine (e.g., a purine analog of guanine and adenine). In some embodiments, a primer comprising a 2- Aminopurine is useful in fluorescence readouts.

[0220] In some embodiments, one of more of the modifications listed herein provides stable primers that are more resistant to nucleases and / or proteases compared to primers or other nucleotides without any modifications.Fluorophores

[0221] In some embodiments, a primer described herein comprises a fluorophore. In some embodiments, a fluorophore is a Cy5, a FAM, a TxRed, a YakYel, or a HEX. In some embodiments, a fluorophore is attached to the 5’ end of the primer. In some embodiments, a fluorophore is attached to the 3’ end of the primer. In some embodiments, a fluorophore is within the primer. In some embodiments, a fluorophore emits a signal when it is separated from a quencher (e.g., the primer is cleaved in such a way that the fluorophore and quencher are separated).Quenchers

[0222] In some embodiments, a primer described herein comprises a quencher. In some embodiments, a quencher is attached to the 5’ end of the primer. In some embodiments, a quencher is attached to the 3’ end of the primer. In some embodiments, a quencher is within the primer. In some embodiments, a quencher is a 3IAbRQsp, a BHQ2, a 3IABkFQ, or a BHQl.Sample

[0223] In some embodiments, technologies (e.g., primers, compositions, kits, amplicons, and methods) of the present disclosure are contacted with a sample. In some embodiments, the present disclosure provides compositions, kits and methods for synthesis and / or amplification and / or detection of target nucleotide sequences in a sample.

[0224] In some embodiments, a sample is or comprises a biological sample. A biological sample typically refers to a sample obtain or derived from a biological source, for example, including a tissue, organism, or cell culture) of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a sample is obtained or derived from an organism (e.g., a mammalian organism, for example, including a human). In some embodiments, a biological sample is or comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and / or excretions; and / or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, obtained cells are or include cells from an individual from whom the sample is obtained. In some embodiments, a sample is a “primary sample’’ obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of certain components, etc.

[0225] In some embodiments, a source of interest comprises a virus or microbe. In some embodiments, a sample is obtained or derived from virus or microbe. In some embodiments, a viral or microbial sample is a “primary sample” obtained directly from a source of interest by any appropriate means. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primarysample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of certain components, etc. In some embodiments, a sample comprises a target nucleotide sequence.

[0226] In some embodiments, a sample is a crude sample (e.g., a primary sample or a sample that has undergone minimal processing).

[0227] In some embodiments, a sample is an environmental sample, such as a food sample (fresh fruits or vegetables, meats), a beverage sample, a paper surface, a fabric surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a wastewater sample, a saline water sample, exposure to atmospheric air or other gas sample, or a combination thereof. In some embodiments, provided technologies are useful for assessment of environmental samples. For example, household / commercial / industrial surfaces made of any materials including, but not limited to, metal, wood, plastic, rubber, or the like, may be swabbed and tested for contaminants.

[0228] In some embodiments, samples (e.g., viral particles and / or cells) are lysed (e.g., processed) using sodium hydroxide (NaOH). In some embodiments, samples are lysed with NaOH at ambient temperature (e.g., room temperature). In some embodiments, the concentration of NaOH is about 1 rnM NaOH to about 200 mM NaOH. In some embodiments, the concentration of NaOH is about 10 mM NaOH to about 100 mM. In some embodiments, samples are lysed with NaOH for about 1 second to about 10 min, such as about 10 seconds to about 8 min, such as about 1 min to about 5 min, such as about 2 min to about 4 min. In some embodiments, samples are treated with NaOH to inhibit or reduce RNase activity. In some embodiments, NaOH releases viral nucleic acids from a viral sample. In some embodiments, NaOH denatures double stranded DNA or RNA (e.g., separates strands).

[0229] In some embodiments, samples (e.g., viral particles and / or cells) are lysed (e.g., processed) using potassium hydroxide (KOH). In some embodiments, samples comprising DNA (e.g., dsDNA) are treated with KOH. In some embodiments, samples are lysed with KOH at ambient temperature (e.g., room temperature). In some embodiments, the concentration of KOH is about 1 mM KOH to about 200 mM KOH. In some embodiments, the concentration of KOH is about 10 mM KOH to about 100 mM. In some embodiments,samples are lysed with KOH for about 1 second to about 10 min, such as about 10 seconds to about 8 min, such as about 1 min to about 5 min, such as about 2 min to about 4 min. In some embodiments, samples are treated with KOH to inhibit or reduce RNase activity. In some embodiments, KOH releases viral nucleic acids from a viral sample. In some embodiments, KOH denatures double stranded DNA or RNA (e.g., separates strands). In some such embodiments, KOH denaturation separates dsDNA and produces ssDNA.Target nucleotide sequence

[0230] Those of ordinary skill in the art will readily appreciate that technologies of the present disclosure are broadly applicable to achieve synthesis and / or amplification and / or detection of a wide range of target nucleotide sequences.

[0231] A target (e.g., target nucleotide sequence) DNA or RNA may be a DNA or RNA or a part of a DNA or RNA to which a contacting nucleic acid or nucleic acids (e.g., primer(s) and / or primer domain(s)) have complementarity. In some embodiments, a target nucleotide sequence be double-stranded. In some embodiments, a template nucleic acid may be single-stranded. In some embodiments, a target nucleotide sequence may be genomic DNA, mitochondrial DNA, viral DNA, plasmid DNA, synthetic dsDNA, or RNA. In some embodiments, a single-stranded nucleic acid comprises single-stranded viral DNA, viral RNA, messenger RNA, ribosomal RNA, transfer RNA, microRNA, short interfering RNA, small nuclear RNA, synthetic RNA, and / or synthetic ssDNA.

[0232] In some embodiments, a target nucleotide sequence is a copied and / or amplified target nucleotide sequence.

[0233] In some embodiments, a useful target nucleotide sequence in accordance with the present disclosure is not limited to a particular length; in some embodiments, a target nucleotide sequence is any length (oligonucleotides or polynucleotides) comprising a sequence to which a guide sequence hybridizes. In some embodiments, a target nucleotide sequence comprises coding and / or non-coding regions. In some embodiments, a target nucleotide sequence comprises exons, introns, mRNA, tRNA, rRNA, siRNA, shRNA, miRNA, ribozymes, cDNA, plasmids, vectors, exogenous nucleotide sequences, and / or endogenous nucleotide sequences. In some embodiments, a target nucleotide sequence comprises modified nucleotides, for example, including methylated nucleotides ornucleotide analogs. In some embodiments, a target nucleotide sequence may be interspersed with non-nucleic acid components. In some embodiments, a target nucleotide is a single-, double-, or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. In some embodiments, a target nucleotide sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, a target nucleotide is ex vivo. In some embodiments, a target nucleotide sequence is present in an in vitro system. In some embodiments, a target nucleotide sequence is present in a sample, e.g., in a biological sample or in an environmental sample.

[0234] In some embodiments, target nucleotide sequences include, for example, nucleic acids from an infectious agent (e.g., a virus, microbe, parasite, etc.), nucleic acids indicative of a particular physiological state or condition (e.g., presence or state of a disease, disorder or condition such as, for example, cancer or an inflammatory or metabolic disease, disorder or condition, etc.), prenatal nucleic acids, etc..

[0235] In some embodiments, a target nucleotide is a viral, a bacterial, a fungal, a protozoan, or a parasitic sequence.

[0236] In some embodiments, a target nucleotide sequence or complement thereof is recognized by CRISPR-Cas technologies (e.g., a guide polynucleotide) and binds a Cas enzyme as described herein. In some embodiments, a target nucleotide sequence or complement thereof comprises a specific, recognizable, protospacer adjacent motif (PAM).In some embodiments, provided technologies are particularly useful or applicable for detection of low- abundance (e.g., less than about 10 fM, or about 1 fM, or about 100 aM, or about 10 aM or about 1 aM) nucleic acids.Polymerases

[0237] In some embodiments, methods and compositions described herein use a polymerase, for example, for target nucleotide sequence synthesis, conversion of one nucleic acid type to another, and / or transcription.

[0238] In some embodiments, compositions, kits and methods of present disclosure comprise a polymerase. In some embodiments, a polymerase is a strand displacing polymerase. A strand displacing polymerase is able to displace downstream DNA during elongation. In some embodiments, wherein a target nucleotide sequence is a RNA, a reverse transcriptase can first be used to copy the RNA target into a cDNA nucleotide sequence suitable for nucleotide synthesis. In some embodiments, a polymerase is a DNA polymerase. In some embodiments, a strand displacing polymerase has elongation activity at ambient temperature. In some embodiments, a strand displacing polymerase has elongation activity at temperatures ranging from about 30°C to about 75°C. In some embodiments, a strand displacing polymerase has elongation activity at room temperature. In some embodiments, a strand displacing polymerase is useful in isothermal amplification.

[0239] In some embodiments, a polymerase has strand displacing activity.

[0240] In some embodiments, a polymerase is a Bsu Polymerase, Bsm Polymerase,Bst Polymerase, or a combination. In some embodiments, a Bst polymerase is a Bst Polymerase 1.0-3.0 (New England Biolabs). In some embodiments, a Bst polymerase is a Bst Polymerase 2.0 (New England Biolabs).

[0241] In some embodiments, a polymerase is a Bsu DNA Polymerase I (Bsu), phi29, Bst 20 DNA Polymerase, Klenow Large Fragment, Klenow Exo -, Bsu Large Fragment, Isopol, and Isopol SD+, or variants thereof. In some embodiments, a strand displacing polymerase is Bsu or a variant thereof. In some embodiments, a strand displacing polymerase is Klenow or a variant thereof.

[0242] In some embodiments, a cLAMP reaction utilizes a DNA polymerase enzyme, preferably a DNA polymerase with high strand displacement activity.Reverse Transcriptase

[0243] In some embodiments, methods and compositions described herein use a reverse transcriptase, for example, for target nucleotide sequence synthesis, conversion of one nucleic acid type to another, and / or transcription.

[0244] In some embodiments, compositions, kits and methods of present disclosure comprise a reverse transcriptase.

[0245] In some embodiments, a target nucleotide sequence consists or comprises of a deoxyribonucleotide sequence. In some embodiments, a primer as described herein (e.g., a FlPt, a BIP, a B3, a F3, and / or a hybrid primer) anneals to a target ribonucleotide sequence and a reverse transcriptase extends the primer hereby synthesizing a DNA strand. In some embodiments, a complement DNA strand is contacted with one or more primers as described herein.

[0246] In some embodiments, synthesis of a nucleotide sequence complementary to a target nucleotide sequence or an amplicon requires a particular type of nucleic acid (e.g., ssDNA, dsDNA, ssRNA) starting material (e.g., substrate). In some embodiments, a target nucleotide sequence may need to be converted to a different type of nucleic acid prior to synthesis of a nucleotide sequence complementary to a target nucleotide sequence or an amplicon. In some embodiments, amplification of a target nucleotide is initiated by conversion of ssRNA to dsDNA by reverse transcription. In some embodiments, methods described herein utilize ssRNA. In some embodiments, methods described herein utilize dsDNA.

[0247] In some embodiments, wherein a target nucleotide sequence is ssDNA, a reaction to convert ssDNA to dsDNA is conducted. In some embodiments, ssDNA is converted to dsDNA by any method known to one of ordinary skill in the art, for example, polymerase chain reaction (PCR) or Klenow reaction.

[0248] In some embodiments, wherein a target nucleotide sequence is a RNA, a RNA is converted to dsDNA by any method known to one of ordinary skill in the art, for example, by a reverse transcription reaction, prior to target nucleotide sequence synthesis and / or amplification. In some embodiments, wherein a target nucleotide sequence is RNA, RNA is converted to dsDNA prior to target nucleotide sequence synthesis and / or amplification. In some embodiments, a primer as described herein is used in a reverse transcription reaction to generate one or more amplicons (e.g., a hairpin-loop amplicon). Insome embodiments, such an amplicon may be a template for a hybrid primer (e.g., a first hybrid primer, a second hybrid primer, or both).

[0249] In some embodiments, technologies described herein uses a reverse transcriptase. In some embodiments, a reverse transcriptase is a Warmstart RTx Reverse Transcriptase, Avian Myeloblastosis Virus (AMV) Reverse Transcriptase and / or derivatives, or Moloney Murine Leukemia Virus (M-MuLV, MMLV) Reverse Transcriptase and / or derivatives, or combinations hereof.Conditions and amplification reagents

[0250] In some embodiments, a target nucleotide sequence, extension products, or a primer is contacted with one or more primers under conditions (e.g., salt, temperature, etc.) that permit annealing to a complementary sequence.

[0251] In some embodiments, amplification reagents comprises one or more dNTPs. In some embodiments, amplification reagents comprises a buffer.

[0252] In some embodiments, the methods of amplification described herein (i.e., cLAMP) is performed under isothermal conditions. In some embodiments, cLAMP is performed at ambient temperatures. In some embodiments, cLAMP is performed at temperatures within a range of about 20°C to about 65°C. In some embodiments, cLAMP is performed at temperatures within a range of about 20°C to about 60°C. In some embodiments, cLAMP is performed at temperatures within a range of about 20°C to about 55°C. In some embodiments, cLAMP is performed at temperatures within a range of about 20°C to about 50°C. In some embodiments, cLAMP is performed at temperatures within a range of about 20°C to about 45°C. In some embodiments, cLAMP is performed at temperatures within a range of about 20°C to about 40°C. In some embodiments, cLAMP is performed at temperatures within a range of about 20°C to about 35°C. In some embodiments, cLAMP is performed at temperatures within a range of about 20°C to about 30°C. In some embodiments, cLAMP is performed at temperatures within a range of about 20°C to about 25°C.

[0253] In some embodiments, technologies described herein (e.g., primers, compositions, kits, amplicons, and methods) comprises sodium, magnesium, salts, orcombinations hereof. In some embodiments, the concentration of sodium is within the range of 0 mM to 100 mM. In some embodiments, the concentration of magnesium is within the range of 0 mM to 20 mM. In some embodiments, a salt is Tris. In some embodiments, the concentration of Tris is 1 mM to 100 mM.

[0254] In some embodiments, compositions and methods described herein uses an inorganic pyrophosphatase (PPase). A PPase may catalyze the hydrolysis of inorganic pyrophosphate and can be used for enhancement of DNA replication. In some embodiments, a PPase is a thermostable pyrophosphatase (TIPP). In some embodiments, a composition as described herein comprises a TIPP. In some embodiments, a kit as described herein comprises a TIPP. In some embodiments, methods described herein utilizes a TIPP. In some embodiments, a step of incubating a target nucleotide sequence with a composition according to the present disclosure, a DNA polymerase, amplification reagents is performed in the presence of TIPP.

[0255] In some embodiments, compositions and methods comprise dNTPs and primers used at any concentration appropriate for the invention, such as including, but not limited to, a concentration of 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, or the like.Hairpin-loop amplicons

[0256] In some embodiments, methods and compositions described herein produce a hairpin-loop amplicon. In some embodiments, a hairpin-loop amplicon is or comprises a nucleotide sequence. In some embodiments, a hairpin-loop amplicon is or comprises a ribonucleotide sequence. In some embodiments, a hairpin-loop amplicon is or comprises a deoxyribonucleotide sequence. In some embodiments, a hairpin-loop amplicon comprises of a 5’ hybrid primer as described herein and a 3’ target sequence or complement thereof.

[0257] In some embodiments, a hairpin-loop amplicon is be formed by primer extension of a target (T) domain (e.g., T1 domain or T2 domain) of a hybrid primer that hybridizes to a target nucleotide. In some embodiments, a hairpin-loop amplicon if formedby primer extension of a target (T) domain (e.g., T1 domain or T2 domain) of a hybrid primer that hybridizes to a complement of a target nucleotide (e.g., an extension product). In some embodiments, a complement of a target nucleotide (e.g., an extension product) is an inner primer extension product.

[0258] In some embodiments, a hairpin-loop amplicon comprises a nucleotide sequence comprising: a) a 3 ’ target nucleotide sequence; b) a 5’ hairpin nucleotide sequence comprising four domains from the 3’ to the 5’1) a Bl domain;2) a LBc domain;3) a B2 domain; and4) a B 1c domain complementary to the B 1 domain; wherein at least one domain is not complementary to the target nucleotide.

[0259] In some embodiments, a hybrid primer exceeds half of the overall size of the hairpin-loop amplicon. In some embodiments, at least 50% of the hairpin-loop amplicon is not complementary to a target nucleotide sequence. In some embodiments, at least 60% of the hairpin-loop amplicon is not complementary to a target nucleotide sequence. In some embodiments, at least 70% of the hairpin-loop amplicon is not complementary to a target nucleotide sequence. In some embodiments, at least 80% of the hairpin-loop amplicon is not complementary to a target nucleotide sequence. In some embodiments, at least 90% of the hairpin-loop amplicon is not complementary to a target nucleotide sequence.

[0260] In some embodiments, one or more target single nucleotide polymorphism(s) (SNP) are incorporated into a hairpin-loop amplicon. In some embodiments, one or more guide polynucleotides are capable of hybridizing to a hairpin-loop amplicon comprising one or more SNPs.

[0261] In some embodiment, a hairpin-loop amplicon is synthesized by a reverse transcriptase. In some embodiments, a hairpin-loop amplicon is synthesized by a polymerase (e.g., a DNA polymerase, such as a DNA polymerase having strand displacement activity). In some embodiments, a hairpin-loop amplicon is synthesized by a reverse transcriptase and a polymerase.Dumbbell-shaped amplicons

[0262] In some embodiments, technologies described herein produces a dumbbellshaped amplicon. In some embodiments, a dumbbell-shaped amplicon can serve as a template for target nucleotide sequence synthesis (e.g., amplification).

[0263] In some embodiments, a dumbbell- shaped amplicon is or comprises a nucleotide sequence. In some embodiments, a dumbbell- shaped amplicon comprises from its 3’ end to its 5’ a first hybrid primer as described herein, a target sequence or complement thereof and a second hybrid primer as described herein.

[0264] The present disclosure provides several non-limiting examples of mechanisms by which a dumbbell-shaped amplicon can be produced. Exemplary variations of producing a dumbbell- shaped amplicon are described in Examples 1-9.

[0265] In some embodiments, a dumbbell- shaped amplicon comprises a nucleotide sequence comprising from its 3’ end to its 5’ end: a) a first hairpin nucleotide acid sequence comprising four domains1) a Bl domain;2) a B2c domain;3) a LB domain; and4) a B 1c domain complementary to the B 1 domain; wherein at least one domain is not complementary to the target nucleotide; b) a target nucleotide sequence or complement thereof; and c) a second hairpin nucleotide acid sequence comprising four domains1) a Fl domain;2) a LFc domain;3) a F2 domain; and4) a Flc domain complementary to the Fl domain.

[0266] In some embodiments, at least two domains of the dumbbell-shaped amplicon are not complementary to a target nucleotide sequence. In some embodiments, at least three domains of the dumbbell-shaped amplicon are not complementary to a target nucleotide sequence. In some embodiments, at least four domains of the dumbbell- shaped amplicon are not complementary to a target nucleotide sequence. In some embodiments, at least five domains of the dumbbell-shaped amplicon are not complementary to a target nucleotide sequence. In some embodiments, at least six domains of the dumbbell-shaped amplicon are not complementary to a target nucleotide sequence. In some embodiments, at least seven domains of the dumbbell-shaped amplicon are not complementary to a target nucleotide sequence. In some embodiments, all domains of the dumbbell-shaped amplicon are not complementary to a target nucleotide sequence.

[0267] In some embodiments, at least 50%, such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% of the dumbbell- shaped amplicon is not complementary to a target nucleotide sequence. In some embodiments, at least 50% of the dumbbell-shaped amplicon is not complementary to a target nucleotide sequence. In some embodiments, at least 55% of the dumbbell-shaped amplicon is not complementary to a target nucleotide sequence. In some embodiments, at least 60% of the dumbbell-shaped amplicon is not complementary to a target nucleotide sequence. In some embodiments, at least 65% of the dumbbell-shaped amplicon is not complementary to a target nucleotide sequence. In some embodiments, at least 70% of the dumbbell-shaped amplicon is not complementary to a target nucleotide sequence. In some embodiments, at least 75% of the dumbbell- shaped amplicon is not complementary to a target nucleotide sequence. In some embodiments, at least 80% of the dumbbell-shaped amplicon is not complementary to a target nucleotide sequence. In some embodiments, at least 85% of the dumbbell-shaped amplicon is not complementary to a target nucleotide sequence. In some embodiments, at least 90% of the dumbbell-shaped amplicon is not complementary to a target nucleotide sequence. In some embodiments, at least 95% of the dumbbell-shaped amplicon is not complementary to a target nucleotide sequence.

[0268] In some embodiments, one or more target SNP are incorporated into a dumbbell-shaped amplicon. In some embodiments, one or more guide polynucleotides are capable of hybridizing to a dumbbell-shaped amplicon comprising one or more SNPs.Uses and methods

[0269] Those skilled in the art, reading the present disclosure, will appreciate that provided compositions and methods may be utilized in numerous and varying contexts including, but not limited to target nucleotide sequence synthesis (e.g., primer extension), amplification, detection, or a combination. For example, methods and / or compositions as provided herein may be utilized in a target nucleotide synthesis reaction, e.g., a cLAMP reaction. In some embodiments, provided compositions and methods may be used to determine or confirm presence or absence of a target nucleotide. In some embodiments, provided compositions and methods may be used to amplify a target nucleotide. In some embodiments, provided compositions and methods may be used to quantify amount of target nucleotide present in a particular sample. In some embodiments, target nucleotide synthesis is combined with other technologies (e.g., detection technologies). In some embodiments, other detection technologies utilize a CRISPR / Cas system, e.g., with collateral activity e.g., SHERLOCK or DETECTR). In some embodiments, compositions, primers, amplicons, methods, kits and uses described herein may be used for diagnostic purposes.

[0270] In some embodiments, the present disclosure provides methods of producing an amplicon, such as a hairpin-loop amplicon, a dumbbell- shaped amplicon, or both. In some embodiments, an amplicon comprises a target nucleotide sequence or a complement or a fragment thereof. Exemplary methods of producing an amplicon are provided herein (e.g., in the Examples).

[0271] In some embodiments, the disclosure provides methods and compositions for producing a hairpin- loop amplicon comprising a target nucleotide sequence or complement thereof. In some embodiments, methods of the present disclosure comprise the steps of:(A) contacting a target nucleotide sequence with a DNA polymerase, amplification reagents and a first hybrid primer whose nucleotide sequence comprises: a) a 3’ T1 domain complementary to a Tic domain of the target nucleotide sequence; b) a first 5’ hairpin nucleotide acid sequence comprising four domains from 3’ to the 5’1) a Bl domain;2) a LBc domain;3) a B2 domain; and4) a B 1c domain complementary to the B 1 domain; wherein at least one domain is not complementary to the target nucleotide; and(B) incubating the target nucleotide sequence with the DNA polymerase, amplification reagents and the first hybrid primer so that the single stranded hairpin-loop product is generated.

[0272] In some embodiments, a method for producing a dumbbell-shaped amplicon, comprises the steps of:(A) contacting a target nucleotide sequence with a DNA polymerase having strand displacement activity, amplification reagents and a plurality of primers comprising: i) a first hybrid primer whose nucleotide sequence comprises: a) a 3’ T1 domain complementary to a Tic domain of the target nucleotide sequence; b) a 5 ’ hairpin nucleotide acid sequence comprising four domains from 3’ to the 5’1) a Bl domain;2) a LBc domain;3) a B2 domain; and4) a Bic domain complementary to the Bl domain; wherein at least one domain is not complementary to the target nucleotide; and ii) a second primer selected from a second hybrid primer whose nucleotide sequence comprises: a) a 3’ T2 domain complementary to a T2c domain of the target nucleotide sequence, wherein Tic and T2c are non-overlappingdomains located on opposite or complementary strands of the target nucleotide sequence; b) a second 5’ hairpin nucleotide acid sequence comprising four domains from 3’ to the 5’1) a Fl domain;2) a LFc domain;3) a F2 domain; and4) a Flc domain complementary to the Fl domain; wherein at least one domain is not complementary to the target nucleotide sequence; or a FlPt primer whose nucleotide sequence comprises: a) a 3’ F2t domain complementary to a F2ct domain of the target nucleotide sequence, wherein F2ct and Tic are non-overlapping domains located on opposite or complementary strands of the target nucleotide sequence; and b) a 5’ Flct domain complementary to a Fit domain of the target nucleotide sequence,(B) incubating the target nucleotide sequence with the DNA polymerase, amplification reagents and the primers so that the dumbbell-shaped target nucleotide structure is generated.

[0273] In some embodiments, the present disclosure provides methods and compositions for amplifying a target nucleotide sequence. In some embodiments, a method for amplifying a target nucleotide sequence utilizes one or more technologies (e.g., primers, compositions, kits, amplicons, methods or a combination) as described herein. In some embodiments, an amplification method according to the present disclosure is a condensed loop mediated isothermal amplification (cLAMP). cLAMP utilizes one or more hairpin primers. Exemplary methods as described herein are shown in Figures 1 -18 and 20-31 and described in the Examples. In some embodiments, cLAMP uses one or more hairpin primers providing several advantages over conventional LAMP and ligation-initiated LAMP methods.

[0274] In some embodiments, a method for amplifying a target nucleotide sequence comprising the steps of:(A) contacting a target nucleotide sequence with a DNA polymerase having strand displacement activity, amplification reagents, and a composition as described herein (e.g., a composition comprising a hybrid primer) and(B) incubating the target nucleotide with the DNA polymerase, amplification reagents and the primers so an amplified nucleic acid comprising the target nucleotide sequence is generated.

[0275] In some embodiments, target nucleotide sequence synthesis and / or amplification by cLAMP can occur in a one -pot method.

[0276] In some embodiments, a downstream reaction (e.g., a nucleic acid processing and / or detection reaction) is performed after nucleotide synthesis and / or amplification.Downstream reactions may or comprise one or more of amplification, cleavage, digestion, hybridization, replication, etc.Detection

[0277] Nucleotides and / or amplicons can be detected by a number of methods. One of skill in the art is aware of various technologies useful in detecting nucleotides. In some embodiments, the present disclosure provides technologies for detecting nucleotides, amplicons, or both.

[0278] In some embodiments, the present disclosure provides methods for detecting a target nucleotide sequence. In some embodiments, a method for detecting a target nucleotide sequence utilizes one or more technologies (e.g., primers, compositions, kits, amplicons, methods, or a combination) as described herein. In some embodiments, a detection method according to the present comprises cLAMP followed by detection of the nucleotide amplicon product. A person skilled in the art is aware of a number of methods to detect nucleotide production (e.g., nucleotide synthesis and / or nucleotide amplification).

[0279] In some embodiments, a method for detecting a target nucleotide sequence comprises the steps of:(a) contacting a target nucleotide sequence with a DNA polymerase having strand displacement activity, amplification reagents, and a composition according to the present disclosure or a kit according to the present disclosure;(b) incubating a target nucleotide, a compositions and amplification reagents so an amplified nucleic acid is generated (e.g. an amplicon); and(c) detecting the amplified nucleotide.

[0280] In some embodiments, detection technologies comprise, for example, absorbance, CRISPR / Cas detection (e.g., CRISPR-SHERLOCK), FRET, gel electrophoresis, lateral flow, mass spectrometry, PCR, real-time PCR, and / or spectrometry. In some embodiments, detection technologies comprise, for example, chemiluminescence, electrochemical technologies, fluorescence, intercalating dye detection, migration, and / or radiation.

[0281] In some embodiments, a step of detecting is performed by detecting a change in florescence as an indication of amplification of the target nucleotide sequence.

[0282] In some embodiments, a wherein the change in the fluorescence is an increase in the intensity of fluorescence emission of the detectably labeled nucleic acid probe.

[0283] In some embodiments, detection technologies comprise, for example, colorimetric, turbidity, other types of catalysts, molecular beacons and other oligonucleotide-based probes, aptamers, or lateral flow.

[0284] In some embodiments, methods according to the present disclosure include non-specific target nucleotide sequence detection. Non-specific nucleotide detection detects nucleotide acid regardless of the particular sequence using a non-specific nucleotide reporter, such as a non-specific fluorescent DNA reporter. Exemplary non-specific nucleic acid reporters include ethidum bromide, propidium iodide, crystal violet, dUTP-conjugated probes, DAIP (4’-,6-diamidino-2-phenylindole), 7-AAD (7 -aminoactinomycin D), Hoechst 33258, Hoechst 33342, Hoechst 34580, PICOGREEN, SYBR dyes, such as SYBR Green I, SYBR Green II, SYBR Gold. In some embodiments, method of detecting a target nucleotide sequence utilize a SYBR dye. In some embodiments, methods of detecting a target nucleotide sequence utilize SYBR Green.

[0285] In some embodiments, a double stranded DNA binding dye is a minor-groove binding dye. In some embodiments, a mino-groove binding dye is SYBR Green I and II, DAPI, PicoGreen, or a combination. In some embodiments, a double stranded DNA binding dye is an intercalating dye. In some embodiments, an intercalating dye is an Ethidium Bromide, Propidium Iodide, EvaGreen, or a combination.

[0286] In some embodiments, detection methods contemplated by the present disclosure include CRISPR based detection methods. Certain CRISPR / Cas enzymes have been identified that exhibit collateral cleavage activity when activated by binding to a target site recognized by the guide polynucleotide with which they are complexed. Exemplary guide polynucleotides are shown in Figure 19. Casl2, Casl3, and Casl4 are non-limiting examples of CRISPR / Cas enzymes that have been shown to have such collateral cleavage activity. Some CRISPR / Cas enzyme having collateral cleavage activity digests or cleaves single strand nucleic acids (e.g., detectably labeled nucleic acid probes). Collateral activity has been harnessed to develop CRISPR / Cas detection (e.g., diagnostic) technologies that achieve detection of nucleic acids containing a relevant target site (e.g., Cas target nucleic acid), or its complement, in biological and / or environmental sample(s).

[0287] In some embodiments, a Cas enzyme has collateral activity.

[0288] CRISPR-SHERLOCK is a detection technology comprising steps of: contacting a CRISPR-Cas complex comprising a Cas enzyme with collateral cleavage activity, a guide polynucleotide selected or engineered to be complementary to a target nucleotide sequence (e.g., a Cas target nucleic acid sequence), and a sample potentially comprising a target nucleotide sequence comprising Cas target nucleic acid. In some embodiments, CRISPR / Cas-based detection may be a CRISPR-Cas 13-based detection system. In some embodiments, a CRISPR / Cas-based detection system is a CRISPR / Casl2- based detection system. In some embodiments, a CRISPR / Casl3- or CRISPR / Casl2-based detection system is a CRISPR-SHERLOCK detection system. In some embodiments, methods according to the present disclosure utilize a CRISPR-SHERLOCK detection system. In some embodiments, an amplified nucleotide comprising a target nucleotide sequence is incubated with a guide polynucleotide capable of binding the target nucleotide sequence, a detectably labeled nucleic acid probe, and a Cas enzyme.

[0289] In some embodiments, a Cas enzyme is a thermostable Cas enzyme. A thermostable Cas enzyme may be a thermostable Cas Enzyme as described in USPublication, US 2023 / 0002811, entitled “APPLICATION OF CAS PROTEIN, METHOD FOR DETECTING TARGET NUCLEIC ACID MOLECULE AND KIT” and published 01 / 05 / 2023; PCT Publication WO 2020 / 142754, entitled “PROGRAMMABLE NUCLEASE IMPROVEMENTS AND COMPOSITIONS AND METHODS FOR NUCLEIC ACID AMPLIFICATION AND DETECTION” and published 07 / 09 / 2021; PCT Publication WO 2021 / 154866, entitled “IMPROVED DETECTION ASSAYS” and published 08 / 05 / 2021; and PCT Publication WO 2023 / 009526, entitled “IMPROVED CRISPR-CAS TECHNOLOGIES” published 02 / 02 / 2023, the content of each which is incorporated herein by reference in its entirety.

[0290] In some embodiments, a Cas enzyme is a Casl2 enzyme. In some embodiments, a Cas enzyme is a Cas 13 enzyme. In some embodiments, a Cas enzyme is a thermostable Cas enzyme. In some embodiments, a Cas enzyme is thermostable within the range of about 4°C to about 65°C.

[0291] In some embodiments, a detectably labeled nucleic acid probe comprises a fluorescent group end and a quenching group. In some embodiments, a detectably labeled nucleic acid probe comprises a fluorescent group at the 5' end and a quenching group at the 3' end.

[0292] In some embodiments, technologies according to the present disclosure utilizes a guide polynucleotide. A guide polynucleotide is, when incubated with a target polynucleotide, capable of binding to a target nucleotide sequence, as an amplified nucleotide comprising a target nucleotide sequence, an amplicon comprising a target nucleotide sequence, or both. In some embodiments, a guide polynucleotide comprises a guide domain (e.g., 3’ SI domain) that is complementary to a target nucleotide sequence or complement thereof. In some embodiments, a guide polynucleotide comprises a guide hairpin. In some embodiments, a guide hairpin comprises a direct repeat sequence. In some embodiments, a direct repeat sequence is not complementary to a target nucleotide sequence.

[0293] In some embodiments, technologies according to the present disclosure utilizes two or more guide polynucleotides. This may be useful for redundancy and / or sensitivity purposes. In some embodiments, two or more guide polynucleotides binds to two different domain of an amplicon (e.g., a dumbbell- shaped amplicon). An example hereof is shown in Example 5.

[0294] In some embodiments, a guide polynucleotide is complementary to a target nucleotide sequence having one or more SNP mutations.

[0295] In some embodiments, a guide polynucleotide is complementary to a target nucleotide sequence having one or more SNP mutations except that it comprises a mismatch. An example hereof is shown in Example 6. This mismatch can be used to lower the binding affinity of the guide toward the target, thereby making it more selective for the SNP sequence over the wild type (WT) sequence. In some embodiments, a guide polynucleotide binds to an amplicon that contains a SNP hereby tolerating the mismatch. In some embodiments, a guide polynucleotide has two mismatches to a WT amplicon sequence and will fail to bind to the WT amplicon sequence, hi some embodiments, a guide polynucleotide comprises two or more mismatches. In some embodiments, a guide polynucleotide comprises three or more mismatches. In some embodiments, a guide polynucleotide comprises four or more mismatches.

[0296] Guide polynucleotides can be designed to straddle a primer nucleotide sequence and a target nucleotide sequence, or target only a primer nucleotide sequence entirely. In some embodiments, a guide polynucleotide is complementary to a target nucleotide sequence. In some embodiments, a first part of a guide polynucleotide is complementary to a target nucleotide sequence and a second part of a guide polynucleotide is not complementary to a target nucleotide sequence. In some embodiments, a guide polynucleotide is not complementary to a target nucleotide sequence. Exemplary guide polynucleotides are shown in Figure 19.

[0297] In some embodiments, generation of increased transcripts can, among other things, decrease the duration of time required for detecting a nucleic acid. In some embodiments, a detection method comprises a CRISPR-Cas based detection method (e.g., CRISPR-SHERLOCK). In some embodiments, a disclosed system for nucleic acid synthesis and / or nucleic acid amplification and / or detection of a nucleic acid occurs in a single reaction vessel (“one-pot” embodiment).

[0298] In some embodiments, methods and compositions provided herein can distinguish between target nucleotides that have sequences comprising only a single nucleotide polymorphism(s) (SNPs) to differentiate between said target nucleotides. In some embodiments, provided technologies can be utilized to detect a SNP-containing nucleic acid. In some embodiments, provided technologies can be utilized to detect SNP-containingnucleic acids in a patient-derived sample or samples. In some embodiments, identification of nucleic acids that have sequences comprising a disease-relevant SNP or disease-relevant SNPs can be utilized for diagnosis and / or informing treatment regimens. In some embodiments, use of multiple guide RNAs in accordance with disclosed technologies may further expand or improve on the number of target nucleic acids that can be distinguished from other target nucleic acids.

[0299] In some embodiments, disclosed technologies can achieve detection of one or more microbial or other infectious agents in a sample. In some embodiments, such a sample may be or comprise a biological sample, for example which may have been obtained from a subject, and / or an environmental sample, for example which may be or comprise soil, water, etc.. In some embodiments, for example, a microbe may be a bacterium, a fungus, a yeast, a protozoa, a parasite, or a virus.

[0300] In some embodiments, disclosed technologies can be used in other methods (or in combination) with other technologies that require identification of a particular microbe species or other infectious agent in a sample or, monitoring the presence of microbe or other infectious agent over time (e.g., by identifying the presence of a particular microbial or infectious proteins (antigens), antibodies, antibody genes, detection of certain phenotypes (e.g., bacterial resistance)), monitoring of disease progression and / or outbreak, and antibiotic screening.

[0301] In some embodiments, provided technologies achieve certain benefits and / or advantages, e.g., relative to alternative technologies, for example, such as technologies that may utilize conventional LAMP reactions. For example, in some embodiments, provided technologies require a reduced the number of target specific domains for nucleotide amplicon production. . Thus, in some embodiments, provided technologies can identify and / or detect highly mutated regions also allowing for reactions requiring fewer multiplexed or degenerated primers than conventional methods.

[0302] Alternatively or additionally, in some embodiments, provided technologies may be particularly amenable to use in point-of-care devices. Thus, in some embodiments, provided technologies can guide therapeutic regimens (e.g., selection of treatment type and / or dose and / or duration of treatment).

[0303] In some embodiments, water samples such as freshwater samples, wastewater samples, or saline water samples can be evaluated for cleanliness and / or safety, and / or potability, to detect the presence of for example, microbial contamination.

[0304] In some embodiments, provided technologies are useful for assessment of environmental samples. For example, household / commercial / industrial surfaces made of any materials including, but not limited to, metal, wood, plastic, rubber, or the like, may be swabbed and tested for contaminants. To give a few examples, soil samples may be tested for the presence of viral particles or fragments thereof, pathogenic bacteria or parasites, or other microbes, both for environmental purposes and / or for human, animal, or plant disease testing. Water samples such as freshwater samples, wastewater samples, or saline water samples can be evaluated for cleanliness and safety, and / or potability, for example to detect the presence of, for example, viral particles, and / or Cryptosporidium parvum, Giardia lamblia, and / or other microbial contamination.

[0305] Identification of microbes may be useful and / or needed for any number of applications, and thus any type of sample from any source deemed appropriate by one of skill in the art may be used in accordance with the invention.

[0306] In some embodiments, technologies of the present inventions are useful in genotyping.Kit

[0307] Provided herein is a kit for performing the present methods. The kit of parts may comprise a composition or a component thereof as described herein.

[0308] In some embodiments, a kit is provided for performing methods of nucleotide synthesis, nucleotide amplification, nucleotide detection, or a combination of a target nucleotide sequence from a sample. In some embodiments, a kit of parts comprises a composition according to the present invention, and / or one or more components thereof. In some embodiments, a kit of parts may comprise a hybrid primer, a BIP, optionally one or more primers, amplification reagents and / or instructions for use.

[0309] In some embodiments, one or more components included in a provided kit is a stock solution as described herein.

[0310] In some embodiments, a kit is provided for performing methods of detecting a target nucleotide sequence in a sample. In some embodiments, a kit of parts comprises a primer composition according to the present invention. In some such embodiments, a kit of parts may also comprise amplification reagents, a nucleic acid sensor system, a sample collection device, and / or instructions for use. For example, in some particular embodiments, a kit of parts may comprise a nucleic acid sensor system useful for detecting a target nucleotide sequence. Non-limiting examples of nucleic acid sensor systems such as an INSPECTR™ nucleic acid detection system, SHERLOCK nucleic acid detection system, etc.

[0311] In some embodiments, a kit of parts also comprises a control nucleic acid, such as may be spiked into a sample as described herein.Exemplification

[0312] Exemplary technologies (e.g., variations) are described in Examples 1-7 herein below. Each of these variations holds a configuration, which requires different domains to be target- specific. Multiple variations can be combined generating a number of cLAMP reaction. Table 1 (below) shows the number of target-specific domains (domains that are complementary to a target nucleotide sequence or its complement) and targetagnostic domains (domains that are not complementary to a target nucleotide sequence or a complement thereof) in each variation described here. Note that all cLAMP variations have fewer target- specific regions, and are therefore less constrained, than conventional LAMP.Table 1

[0313] In some embodiments, at the most 8 primer domains are complementary to a target nucleotide sequence. In some embodiments, at the most 7 primer domains are complementary to a target nucleotide sequence. In some embodiments, at the most 6 primer domains are complementary to a target nucleotide sequence. In some embodiments, at the most 5 primer domains are complementary to a target nucleotide sequence. In some embodiments, at the most 4 primer domains are complementary to a target nucleotide sequence. In some embodiments, at the most 3 primer domains are complementary to a target nucleotide sequence. In some embodiments, at the most 2 primer domains are complementary to a target nucleotide sequence.Example 1: Variation 1

[0314] The present demonstrates an exemplary variation of nucleotide synthesis and amplification using a single hybrid primer. The present Example demonstrates that a single hybrid primer is useful in an improved LAMP (cLAMP) requiring fewer target specific sequences compared to conventional LAMP.

[0315] Figure 1 shows the components required for a cLAMP variation that comprises a single hybrid primer. Here, each domain represents a 9-30-base nucleic acid region, which is composed of a target- specific sequence (green), a target- agonistic (blue), or a guide RNA direct repeat sequence (purple). The reaction uses the standard target- specific Forward Loop Primer, Forward Inner Primer, and Forward Outer Primer found in conventional LAMP assays. The Backward Outer Primer is omitted in this example, but may also be present as a target-specific primer in other examples (Figs. 3 and 4). Unlike a conventional LAMP reaction, the Backward Inner Primer and Backward Loop Primer comprises target-agnostic sequences. The cLAMP method also includes hybrid primer having target-agnostic domains and a target- specific domain. This primer represents one of the two stem-loop structures found in a cLAMP amplicon dumbbell, with an additional 3’ domain complementary to the target nucleotide sequence.

[0316] Figure 2 shows the cLAMP mechanism in detail. In step 1, the “t” domain of the hybrid primer hinds to the “tc” domain of the template strand, and is extended from the3’ end by a polymerase. In step 2, the FlPt primer and F3 primer invade into the resulting duplex structure and bind to the “F2c” and “F3c” domains, respectively, of the initiation primer’s extension product. In step 3, the FlPt primer is extended by a polymerase to form a complement of the initiation primer’ s extension product. The F3 primer is then extended, and the strand-displacing activity of the polymerase releases a dumbbell amplicon suitable for exponential amplification. In step 4, this dumbbell structure is amplified by BIP, LB, FlPt, and LFt primers to form many amplicon copies, including the concatemer products found in a conventional LAMP reaction.Example 2: Variation 2

[0317] The present Example demonstrates an exemplary variation of nucleic acid synthesis and amplification using a single hybrid primer and a Backward Outer Primer (B3). The present Example demonstrates that a single hybrid primer is useful in an improved LAMP (cLAMP) requiring fewer target specific sequences compared to conventional LAMP.

[0318] Figure 3 demonstrates the components required for a cLAMP variation, which includes both a target -specific Backward Outer Primer (B3), and a target- specific Forward Outer Primer (F3). This strategy requires an additional target- specific domain, but alters the cLAMP initiation steps in a favorable manner. Figure 4 shows exemplary reaction mechanism for cLAMP variation 2. In step 1, the hybrid primer binds to the “tc” domain of the target nucleotide sequence, and the B3 primer binds to the “B3c” domain directly upstream. In step 2a, the hybrid primer is extended by a polymerase to form a complement to the target polynucleotide sequence. The B3 primer is then extended, and the stranddisplacement activity of the polymerase releases the hybrid primer extension product. In step 2b, the FlPt and F3 primers bind to this extension product directly. Unlike the mechanism in Figure 2, the FlPt and F3 primers do not need to invade into a duplex region during this step. This is an advantage of including both the F3 and B3 primers in variation 2. Steps 3-4 of the mechanism proceed as demonstrated in Figure 2.Example 3: Variation 3

[0319] The present Example demonstrates an exemplary variation of nucleic acid synthesis and amplification using a single hybrid primer. The present Example demonstrates that the hybrid initiation primer can hybridize to the target nucleotide sequence or its complement without affecting nucleic acid synthesis and / or amplification.

[0320] Variation 3 (Figs. 5-6) demonstrates a hybrid primer can be used in more than one orientation. A hybrid initiation primer can hybridize to a target nucleotide sequence or complement thereof. In this example, cLAMP is performed in the reverse orientation from previous examples. Here, the Backward Loop Primer, Backward Inner Primer, and Backward Outer Primer are target specific, the Forward Inner Primer and Forward Loop Primer are target-agnostic, and the Hybrid Primer contains the forward domains “Fl”, “LFc”, “F2”, and “Flc”. Figure 6 shows the resulting mechanism. This is equivalent to previously presented mechanisms, but in the opposite orientation.Example 4: Variation 4

[0321] The present Example demonstrates an exemplary variation of nucleic acid synthesis and amplification using a single hybrid primer. The present Example demonstrates that the hybrid primer can hybridize to a target domain that is identical to the Flc domain of the Forward Inner Primer, hereby using fewer target-specific domains, without affecting nucleic acid synthesis and / or amplification.

[0322] Variation 4 (Figs. 7-8) shows a version of cLAMP where the hybrid primer binds to a “Fl” domain of the target nucleotide sequence, rather than the inter-stem region. This results in a shortened dumbbell with a reduced or nonexistent inter-stem region between “Bl” and “Flc” domains. It also results in a reduced number of target-specific domains, which is an advantageous in terms of reducing LAMP primer design constraints and complexity. Figure 8 shows the amplification mechanism in more detail. Steps 1 and 2 are similar to those in Figure 2, but the hybrid primer binds and extends from the “Fl” region of the target nucleotide sequence, rather than the “tc” domain in the inter-stem region. Step 3, while similar to that in Figure 2, produces a shorter dumbbell amplicon lacking an inter-stem region. Step 4 proceeds through standard exponential amplification of this amplicon.Example 5: Variation 5

[0323] The present Example demonstrates an exemplary variation of nucleic acid synthesis and amplification using a single hybrid primer followed by detection using multiple RNA guides.

[0324] Variation 5 (Figs. 9-10) shows an exemplary method where multiple guide RNAs are used for Cas-mediated readout of cLAMP. This may be useful for redundancy and / or sensitivity purposes. While the amplification mechanism for this variation (Fig. 10) is unchanged from Figure 2 in steps 1-4, Step 5 shows both guide RNAs binding simultaneously to the amplicon product. One of these guides binds to the inter-stem region through its “ t’ “ and “si” domains, while the other guide binds to the loop region through its “LF” domain. Either guide may be sufficient for Cas-mediated readout in this example.Example 6: Variation 6

[0325] The present Example demonstrates an exemplary variation of nucleic acid synthesis and amplification using a single hybrid primer, wherein the target nucleotide sequence has a SNP mutation in a detection domain.

[0326] Variation 6 (Figs. 11-12) demonstrates an exemplary method where cLAMP is used in combination with a Cas-mediated readout to detect SNPs in the target strand. The components are similar to those in Figure 1, but there is a SNP mutation in the “sic” domain of the target (red star) and the SNP complement in the “si” domain of the guide. An optional, mismatch in the Guide RNA “t’“ domain can also be added (blue star). This mismatch can be used to lower the binding affinity of the guide toward the target, thereby making it more selective for the SNP sequence over the WT sequence. Although one mismatch is shown here, additional mismatches could be added in principle, and / or positioned differently in the guide’s spacer domain. The mechanism (Fig. 12) is similar to that in Figure 1, but the SNP mutation is incorporated into the final amplicon. In step 5, the guide will bind to an amplicon which contains the SNP, tolerating the mismatch (blue star). However, the guide will have two mismatches to the WT amplicon sequence, and will fail to bind. This provides a SNP-selective Cas readout for the cLAMP assay.Example 7: Variation 7

[0327] The present Example demonstrates an exemplary variation of nucleic acid synthesis and amplification using a single hybrid primer, wherein the target nucleotide sequence has a SNP mutation in a detection domain. The present Example also demonstrates that multiple guides can be used for detection.

[0328] Variation 7 (Figs. 13-14) is a cLAMP exemplary method combining principles from variations 5 and 6 to achieve SNP detection with multiple guide RNAs. The inter-stem targeting guide RNA contains the “t’“ and “si” domains with two SNPs, and has SNP-specific binding and activation (Fig. 14, Step 5). The other guide RNA binds to the “LFc” domain of the loop and does not contain any mismatches, meaning that it will always bind to the amplicon regardless of its SNP status. This loop-specific guide can serve as a positive control for amplification, especially when each guide triggers a different fluorogenic reporter in Cas-mediated assays.Example 8: Variation 8

[0329] The present demonstrates an exemplary variation of nucleotide synthesis and amplification using two hybrid primers. The present Example demonstrates that two hybrid primers are useful in an improved LAMP (cLAMP) requiring fewer target specific sequences compared to conventional LAMP. The Example demonstrates that only two target domains are required for producing a dumbbell- shaped amplicon.

[0330] Variation 8 (Figs. 15-16) uses the standard cLAMP mechanism, but employs two hybrid primers: a first hybrid primer and a second hybrid primer. This greatly reduces the number of target- specific domains that are required in cLAMP. In this cLAMP mechanism (Fig. 16), the first hybrid primer first binds to the “tic” domain of the target nucleotide sequence and is extended by a polymerase in Step 1. The second hybrid primer then binds to the “t2c” domain of this extension product in Step 2, partially displacing the complementary strand. In Step 3, the second hybrid primer is extended by a polymerase to form a complete dumbbell-shaped structure / amplicon. This dumbbell structure is similar to that presented in Figure 2, but comprises two hybrid primer loops instead of one. Step 4 proceed nearly identically to the original cLAMP mechanism in Figure 2, with exponential amplification of the dumbbell amplicon.Example 9: V riation 9

[0331] The present Example demonstrates an exemplary variation of nucleotide synthesis and amplification using a single hybrid primer. In this variation, the target (T) domain of the hybrid primer is identical to the target nucleotide sequence and the F2 domain of the inner primer is complementary to a target nucleotide sequence (i.e., the F2c domain of the target nucleotide). The inner primer initiates primer extension and the hybrid primer hybridizes to the inner primer extension product.

[0332] cLAMP Variation 9 (Figs. 17-18) demonstrates that the first step of cLAMP can be initiated either from a hairpin-shaped initiation primer, or by one of the Inner Primers (Forward or Backward). In the example mechanism in Figure 18, the FIP primer first binds to the “F2c” domain of the target nucleotide sequence and is extended by the polymerase. The Backward Initiation Primer and Backward Outer Primer then invade into the resulting duplex and bind to the “tc” and “B3c” domain of the extension product, respectively. In Step 3, the Backward Initiation Primer is extended by the polymerase, followed by extension of the Backward Outer Primer upstream. The strand displacing activity of the polymerase releases the final amplicon, and step 4 proceed similarly to step 4 in Figure 2.Example 10: Amplification using cLAMP hybrid primer

[0333] The present Example demonstrates that cLAMP functions as an amplification process using a hybrid primer containing both Influenza A (non-target-specific) and Influenza B (target-specific) regions. Using this cLAMP composition 20,000 copies of an Influenza B target were successfully amplified and distinguished from a non-target control.(A) Influenza B Segment 8

[0334] In this Example, the hybrid primer comprises an off-target polynucleotide sequence based on Influenza A segment 1 (black portion in Figure 32D) and a target polynucleotide sequence based on Influenza B segment 8 (red dotted portion in Figure 32D). Influenza B segment 8 is the amplified region of the target.

[0335] cLAMP amplification of Influenza B target using a hybrid primer - allows distinction of target (10,000 copies per reaction) from non-target material (NTC). Influenza B genomic segment 8 is targeted for amplification. Primer concentrations used in reaction are shown in Table 2.Table 2

[0336] In the absence of the hybrid primer, Influenza B target (10,000 copies per reaction) is no longer distinguishable from non-target material (NTC) (Figure 32B). The reaction conditions are the same as those in (Figure 32A), with the exception that there is no hybrid primer present and an additional loop primer was included here (400nM), which would target the hybrid primer, if it were present.

[0337] Conventional LAMP amplification of the same Influenza B target region as that amplified in (Figure 32A). Reaction includes 6 typical LAMP primers. The BIP, B3, F3, and LB primers shown in Table 2 were used in this reaction at the displayed concentration. Additionally, the reaction included the opposing Influenza B-targeting FIP (1.6uM) and FL (400nM) primers (Figure 32C).

[0338] Sequence of the hybrid primer used in (Figure 32A), highlighting regions that are based on off-target Influenza A sequences (segment 1) and regions complementary to Influenza B (segment 8) - the target of interest.(B) - Influenza B Segment 5

[0339] In this Example, the hybrid primer comprises an off-target polynucleotide sequence based on Influenza A segment 1 (black portion in Figure 33D) and a target polynucleotide sequence based on influenza B segment 5 (red dotted portion in Figure 33D). Influenza B segment 8 is the amplified region of the target.

[0340] cLAMP amplification of Influenza B target using a hybrid primer - allows distinction of target (10,000 copies per reaction) from non-target material (NTC). Influenza B genomic segment 5 is targeted for amplification. Primer used in reaction are shown in Table 3.Table 3

[0341] In the absence of the hybrid primer, Influenza B target (10,000 copies per reaction) is no longer distinguishable from non-target material (NTC) (Figure 33B). The reaction conditions are the same as those in (Figure 33A), with the exception that there is no hybrid primer present.

[0342] Conventional LAMP amplification of the same Influenza B target region as that amplified in (Figure 33A). Reaction includes 6 typical LAMP primers. The BIP, B3, F3, and LB primers shown in Table 3 were used in this reaction at the displayed concentration. Additionally, the reaction included the opposing Influenza B-targeting FIP (1.6uM) and FL (400nM) primers (Figure 33C).

[0343] Sequence of the hybrid primer used in (Figure 33A), highlighting regions that are based on off-target Influenza A sequences (segment 1) and regions complementary to Influenza B (segment 5) - the target of interest.(C) - Influenza B Segment 5

[0344] In Example 10C, the hybrid primer comprises an off-target polynucleotide sequence based on Influenza A segment 2 (black portion in Figure 34D) and a target polynucleotide sequence based on influenza B segment 5 (red dotted portion in Figure 34D). Influenza B segment 8 is the amplified region of the target.

[0345] cLAMP amplification of Influenza B target using a hybrid primer - allows distinction of target (10,000 copies per reaction) from non-target material (NTC). Influenza B genomic segment 5 is targeted for amplification. Primer sequences and concentrations used in reaction are shown in Table 4.Table 4

[0346] In the absence of the hybrid primer, Influenza B target (10,000 copies per reaction) is no longer distinguishable from non-target material (NTC) (Figure 34B). The reaction conditions are the same as those in (Figure 34A), with the exception that there is no hybrid primer present.

[0347] Regular LAMP amplification of the same Influenza B target region as that amplified in (Figure 34A). Reaction includes 6 typical LAMP primers. The BIP, B3, F3, and LB primers shown in Table 4 were used in this reaction at the displayed concentration. Additionally, the reaction included the opposing Influenza B-targeting FIP (1.6uM) and FL (400nM) primers (Figure 34C).

[0348] Sequence of the hybrid primer used in (Figure 34 A), highlighting regions that are based on off-target Influenza A sequences (segment 2) and regions complementary to Influenza B (segment 5) - the target of interest.Example 11: Variation 1 followed by detection.

[0349] The present Example demonstrates an exemplary method of nucleotide synthesis, amplification and detection using a single hybrid primer and a single guide RNA.

[0350] Figure 21 shows the components required for a cLAMP variation that comprises a single hybrid primer. Here, each domain represents a 9-30-base nucleic acid region, which is composed of a target- specific sequence (green), a target- agonistic (blue), or a guide RNA direct repeat sequence (purple). The reaction uses the standard target- specific Forward Loop Primer, Forward Inner Primer, and Forward Outer Primer found in conventional LAMP assays. The Backward Outer Primer is omitted in this example, but may also be present as a target-specific primer in other examples (Figs. 3 and 4). Unlike a conventional LAMP reaction, the Backward Inner Primer and Backward Loop Primer comprises target-agnostic sequences. The cLAMP method also includes hybrid primer having target-agnostic domains and a target- specific domain. This primer represents one of the two stem-loop structures found in a cLAMP amplicon dumbbell, with an additional 3’domain complementary to the target nucleotide sequence. The reaction includes one or more guide RNA components for Cas-mediated readout. The guide RNA contains targetspecific spacer domains recognizing the inter-stem region of the final amplicon. Guide RNA(s) may target other target- specific and / or target-agnostic regions of the amplicon in other cLAMP examples.

[0351] Figure 22 shows the cLAMP and detection mechanism in detail. In step 1, the “t” domain of the hybrid primer binds to the “tc” domain of the template strand, and is extended from the 3’ end by a polymerase. In step 2, the FIP primer and F3 primer invade into the resulting duplex structure and bind to the “F2c” and “F3c” domains, respectively, of the initiation primer’s extension product. In step 3, the FIP primer is extended by a polymerase to form a complement of the initiation primer’s extension product. The F3 primer is then extended, and the strand-displacing activity of the polymerase releases a dumbbell amplicon suitable for exponential amplification. In step 4, this dumbbell structure is amplified by BIP, LB, FIP, and LF primers to form many amplicon copies, including the concatemer products found in a conventional LAMP reaction. Finally, in step 5, the templ te-specific spacer domain(s) of the guide RNA molecule (“ t’ ” and “si”) bind to the inter-stem regions of the cLAMP amplicon (dumbbell- shaped amplicon). This final guide: amplicon complex is suitable for Cas enzyme-mediated readout methods. However, if a Cas-mediated readout is not necessary, the cLAMP method could also omit the guide RNA and rely on a general dsDNA-binding dye such as SYBR green to indicate amplification.Example 12: Variation 2 followed by detection

[0352] The present Example demonstrates an exemplary method of nucleotide synthesis, amplification and detection using a single hybrid primer, a Backward Outer Primer (B3) and a single guide RNA.

[0353] Figure 22 demonstrates the components required for a cLAMP variation, which includes both a target -specific Backward Outer Primer (B3), and a target- specific Forward Outer Primer (F3). This strategy requires an additional target- specific domain, but alters the cLAMP initiation steps in a favorable manner. Figure 23 shows exemplary reaction mechanism for cLAMP variation 2 followed by detection. In step 1, the hybrid primer binds to the “tc” domain of the target nucleotide sequence, and the B3 primer bindsto the “B3c” domain directly upstream. In step 2a, the hybrid primer is extended by a polymerase to form a complement to the target polynucleotide sequence. The B3 primer is then extended, and the strand-displacement activity of the polymerase releases the hybrid primer extension product. In step 2b, the FIP and F3 primers bind to this extension product directly. Unlike the mechanism in Figure 21, the FIP and F3 primers do not need to invade into a duplex region during this step. This is an advantage of including both the F3 and B3 primers in variation 2. Steps 3-5 of the mechanism proceed as demonstrated in Figure 20.Example 13: Variation 3 followed by detection

[0354] The present Example demonstrates an exemplary variation of nucleic acid synthesis and amplification using a single hybrid primer followed by detection.

[0355] Variation 3 followed by detection (Figs. 24-25) demonstrates that a hybrid primer can be used in more than one orientation and that the amplification product can be detected. A hybrid initiation primer can hybridize to a target nucleotide sequence or complement thereof. In this example, cLAMP is performed in the reverse orientation from previous examples. Here, the Backward Loop Primer, Backward Inner Primer, and Backward Outer Primer are target specific, the Forward Inner Primer and Forward Loop Primer are target-agnostic, and the Hybrid Primer contains the forward domains “Fl”, “LFc”, “F2”, and “Flc”. Figure 25 shows the resulting mechanism. This is equivalent to previously presented mechanisms, but in the opposite orientation. In step 5, the templatespecific spacer domain(s) of the guide RNA molecule (“t”’ and “si”) bind to the inter-stem regions of the cLAMP amplicon (dumbbell- shaped amplicon). This final guide: amplicon complex is suitable for Cas enzyme-mediated readout methods. However, if a Cas-mediated readout is not necessary, the cLAMP method could also omit the guide RNA and rely on a general dsDNA-binding dye such as SYBR green to indicate amplification.Example 14: Variation 4 followed by detection.

[0356] The present Example demonstrates an exemplary variation of nucleic acid synthesis and amplification using a single hybrid primer followed by detection. The present Example demonstrates that guide RNA can hybridize to a target domain within the hairpin-loop structure of the dumbbell- shaped amplicon, hereby using fewer target- specific domains, without affecting nucleic acid synthesis, amplification, and / or detection.

[0357] Variation 4 followed by detection (Figs. 26-27) shows a version of cLAMP where the hybrid primer binds to a “Fl” domain of the target nucleotide sequence, rather than the inter-stem region. This results in a shortened dumbbell with a reduced or nonexistent inter-stem region between “Bl” and “Flc” domains. It also results in a reduced number of target-specific domains, which is an advantageous in terms of reducing LAMP primer design constraints and complexity. Figure 27 shows the amplification and detection mechanism in more detail. Steps 1 and 2 are similar to those in Figure 21, but the hybrid primer binds and extends from the “Fl” region of the target nucleotide sequence, rather than the “tc” domain in the inter-stem region. Step 3, while similar to that in Figure 21, produces a shorter dumbbell amplicon lacking an inter-stem region. Step 4 proceeds through standard exponential amplification of this amplicon. Finally, in Step 5, the guide RNA binds to the target- specific “LFc” domain of the amplicon for Cas-mediated readout. Since the inter-stem region has been eliminated from the duplex, a guide RNA must target the loop region(s) of the duplex if Cas-mediated readout is desired. In this Example, the guide RNA contains a spacer sequence identical to the “LF’ domain, and binds the “LFc” domain on the loop of the final amplicon.Example 15: Variation 8 followed by detection

[0358] The present demonstrates an exemplary variation of nucleotide synthesis, amplification and detection using two hybrid primers and a guide RNA. The present Example demonstrates that two hybrid primers are useful in an improved LAMP (cLAMP) requiring fewer target specific sequences compared to conventional LAMP.

[0359] Variation 8 followed by detection (Figs. 28-29) uses the standard cLAMP mechanism, but employs two hybrid primers: a first hybrid primer and a second hybrid primer. This greatly reduces the number of target- specific domains that are required in cLAMP. In this cLAMP mechanism (Fig. 28), the first hybrid primer first binds to the “tic” domain of the target nucleotide sequence and is extended by a polymerase in Step 1. The second hybrid primer then binds to the “t2c” domain of this extension product in Step 2, partially displacing the complementary strand. In Step 3, the second hybrid primer isextended by a polymerase to form a complete dumbbell-shaped structure / amplicon. This dumbbell structure is similar to that presented in Figure 21, but comprises two hybrid primer loops instead of one. Steps 4 and 5 proceed nearly identically to the original cLAMP mechanism in Figure 21, with exponential amplification of the dumbbell amplicon and subsequent guide RNA binding.Example 16: Variation 9 followed by detection

[0360] The present Example demonstrates an exemplary variation of nucleotide synthesis, amplification and detection using a single hybrid primer and a guide RNA. In this variation, the target (T) domain of the hybrid primer is identical to the target nucleotide sequence and the F2 domain of the inner primer is complementary to a target nucleotide sequence (i.e., the F2c domain of the target nucleotide). The inner primer initiates primer extension and the hybrid primer hybridizes to the inner primer extension product.

[0361] cLAMP Variation 9 followed by detection (Figs. 30-31) demonstrates that the first step of cLAMP can be initiated either from a hairpin- shaped initiation primer, or by one of the Inner Primers (Forward or Backward). In the example mechanism in Figure 18, the FIP primer first binds to the “F2c” domain of the target nucleotide sequence and is extended by the polymerase. The Backward Initiation Primer and Backward Outer Primer then invade into the resulting duplex and bind to the “tc” and “B3c” domain of the extension product, respectively. In Step 3, the Backward Initiation Primer is extended by the polymerase, followed by extension of the Backward Outer Primer upstream. The strand displacing activity of the polymerase releases the final amplicon, and the remaining steps (4 and 5) proceed similarly to those in Figure 21.Example 17: Ambient temperature viral particle lysis

[0362] This example demonstrates effective viral particle lysis and effective inhibition of RNase at ambient temperature, through use of lysis technologies provided herein.Viral lysis

[0363] Frozen titered viral stocks of FluA and SARS-CoV-2 were added to a 10 mM Tris-HCl, 1 mM EDTA (pH 8.0) buffer (TE buffer) or one of 3 nasal swab matrices (created by eluting one anterior nasal swab in 1 mL of TE buffer). These contrived specimens were treated with 20 mM Sodium hydroxide (NaOH) or a 95°C / 3 minute heat lysis step with 5 mM TCEP present (a reducing agent) was used as a positive control. A portion of the lysed material was added to a room temperature (22°C) reverse transcriptase (RT) reaction, to convert released viral RNA to cDNA in a 1:1 ratio. The RT reaction was stopped by heat inactivating the RT reaction. A portion of the RT reaction was added to a standard qPCR reaction with primers and taqman probes specific for the appropriate virus.

[0364] Viral particle lysis preparations were assessed by measuring Cq value of the samples. Poor to no viral particle lysis was indicated by high Cq values, while optimum viral particle lysis was indicated by lower Cp values.

[0365] The results show that FluA (Figure 35A) and SARS-CoV-2 (SCV2) (Figure 35B) virus can be effectively lysed in and detected from nasal swab matrix with 20 mM NaOH at room temperature comparable to or better than heat lysis control condition.RNase inhibition

[0366] A nasal swab matrix was created by eluting one anterior nasal swab in 1 mL of TE buffer. The nasal swan matrix was subsequent treated with a commercially available RNase inhibitor or 15 mM NaOH. Remaining RNase activity was analyzed using a standard RNase Alert protocol.

[0367] The results show that NaOH treatment effectively reduced the amount of RNase activity present in the sample (Figure 36). NaOH has the additional benefit of inhibiting RNases present in the clinical matrix, protecting the released viral genomes, both from pre-lysed virions as well as virions lysed by the treatments. pH solutions

[0368] Samples were generated by diluting a SARS-CoV-2 (SCV2) viral stock with TE buffer. The samples were treated with KOH concentrations of 10 mM, 25 mM, 50 mM, 75 mM or 100 mM, or NaOH concentrations of 10 mM, 25 mM, 50 mM, 75 mM or 100 mM. 95°C / 3 minute heat lysis step was used as a positive lysis control. A portion of the lysed material was added to a room temperature (in this case 22°C) reverse transcriptase (RT) reaction, to convert released viral RNA to cDNA in a 1:1 ratio. The RT reaction wasstopped by heat inactivating the RT. A portion of the RT reaction was then added to a standard qPCR reaction with primers and taqman probes specific for the appropriate virus.

[0369] Viral particle lysis preparations were assessed by measuring Cq value of the samples. Poor to no viral particle lysis was indicated by high Cq values, while optimum viral particle lysis was indicated by lower Cp values.

[0370] The results show that both NaOH and KOH are effective at releasing viral nucleic acids over a range of concentrations (Figure 37).Hydroxide -based chemical lysis of non-enveloped virus

[0371] Samples were prepared by diluting a human adenoviral stock in TE buffer. Samples were left at room temp (22°C), heated 195°C (heat lysis), or treated with increasing concentrations (20 mM, 40 mM, 60 mM, 80 mM, 100 mM, or 200 mM) of NaOH for 5 minutes.

[0372] After treatment, the solutions were analyzed by qPCR to determine viral nucleic acid release using human adenoviral specific PCR primers and a taqman probe

[0373] The results show that NaOH lysis at room temperature of non-enveloped human adenoviral releases more DNA than heat lysis (Figure 38).Example 18: Ambient temperature bacterial lysis

[0374] This example demonstrates effective bacterial lysis at ambient temperature through use of lysis technologies provided herein.Bacteria lysis

[0375] Samples were generated by diluting freshly grown N. gonorrhoeae or C. Trachomatis (from frozen stock) with TE buffer. Samples were hereafter treated with KOH concentrations of 10 mM, 25 mM, 50 mM, 75 mM or 100 mM. Bead beating (bead lysis) was used as a positive control. A portion of the lysate was then added to a standard qPCR reaction with primers and taqman probes specific for the appropriate bacterium.

[0376] Bacterial lysis preparations were assessed by measuring Cq value of the samples. Poor to no bacterial lysis was indicated by high Cq values, while optimum viral particle lysis was indicated by lower Cq values.

[0377] The results show that KOH lysis with high KOH concentrations, specifically above 25 mM, are efficient at releasing bacterial nucleic acids over a range of concentrations (Figure 39A-B). In particular, concentrations at 100 mM and 200 mM showed similar or better lysis than bead lysis (Figure 39A-B).Addition of detergents

[0378] Samples were generated by diluting N. gonorrhoeae with TE buffer. Samples were hereafter treated with 50 mM KOH and an additional detergent as shown in Figure 40. Bead beating (bead lysis) was used as a positive control.

[0379] Addition of a detergent allows for higher lysis efficiency at lower concentrations of KOH.Example 19: Lysis at varying incubation temperatures

[0380] This example demonstrates effective bacterial lysis at varying temperatures through use of lysis technologies provided herein.

[0381] Samples were prepared by diluting N. gonorrhoeae in TE buffer. Samples were treated with KOH or additives as indicated below and incubates for 3-5 minutes at indicated temperatures. 95°C heat lysis was used as a positive control. A portion of the lysates were added to a PCR reaction and the nucleic acid concentration, reflecting the nucleic acid release, was measured for each sample. Results are normalized to heat lysis (95°C) at 100%.PI64 9

[0382] Samples were treated with KOH or HC1 or PI64 9 0.5% and incubated at room temperature (22°C), 50°C or 65°C, see the table below.Table 5:

[0383] The results show that P164 alone is ineffective and that 50 mM KOH achieve effective bacteria lysis, regardless of the temperature or addition of an additional detergent (Figure 41). Treatment with 13.5 mM HC1 demonstrated better lysis at higher temperatures and with the addition of an additional detergent, but not as well as KOH (Figure 41).ESH9

[0384] Samples were treated with KOH or HC1 and 3% ESH9 and incubated for 3- 5 minutes at room temperature (22°C), 50°C or 70°C.

[0385] Table 6:

[0386] The results show effective N. gonorrhoeae lysis using 50 mM KOH regardless of lysis temperature. Treatment with HC1 + Ecosurf demonstrated better lysis a higher temperatures, specifically at 50°C and 65°C lysis, but less effective than KOH (Figure 42). Additionally, no lysis was observed at 50°C or 70°C incubation alone, without any detergents or additives (Figure 42).NP40 — constant KOH concentration

[0387] Samples were treated with 50mM KOH or HC1 and l%-3% NP40 and incubated for 3- 5 minutes at room temperature (22°C) or 50°C.Table 7:

[0388] The results show that NP40 alone is ineffective for gDNA release and that 50 mM KOH achieve effective bacteria lysis, regardless of the temperature or addition of an additional detergent (Figure 43). Treatment with 13.5 mM HC1 demonstrated better lysis at higher temperatures and with the addition of an additional detergent, but not as well as KOH (Figure 43).NP40 - various KOH concentrations

[0389] Samples were treated with 0 mM to 50 mM KOH or HC1 and 3% NP40 and incubated for 3- 5 minutes at room temperature (22°C) or 50°C.Table 8:

[0390] The results show that in the presence of 3% NP40 lower concentrations of KOH can be used to achieve the same lysis as 50 mM KOH alone (Figure 44).Improved LAMP-Cas detection

[0391] Samples were treated with 50 mM KOH and incubated for 3- 10 minutes at room temperature (22°C). Lysed samples comprising released nucleic acids were amplified by LAMP and detected by a Cas detection system.

[0392] The results show an improvement in LoD when samples were lysed with either KOH or heat lysis compared to no lysis control (Figure 45).Example 20: KOH lysis of N. gonorrhoeae in vaginal matrix in LAMP-Cas

[0393] This example demonstrates effective bacterial lysis in a vaginal matrix through use of lysis technologies provided herein.

[0394] Samples were prepared by diluting N. gonorrhoeae in vaginal swab matrix (1 swab eluted in 3 mL buffer). Samples were treated with OmM, 15 mM, 25 mM, 50 mM, or 85 mM KOH and DNA detected by LAMP-Cas reactions (run at 60C in ABIQS5).

[0395] The results show that a low KOH concentration, specifically 15 mM KOH, do not improve the sensitivity for the LAMP Cas reaction over no KOH treatment. When the bacterial cells in vaginal matrix are treated with higher concentrations of KOH, specifically 25 mM or higher, the sensitivity of the LAMP Cas reactions are improved (Figure 46). Treatment of the bacterial cells with >25 mM KOH results in more released genomic DNA for downstream amplifications.Equivalents

[0396] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:I

Claims

ClaimsWe claim:

1. A composition comprising(i) a first hybrid primer whose nucleotide sequence comprises: a) a 3’ T1 domain complementary to a Tic domain of a target nucleotide sequence; b) a first 5’ hairpin nucleotide sequence comprising four domains from 3’ to the 5’1) a Bl domain;2) a LBc domain;3) a B2 domain; and4) a Bic domain complementary to the Bl domain, wherein at least one domain is not complementary to the target nucleotide sequence; and(ii) a backward inner primer (BIP) whose nucleotide sequence comprises a 3’ B2 domain and a 5’ Bic domain complementary to the Bl domain.

2. The composition of claim 1, wherein at least two domains from the first 5’ hairpin nucleotide sequence are not complementary to the target nucleotide sequence.

3. The composition of claim 2, wherein at least three domains from the first 5’ hairpin nucleotide sequence are not complementary to the target nucleotide sequence.

4. The composition of claim 3, wherein the first hairpin nucleotide acid sequence is not complementary to the target nucleotide sequence.

5. The composition of claim 1, wherein the BIP is not complementary to the target nucleotide sequence.

6. The composition of any one of the previous claims, wherein the composition further comprises:a LB primer whose nucleic acid sequence comprises a LB domain that is complementary to the LBc domain.

7. The composition of claim 6, wherein the LB primer is not complementary to the target nucleotide sequence.

8. The composition of any one of the previous claims, wherein the composition further comprises: a second hybrid primer whose nucleotide sequence comprises: a) a 3’ T2 domain complementary to a T2c domain of the target nucleotide sequence, wherein Tic and T2c are non-overlapping domains located on opposite or complementary strands of the target nucleotide sequence; b) a second 5 ’ hairpin nucleotide sequence comprising four domains from 3’ to the 5’1) a Fl domain;2) a LFc domain;3) a F2 domain; and4) a Flc domain complementary to the Fl domain, wherein at least one domain is not complementary to the target nucleotide sequence.

9. The composition of claim 8, wherein at least two domains from the second 5’ hairpin nucleotide sequence are not complementary to the target.

10. The composition of claim 9, wherein at least three domains from the second 5’ hairpin nucleotide sequence are not complementary to the target.

11. The composition of claim 8, wherein the second hairpin nucleotide acid sequence is not complementary to the target nucleotide sequence.

12. The composition of claim 8, wherein the composition further comprisesa forward inner primer (FIP) whose nucleotide sequence comprises a 3’ F2 domain and a 5’ Flc domain complementary to the Fl domain.

13. The composition of claim 12, wherein the FIP is not complementary to the target nucleotide sequence.

14. The composition of claim 12, wherein the FIP is a FIPp.

15. The composition of claims 12-14, wherein the composition further comprises an LFp primer whose nucleotide sequence is or comprises a LFp domain that is complementary to the LFc domain.

16. The composition of claim 15, wherein the LF primer is not complementary to the target nucleotide sequence.

17. The composition of claims 1-8, wherein the composition further comprises a FlPt primer whose nucleotide sequence comprises: a) a 3’ F2t domain complementary to a F2ct domain of the target nucleotide sequence, wherein F2ct and Tic are non-overlapping domains located on opposite or complementary strands of the target nucleotide sequence; and b) a 5’ Flct domain complementary to a Fit domain of the target nucleotide sequence.

18. The composition of claim 17, wherein the Flct domain is located 5’ to the F2ct domain on the same strand.

19. The composition according to any one of the preceding claims, wherein the composition further comprises a F3 primer whose nucleotide sequence is complementary to a F3c domain of the target nucleotide sequence.

20. The composition of claim 19, wherein the F3c domain is located 3’ to the F2c domain on the same target strand.

21. The composition according to any one of the preceding claims, wherein the composition further comprises an LFt primer whose nucleotide sequence is complementary to an LFct domain of the target nucleotide sequence.

22. The composition of claim 17, wherein the LFct domain is located between Fit and F2t on the same target strand.

23. The composition of any one of the preceding claims, wherein the composition further comprises a B3 primer whose nucleotide sequence is complementary to a B3c domain of the target nucleotide sequence.

24. The composition of claim 19, wherein the B3c domain is located 3’ to the Tic domain on the same strand.

25. The composition of any one of the previous claims, wherein the primer nucleotide sequence is a ribonucleotide sequences.

26. The composition of any one of the previous claims, wherein the primer nucleotide sequence is a deoxyribonucleotide sequence.

27. The composition of any one of the previous claims, wherein the primer nucleotide sequence is a mixture of deoxyribonucleotides and polyribonucleotides.

28. The composition of claims 25-27, wherein the primer nucleotide sequence includes peptide nucleic acids or locked nucleic acids.

29. The composition of claims 25-27, wherein the primer nucleotide sequence includes peptide nucleic acids or locked nucleic acids.

30. The composition of claims 25-27, wherein the primer nucleotide sequence includes 2’ Fluoro modifications, 2’-O-methyl modifications, or a combination thereof.

31. The composition of claim 25, wherein the primer ribonucleotide sequence includes 2- Aminopurine.

32. The composition of claim 26, wherein the primer deoxyribonucleotide sequence includes a phosphodiester deoxyribonucleotide.

33. The composition of claim 26, wherein the primer deoxyribonucleotide sequence includes a phosphorothioated deoxyribonucleotide.

34. The composition according to any one of the preceding claims, wherein one or more of the primer domains are about 9 to about 30 nucleotides.

35. The composition according to any one of the preceding claims, wherein the primer domain complementary to the target nucleotide sequence is at least 80% complementary to its hybridization site in the target nucleotide sequence or complement thereof.

36. The composition according to any one of the preceding claims, wherein the domain complementary to the target nucleotide sequence comprises 1, 2, 3, or 4 mismatches.

37. The composition according to any one of the preceding claims, wherein one or more of the primers comprises a fluorophore.

38. The composition according to claim 37, wherein one or more of the primers comprises a quencher.

39. A kit comprising a composition according to any one of claims 1-38 and a DNA polymerase.

40. A kit of claim 39, wherein the DNA polymerase has strand displacement activity.

41. The kit of claim 40, wherein the DNA polymerase is a Bsu Polymerase, Bsm Polymerase, Bst Polymerase, or a combination thereof.

42. The kit of claim 39, wherein the kit further comprises amplification reagents.

43. The kit of claim 39, wherein the kit further comprises a reverse transcriptase.

44. The kit of claim 39, wherein the kit further comprises a guide RNA.

45. The kit of claim 39, wherein the kit further comprises a Cas enzyme.

46. The kit of claim 45, wherein the Cas enzyme is a Cas 12 or Cas 13 enzyme.

47. The kit of claims 45-46, wherein the Cas enzyme has collateral cleavage activity.

48. The kit of claims 45-47, wherein the Cas enzyme is a thermostable Cas enzyme.

49. The kit of claim 48, wherein the Cas enzyme is thermostable within a range of about 20°C to about 65°C.

50. The kit of claims 39-49, wherein the kit further comprises a delectably labeled nucleic acid probe.

51. The kit of claim 39-50, wherein the kit further comprises a double stranded DNA binding dye.

52. The kit of claim 51, wherein the double stranded DNA binding dye is a SYBR Green I and II, DAPI, PicoGreen, Ethidium Bromide, Propidium Iodide, EvaGreen, or a combination.

53. The kit of claim 39-52, wherein the kit further comprises a single stranded DNA binding dye.

54. The kit according to claims 39-53, wherein the kit further comprises Thermostable Inorganic Pyrophosphatase (TIPP).

55. A method for producing a hairpin-loop amplicon comprising a target nucleotide sequence or complement thereof, comprising the steps of:(A) contacting a target nucleotide sequence with a DNA polymerase or a reverse transcriptase, amplification reagents and a first hybrid primer whose nucleotide sequence comprises: a) a 3’ T1 domain complementary to a Tic domain of the target nucleotide sequence; b) a first 5’ hairpin nucleotide acid sequence comprising four domains from 3’ to the 5’1) a Bl domain;2) a LBc domain;3) a B2 domain; and4) a Bic domain complementary to the Bl domain; wherein at least one domain is not complementary to the target nucleotide; and(B) incubating the target nucleotide sequence with the DNA polymerase or the reverse transcriptase, amplification reagents and the first hybrid primer so that the hairpinloop product is generated.

56. The method of claim 55, wherein the step of incubating is performed in the presence of TIPP.

57. The method of claims 55-56, wherein the target nucleotide sequence is in a sample.

58. The method of claims 57, wherein the sample is a crude sample.

59. The method of claims 57-58, wherein the sample is a biological sample or environmental sample.

60. The method of claim 59, wherein the biological sample is obtained from a subject.

61. The method of claims 55-60, wherein the method further comprises a step of: isolating the target nucleotide sequence.

62. The method of claim 59, wherein the biological sample is saliva, blood, plasma, buffy coat, serum, teeth, urine, nasal fluid, nasopharyngeal swab, oropharyngeal swab, nasal aspirate, sputum, bronchoalveolar lavage, buccal swab, vaginal swab, rectal swab, wound swab, skin swab, bone, muscle, tissue, CSF, semen, fecal matter, hair follicle, skin sample, or a combination hereof.

63. The method of claims 55-62, wherein the target nucleotide sequence is a viral, a bacterial, a fungal, a protozoan, or a parasitic sequence.

64. A method for producing a dumbbell-shaped amplicon, comprising the steps of:(A) contacting a target nucleotide sequence with a DNA polymerase having strand displacement activity, amplification reagents and a plurality of primers comprising: i) a first hybrid primer whose nucleotide sequence comprises: a) a 3’ T1 domain complementary to a Tic domain of the target nucleotide sequence; b) a 5 ’ hairpin nucleotide acid sequence comprising four domains from 3’ to the 5’1) a Bl domain;2) a LBc domain;3) a B2 domain; and4) a Bic domain complementary to the Bl domain; wherein at least one domain is not complementary to the target nucleotide; and ii) a second primer selected from a second hybrid primer whose nucleotide sequence comprises: a) a 3’ T2 domain complementary to a T2c domain of the target nucleotide sequence, wherein Tic and T2c are non-overlapping domains located on opposite or complementary strands of the target nucleotide sequence;b) a second 5 ’ hairpin nucleotide acid sequence comprising four domains from 3’ to the 5’1) a Fl domain;2) a LFc domain;3) a F2 domain; and4) a Flc domain complementary to the Fl domain; wherein at least one domain is not complementary to the target nucleotide sequence; or a FlPt primer whose nucleotide sequence comprises: a) a 3’ F2t domain complementary to a F2ct domain of the target nucleotide sequence, wherein F2ct and Tic are non-overlapping domains located on opposite or complementary strands of the target nucleotide sequence; and b) a 5’ Flct domain complementary to a Fit domain of the target nucleotide sequence,(B) incubating the target nucleotide sequence with the DNA polymerase, amplification reagents and the primers so that the dumbbell- shaped target nucleotide structure is generated.

65. The method of claim 64, wherein the target nucleotide sequence is also contacted with a reverse transcriptase.

66. The method of claims 64-65, wherein the step of incubating is performed in the presence of TIPP.

67. The method of claims 64-66, wherein the target nucleotide sequence is in a sample.

68. The method of claim 67, wherein the sample is a crude sample.

69. The method of claims 67-68, wherein the sample is a biological sample or environmental sample.

70. The method of claim 69, wherein the biological sample is obtained from a subject.

71. The method of claims 64-70, wherein the method further comprises a step of: isolating the target nucleotide sequence.

72. The method of claims 69-70, wherein the biological sample is saliva, blood, plasma, buffy coat, serum, teeth, urine, nasal fluid, nasopharyngeal swab, oropharyngeal swab, nasal aspirate, sputum, bronchoalveolar lavage, buccal swab, vaginal swab, rectal swab, wound swab, skin swab, bone, muscle, tissue, CSF, semen, fecal matter, hair follicle, skin sample, or a combination hereof.

73. The method of claims 64-72, wherein the target nucleotide sequence is a viral, a bacterial, a fungal, a protozoan, or a parasitic sequence.

74. The method of claims 64-73, wherein the plurality of primers further comprises a F3 primer whose nucleotide sequence is complementary to an F3c domain of the target nucleotide sequence.

75. A method for synthesizing a deoxyribonucleotide sequence comprising the steps of:(A) contacting a target ribonucleotide sequence with a reverse transcriptase, amplification reagents, and the composition according to any one of claims 1-38; and(B) incubating the target ribonucleotide with the reverse transcriptase, amplification reagents and the primers so a deoxyribonucleotide sequence comprising the target nucleotide sequence or a complement thereof is generated.

76. A method for amplifying a target nucleotide sequence comprising the steps of:(A) contacting a target nucleotide sequence with a DNA polymerase having strand displacement activity, amplification reagents, and the composition according to any one of claims 1-38; and(B) incubating the target nucleotide with the DNA polymerase, amplification reagents and the primers so an amplified nucleotide sequence comprising the target nucleotide sequence is generated.

77. The method of claim 76, wherein the method further comprises the steps of:(a) contacting a target ribonucleotide sequence with a reverse transcriptase, amplification reagents, and the composition according to any one of claims 1-38;(b) incubating the target ribonucleotide with the reverse transcriptase, amplification reagents and the primers so a deoxyribonucleotide sequence comprising the target nucleotide sequence is generated; before performing step (A) and (B).

78. The method of claims 76-77, wherein the step of incubating is performed in the presence of TIPP.

79. The method of claims 76-78, wherein the method for amplifying a target nucleotide sequence is conducted in a single vessel.

80. The method of claims 76-79, wherein the amplification is an isothermal amplification reaction.

81. The method of claims 76-80, wherein the amplification is performed at ambient temperature.

82. The method of claims 76-81, wherein the target nucleotide sequence is in a sample.

83. The method of claim 82, wherein the sample is a crude sample.

84. The method of claims 82-83, wherein the sample is a biological sample or environmental sample.

85. The method of claim 76-84, wherein the method further comprises obtaining a sample from a subject.

86. The method of claims 76-85, wherein the method further comprises a step of: isolating the target nucleotide sequence.

87. The method of claims 82-84, wherein the sample is saliva, blood, plasma, buffy coat, serum, teeth, urine, nasal fluid, nasopharyngeal swab, oropharyngeal swab, nasal aspirate, sputum, bronchoalveolar lavage, buccal swab, vaginal swab, rectal swab, wound swab, skin swab, bone, muscle, tissue, CSF, semen, fecal matter, hair follicle, skin sample, or a combination hereof.

88. A method for detecting a target nucleotide sequence comprising the steps of:(a) contacting the target nucleotide sequence with a DNA polymerase having strand displacement activity, amplification reagents, and the composition according to any one of claims 1-38 or kit according to any one of claims 39-54;(b) incubating the target nucleotide, the compositions and amplification reagents so an amplified nucleic acid is generated; and(c) detecting the amplified nucleic acid.

89. The method of claim 88, wherein the step of incubating is performed in the presence of TIPP.

90. The method of claims 88-89, wherein the target nucleotide sequence is in a sample.

91. The method of claim 90, wherein the sample is a crude sample.

92. The method of claims 90-91, wherein the sample is a biological sample or environmental sample.

93. The method of claim 92, wherein the biological sample is obtained from a subject.

94. The method of claims 88-93, wherein the method further comprises a step of: isolating the target nucleotide sequence.

95. The method of claims 91-93, wherein the sample is saliva, blood, plasma, huffy coat, serum, teeth, urine, nasal fluid, nasopharyngeal swab, oropharyngeal swab, nasal aspirate, sputum, bronchoalveolar lavage, buccal swab, vaginal swab, rectal swab, wound swab, skin swab, bone, muscle, tissue, CSF, semen, fecal matter, hair follicle, skin sample, or a combination hereof.

96. The method of claims 88-95, wherein the step of detecting the amplified nucleotide is performed by incubating the amplified nucleotide with a double DNA stranded binding dye.

97. The method of claim 96, wherein when the double stranded DNA binding dye binds to the amplified nucleotide it transits from a first undetectable state to a second detectable state.

98. The method of claims 96-97, wherein the double stranded DNA binding dye emits fluorescence in its second detectable state.

99. The method of claims 88-98, wherein the amplified nucleotide is incubated with a guide polynucleotide capable of binding the target nucleotide sequence, a detectably labeled nucleic acid probe, and a Cas enzyme.

100. The method of claim 99, wherein the Cas enzyme is a Cas 13 enzyme.

101. The method of claim 99, wherein the Cas enzyme is a Casl2 enzyme.

102. The method of claims 99-101, wherein the Cas enzyme is a thermostable Cas enzyme.

103. The method of claims 99-102, wherein the delectably labeled nucleic acid probe comprises a fluorescent group end and a quenching group.

104. The method of claims 99-103, wherein the detectably labeled nucleic acid probe comprises a fluorescent group at the 5' end and a quenching group at the 3' end.

105. The method of claims 88-104, the step of detecting is performed by detecting a change in florescence as an indication of amplification of the target nucleotide sequence.

106. The method of claim 105, wherein the change in the fluorescence is an increase in the intensity of fluorescence emission of the detectably labeled nucleic acid probe.

107. A hairpin-loop amplicon comprising a nucleotide sequence comprising: a) a 3’ target nucleotide sequence; b) a 5 ’ hairpin nucleotide sequence comprising four domains from the3’ to the 5’1) a Bl domain;2) a LBc domain;3) a B2 domain; and4) a Bic domain complementary to the Bl domain; wherein at least one domain is not complementary to the target nucleotide.

108. A dumbbell-shaped amplicon comprising a nucleotide sequence comprising from its 3’ end to its 5’ end: a) a first hairpin nucleotide sequence comprising four domains1) a Bl domain;2) a B2c domain;3) a LB domain; and4) a Bic domain complementary to the Bl domain; wherein at least one domain is not complementary to the target nucleotide; b) a target nucleotide sequence or complement thereof; andc) a second hairpin nucleotide sequence comprising four domains1) a Fl domain;2) a LFc domain;3) a F2 domain; and4) a Flc domain complementary to the Fl domain.

109. The amplicon of claim 108, wherein at least two domains are not complementary to the target nucleotide sequence.

110. The amplicon of claims 108-109, wherein at least three domains are not complementary to the target nucleotide sequence.

111. The amplicon of claims 108- 110, wherein at least four domains are not complementary to the target nucleotide sequence.

112. The amplicon of claims 108-111, wherein the two hairpin nucleotide acid sequences are not complementary to the target nucleotide sequence.

113. The amplicon of claims 108-112, wherein at least half of the product is not complementary to the target nucleotide sequence.

114. The amplicon of claims 108-113, wherein the target nucleotide sequence is at the most 100 nucleotides.

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