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WO2024249878A1 - Amplification isotherme à médiation par boucle condensée - Google Patents

Amplification isotherme à médiation par boucle condensée Download PDF

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Publication number
WO2024249878A1
WO2024249878A1 PCT/US2024/032026 US2024032026W WO2024249878A1 WO 2024249878 A1 WO2024249878 A1 WO 2024249878A1 US 2024032026 W US2024032026 W US 2024032026W WO 2024249878 A1 WO2024249878 A1 WO 2024249878A1
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Prior art keywords
domain
nucleotide sequence
complementary
primer
target
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English (en)
Inventor
Leland HYMAN
Pradeep Ramesh
Heike Boisvert
Kristine WERLING
Mary Katherine WILSON
Mary Elizabeth NATOLI
Deborah Paola Almeida
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Sherlock Biosciences Inc
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Sherlock Biosciences Inc
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Publication of WO2024249878A1 publication Critical patent/WO2024249878A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • PCR Polymerase Chain Reaction
  • LAMP Loop-mediated isothermal amplification
  • 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.
  • nucleotide amplicon production e.g., nucleotide synthesis and/or nucleotide amplification.
  • the present disclosure provides particular technologies (e.g., primers, compositions, kits, amplicons, and methods) for nucleotide amplicon production.
  • 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.
  • 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.
  • F3 and FIP forward-inner
  • B3 and BIP backward- inner primers on the template reverse-complement.
  • 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.
  • ligation-initiated LAMP process carries some inherent disadvantages.
  • a ligase enzyme is an additional point of failure in the reaction, and would require additional optimization of the buffer composition, lyophilization conditions, and other factors before the assay could be deployed into a point-of-need test.
  • 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.
  • particularly useful technologies e.g., primers, compositions, kits, amplicons, and methods
  • Condensed loop-mediated isothermal amplification 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.
  • compositions and methods of the present disclosure use at least one hybrid primer.
  • 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).
  • the present disclosure surprisingly demonstrates that a relatively small target nucleotide sequence is required for amplifying a target nucleotide sequence.
  • cLAMP as described in the present disclosure, fewer target-specific domains are required than in standard LAMP.
  • 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.
  • 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.
  • 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.
  • cLAMP primer design is less constrained than conventional LAMP primer design.
  • 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.
  • 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.
  • the present disclosure provides solutions that do not include ligation-initiated LAMP strategies.
  • cLAMP initiates its amplicon formation purely through polymerase-mediated mechanisms.
  • 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).
  • cLAMP avoids the time-intensive ligation step (-20-30 minutes or more) and therefore reduces time-to-result.
  • 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.
  • the present disclosure provides particular technologies (e.g., primers, compositions, kits, amplicons, and methods) for target nucleotide detection.
  • the present disclosure provides a composition
  • 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.
  • BIP backward inner primer
  • 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 hairpin nucleotide acid sequence is not complementary to the target nucleotide sequence. In some embodiments, a BIP is not complementary to the target nucleotide sequence.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIP forward inner primer
  • a FIP is not complementary to the target nucleotide sequence.
  • a FIP is a FIPp.
  • 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.
  • 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.
  • a Flct domain is located 5’ to the F2ct domain on the same strand.
  • a composition provided herein further comprises a F3 primer whose nucleotide sequence is complementary to a F3c domain of the target nucleotide sequence.
  • a F3c domain is located 3’ to the F2c domain on the same target strand.
  • a composition provided herein further comprises an LFt primer whose nucleotide sequence is complementary to an LFct domain of the target nucleotide sequence.
  • a LFct domain is located between Fit and F2t on the same target strand.
  • a composition provided herein further comprises a B3 primer whose nucleotide sequence is complementary to a B3c domain of the target nucleotide sequence.
  • a B3c domain is located 3’ to the Tic domain on the same strand.
  • 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.
  • 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.
  • a domain complementary to the target nucleotide sequence comprises 1, 2, 3, or 4 mismatches.
  • one or more of the primers comprises a fluorophore.
  • one or more of the primers comprises a quencher.
  • kits comprising a composition as provided herein and a DNA polymerase.
  • a DNA polymerase has strand displacement activity.
  • a DNA polymerase is a Bsu Polymerase, Bsm Polymerase, Bst Polymerase, or a combination thereof.
  • a kit further comprises amplification reagents.
  • a kit further comprises a reverse transcriptase.
  • a kit further comprises a guide RNA.
  • a kit further comprises a Cas enzyme.
  • a Cas enzyme is a Casl2 or Casl3 enzyme.
  • a Cas enzyme has collateral cleavage activity.
  • a Cas enzyme is a thermostable Cas enzyme.
  • a Cas enzyme is thermostable within a range of about 20°C to about 65°C.
  • a kit further comprises a detectably labeled nucleic acid probe.
  • a kit further comprises a double stranded DNA binding dye.
  • a double stranded DNA binding dye is a SYBR Green I and II, DAPI, PicoGreen, Ethidium Bromide, Propidium Iodide, EvaGreen, or a combination.
  • a kit further comprises a single stranded DNA binding dye.
  • a kit further comprises Thermostable Inorganic Pyrophosphatase (TIPP).
  • TIPP Thermostable Inorganic Pyrophosphatase
  • 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
  • the step of incubating is performed in the presence of TIPP.
  • a target nucleotide sequence is in a sample.
  • a sample is a crude sample.
  • a sample is a biological sample or environmental sample.
  • a biological sample is obtained from a subject.
  • a method provided herein further comprises a step of: isolating the target nucleotide sequence.
  • 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.
  • a target nucleotide sequence is a viral, a bacterial, a fungal, a protozoan, or a parasitic sequence.
  • 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 target nucleotide sequence is also contacted with a reverse transcriptase.
  • a step of incubating is performed in the presence of TIPP.
  • a target nucleotide sequence is in a sample.
  • a sample is a crude sample.
  • a sample is a biological sample or environmental sample.
  • a biological sample is obtained from a subject.
  • a method provided herein further comprises a step of: isolating the target nucleotide sequence.
  • 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.
  • a target nucleotide sequence is a viral, a bacterial, a fungal, a protozoan, or a parasitic sequence.
  • a plurality of primers further comprises a F3 primer whose nucleotide sequence is complementary to an F3c domain of the target nucleotide sequence.
  • 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.
  • 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.
  • 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.
  • a method for amplifying a target nucleotide sequence is conducted in a single vessel.
  • amplification is an isothermal amplification reaction.
  • amplification is performed at ambient temperature.
  • a target nucleotide sequence is in a sample.
  • a sample is a crude sample.
  • a sample is a biological sample or environmental sample.
  • 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.
  • 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.
  • 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.
  • a step of incubating is performed in the presence of TIPP.
  • a nucleotide sequence is in a sample.
  • a sample is a crude sample.
  • a sample is a biological sample or environmental sample.
  • biological sample is obtained from a subject.
  • a method further comprises a step of: isolating the target nucleotide sequence.
  • 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.
  • a step of detecting the amplified nucleotide is performed by incubating the amplified nucleotide with a double DNA stranded binding dye.
  • a double stranded DNA binding dye binds to the amplified nucleotide it transits from a first undetectable state to a second detectable state.
  • a double stranded DNA binding dye emits fluorescence in its second detectable state.
  • 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.
  • 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 the fluorescence is an increase in the intensity of fluorescence emission of the detectably labeled nucleic acid probe.
  • 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.
  • 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.
  • 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.
  • 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.
  • Figure 2 shows exemplary steps of an amplification process as described herein.
  • Dotted line target- specific sequences
  • Solid line target- agonistic sequence.
  • 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.
  • Figure 4 shows exemplary steps of an amplification process as described herein.
  • Dotted line target- specific sequences
  • Solid line target-agonistic sequence.
  • FIG. 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.
  • Figure 6 shows exemplary steps of an amplification process as described herein.
  • Dotted line target- specific sequences
  • Solid line target-agonistic sequence.
  • FIG. 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.
  • Figure 8 shows exemplary steps of an amplification process as described herein. Dotted line: target- specific sequences; Solid line: target- agonistic sequence.
  • FIG. 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’).
  • 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).
  • 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).
  • 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’).
  • sic Single nucleotide polymorphism (SNP) sites within each domain are indicated with a single asterisk. Mismatch(es) are indicated with two asterisks.
  • 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).
  • 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).
  • FIG 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.
  • 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).
  • 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) .
  • 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 a lowercase ‘c’ (e.g., “Flc”).
  • Dotted line target-specific sequences
  • Solid line target- agonistic sequence.
  • Figure 16 shows exemplary steps of an amplification process as described herein.
  • Dotted line target- specific sequences
  • Solid line target-agonistic sequence.
  • 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.
  • Figure 18 shows exemplary steps of an amplification process as described herein.
  • Dotted line target- specific sequences
  • Solid line target-agonistic sequence.
  • 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).
  • 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).
  • Figure 21 shows exemplary steps of an amplification and detection process as described herein.
  • Dotted line target-specific sequences
  • Solid line target-agonistic sequence.
  • FIG 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 for detection.
  • Dotted line target-specific sequences
  • Solid line target-agonistic sequence
  • Dashed line direct repeat sequence (template-agonistic).
  • 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).
  • 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) .
  • 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).
  • 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”).
  • 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).
  • 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).
  • 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).
  • 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).
  • FIG 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).
  • 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).
  • FIGS 32 A-D show 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.
  • FIG. 33A-D 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.
  • Figures 34 A-D show 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 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.
  • Figure 35 shows lysis of respiratory viruses.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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 of detergent 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 3’-end and 5’-end‘ 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.
  • agent in general, 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.).
  • 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
  • phenomenon e.g., heat, electric current or field, magnetic force or field, etc.
  • the term may be used to refer to a natural product in that it is found in and/or is obtained from nature.
  • 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.
  • an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form.
  • potential agents may be provided as collections or libraries, for example that may be screened to identify or characterize active agents within them.
  • 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.
  • Ambient temperature is the temperature of surroundings.
  • 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.
  • 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°
  • 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.
  • 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.
  • an amino acid has the general structure H2N-C(H)(R)-COOH.
  • an amino acid is a naturally- occurring amino acid.
  • 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.
  • an amino acid including a carboxy- and/or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above.
  • 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.
  • 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.
  • 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.
  • 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.
  • a particular entity e.g., polypeptide, genetic signature, metabolite, microbe, etc
  • 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.
  • 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.
  • Binding 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).
  • 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.
  • a source of interest is or comprises an organism, such as an animal or human.
  • a biological sample is or comprises biological tissue or fluid.
  • 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.
  • a biological sample is or comprises cells obtained from an individual.
  • obtained cells are or include cells from an individual from whom the sample is obtained.
  • a sample is a “primary sample” obtained directly from a source of interest by any appropriate means.
  • 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.
  • 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.
  • 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.
  • a cellular lysate includes predominantly hydrophilic components; in some embodiments, a cellular lysate includes predominantly hydrophobic components.
  • 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.
  • a cellular lysate is a lysate of one or more abnormal cells, such as cancer cells.
  • 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.
  • composition may be used to refer to a discrete physical entity that comprises one or more specified components.
  • a composition may be of any form - e.g., gas, gel, liquid, solid, etc.
  • Complementary refers to the overall relatedness between polymeric molecules, e.g., between polynucleotides.
  • 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.
  • 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.
  • 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 composition or method.
  • 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.
  • 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.
  • known or disclosed equivalents of any named essential element or step may be substituted for that element or step.
  • 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.
  • a monomeric residue in a polymer e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide
  • corresponding to a residue in an appropriate reference polymer.
  • 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 190 th amino 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.
  • 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.
  • 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, ScalaBL
  • 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.
  • Detectable entity 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.
  • detectable entities include, but are not limited to: various ligands, radionuclides (e.g., 3 H, 14 C, 18 F, 19 F, 32 P, 35 S, 135 I, 125 I, 123 I, M Cu, 187 Re, 11 ] In, 90 Y, 99m Tc, 177 LU, 89 Zr 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, dioxigen
  • determining involves manipulation of a physical sample.
  • 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.
  • determining involves receiving relevant information and/or materials from a source.
  • determining involves comparing one or more features of a sample or entity to a comparable reference.
  • expression 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.
  • GeV As used herein, the term “gel” refers to viscoelastic materials whose rheological properties distinguish them from solutions, solids, etc.
  • 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.
  • homology refers to the overall relatedness between polymeric molecules, e.g., between polypeptide molecules.
  • 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.
  • polymeric molecules are considered to be “homologous” to one another if their sequences are at least 80%, 85%, 90%, 95%, or 99% similar.
  • 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.
  • 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 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).
  • 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.
  • 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 percent identity 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).
  • 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.
  • in vitro 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.
  • Isolated 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.
  • 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.
  • a substance is "pure” if it is substantially free of other components.
  • 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.
  • 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 system that is not of the species that produces it in nature.
  • 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.
  • 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.
  • Nucleic acid' refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
  • a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage.
  • 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.
  • a "nucleic acid” is or comprises RNA; in some embodiments, a “nucleic acid” is or comprises DNA.
  • a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues.
  • a nucleic acid is, comprises, or consists of one or more nucleic acid analogs.
  • a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone.
  • 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.
  • a nucleic acid has one or more phosphorothioate and/or 5'-N-phosphoramidite linkages rather than phosphodiester bonds.
  • 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).
  • adenosine thymidine, guanosine, cytidine
  • uridine deoxy adenosine
  • deoxythymidine deoxy guanosine
  • deoxycytidine deoxycytidine
  • 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, methyl
  • 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.
  • a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein.
  • a nucleic acid includes one or more introns.
  • 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.
  • 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.
  • a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded.
  • 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.
  • Polypeptide As used herein refers to any polymeric chain of amino acids.
  • a polypeptide has an amino acid sequence that occurs in nature.
  • a polypeptide has an amino acid sequence that does not occur in nature.
  • 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.
  • a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both.
  • a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids.
  • a polypeptide may comprise D-amino acids, L-amino acids, or both.
  • 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.
  • a polypeptide is not cyclic and/or does not comprise any cyclic portion.
  • a polypeptide is linear.
  • a polypeptide may be or comprise a stapled polypeptide.
  • 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.
  • 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.
  • 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).
  • 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%.
  • a conserved region that may in some embodiments be or comprise a characteristic sequence element
  • 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.
  • a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide.
  • 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.
  • 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.
  • 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.
  • proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.
  • 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.
  • sample typically refers to an aliquot of material obtained or derived from a source of interest, as described herein.
  • a source of interest is a biological or environmental source.
  • 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).
  • a source of interest is or comprises biological tissue or fluid.
  • a biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, 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.
  • 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.
  • a biological fluid may be or comprise a plant exudate.
  • 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).
  • a biological sample is or comprises cells obtained from an individual.
  • a sample is a “primary sample” obtained directly from a source of interest by any appropriate means.
  • 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.
  • processing e.g., by removing one or more components of and/or by adding one or more agents to
  • a primary sample e.g., 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.
  • an agent 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).
  • 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 target entity. 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).
  • Specificity is a measure of the ability of a particular ligand to distinguish its binding partner from other potential binding partners.
  • 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).
  • a human subject is an adult, adolescent, or pediatric subject.
  • 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.
  • 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.
  • a subject displays one or more symptoms of a disease, disorder or condition.
  • a subject does not display a particular symptom (e.g,. clinical manifestation of disease) or characteristic of a disease, disorder, or condition.
  • a subject does not display any symptom or characteristic of a disease, disorder, or condition.
  • a subject is a patient.
  • a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
  • 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.
  • 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.
  • F3 Forward Primer
  • B3 Backward Primer
  • FIP Forward Inner Primer
  • BIP Backward Inner Primer
  • an additional pair of primers e.g., loop primers
  • stem-loops e.g., as discussed below, step 5
  • loop primers can increase LAMP product generated and/or decrease duration of a reaction required to achieve a detection limit.
  • the present disclosure 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.
  • a conventional LAMP reaction often involves steps such as primer annealing and initiation of nucleotide synthesis.
  • a representative LAMP reaction A representative LAMP reaction in described in e.g., W02002024902A.
  • technologies disclosed herein are useful in condensed Loop-mediated Isothermal Amplification (cLAMP).
  • cLAMP condensed Loop-mediated Isothermal Amplification
  • 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.
  • 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.
  • a primer as described herein comprises a primer domain.
  • a primer comprises at least one primer domain.
  • a primer comprises one or more primer domains.
  • a primer domain is a domain present within a primer as described herein below.
  • a primer comprises a plurality of primer domains.
  • primer domains are directly linked (e.g., covalently).
  • primer domains are separated by one or more nucleotides.
  • primer domains are separated by 2 to 10 nucleotides, such as 2 to 5 nucleotides, such as 4 nucleotides.
  • 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 thymine one or more thymine (T). In some embodiments, a linker is a TTTT linker. A linker may improve spacing and flexibility.
  • 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.
  • 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).
  • a primer comprises a first primer domain and a second primer domain, wherein the two domains are complementary.
  • 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.
  • a primer comprises a first primer domain that is complementary to a second primer domain.
  • a primer comprises two primer domains that are complementary to each other.
  • a primer comprises a primer domain that is complementary to a primer domain in another primer.
  • 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.
  • the entire primer is complementary to a target nucleotide sequence or complementary target domain(s), or complements thereof.
  • a primer domain that is complementary to a target nucleotide sequence is capable of hybridizing to the target nucleotide sequence, i.e., hybridization site.
  • a primer domain that is complementary to a primer nucleotide sequence is capable of hybridizing to the primer nucleotide sequence, i.e., hybridization site.
  • 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%.
  • 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.
  • composition and methods described herein comprise one or more hybrid primer(s) (e.g., a first hybrid primer, a second hybrid primer, etc.).
  • a primer as described herein is a hybrid primer.
  • a hybrid primer is a first hybrid primer.
  • a hybrid primer is a second hybrid primer.
  • 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.
  • 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.
  • 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.
  • a hybrid primer comprises one or more 3’ Target (T) domain(s) and one or more 5’ hairpin nucleotide sequence(s).
  • a T domain is a T1 domain.
  • a T domain is a T2 domain.
  • a T1 domain and a T2 domain are non-overlapping.
  • a T1 domain and a T2 domain hybridize to opposite or complementary strands of the target nucleotide sequence.
  • a first hybrid primer comprises a 3’ T1 domain and a 5’ first hairpin nucleotide sequence.
  • a second hybrid primer comprises a 3’ T2 domain and a 5’ second hairpin nucleotide sequence.
  • a 3’ Target (T) domain (e.g., a 3’ T1 domain, a 3’ T2 domain, or both) is complementary to a target nucleotide sequence.
  • a 3’ T domain is complementary to a Tc domain of the target nucleotide sequence.
  • a 3’ T1 domain is complementary to a Tic domain of the target nucleotide sequence.
  • a 3’ T2 domain is complementary to a T2c domain of the target nucleotide sequence.
  • a 3’ T domain of a hybrid primer is capable of hybridizing to a Tc domain of a target nucleotide sequence.
  • one or more primer domains are capable of hybridizing to a Tc domain of the target nucleotide sequence.
  • a 3’ T domain of a hybrid primer and an inner primer domain are both capable of hybridizing to the same Tc domain of the target nucleotide sequence.
  • An example hereof is shown in Example 4.
  • 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
  • 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.
  • 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.
  • 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.
  • a 3’ T1 domain is complementary to a Tic domain of a target nucleotide sequence.
  • a 3’ T2 domain is complementary to a T2c domain of a target nucleotide sequence.
  • a Tic domain and a T2c domain are non-overlapping domains located on opposite or complementary strands of the target nucleotide sequence.
  • 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.
  • 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.
  • 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 nucleotide sequence.
  • 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.
  • 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.
  • a first 5’ hairpin nucleotide sequence comprises four domains from 3’ to the 5’
  • a second 5’ hairpin nucleotide sequence comprises four domains from 3’ to the 5’
  • At least one domain of the first 5’ hairpin nucleotide sequence is not complementary to a target nucleotide sequence.
  • 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
  • at least two domains of the first 5’ hairpin nucleotide sequence are not complementary to a target nucleotide sequence.
  • 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, at least 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.
  • 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.
  • a 5’ hairpin nucleotide sequence (e.g., a first 5’ hairpin nucleotide sequence and/or second 5 ’ hairpin nucleotide sequence) adapts a folded form.
  • a folded form comprises a hybrid primer stem portion and hybrid primer loop.
  • a 1) 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.
  • 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).
  • a hybrid primer loop is single stranded.
  • a hybrid primer stem is double stranded.
  • a Bl domain and a Bic domain hybridize to form a first hybrid primer stem.
  • an LBc domain and a B2 domain form a first hybrid primer loop.
  • a Fl domain and an Flc domain hybridize to form a second hybrid primer stem.
  • an LFc domain and a F2 domain form a second hybrid primer loop.
  • a Bl domain comprises or consists of a nucleotide sequence.
  • a Bl domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof.
  • a Bl domain is complementary to another primer domain (e.g., hybrid primer domain).
  • a Bl domain is complementary to a Bic domain.
  • a Bl domain is characterized in that it hybridizes to a Bic domain.
  • a Bl domain and a B 1c domain hybridize to form a first hybrid primer stem.
  • 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.
  • 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.
  • an LBc domain comprises or consists of a nucleotide sequence.
  • an LBc domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof.
  • an LBc domain is a part of a hybrid primer loop (e.g., a first hybrid primer loop).
  • an LBc domain is not complementary to any of the first 5’ hairpin nucleotide sequence domains.
  • an LBc domain is characterized in that it does not hybridize to any of the first 5’ hairpin nucleotide sequence domains.
  • a hybrid primer loop comprises an LBc domain and a B2 domain.
  • a hybrid primer loop comprises an LBc domain, a B2 domain, and one or more primer domains.
  • an LBc domain and a B2 domain form a first hybrid primer loop (e.g., a loop without any pairing nucleotides).
  • an LBc domain is complementary to a LB domain of a LB primer (e.g., a loop primer).
  • a LBc domain comprises a nucleotide sequence of about 9 to about 30 nucleotides that is complementary to a LB primer.
  • a hybrid primer loop (e.g., a first hybrid primer loop) is complementary to a LB primer.
  • 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.
  • 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.
  • 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. In some 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.
  • 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.
  • a B2 domain comprises or consists of a nucleotide sequence.
  • B2 domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof.
  • a B2 domain is not complementary to any of the first 5 ’ hairpin nucleotide sequence domains.
  • a B2 domain is complementary to a B2c domain.
  • a B2 domain of a first hybrid primer is identical to a B2 domain of a backward inner primer (BIP).
  • 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.
  • a Bic domain comprises or consists of a nucleotide sequence.
  • a Bic domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof.
  • a Bic domain is complementary to a primer domain (e.g., hybrid primer domain).
  • a Bic domain is complementary to a Bl domain.
  • a Bic domain is characterized in that it hybridizes to a Bl domain.
  • a Bic domain and a Bl domain hybridize to form a first hybrid primer stem.
  • 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.
  • a Bic domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides. In some embodiments, a Bic domain is or comprises a nucleotide sequence of about 10 nucleotides to about 40 nucleotides, such as about 15 nucleotides to about 35 nucleotides.
  • a composition comprises
  • 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’
  • 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.
  • a Fl domain comprises or consists of a nucleotide sequence.
  • a Fl domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof.
  • a Fl domain is complementary to another primer domain (e.g., hybrid primer domain).
  • a Fl domain is complementary to an Flc domain.
  • a Fl domain is characterized in that it hybridizes to an Flc domain.
  • a Fl domain and an Flc domain hybridize to form a second hybrid primer stem.
  • a Fl domain is directly connected to a T2 domain or linked (e.g., covalently) by one or more nucleotides to a T2 domain.
  • a Fl domain is directly connected to an LFc domain or linked by one or more nucleotides to an LFc domain.
  • a Fl domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides.
  • 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.
  • an LFc domain comprises or consists of a nucleotide sequence.
  • an LFc domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof.
  • an LFc domain is a part of a hybrid primer loop (e.g., a second hybrid primer loop).
  • an LFc domain is not complementary to any of the second 5’ hairpin nucleotide sequence domains.
  • an LFc domain is characterized in that it does not hybridize to any of the second 5’ hairpin nucleotide sequence domains.
  • a hybrid primer loop comprises an LFc domain and a F2 domain.
  • a hybrid primer loop comprises an LFc domain, a F2 domain, and one or more primer domains.
  • an LFc domain and a F2 domain form a first hybrid primer loop (e.g., a loop without any pairing nucleotides).
  • an LFc domain is complementary to a LF domain of a LF primer (e.g., a loop primer).
  • a LFc domain comprises a nucleotide sequence of about 9 to about 30 nucleotides that is complementary to a LF primer.
  • about 20% to about 50% of the hybrid primer loop is complementary to a LF primer.
  • 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.
  • 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.
  • 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.
  • an LFc domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides.
  • a 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.
  • a F2 domain comprises or consists of a nucleotide sequence.
  • F2 domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof.
  • a F2 domain is not complementary to any of the first 5 ’ hairpin nucleotide sequence domains.
  • a F2 domain of a second hybrid primer is the same as an F2p domain of a forward inner primer (FIPp).
  • 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.
  • an Flc domain comprises or consists of a nucleotide sequence.
  • an Flc domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof.
  • an Flc domain is complementary to a primer domain (e.g., hybrid primer domain).
  • an Flc domain is complementary to a Fl domain.
  • an Flc domain is characterized in that it hybridizes to a Fl domain.
  • an Flc domain and a Fl domain hybridize to form a second hybrid primer stem.
  • an Flc domain is directly connected to an F2 domain or linked (e.g., covalently) by one or more nucleotides to an F2 domain.
  • 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.
  • a composition comprises
  • 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’
  • 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.
  • compositions and methods of the present disclosure comprise an inner primer (e.g., a backward inner primer (BIP) and/or forward inner primer (FIP)).
  • a primer as described herein is an inner primer.
  • an inner primer is a BIP.
  • an inner primer is a FIP (e.g., a FlPt or a FIPp).
  • 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.
  • 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 primer nucleotide 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.
  • 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.
  • 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.
  • 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.
  • a BIP comprises a B2 domain and a Bic domain.
  • the B2 domain may be located 3’ to the Bic domain.
  • a BIP comprises a 3’ B2 domain and a 5’ Bic domain.
  • a B2 domain and a Bic domain are separated by a linker.
  • a linker is a TTTT linker.
  • a B2 domain comprises or consists of a nucleotide sequence.
  • a B2 domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof.
  • a B2 domain is not complementary to any of the first 5 ’ hairpin nucleotide sequence domains.
  • a B2 domain is complementary to a B2c domain.
  • 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).
  • a B2 domain of a BIP is the same as a B2 domain of a first hybrid primer.
  • a B2 domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides.
  • 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.
  • a Bic domain comprises or consists of a nucleotide sequence.
  • Bic domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof.
  • a Bic domain is complementary to a primer domain.
  • a B 1c domain is complementary to a Bl domain (e.g., a first hybrid primer Bl domain).
  • a Bic domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides.
  • 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.
  • 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).
  • 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).
  • 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.
  • a composition comprises a BIP whose nucleotide sequence comprises a 3’ B2 domain and a 5’ Bic domain complementary to the Bl domain.
  • a composition comprises
  • 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;
  • a backward inner primer (BIP) whose nucleotide sequence comprises a 3’ B2 domain and a 5’ Bic domain complementary to the Bl domain
  • a forward inner primer comprises a nucleotide sequence.
  • a FIP comprises one or more primer domains.
  • 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.
  • a FIP capable of hybridizing to a hybrid primer is a FIPp.
  • a FIP capable of hybridizing to a target nucleotide sequence is a FlPt.
  • a composition comprises a FIP primer whose nucleotide sequence comprises a 3’ F2 domain and a 5’ Flc domain complementary to the Fl domain.
  • 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).
  • a FIPp comprises an F2p domain and an Flcp domain.
  • the F2p domain may be located 3’ to the Flcp domain.
  • a FIPp comprises a 3’ F2p domain and a 5’ Flcp domain.
  • an F2p domain and an Flcp domain are separated by a linker.
  • a linker is a TTTT linker.
  • 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).
  • 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.
  • an Flcp domain comprises or consists of a nucleotide sequence.
  • an Flcp domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof.
  • an Flcp domain is complementary to a primer domain.
  • an Flcp domain is complementary to an Fl domain (e.g., a second hybrid primer Fl domain).
  • an Flcp domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides.
  • 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.
  • 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).
  • 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 hybrid primer permits extension of the FIPp by a polymerase enzyme (e.g., with stand displacement activity) thereby synthesizing a new strand.
  • a composition comprises a FTPp whose nucleotide sequence comprises a 3’ F2p domain and a 5’ Flcp domain complementary to a Fl domain.
  • one or more FlPt primer domains are complementary to a target nucleotide sequence (e.g., target domain) or complement thereof.
  • two or more FlPt primer domains are complementary to a target nucleotide sequence (e.g., target domain) or complement thereof.
  • a FlPt primer consists or comprises of a ribonucleotide sequence.
  • a FlPt ribonucleotide primer can be used to convert an ssRNA into an ssDNA and/or dsDNA by reverse transcription.
  • a FlPt primer consists or comprises of a deoxyribonucleotide sequence.
  • a FlPt deoxyribonucleotide primer can be used to synthesize a complementary DNA strand a target deoxyribonucleotide sequence by a polymerase.
  • a FlPt comprises an F2t domain and an Flct domain.
  • the F2t domain may be located 3’ to the Flct domain.
  • a FlPt comprises a 3’ F2t domain and a 5’ Flct domain.
  • an F2t domain and an Flct domain are separated by a linker.
  • a linker is a TTTT linker.
  • a FlPt-linked nucleotide strand comprises complementary regions at the 5’ end, resulting in formation of a stem-loop structure at said 5’ end.
  • a FlPt-linked nucleotide strand may be released as a result of DNA synthesized initiated by a F3 primer.
  • an F2t domain comprises or consists of a nucleotide sequence.
  • an F2t domain is complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof.
  • an F2t domain is complementary to an F2ct domain of a target sequence.
  • an F2ct domain is formed as a result of synthesis of a DNA strand that is complementary to an F2t domain of the target sequence.
  • 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.
  • an Flct domain comprises or consists of a nucleotide sequence.
  • an Flct domain is complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof.
  • an Flcp domain is or comprises a nucleotide sequence of about 9 nucleotides to about 30 nucleotides.
  • 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.
  • 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.
  • a polymerase enzyme e.g., with stand displacement activity
  • a composition comprises a FlPt whose nucleotide sequence comprises a 3’ F2t domain and a 5’ Flct domain complementary to the Fit domain.
  • 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.
  • an Flct domain is located 5’ to an F2ct domain of the same strand, i.e., target stand or complement thereof.
  • an outer primer e.g., a B3 primer, F3 primer, or both.
  • a primer as described herein is an outer primer.
  • an outer primer is a B3 primer.
  • an outer primer is a F3 primer.
  • a hybrid primer is capable of annealing to a Tc domain of a target nucleotide sequence.
  • 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.
  • a polymerase 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.
  • an outer primer e.g., a B3 primer or F3 primer
  • a hybrid primer or inner e.g., FIP or BIP
  • 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.
  • 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
  • a composition comprises a B3 primer whose nucleotide sequence is complementary to a B3c domain of a target nucleotide sequence.
  • F3 primers whose nucleotide sequence is complementary to a B3c domain of a target nucleotide sequence.
  • 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.
  • 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
  • a composition comprises a F3 primer whose nucleotide sequence is complementary to an F3c domain of a target nucleotide sequence.
  • 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.
  • 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.
  • a hairpin-loop amplicon e.g., amplicons produced by methods and compositions described herein, such as a hairpin-loop amplicon, a dumbbell-shaped amplicon, or a combination
  • Loop primers are particular useful in facilitating subsequent rounds of amplification through extension on the loops and annealing of the primers.
  • a primer as described herein is a loop-primer.
  • a loop-primer is a LB primer.
  • a loop-primer is a LF primer.
  • 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).
  • 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.
  • 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.
  • a polymerase enzyme e.g., with stand displacement activity
  • a composition comprises a LB primer whose nucleic acid sequence comprises a LB domain that is complementary to the LBc domain.
  • a LF primer comprises a nucleotide sequence.
  • a LF primer is or comprises a primer domain.
  • a LF primer is or comprises a LF domain.
  • a LF domain is not complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof.
  • a LB domain capable of hybridizing to a hybrid primer is an LBp.
  • a LF domain is complementary to a target nucleotide sequence (e.g., a target domain) or complement thereof.
  • a LF domain capable of hybridizing to a target nucleotide sequence or complement thereof is an LFt.
  • a composition comprises a LF primer whose nucleotide sequence is or comprises a LF domain that is complementary to an LFc domain.
  • an LFp primer comprises a nucleotide sequence complementary to the single stranded loop region of the second hybrid primer.
  • an LFp domain is complementary to an LFc domain (e.g., an LFc domain of a second hybrid primer).
  • 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.
  • 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.
  • a polymerase enzyme e.g., with stand displacement activity
  • a composition comprises an LFp primer whose nucleotide sequence is or comprises an LFp domain that is complementary to an LFc domain.
  • an LFt primer comprises a nucleotide sequence complementary to a target nucleotide sequence.
  • an LFt domain is complementary to an LFc domain (e.g., an LFc domain of a target sequence).
  • 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.
  • 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 an LFc domain permits extension of the LFt by a polymerase enzyme (e.g., with stand displacement activity) hereby synthesizing a new strand.
  • a polymerase enzyme e.g., with stand displacement activity
  • a composition comprises an LFt primer whose nucleotide sequence is or comprises an LFt domain that is complementary to an LFct domain.
  • a primer described herein comprises one or more modified nucleotides (e.g., modified ribonucleotides, modified deoxyribonucleotides, or a combination hereof).
  • modified nucleotides e.g., modified ribonucleotides, modified deoxyribonucleotides, or a combination hereof.
  • a modified nucleotide is a peptide nucleic acid (PNA).
  • 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.
  • 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).
  • 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.
  • a nucleic acid comprising a modified deoxyribonucleotide can be amplified at lower temperatures relative to a control without modified deoxyribonucleotide.
  • a primer comprises a modified nucleotide in place of at least one guanine or adenine.
  • a modified nucleotide is a 2- Aminopurine (e.g., a purine analog of guanine and adenine).
  • a primer comprising a 2- Aminopurine is useful in fluorescence readouts.
  • 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.
  • a primer described herein comprises a fluorophore.
  • a fluorophore is a Cy5, a FAM, a TxRed, a YakYel, or a HEX.
  • a fluorophore is attached to the 5’ end of the primer.
  • a fluorophore is attached to the 3’ end of the primer.
  • a fluorophore is within the primer.
  • 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).
  • a primer described herein comprises a quencher.
  • a quencher is attached to the 5’ end of the primer.
  • a quencher is attached to the 3’ end of the primer.
  • a quencher is within the primer.
  • a quencher is a 3IAbRQsp, a BHQ2, a 3IABkFQ, or a BHQl.
  • 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.
  • a source of interest comprises an organism, such as an animal or human.
  • a sample is obtained or derived from an organism (e.g., a mammalian organism, for example, including a human).
  • a biological sample is or comprises biological tissue or fluid.
  • 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.
  • a biological sample is or comprises cells obtained from an individual.
  • obtained cells are or include cells from an individual from whom the sample is obtained.
  • a sample is a “primary sample’’ obtained directly from a source of interest by any appropriate means.
  • 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.
  • 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.
  • 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.
  • a source of interest comprises a virus or microbe.
  • a sample is obtained or derived from virus or microbe.
  • a viral or microbial sample is a “primary sample” obtained directly from a source of interest by any appropriate means.
  • 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.
  • a sample comprises a target nucleotide sequence.
  • a sample is a crude sample (e.g., a primary sample or a sample that has undergone minimal processing).
  • 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.
  • 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.
  • a food sample fresh fruits or vegetables, meats
  • a beverage sample such as 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
  • samples are lysed (e.g., processed) using sodium hydroxide (NaOH).
  • NaOH sodium hydroxide
  • samples are lysed with NaOH at ambient temperature (e.g., room temperature).
  • the concentration of NaOH is about 1 rnM NaOH to about 200 mM NaOH.
  • the concentration of NaOH is about 10 mM NaOH to about 100 mM.
  • 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.
  • samples are treated with NaOH to inhibit or reduce RNase activity.
  • NaOH releases viral nucleic acids from a viral sample.
  • NaOH denatures double stranded DNA or RNA (e.g., separates strands).
  • samples e.g., viral particles and/or cells
  • KOH potassium hydroxide
  • samples comprising DNA e.g., dsDNA
  • samples are lysed with KOH at ambient temperature (e.g., room temperature).
  • 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.
  • 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.
  • samples are treated with KOH to inhibit or reduce RNase activity.
  • KOH releases viral nucleic acids from a viral sample.
  • KOH denatures double stranded DNA or RNA (e.g., separates strands).
  • KOH denaturation separates dsDNA and produces ssDNA.
  • 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.
  • a target nucleotide sequence be double-stranded.
  • a template nucleic acid may be single-stranded.
  • a target nucleotide sequence may be genomic DNA, mitochondrial DNA, viral DNA, plasmid DNA, synthetic dsDNA, or RNA.
  • 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.
  • a target nucleotide sequence is a copied and/or amplified target nucleotide sequence.
  • 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.
  • 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.
  • a target nucleotide sequence comprises modified nucleotides, for example, including methylated nucleotides or nucleotide analogs.
  • a target nucleotide sequence may be interspersed with non-nucleic acid components.
  • 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.
  • a target nucleotide sequence is located in the nucleus or cytoplasm of a cell.
  • a target nucleotide is ex vivo.
  • a target nucleotide sequence is present in an in vitro system.
  • a target nucleotide sequence is present in a sample, e.g., in a biological sample or in an environmental sample.
  • 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.
  • 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 e.g., prenatal nucleic acids, etc.
  • a target nucleotide is a viral, a bacterial, a fungal, a protozoan, or a parasitic sequence.
  • 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.
  • a target nucleotide sequence or complement thereof comprises a specific, recognizable, protospacer adjacent motif (PAM).
  • 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.
  • polymerase for example, for target nucleotide sequence synthesis, conversion of one nucleic acid type to another, and/or transcription.
  • compositions, kits and methods of present disclosure comprise a polymerase.
  • a polymerase is a strand displacing polymerase.
  • a strand displacing polymerase is able to displace downstream DNA during elongation.
  • 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.
  • a polymerase is a DNA polymerase.
  • a strand displacing polymerase has elongation activity at ambient temperature.
  • 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.
  • a polymerase has strand displacing activity.
  • a polymerase is a Bsu Polymerase, Bsm Polymerase,
  • 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).
  • 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.
  • a strand displacing polymerase is Bsu or a variant thereof.
  • a strand displacing polymerase is Klenow or a variant thereof.
  • a cLAMP reaction utilizes a DNA polymerase enzyme, preferably a DNA polymerase with high strand displacement activity.
  • 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.
  • compositions, kits and methods of present disclosure comprise a reverse transcriptase.
  • a target nucleotide sequence consists or comprises of a deoxyribonucleotide sequence.
  • a primer as described herein e.g., a FlPt, a BIP, a B3, a F3, and/or a hybrid primer
  • a reverse transcriptase extends the primer hereby synthesizing a DNA strand.
  • a complement DNA strand is contacted with one or more primers as described herein.
  • 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).
  • 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.
  • amplification of a target nucleotide is initiated by conversion of ssRNA to dsDNA by reverse transcription.
  • methods described herein utilize ssRNA.
  • methods described herein utilize dsDNA.
  • a reaction to convert ssDNA to dsDNA is conducted.
  • 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.
  • PCR polymerase chain reaction
  • 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.
  • a target nucleotide sequence is RNA
  • RNA is converted to dsDNA prior to target nucleotide sequence synthesis and/or amplification.
  • a primer as described herein is used in a reverse transcription reaction to generate one or more amplicons (e.g., a hairpin-loop amplicon).
  • such an amplicon may be a template for a hybrid primer (e.g., a first hybrid primer, a second hybrid primer, or both).
  • 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.
  • AMV Avian Myeloblastosis Virus
  • M-MuLV Moloney Murine Leukemia Virus
  • 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.
  • conditions e.g., salt, temperature, etc.
  • amplification reagents comprises one or more dNTPs. In some embodiments, amplification reagents comprises a buffer.
  • the methods of amplification described herein is performed under isothermal conditions.
  • cLAMP is performed at ambient temperatures.
  • cLAMP is performed at temperatures within a range of about 20°C to about 65°C.
  • cLAMP is performed at temperatures within a range of about 20°C to about 60°C.
  • cLAMP is performed at temperatures within a range of about 20°C to about 55°C.
  • cLAMP is performed at temperatures within a range of about 20°C to about 50°C.
  • cLAMP is performed at temperatures within a range of about 20°C to about 45°C.
  • 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.
  • technologies described herein comprises sodium, magnesium, salts, or combinations hereof.
  • the concentration of sodium is within the range of 0 mM to 100 mM.
  • the concentration of magnesium is within the range of 0 mM to 20 mM.
  • a salt is Tris.
  • the concentration of Tris is 1 mM to 100 mM.
  • compositions and methods described herein uses an inorganic pyrophosphatase (PPase).
  • PPase may catalyze the hydrolysis of inorganic pyrophosphate and can be used for enhancement of DNA replication.
  • a PPase is a thermostable pyrophosphatase (TIPP).
  • a composition as described herein comprises a TIPP.
  • a kit as described herein comprises a TIPP.
  • methods described herein utilizes a TIPP.
  • 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.
  • 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
  • a hairpin-loop amplicon is or comprises a nucleotide sequence.
  • a hairpin-loop amplicon is or comprises a ribonucleotide sequence.
  • a hairpin-loop amplicon is or comprises a deoxyribonucleotide sequence.
  • a hairpin-loop amplicon comprises of a 5’ hybrid primer as described herein and a 3’ target sequence or complement thereof.
  • 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.
  • a hairpin-loop amplicon if formed by 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).
  • a complement of a target nucleotide is an inner primer extension product.
  • 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’
  • a hybrid primer exceeds half of the overall size of the hairpin-loop amplicon.
  • at least 50% of the hairpin-loop amplicon is not complementary to a target nucleotide sequence.
  • at least 60% of the hairpin-loop amplicon is not complementary to a target nucleotide sequence.
  • at least 70% of the hairpin-loop amplicon is not complementary to a target nucleotide sequence.
  • at least 80% of the hairpin-loop amplicon is not complementary to a target nucleotide sequence.
  • at least 90% of the hairpin-loop amplicon is not complementary to a target nucleotide sequence.
  • one or more target single nucleotide polymorphism(s) are incorporated into a hairpin-loop amplicon.
  • one or more guide polynucleotides are capable of hybridizing to a hairpin-loop amplicon comprising one or more SNPs.
  • a hairpin-loop amplicon is synthesized by a reverse transcriptase.
  • a hairpin-loop amplicon is synthesized by a polymerase (e.g., a DNA polymerase, such as a DNA polymerase having strand displacement activity).
  • a hairpin-loop amplicon is synthesized by a reverse transcriptase and a polymerase. Dumbbell-shaped amplicons
  • a dumbbell-shaped amplicon can serve as a template for target nucleotide sequence synthesis (e.g., amplification).
  • 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.
  • dumbbell-shaped amplicon 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.
  • 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 domains
  • At least two domains of the dumbbell-shaped amplicon are not complementary to a target nucleotide sequence.
  • at least three domains of the dumbbell-shaped amplicon are not complementary to a target nucleotide sequence.
  • at least four domains of the dumbbell- shaped amplicon are not complementary to a target nucleotide sequence.
  • at least five domains of the dumbbell-shaped amplicon are not complementary to a target nucleotide sequence.
  • at least six domains of the dumbbell-shaped amplicon are not complementary to a target nucleotide sequence.
  • 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.
  • 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.
  • at least 50% of the dumbbell-shaped amplicon is not complementary to a target nucleotide sequence.
  • at least 55% of the dumbbell-shaped amplicon is not complementary to a target nucleotide sequence.
  • at least 60% of the dumbbell-shaped amplicon is not complementary to a target nucleotide sequence.
  • 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.
  • one or more target SNP are incorporated into a dumbbell-shaped amplicon.
  • one or more guide polynucleotides are capable of hybridizing to a dumbbell-shaped amplicon comprising one or more SNPs.
  • 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.
  • methods and/or compositions as provided herein may be utilized in a target nucleotide synthesis reaction, e.g., a cLAMP reaction.
  • provided compositions and methods may be used to determine or confirm presence or absence of a target nucleotide.
  • provided compositions and methods may be used to amplify a target nucleotide.
  • compositions and methods may be used to quantify amount of target nucleotide present in a particular sample.
  • target nucleotide synthesis is combined with other technologies (e.g., detection technologies).
  • other detection technologies utilize a CRISPR/Cas system, e.g., with collateral activity e.g., SHERLOCK or DETECTR).
  • compositions, primers, amplicons, methods, kits and uses described herein may be used for diagnostic purposes.
  • an amplicon such as a hairpin-loop amplicon, a dumbbell- shaped amplicon, or both.
  • 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).
  • the disclosure provides methods and compositions for producing a hairpin- loop amplicon comprising a target nucleotide sequence or complement thereof.
  • 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’
  • 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’
  • 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,
  • the present disclosure provides methods and compositions for amplifying a target nucleotide sequence.
  • 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.
  • 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.
  • cLAMP uses one or more hairpin primers providing several advantages over conventional LAMP and ligation-initiated LAMP methods.
  • a method for amplifying a target nucleotide sequence comprising the steps of:
  • target nucleotide sequence synthesis and/or amplification by cLAMP can occur in a one -pot method.
  • a downstream reaction e.g., a nucleic acid processing and/or detection reaction
  • a downstream reaction is performed after nucleotide synthesis and/or amplification.
  • Downstream reactions may or comprise one or more of amplification, cleavage, digestion, hybridization, replication, etc.
  • 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.
  • the present disclosure provides technologies for detecting nucleotides, amplicons, or both.
  • the present disclosure provides methods for detecting a target nucleotide sequence.
  • 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.
  • 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).
  • 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;
  • 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.
  • detection technologies comprise, for example, chemiluminescence, electrochemical technologies, fluorescence, intercalating dye detection, migration, and/or radiation.
  • a step of detecting is performed by detecting a change in florescence as an indication of amplification of the target nucleotide sequence.
  • a wherein the change in the fluorescence is an increase in the intensity of fluorescence emission of the detectably labeled nucleic acid probe.
  • detection technologies comprise, for example, colorimetric, turbidity, other types of catalysts, molecular beacons and other oligonucleotide-based probes, aptamers, or lateral flow.
  • 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.
  • method of detecting a target nucleotide sequence utilize a SYBR dye.
  • methods of detecting a target nucleotide sequence utilize SYBR Green.
  • a double stranded DNA binding dye is a minor-groove binding dye.
  • a mino-groove binding dye is SYBR Green I and II, DAPI, PicoGreen, or a combination.
  • a double stranded DNA binding dye is an intercalating dye.
  • an intercalating dye is an Ethidium Bromide, Propidium Iodide, EvaGreen, or a combination.
  • 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).
  • CRISPR/Cas detection e.g., diagnostic
  • nucleic acids containing a relevant target site e.g., Cas target nucleic acid
  • a relevant target site e.g., Cas target nucleic acid
  • a Cas enzyme has collateral activity.
  • 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.
  • CRISPR/Cas-based detection may be a CRISPR-Cas 13-based detection system.
  • a CRISPR/Cas-based detection system is a CRISPR/Casl2- based detection system.
  • a CRISPR/Casl3- or CRISPR/Casl2-based detection system is a CRISPR-SHERLOCK detection system.
  • methods according to the present disclosure utilize a CRISPR-SHERLOCK detection system.
  • 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.
  • a Cas enzyme is a thermostable Cas enzyme.
  • a thermostable Cas enzyme may be a thermostable Cas Enzyme as described in US Publication, US 2023/0002811, entitled “APPLICATION OF CAS PROTEIN, METHOD FOR DETECTING TARGET NUCLEIC ACID MOLECULE AND KIT” and published 01/01/2023; PCT Publication WO 2020/142754, entitled “PROGRAMMABLE NUCLEASE IMPROVEMENTS AND COMPOSITIONS AND METHODS FOR NUCLEIC ACID AMPLIFICATION AND DETECTION” and published 07/03/2021; PCT Publication WO 2021/154866, entitled “IMPROVED DETECTION ASSAYS” and published 08/01/2021; 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.
  • 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.
  • 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.
  • 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.
  • a guide polynucleotide comprises a guide domain (e.g., 3’ SI domain) that is complementary to a target nucleotide sequence or complement thereof.
  • a guide polynucleotide comprises a guide hairpin.
  • a guide hairpin comprises a direct repeat sequence.
  • a direct repeat sequence is not complementary to a target nucleotide sequence.
  • technologies according to the present disclosure utilizes two or more guide polynucleotides. This may be useful for redundancy and/or sensitivity purposes.
  • 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.
  • a guide polynucleotide is complementary to a target nucleotide sequence having one or more SNP mutations.
  • 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.
  • WT wild type
  • a guide polynucleotide binds to an amplicon that contains a SNP hereby tolerating the mismatch.
  • 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.
  • Guide polynucleotides can be designed to straddle a primer nucleotide sequence and a target nucleotide sequence, or target only a primer nucleotide sequence entirely.
  • a guide polynucleotide is complementary to a target nucleotide sequence.
  • 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.
  • a guide polynucleotide is not complementary to a target nucleotide sequence. Exemplary guide polynucleotides are shown in Figure 19.
  • a detection method comprises a CRISPR-Cas based detection method (e.g., CRISPR-SHERLOCK).
  • 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).
  • 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.
  • SNPs single nucleotide polymorphism(s)
  • provided technologies can be utilized to detect a SNP-containing nucleic acid.
  • provided technologies can be utilized to detect SNP-containing nucleic acids in a patient-derived sample or samples.
  • 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.
  • 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.
  • 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..
  • a microbe may be a bacterium, a fungus, a yeast, a protozoa, a parasite, or a virus.
  • 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.
  • 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.
  • provided technologies require a reduced the number of target specific domains for nucleotide amplicon production.
  • provided technologies can identify and/or detect highly mutated regions also allowing for reactions requiring fewer multiplexed or degenerated primers than conventional methods.
  • provided technologies may be particularly amenable to use in point-of-care devices.
  • provided technologies can guide therapeutic regimens (e.g., selection of treatment type and/or dose and/or duration of treatment).
  • 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.
  • provided technologies are useful for assessment of environmental samples.
  • 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.
  • 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.
  • 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.
  • technologies of the present inventions are useful in genotyping.
  • kit for performing the present methods may comprise a composition or a component thereof as described herein.
  • kits for performing methods of nucleotide synthesis, nucleotide amplification, nucleotide detection, or a combination of a target nucleotide sequence from a sample.
  • a kit of parts comprises a composition according to the present invention, and/or one or more components thereof.
  • a kit of parts may comprise a hybrid primer, a BIP, optionally one or more primers, amplification reagents and/or instructions for use.
  • kits for performing methods of detecting a target nucleotide sequence in a sample.
  • a kit of parts comprises a primer composition according to the present invention.
  • a kit of parts may also comprise amplification reagents, a nucleic acid sensor system, a sample collection device, and/or instructions for use.
  • a kit of parts may comprise a nucleic acid sensor system useful for detecting a target nucleotide sequence.
  • nucleic acid sensor systems such as an INSPECTRTM nucleic acid detection system, SHERLOCK nucleic acid detection system, etc.
  • a kit of parts also comprises a control nucleic acid, such as may be spiked into a sample as described herein.
  • Exemplary technologies 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 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.
  • primer domains are complementary to a target nucleotide sequence.
  • at the most 7 primer domains are complementary to a target nucleotide sequence.
  • at the most 6 primer domains are complementary to a target nucleotide sequence.
  • at the most 5 primer domains are complementary to a target nucleotide sequence.
  • at the most 4 primer domains are complementary to a target nucleotide sequence.
  • at the most 3 primer domains are complementary to a target nucleotide sequence.
  • at the most 2 primer domains are complementary to a target nucleotide sequence.
  • 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.
  • Figure 1 shows the components required for a cLAMP variation that comprises a single hybrid primer.
  • 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).
  • 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.
  • FIG. 2 shows the cLAMP mechanism in detail.
  • step 1 the “t” domain of the hybrid primer hinds to the “tc” domain of the template strand, and is extended from the 3’ end by a polymerase.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • B3 Backward Outer Primer
  • F3 Target-specific Forward Outer Primer
  • FIG 4 shows exemplary reaction mechanism for cLAMP variation 2.
  • 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.
  • step 2a the hybrid primer is extended by a polymerase to form a complement to the target polynucleotide sequence.
  • 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.
  • 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.
  • cLAMP is performed in the reverse orientation from previous examples.
  • 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
  • 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.
  • 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.
  • Variation 4 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
  • 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.
  • Variation 5 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.
  • 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.
  • 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.
  • additional mismatches could be added in principle, and/or positioned differently in the guide’s spacer domain.
  • Fig. 12 The mechanism (Fig. 12) is similar to that in Figure 1, but the SNP mutation is incorporated into the final amplicon.
  • 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
  • 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.
  • Variation 7 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.
  • 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.
  • Variation 8 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.
  • 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.
  • 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
  • the present Example demonstrates an exemplary variation of nucleotide synthesis and amplification using a single hybrid primer.
  • 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.
  • 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).
  • 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.
  • 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
  • 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.
  • cLAMP composition 20,000 copies of an Influenza B target were successfully amplified and distinguished from a non-target control.
  • 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.
  • 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.
  • Influenza B target (10,000 copies per reaction) is no longer distinguishable from non-target material (NTC) ( Figure 32B).
  • NTC non-target material
  • Figure 32A 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Influenza B target (10,000 copies per reaction) is no longer distinguishable from non-target material (NTC) ( Figure 34B).
  • NTC non-target material
  • Example 11 Variation 1 followed by detection.
  • the present Example demonstrates an exemplary method of nucleotide synthesis, amplification and detection using a single hybrid primer and a single guide RNA.
  • Figure 21 shows the components required for a cLAMP variation that comprises a single hybrid primer.
  • 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).
  • 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.
  • FIG. 22 shows the cLAMP and detection mechanism in detail.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • B3 Backward Outer Primer
  • F3 Target-specific Forward Outer Primer
  • FIG 23 shows exemplary reaction mechanism for cLAMP variation 2 followed by detection.
  • 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.
  • step 2a the hybrid primer is extended by a polymerase to form a complement to the target polynucleotide sequence.
  • 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.
  • the present Example demonstrates an exemplary variation of nucleic acid synthesis and amplification using a single hybrid primer followed by detection.
  • Variation 3 followed by detection 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.
  • cLAMP is performed in the reverse orientation from previous examples.
  • 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
  • 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.
  • 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.
  • 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.
  • Variation 4 followed by detection 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.
  • 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.
  • 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.
  • Variation 8 followed by detection 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.
  • 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.
  • 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 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.
  • the present Example demonstrates an exemplary variation of nucleotide synthesis, amplification and detection using a single hybrid primer and a guide RNA.
  • 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.
  • 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).
  • 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.
  • 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
  • This example demonstrates effective viral particle lysis and effective inhibition of RNase at ambient temperature, through use of lysis technologies provided herein.
  • RT room temperature reverse transcriptase
  • 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.
  • 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.
  • 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.
  • 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 was stopped 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.
  • 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.
  • 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.
  • Example 18 Ambient temperature bacterial lysis
  • 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.
  • 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.
  • Example 19 Lysis at varying incubation temperatures
  • 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%.
  • Example 20 KOH lysis of N. gonorrhoeae in vaginal matrix in LAMP-Cas
  • This example demonstrates effective bacterial lysis in a vaginal matrix through use of lysis technologies provided herein.
  • 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).

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Abstract

La présente divulgation concerne des procédés et des compositions pour l'amplification de nucléotides.
PCT/US2024/032026 2023-06-01 2024-05-31 Amplification isotherme à médiation par boucle condensée Pending WO2024249878A1 (fr)

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US20220348990A1 (en) * 2016-02-17 2022-11-03 President And Fellows Of Harvard College Molecular programming tools
US20210254139A1 (en) * 2016-11-10 2021-08-19 Talis Biomedical Corporation Probe detection of loop-mediated amplification products
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