Methods for detecting polynucleotide analytes

Abstract

The present invention typically relates to the field of nucleic acid detection. In particular, the present specification teaches a method for detecting a polynucleotide analyte in a sample.

Description

[Technical field]

[0001] The present invention typically relates to the field of nucleic acid detection. In particular, the present specification teaches a method for detecting a polynucleotide analyte in a sample. [Background technology]

[0002] background In vitro amplification and detection of nucleic acids has a variety of clinical applications, particularly in the diagnosis of acute and chronic pathologies, including those caused by infectious diseases. Quantitative polymerase chain reaction (qPCR) and direct sequencing are reliable methods for nucleic acid detection, but these techniques require sophisticated equipment and can be costly and time-consuming to implement. New generations of detection assays, including the Invader assay and CRISPR-based methods, aim to combine ease of use and cost-effectiveness without compromising the specificity and sensitivity of the assay.

[0003] The invader assay is a method for detecting and quantitatively analyzing DNA or RNA. The assay does not amplify the target of interest, but instead generates and amplifies an unrelated signal only in the presence of the correct target sequence. The assay involves generating an invasion cleavage structure in a target-dependent manner, allowing a structure-specific enzyme to cleave the invasion cleavage structure to release a signal, and then allowing this signal to be detected. The assay involves the use of two primers, an invasion primer and a flap primer. The flap primer has a 3' portion that is complementary to the target, and a 5' portion that is usually unrelated to the target sequence. The 5' portion of the flap primer that does not hybridize to the target forms a 5' flap. The invasion primer anneals to the target 5' of the 5' portion of the flap primer annealed to the target, and the flap primer and invasion primer overlap to form a branched overlapping structure that is believed to resemble the structure generated during strand displacement DNA synthesis. The invasion primer and flap primer often overlap by one nucleotide, but the use of longer overlaps is also possible. The branched structure is cleaved to release the 5' flap of the 5' primer, which then serves as a detectable signal. Disadvantages of the Invader assay include its slow detection rate, the need for an initial denaturation step, and the need for a fluorescent detection system.

[0004] CRISPR-based methods include DETECTR and SHERLOCK assays. DETECTR assays rely on Cas12 nuclease to detect DNA targets, while SHERLOCK assays rely on Cas13 nuclease to detect RNA targets. In both methods, recognition of the nucleic acid target results in off-target cleavage that generates a signal. Cas12-based assays suffer from the limitation of the sequences that can be detected, since a PAM sequence is required near the target sequence for recognition by Cas nuclease. Cas13-based assays require in vitro transcription of DNA targets into RNA for detection, which makes detection more complex. In both cases, guide RNA activity must also be optimized for the detection of each novel target.

[0005] It would be desirable to overcome or ameliorate at least one of the above problems, or at least provide a useful alternative. Summary of the Invention

[0006] Disclosed herein is a method of detecting a polynucleotide analyte in a sample, comprising the steps of: a) contacting a sample containing the polynucleotide analyte with: i) a first nucleic acid probe comprising a 3' portion that is complementary to a first portion of the polynucleotide analyte and a 5' portion that is complementary to and does not hybridize to the polynucleotide analyte; ii) a second nucleic acid probe comprising a 5' portion that is complementary to a second portion of the polynucleotide analyte and a 3' portion that is complementary to and does not hybridize to the polynucleotide analyte, wherein the first portion of the polynucleotide analyte is 5' to and adjacent to the second portion of the polynucleotide analyte; and iii) a structure-specific nucleic acid cleavage agent, wherein hybridization of the first nucleic acid probe to the first portion of the polynucleotide analyte and hybridization of the second nucleic acid probe to the second portion of the polynucleotide analyte form a cleavage structure; and wherein formation of the cleavage structure results in cleavage of the first nucleic acid probe with the cleavage agent to form a structure-specific nucleic acid cleavage agent that is complementary to and does not hybridize to the polynucleotide analyte. a) releasing a 5' flap comprising a 5' portion of the first nucleic acid probe that does not bind to the 5' flap; b) ligating the 5' flap to a nucleic acid adapter to form an adapter ligation product, the nucleic acid adapter comprising a double-stranded region and a 3' overhang extension that is complementary to the 5' flap, the 3' overhang extension hybridizing to the 5' flap; and c) coupling the adapter ligation product to: i) a type V CRISPR / Cas effector protein; ii) a type V CRISPR / Cas effector protein; a guide RNA comprising a region that binds to an effector protein and a guide sequence that is complementary to a portion of a 5' flap and a portion of a nucleic acid adapter ligated to and adjacent to the 5' flap; and iii) contacting with single-stranded detector DNA; and d) measuring a detectable signal resulting from cleavage of the single-stranded detector DNA by a Type V CRISPR / Cas effector protein to detect the adapter ligation product, thereby detecting a polynucleotide analyte in the sample.

[0007] Disclosed herein is a method for detecting a single nucleotide polymorphism (SNP) in a polynucleotide analyte in a sample, comprising the steps of: a) contacting a sample comprising a polynucleotide analyte with: i) a first nucleic acid probe comprising a 3' portion that is complementary to a first portion of the polynucleotide analyte and a 5' portion that is complementary to and does not hybridize with the polynucleotide analyte; ii) a second nucleic acid probe comprising a 5' portion that is complementary to a second portion of the polynucleotide analyte and a 3' portion that is complementary to and does not hybridize with the polynucleotide analyte, wherein the first portion of the polynucleotide analyte is 5' to and adjacent to the second portion of the polynucleotide analyte; and iii) a structure-specific nucleic acid cleavage agent, wherein hybridization of the first nucleic acid probe to the first portion of the polynucleotide analyte and hybridization of the second nucleic acid probe to the second portion of the polynucleotide analyte form a cleavage structure; and wherein formation of the cleavage structure results in cleavage of the first nucleic acid probe with the cleavage agent to form a structure-specific nucleic acid cleavage agent that is complementary to and does not hybridize with the polynucleotide analyte. a) releasing a 5' flap comprising a 5' portion of the first nucleic acid probe that does not hybridize to the 5' flap; b) ligating the 5' flap to a nucleic acid adapter to form an adapter ligation product, the nucleic acid adapter comprising a double-stranded region and a 3' overhang extension that is complementary to the 5' flap, the 3' overhang extension hybridizing to the 5' flap; and c) coupling the adapter ligation product to: i) a type V CRISPR / Cas effector protein; ii) a type V CRISPR / Cas effector protein; and iii) a guide RNA comprising a region that binds to an effector protein and a guide sequence that is complementary to a portion of a 5' flap and a portion of a nucleic acid adapter ligated to and adjacent to the 5' flap; and iii) contacting with single-stranded detector DNA; and d) measuring a detectable signal resulting from cleavage of the single-stranded detector DNA by a Type V CRISPR / Cas effector protein to detect the adapter ligation product, thereby detecting a SNP in a polynucleotide analyte in the sample.

[0008] Disclosed herein is a method for detecting a polynucleotide analyte in a sample, the method comprising: a) providing a sample containing a polynucleotide analyte; i) a first nucleic acid probe and a second nucleic acid probe configured to form a cleavage structure in the presence of a polynucleotide analyte; and ii) Structure-specific nucleic acid cleavage agents contacting the Upon formation of the cleavage structure, the first nucleic acid probe is cleaved with a cleavage agent to release a 5' flap from the first nucleic acid probe; b) ligating the 5' flap to a nucleic acid adaptor to form an adaptor ligation product, the nucleic acid adaptor comprising a double-stranded region and a 3' overhang extension that is complementary to the 5' flap, the 3' overhang extension hybridizing to the 5' flap; c) ligating the adapter ligation product to i) Type V CRISPR / Cas effector proteins; ii) a guide RNA comprising a region that binds to a type V CRISPR / Cas effector protein and a guide sequence that is complementary to a portion of a 5' flap and a portion of a nucleic acid adapter ligated to and adjacent to the 5' flap; and iii) Single-stranded detector DNA and contacting the d) detecting the adaptor ligation product by measuring a detectable signal resulting from cleavage of the single-stranded detector DNA by the type V CRISPR / Cas effector protein, thereby detecting the polynucleotide analyte in the sample; The method includes:

[0009] Disclosed herein is a kit for detecting a polynucleotide analyte in a sample, the kit comprising a structure-specific nucleic acid cleavage agent, a nucleic acid ligase and a type V CRISPR / Cas effector protein.

[0010] Embodiments of the invention will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is an overall schematic diagram of the use of flap endonuclease, Taq ligase and CRISPR-Cas12 in the disclosed methods. [Diagram 2] Schematic of the workflow for polynucleotide detection using FELICX (flap endonuclease, Taq ligase and CRISPR-Cas for diagnostics(X)). (a) Genetic material is extracted from clinical samples and isothermally amplified. (b) Target nucleotide sequences are detected and a signal is generated by the sequential action of FEN, ligase and Cas12-sgRNA. The output of FEN is an oligo that ligates with an adapter to form a substrate for Cas12-sgRNA, which binds to Cas12 resulting in cleavage of the ssDNA reporter. (c) The output can be a fluorescent signal or readout on a lateral flow strip. [Diagram 3] Binding of the invading primer and flap probe to a DNA target. The underlined sequence is the flap that remains constant for any target. The red arrow indicates the point where FEN cleaves the flap probe. [Figure 4]Figure 1 shows the flap endonuclease activity of commonly used Tth pol and Tth pol v2 using DNA as a target. Activity was determined using a flap probe labeled with a fluorophore and a quencher. Fluorescence signals are observed after cleavage by the purified enzyme. Reactions were performed in a buffer containing Tris-Cl, NaCl, MgCl2, Triton-X-100 and DTT. [Diagram 5] Figure 1 shows ligation of a flap to a dsDNA adapter containing a 5'p adapter strand and a ds converter strand. The flap product is shaded. The box on the top strand of the ligation product is the binding region of the gRNA, and the box on the bottom strand is the PAM sequence for CRISPR-Cas12. [Figure 6] Graph showing detection of (a) LAMP amplified orf1ab copies; (b) LAMP amplified E. coli genome copies; (c) HAD amplified orf1ab copies; (d) HDA amplified E. coli genome copies. NTC-No Template Control. All samples were prepared by spiking target DNA into HEK293t total DNA to simulate clinical samples. [Figure 7] Photographs showing lateral flow readout of orf1ab (mock sample) after detection using HDA and the method described herein. (a) Readout after 1hr HDA, 1hr FEN+Taq reaction and 30min cas12 reaction. (b) Readout after 20min HDA, 20min FEN+Taq reaction and 20min cas12 reaction. 5'6-FAM-TTATTATTAT-3'Bio ssDNA was used as a reporter for the orf1ab gene corresponding to T1. C-control, T2-second test line not used in this experiment. [Figure 8]Figure 8a shows the amplification of orf1ab RNA using RTx and HDA in a two-step or one-step method. Blue triangles indicate increasing volumes of RTx mix added to HDA. Figure 8b shows the lateral flow readout of orf1ab RNA (mock sample) at 90 min (45 min HDA, 20 min FEN+Taq reaction, and 20 min cas12 reaction). 5'6-FAM-TTATTATTAT-3'Bio ssDNA was used as a reporter for orf1ab RNA corresponding to T1. C-control, T2-second test line not used in this experiment. NTC-no template control. [Figure 9] Graph showing detection of EBV and gapdh in C666-1 and HK-1 cells. NTC-No template control. The graph shows the fluorescence readout of the Cas12 step of the detection method. [Figure 10] Graph showing detection of blaKPC and khe in Klebseilla pneumoniae spiked into HK-1 cells. NTC-No template control. Graph shows fluorescence readout of the Cas12 step of the detection method. [Figure 11] 1 is a graph showing detection of scgb2a2 spiked into FBS. The graph shows the fluorescence readout of the Cas12 step of the detection method. [Figure 12] Graph showing detection of WT or mut DNA corresponding to the spike protein of SARS-CoV-2 with (a) wild type flap probe (b) mut flap probe. The graph shows the fluorescence readout of the Cas12 step of the detection method. [Figure 13-1]Figure 1 shows a comparison of FEN activity of various enzymes and their engineered variants. (a) Schematic of the fluorescent assay to quantify FEN activity. (b) Activity of various FEN enzymes on 1 nM orf1ab DNA / RNA substrate with various flap and invading primer configurations including: the configuration shown in (a) (first column), a double flap configuration with flaps on both the invading and flap primers (second column), a configuration without an invading primer (third column), and a configuration with only a flap primer (fourth column). No FEN refers to reactions without enzyme (fifth column). Data on the left are for DNA substrates and data on the right are for RNA substrates. Data represent the average (n=3 biological replicates). Thermo FEN was obtained from NEB (catalog no. M0645S). (c) FEN activity of Tth pol v2 after 60 min incubation at 65°C with different enzyme concentrations against probe (flap primer only), DNA substrate (1 nM), and RNA substrate (1 nM). Black horizontal line represents the mean (n=3 biological replicates). p-value <0.05 is calculated using a two-sample t-test. (d) and (e) Detection limits for orf1ab DNA (left) and RNA (right) after 30 min incubation at 65°C with 10 nM Tth pol v2. Data represent the mean ± SD (n=3 biological replicates). p-value <0.05 compared to NTC is calculated using a two-sample t-test. NTC: no target control. [Figure 13-2]Figure 1 shows a comparison of FEN activity of various enzymes and their engineered variants. (a) Schematic of the fluorescent assay to quantify FEN activity. (b) Activity of various FEN enzymes on 1 nM orf1ab DNA / RNA substrate with various flap and invading primer configurations including: the configuration shown in (a) (first column), a double flap configuration with flaps on both the invading and flap primers (second column), a configuration without an invading primer (third column), and a configuration with only a flap primer (fourth column). No FEN refers to reactions without enzyme (fifth column). Data on the left are for DNA substrates and data on the right are for RNA substrates. Data represent the average (n=3 biological replicates). Thermo FEN was obtained from NEB (catalog no. M0645S). (c) FEN activity of Tth pol v2 after 60 min incubation at 65°C with different enzyme concentrations against probe (flap primer only), DNA substrate (1 nM), and RNA substrate (1 nM). Black horizontal line represents the mean (n=3 biological replicates). p-value <0.05 is calculated using a two-sample t-test. (d) and (e) Detection limits for orf1ab DNA (left) and RNA (right) after 30 min incubation at 65°C with 10 nM Tth pol v2. Data represent the mean ± SD (n=3 biological replicates). p-value <0.05 compared to NTC is calculated using a two-sample t-test. NTC: no target control. [Figure 14-1]Optimization of adapter ligation and the nucleic acid detection limit using FELICX. (a) Sequence of the flap product ligated to a dsDNA adapter. The PAM is underlined and variable base pairs are shaded. (b) Schematic showing ligation of an oligo similar to the flap product formed by FEN to a dsDNA adapter by ligase. The ligation product forms a substrate for Cas12-sgRNA, which upon activation cleaves the ssDNA reporter, resulting in a fluorescent signal. (c) Fluorescent signal was observed after 30 min ligation of synthetic flap oligos to the dsDNA adapter using different ligases, followed by detection with Cas12-sgRNA (10 min or 30 min of cleavage). The ligation volume was 15 μL, to which 5 μL of Cas12-sgRNA was then added. Data represent the mean ± SD (n=3 biological replicates). NTC: no target control. Ligated: pre-ligated substrate. (d) Optimization of FEN concentration and incubation time for FEN+Taq ligase reaction (15 μL) using orf1ab DNA (1 nM). After ligation, Cas12-sgRNA reaction (20 μL) was performed for 30 min. Data represent mean ± SD (n = 2 biological replicates). (e) and (f) Detection limit of orf1ab DNA or RNA spiked into purified HEK293T total DNA or RNA, respectively, using the present detection system. FEN+Taq ligase reaction (20 μL) was performed for 4 h and Cas12-sgRNA reaction (additional 7 μL) was performed for 30 min. Data represent mean ± SD (n = 3 biological replicates). p value < 0.05 compared to NTC calculated using a two-sample t-test. (g) and (h) Detection limits of HDA+FELICX for orf1ab DNA and E. coli genome spiked into HEK293T total DNA. HDA was performed for 1 h (in 10 μL reactions), FEN+Taq ligase reactions for 1 h (total volume 20 μL), and Cas12-sgRNA reactions for 30 min (total volume 27 μL). Data represent the mean ± SD (n=3 biological replicates).A p-value <0.05 compared to NTC was calculated using a two-sample t-test. [Figure 14-2]Optimization of adapter ligation and the nucleic acid detection limit using FELICX. (a) Sequence of the flap product ligated to a dsDNA adapter. The PAM is underlined and variable base pairs are shaded. (b) Schematic showing ligation of an oligo similar to the flap product formed by FEN to a dsDNA adapter by ligase. The ligation product forms a substrate for Cas12-sgRNA, which upon activation cleaves the ssDNA reporter, resulting in a fluorescent signal. (c) Fluorescent signal was observed after 30 min ligation of synthetic flap oligos to the dsDNA adapter using different ligases, followed by detection with Cas12-sgRNA (10 min or 30 min of cleavage). The ligation volume was 15 μL, to which 5 μL of Cas12-sgRNA was then added. Data represent the mean ± SD (n=3 biological replicates). NTC: no target control. Ligated: pre-ligated substrate. (d) Optimization of FEN concentration and incubation time for FEN+Taq ligase reaction (15 μL) using orf1ab DNA (1 nM). After ligation, Cas12-sgRNA reaction (20 μL) was performed for 30 min. Data represent mean ± SD (n = 2 biological replicates). (e) and (f) Detection limit of orf1ab DNA or RNA spiked into purified HEK293T total DNA or RNA, respectively, using the present detection system. FEN+Taq ligase reaction (20 μL) was performed for 4 h and Cas12-sgRNA reaction (additional 7 μL) was performed for 30 min. Data represent mean ± SD (n = 3 biological replicates). p value < 0.05 compared to NTC calculated using a two-sample t-test. (g) and (h) Detection limits of HDA+FELICX for orf1ab DNA and E. coli genome spiked into HEK293T total DNA. HDA was performed for 1 h (in 10 μL reactions), FEN+Taq ligase reactions for 1 h (total volume 20 μL), and Cas12-sgRNA reactions for 30 min (total volume 27 μL). Data represent the mean ± SD (n=3 biological replicates).A p-value <0.05 compared to NTC was calculated using a two-sample t-test. [Figure 14-3]Optimization of adapter ligation and the nucleic acid detection limit using FELICX. (a) Sequence of the flap product ligated to a dsDNA adapter. The PAM is underlined and variable base pairs are shaded. (b) Schematic showing ligation of an oligo similar to the flap product formed by FEN to a dsDNA adapter by ligase. The ligation product forms a substrate for Cas12-sgRNA, which upon activation cleaves the ssDNA reporter, resulting in a fluorescent signal. (c) Fluorescent signal was observed after 30 min ligation of synthetic flap oligos to the dsDNA adapter using different ligases, followed by detection with Cas12-sgRNA (10 min or 30 min of cleavage). The ligation volume was 15 μL, to which 5 μL of Cas12-sgRNA was then added. Data represent the mean ± SD (n=3 biological replicates). NTC: no target control. Ligated: pre-ligated substrate. (d) Optimization of FEN concentration and incubation time for FEN+Taq ligase reaction (15 μL) using orf1ab DNA (1 nM). After ligation, Cas12-sgRNA reaction (20 μL) was performed for 30 min. Data represent mean ± SD (n = 2 biological replicates). (e) and (f) Detection limit of orf1ab DNA or RNA spiked into purified HEK293T total DNA or RNA, respectively, using the present detection system. FEN+Taq ligase reaction (20 μL) was performed for 4 h and Cas12-sgRNA reaction (additional 7 μL) was performed for 30 min. Data represent mean ± SD (n = 3 biological replicates). p value < 0.05 compared to NTC calculated using a two-sample t-test. (g) and (h) Detection limits of HDA+FELICX for orf1ab DNA and E. coli genome spiked into HEK293T total DNA. HDA was performed for 1 h (in 10 μL reactions), FEN+Taq ligase reactions for 1 h (total volume 20 μL), and Cas12-sgRNA reactions for 30 min (total volume 27 μL). Data represent the mean ± SD (n=3 biological replicates).A p-value <0.05 compared to NTC is calculated using a two-sample t-test. [Figure 14-4]Optimization of adapter ligation and the nucleic acid detection limit using FELICX. (a) Sequence of the flap product ligated to a dsDNA adapter. The PAM is underlined and variable base pairs are shaded. (b) Schematic showing ligation of an oligo similar to the flap product formed by FEN to a dsDNA adapter by ligase. The ligation product forms a substrate for Cas12-sgRNA, which upon activation cleaves the ssDNA reporter, resulting in a fluorescent signal. (c) Fluorescent signal was observed after 30 min ligation of synthetic flap oligos to the dsDNA adapter using different ligases, followed by detection with Cas12-sgRNA (10 min or 30 min of cleavage). The ligation volume was 15 μL, to which 5 μL of Cas12-sgRNA was then added. Data represent the mean ± SD (n=3 biological replicates). NTC: no target control. Ligated: pre-ligated substrate. (d) Optimization of FEN concentration and incubation time for FEN+Taq ligase reaction (15 μL) using orf1ab DNA (1 nM). After ligation, Cas12-sgRNA reaction (20 μL) was performed for 30 min. Data represent mean ± SD (n = 2 biological replicates). (e) and (f) Detection limit of orf1ab DNA or RNA spiked into purified HEK293T total DNA or RNA, respectively, using the present detection system. FEN+Taq ligase reaction (20 μL) was performed for 4 h and Cas12-sgRNA reaction (additional 7 μL) was performed for 30 min. Data represent mean ± SD (n = 3 biological replicates). p value < 0.05 compared to NTC calculated using a two-sample t-test. (g) and (h) Detection limits of HDA+FELICX for orf1ab DNA and E. coli genome spiked into HEK293T total DNA. HDA was performed for 1 h (in 10 μL reactions), FEN+Taq ligase reactions for 1 h (total volume 20 μL), and Cas12-sgRNA reactions for 30 min (total volume 27 μL). Data represent the mean ± SD (n=3 biological replicates).A p-value <0.05 compared to NTC is calculated using a two-sample t-test. [Figure 15] Figure 1. Combination of FELICX and lateral flow strips for nucleic acid detection. (a) Schematic of the lateral flow strips used in this study. Test line T1 corresponds to the reporter with FAM and biotin, while T2 corresponds to the reporter with digoxigenin (DIG) and biotin. C is the lateral flow control. (b) Schematic of nucleic acid detection using HDA+FELICX with lateral flow strips. The lateral flow output in the presence and absence of target in the sample is depicted. (c) Lateral flow for detection of orf1ab DNA spiked into HEK293T total DNA. HDA was performed for 1 h (25 μL reaction followed by rapid purification), FEN+Taq reaction for 1 h (total volume 20 μL), and Cas12-sgRNA reaction for 30 min (total volume 27 μL). In this experiment, only T1 was used. The numbers below the strips indicate the fold change in T1 band intensity compared to NTC. (d) As in (c), but HDA, FEN+Taq, and Cas12-sgRNA reactions were each performed at 20 min. Numbers below the strips indicate fold change in T1 band intensity compared to NTC. (e) Lateral flow for detection of orf1ab RNA spiked into HEK293T total RNA. HDA was performed for 45 min (25 μL reaction volume followed by rapid purification), FEN+Taq reactions for 20 min (20 μL total volume), and Cas12-sgRNA reactions for 20 min (27 μL total volume). In this experiment, only T1 was used. Numbers below the strips indicate fold change in T1 band intensity compared to NTC. [Figure 16-1]Figure 1. SNP detection using FELICX. (a) Schematic showing cleavage of the flap primer in the case of wild type (WT) target and no cleavage of the flap primer in the case of SNP by FEN. The cleavage site of the flap primer is marked with an arrow. The last nucleotide of the invasion primer is mismatched with the target, preventing cleavage by FEN. (b) Detection with FELICX of WT target, target with SNP / mutation, or no target, and the corresponding lateral flow strip patterns observed in each case. (c)-(e) Detection of the WT receptor binding domain sequence of SARS-CoV-2 or its variant (T478K) by FELICX using WT and mut probes. Panel (c) shows the results of the fluorescent signal, while panel (d) shows the results of the lateral flow, after FELICX was performed on pre-amplified substrate using both probes. Pre-amplification was performed by PCR, followed by purification of DNA to ensure equal masses of both WT and mutant DNA were used to ensure accurate comparison. FEN+Taq reactions were performed for 60 min (total volume 20 μL) and Cas12-sgRNA reactions were performed for 30 min (total volume 27 μL). In (c), data represent the mean ± SD (n=3 biological replicates). p-values ​​<0.05 compared to NTC were calculated using a two-sample t-test. (e) Percentage change in band intensity corresponding to WT and mut probes compared to NTC in (d). NTC: no target control. [Figure 16-2]Figure 1. SNP detection using FELICX. (a) Schematic showing cleavage of the flap primer in the case of wild type (WT) target and no cleavage of the flap primer in the case of SNP by FEN. The cleavage site of the flap primer is marked with an arrow. The last nucleotide of the invasion primer is mismatched with the target, preventing cleavage by FEN. (b) Detection with FELICX of WT target, target with SNP / mutation, or no target, and the corresponding lateral flow strip patterns observed in each case. (c)-(e) Detection of the WT receptor binding domain sequence of SARS-CoV-2 or its variant (T478K) by FELICX using WT and mut probes. Panel (c) shows the results of the fluorescent signal, while panel (d) shows the results of the lateral flow, after FELICX was performed on pre-amplified substrate using both probes. Pre-amplification was performed by PCR, followed by purification of DNA to ensure equal masses of both WT and mutant DNA were used to ensure accurate comparison. FEN+Taq reactions were performed for 60 min (total volume 20 μL) and Cas12-sgRNA reactions were performed for 30 min (total volume 27 μL). In (c), data represent the mean ± SD (n=3 biological replicates). p-values ​​<0.05 compared to NTC were calculated using a two-sample t-test. (e) Percentage change in band intensity corresponding to WT and mut probes compared to NTC in (d). NTC: no target control. [Figure 17]Figure 1 demonstrates the versatility of FELICX. FELICX was used to detect targets in more complex samples, e.g., bacterial and mammalian whole cells as well as serum. Fluorescence signal fold change for detection of (a) EBV and gapdh in C666-1 and HK-1 whole cells, (b) blaKPC and khe in K. pneumoniae (Kp) cells spiked into HK-1 whole cells, and (c) SCGB2A2 RNA spiked into FBS. Samples were lysed and rapidly purified as described in the methods section. HDA was performed for 1h (in 10μL reaction), FEN+Taq ligase reaction for 1h (total volume 20μL), and Cas12-sgRNA reaction for 30min (total volume 27μL). NTC: no target control. [Figure 18] Figure 18a is a schematic showing the various FEN enzymes used in this study. The boxes indicate the domains of the enzymes and the lines represent the positions of the point mutations introduced into the enzymes. Figure 18b is a table showing the point mutations introduced into Tth pol v1, v2 and v3. Figure 18c is a photograph showing SDS-PAGE showing the purification of the various FEN enzymes. The proteins of interest are marked with arrowheads. Figure 18d is a schematic of the fluorescent assay used to quantitate FEN activity. In this configuration, the 5'-flap to the flap primer and the 3'-flap to the invading primer are shown. [Figure 19] Figure 19a shows the structure of T5 exonuclease (PDB ID: 1EXN) with arrows on the helical arches. Figure 19b shows the predicted structure of Tth pol with arrows on the irregular arches. The structures were predicted using SWISS-MODEL5. Figure 19c shows a graph of the FEN activity of the different enzymes using 1 nM DNA (left panel) and 1 nM RNA (right panel) as substrates. Data represent the mean ± SD (n = 3 biological replicates). [Figure 20]Figures 20a and 20b are graphs showing the detection limits of orf1ab DNA and RNA, respectively, after 30 min incubation at 65°C with 50 nM Tth pol v2. Data represent the mean ± SD (n = 3 biological replicates). p values ​​< 0.05 compared to NTC are calculated using a two-sample t-test. NTC: no target control. [Figure 21] Graph showing Cas12-sgRNA activity using either ssDNA (1 nM) or dsDNA (1 nM) as substrates, which was determined by co-incubation in 20 μL reactions at 37° C. and measuring the fluorescent signal generated by reporter cleavage. Data represent the mean (n=3 biological replicates). [Figure 22] Graph showing the fluorescence signal after 30 min incubation at 37 °C with dsDNA substrate (1 nM) observed with varying Cas12-sgRNA complexes (RNP) and their DNA reporters (rep). RNP (1x) = 120 nM Cas12 and 100 nM sgRNA, RNP (2x) = 200 nM Cas12 and 167 nM sgRNA, rep (1x) = 80 nM DNA reporter and rep (2x) = 133.3 nM DNA reporter. NTC: no target control. Data represent the mean ± SD (n = 2 biological replicates). [Diagram 23] Figures 23a and 23b are schematic diagrams of pre-ligated dsDNA adapters with N=A, C, G, or T (shaded boxes) hybridizing with sgRNA and sgRNA-7 DNA, respectively. Both gRNAs are complementary to dsDNA adapters with N=T. PAM sequences are underlined. Figure 23c is a graph showing the activity of Cas12-sgRNA and Cas12-sgRNA-7 DNA complexes against four possible pre-ligated dsDNA adapters (5 nM) (determined by co-incubation in 20 μL reactions at 37° C. and measuring the fluorescent signal generated by reporter cleavage). Data represent the mean (n=3 biological replicates). NTC: no target control. [Figure 24] Figures 24a and 24b are graphs showing the detection limit of orf1ab DNA or E. coli genome spiked into purified HEK293tT total DNA using LAMP+FELICX. LAMP reactions were performed for 30 min (in 10 μL reaction), FEN+Taq reactions for 1 hr (total volume 20 μL), and Cas12-sgRNA reactions for 30 min (total volume 27 μL). Data represent the mean ± SD (n=3 biological replicates). p-values ​​<0.05 compared to NTC calculated using a two-sample t-test. NTC: no target control. [Diagram 25] FIG. 1 is a graph showing Cq values ​​of LAMP performed at 65° C. using various primer sets according to the manufacturer's instructions. DNA: 60,000 copies of orf1ab DNA. NTC: no target control. In set 1, the biological replicates of NTC did not show any amplification, so Cq values ​​were not determined. The black horizontal line represents the mean (n=3 biological replicates). [Figure 26] Figure 26a is a photograph showing an agarose gel showing the amplification of orf1ab RNA by two-step or one-step RTx+HDA. In the two-step method, reverse transcription was performed using RTx for 10 min, followed by HDA at 65°C for 50 min. In the one-step method, RTx+HDA was performed simultaneously by incubating at 65°C for 60 min. No amplification was observed in NTC (no target control) samples. Figure 26b is a graph showing quantification of band intensities corresponding to the amplification products seen in (a). Quantification was performed using ImageJ. Data represent the mean ± SD (n=3 biological replicates). [Figure 27]Figure 1 shows the fold change in fluorescent signal for detection of EBV and gapdh in C666-1 and HK-1 cell lines (upper panel), and blaKPC and khe in K. pneumoniae (Kp) spiked into HK-1 cells (lower panel). Samples were lysed and rapidly purified as described in the methods section. HDA was performed for 1hr (in 10μL reaction), FEN+Taq ligase reaction for 1hr (total volume 20μL), and Cas12-sgRNA reaction for 30min (total volume 27μL). NTC: no target control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description The present specification teaches methods for detecting a polynucleotide analyte in a sample. The method can include providing a first nucleic acid probe and a second nucleic acid probe configured to form a cleavage structure in the presence of a polynucleotide analyte. The method may include a) contacting a sample containing a polynucleotide analyte with: i) a first nucleic acid probe comprising a 3' portion that is complementary to a first portion of the polynucleotide analyte and a 5' portion that is complementary to and does not hybridize to the polynucleotide analyte; ii) a second nucleic acid probe comprising a 5' portion that is complementary to a second portion of the polynucleotide analyte and a 3' portion that is complementary to and does not hybridize to the polynucleotide analyte, wherein the first portion of the polynucleotide analyte is 5' to and adjacent to the second portion of the polynucleotide analyte; and iii) a structure-specific nucleic acid cleaving agent, wherein hybridization of the first nucleic acid probe to the first portion of the polynucleotide analyte and hybridization of the second nucleic acid probe to the second portion of the polynucleotide analyte form a cleavage structure; and formation of the cleavage structure results in cleavage of the first nucleic acid probe with the cleavage agent to release a 5' flap that comprises the 5' portion of the first nucleic acid probe that is complementary to and does not hybridize to the polynucleotide analyte. The above method may further comprise the step of b) ligating the 5' flap to a nucleic acid adapter to form an adapter ligation product, wherein the nucleic acid adapter comprises a double-stranded region and a 3' overhang extension that is complementary to the 5' flap, and wherein the 3' overhang extension of the nucleic acid adapter hybridizes to the 5' flap.The method may further comprise c) contacting the adapter ligation product with i) a type V CRISPR / Cas effector protein; ii) a guide RNA comprising a region that binds to the type V CRISPR / Cas effector protein and a guide sequence that is complementary to a portion of the 5' flap and a portion of the nucleic acid adapter ligated to and adjacent to the 5' flap; and iii) single-stranded detector DNA; and d) measuring a detectable signal resulting from cleavage of the single-stranded detector DNA by the type V CRISPR / Cas effector protein to detect the adapter ligation product, thereby detecting the polynucleotide analyte in the sample.

[0013] Without being bound by theory, the present invention involves the use of a structure-specific nucleic acid cleavage agent (e.g., flap endonuclease), a nucleic acid ligase and a type V CRISPR-Cas nuclease for rapid, sensitive and specific detection of nucleic acids (either DNA or RNA). First, a sample is incubated with a pair of DNA oligonucleotides that bind to the target DNA / RNA to form a flap structure, which is recognized and then cleaved by the structure-specific nucleic acid cleavage agent (FIG. 1). The cleaved flap product is ligated with a double-stranded DNA (dsDNA) adapter and a nucleic acid ligase to form a substrate for the CRISPR-Cas nuclease. In the presence of the substrate, the Cas nuclease bound to its guide RNA (gRNA) cleaves a single-stranded DNA (ssDNA) reporter to generate a detectable signal, which can be, for example, a visible band on a lateral flow detection system, or a fluorescent signal. Different nucleic acids, even those differing by a single nucleotide, can be distinguished by the use of DNA oligonucleotides and adapters specific for each nucleic acid. The sensitivity of the above method can be improved by incorporating an isothermal amplification system, such as loop-mediated isothermal amplification (LAMP) or helicase-dependent amplification (HDA), prior to the binding of the DNA oligonucleotides, and using the product of this amplification as a substrate for the series of binding and enzymatic reactions of the present disclosure.

[0014] As used herein, the term "nucleic acid" and equivalent terms, such as "polynucleotide", refer to a polymeric form of nucleotides of any length, such as ribonucleotides, deoxyribonucleotides, or peptide nucleic acids (PNAs), containing purine and pyrimidine bases, or other naturally occurring, chemically or biochemically modified, non-naturally occurring, or derivatized nucleotide bases. Nucleic acids can be double-stranded or single-stranded. Reference to a single-stranded nucleic acid includes reference to the sense or antisense strand. The backbone of a polynucleotide can include sugar and phosphate groups as may typically be found in RNA or DNA, or modified or substituted sugar or phosphate groups. A polynucleotide can include modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. The terms nucleoside, nucleotide, deoxynucleoside, and deoxynucleotide generally include complements, fragments, and variants of nucleosides, nucleotides, deoxynucleosides, and deoxynucleotides, or analogs thereof.

[0015] An "oligonucleotide," as used herein, is a single-stranded molecule that can be used in hybridization or amplification techniques. Generally, oligonucleotides can be any integer from about 15 to about 100 nucleotides in length, but can be longer.

[0016] The term "complementary" refers to base pairing between nucleotides or nucleic acids, such as between the two strands of a double-stranded DNA molecule, or between an oligonucleotide primer and a primer binding site on a single-stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are generally A and T (or A and U), or C and G. Two single-stranded RNA or DNA molecules are said to be complementary when, when optimally aligned and compared with appropriate nucleotide insertions or deletions, the nucleotides of one strand pair with at least about 80%, usually at least about 90%-95%, and more preferably about 98%-100% of the nucleotides of the other strand.

[0017] The term "not complementary" or "non-complementary" can refer to the lack of base pairing between nucleotides or nucleic acids, such as between the two strands of a double-stranded DNA molecule, or between an oligonucleotide primer and a primer binding site on a single-stranded nucleic acid to be sequenced or amplified. Two single-stranded RNA or DNA molecules are considered to be non-complementary when the nucleotides of one strand pair with less than 50% of the nucleotides of the other strand, and optionally less than 40%, 30%, 20% or 10% of the nucleotides of the other strand.

[0018] As used herein, the term "hybridization" or "hybridize" refers to the process in which two single-stranded polynucleotides non-covalently bind to form a stable double-stranded polynucleotide, i.e., a duplex. The term "hybridization" can also refer to triple-stranded hybridization. The resulting double-stranded polynucleotide (usually) is a "hybrid." The percentage of the polynucleotide population that forms stable hybrids is referred to herein as the "degree of hybridization."

[0019] Hybridization conditions typically include salt concentrations of less than about 1M, more typically less than about 500 mM and less than about 200 mM. Hybridization temperatures are typically greater than 22°C, more typically greater than about 30°C, and preferably greater than about 37°C. In one embodiment, hybridization occurs at about 65°C. Hybridization is usually performed under stringent conditions, i.e., conditions under which the probe hybridizes to its target. Stringent conditions are sequence-dependent and will be different under different circumstances. Longer fragments may require higher hybridization temperatures for specific hybridization. Other factors, including base composition and length of complementary strands, the presence of organic solvents, and the degree of base mismatching, can affect the stringency of hybridization, so the combination of parameters is more important than the absolute amount of any one alone. In general, stringent conditions are determined by the thermal melting point (T) for a particular sequence at a defined ionic strength and pH. m ) is chosen to be approximately 5°C lower than T m is the temperature (under defined ionic strength, pH and nucleic acid composition) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium.

[0020] The term "probe" refers to any molecule capable of selectively binding to a specifically intended target nucleic acid, such as genomic DNA, a polynucleotide transcript, viral DNA or RNA. Probes can be synthesized by those skilled in the art or derived from appropriate biological preparations.

[0021] As used herein, the term "cleavage structure" refers to the structure formed by the interaction of at least one nucleic acid probe with a target polynucleotide, the structure comprises a duplex with adjacent single-stranded nucleic acid flaps, which structure can be cleaved by a structure-specific nucleic acid cleavage agent.The cleavage structure is a suitable substrate for specific cleavage by a structure-specific nucleic acid cleavage agent due to its secondary structure.

[0022] As used herein, the term "flap probe" or "flap oligonucleotide" refers to an oligonucleotide that interacts with a target polynucleotide to form a cleavage structure, with or without an invader oligonucleotide. Upon hybridization with a target polynucleotide, the flap probe and the target form a cleavage structure, and cleavage occurs within the flap probe.

[0023] As used herein, the term "invader probe" or "invader oligonucleotide" refers to an oligonucleotide that hybridizes to a target polynucleotide at a position proximate to the region of hybridization between the flap probe and the target nucleic acid, and the invader probe includes a portion (e.g., a chemical moiety or a nucleotide, which may or may not be complementary to its target) that overlaps with the region of hybridization between the flap probe and the target.

[0024] The first nucleic acid probe (or flap probe) of the present disclosure may include a 3' portion that is complementary to a first portion of a polynucleotide analyte. The 3' portion may include or consist of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleobases. The first nucleic acid probe may include a 5' portion that is not complementary to and does not hybridize to the polynucleotide analyte. The 5' portion may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleobases.

[0025] The second nucleic acid probe (or invader probe) of the present disclosure may include a 5' portion that is complementary to the second portion of the polynucleotide analyte, and a 3' portion that is not complementary to and does not hybridize with the polynucleotide analyte. The 5' portion may comprise or consist of about 20-50 nucleobases. The 5' portion may comprise or consist of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases. The 3' portion may comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleobases. In one embodiment the 3' portion consists of 1 nucleobase.

[0026] In one embodiment, the first and second nucleic acid probes overlap each other by at least one nucleobase when hybridized to a polynucleotide analyte. The first and second nucleic acid probes can overlap each other by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobases.

[0027] In one embodiment, a cleavage structure is formed from hybridization of a first and a second nucleic acid probe to a target polynucleotide. The cleavage structure may include a duplex formed between the first nucleic acid probe and the polynucleotide, and a 5' flap including a 5' portion of the first nucleic acid probe (not hybridized to the polynucleotide). The cleavage structure may further include a 3' flap including a 3' portion of the second nucleic acid probe (not hybridized to the polynucleotide). The 5' flap may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides in length. The 3' flap may be 1, 2, 3, 4 or 5 nucleotides in length.

[0028] As used herein, the term "structure-specific nucleic acid cleavage agent" or "cleavage agent" refers to any agent that can cleave a cleavage structure, including but not limited to enzymes. "Structure-specific nuclease" or "structure-specific enzyme" is an enzyme that recognizes specific secondary structures in nucleic acid molecules and cleaves these structures. The structure-specific nucleic acid cleavage agent of the present disclosure cleaves a nucleic acid molecule upon the formation of a cleavage structure; it is not necessary for the cleavage agent to cleave the cleavage structure at any specific position within the cleavage structure. The cleavage agent can include nuclease activity provided by various sources, including flap endonucleases (FENs) from prokaryotes and eukaryotes, DNA polymerases (e.g., Taq DNA polymerase, DNA polymerase from Thermus species and DNA polymerase I of E. coli), and exonucleases (e.g., bacteriophage T5 exonuclease). Cleavage agents can include enzymes with 5' nuclease activity, such as Taq DNA polymerase, E. coli DNA polymerase I, bacteriophage T5 exonuclease, Thermus species DNA polymerase, etc. Cleavage agents can also include modified or engineered DNA polymerases, where the DNA polymerase has 5' nuclease activity but lacks or has reduced synthetic activity.

[0029] As used herein, the term "cleavage product" or "5' cleavage product" refers to a product generated by the action of a structure-specific nucleic acid cleavage agent on a cleavage structure, for example, a 5' flap that is cleaved from a flap probe.

[0030] In one embodiment, formation of the cleavage structure results in cleavage of the first nucleic acid probe with a cleavage agent to release a 5' flap that includes a 5' portion of the first nucleic acid probe that is not complementary to and does not hybridize to the polynucleotide analyte.

[0031] The cleavage agent is capable of cleaving the first nucleic acid probe at a position one nucleobase 3' to the portion of the first nucleic acid probe that overlaps with the second nucleic acid probe. In some embodiments, the structure-specific nucleic acid cleavage agent is an enzyme having flap endonuclease activity.

[0032] As used herein, the term "flap endonuclease (FEN)" refers to an enzyme with 5' exonuclease activity and structure-specific endonuclease activity, which recognizes and cleaves a cleavage structure to generate a 5' cleavage product. The enzyme with FEN activity herein can be a wild-type enzyme or an engineered enzyme from a prokaryotic or eukaryotic organism, where the enzyme is, for example, a flap endonuclease from the FEN1 protein family, a bacterial DNA polymerase, a bacteriophage T5 and T7 exonuclease, and a nuclease from the XPG / Rad2 superfamily. Such an enzyme can include one or more additional enzyme activities in addition to FEN activity, such as a nucleic acid polymerase activity. The enzyme can be engineered to reduce or eliminate non-FEN activity.

[0033] In some embodiments, the enzyme having FEN activity is a thermostable enzyme. The term "thermostable", when used with respect to an enzyme, such as, for example, an enzyme with FEN activity or a nucleic acid ligase, indicates that the enzyme functions or is active (i.e., capable of performing catalysis) at elevated temperatures, e.g., 45°C or higher. In some embodiments, the thermostable enzymes herein function at temperatures between about 45°C and about 80°C, between about 50°C and about 75°C, between about 55°C and about 70°C, or between about 60°C and about 70°C. In some embodiments, the thermostable enzymes herein function at temperatures of 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, or 75°C or higher. In one embodiment, the thermostable enzymes herein function at temperatures between about 45°C and about 80°C.

[0034] In some embodiments, the enzyme is a DNA polymerase from Thermus thermophiles (ie, Tth polymerase). The enzyme can be a wild-type Tth polymerase, a mutant isoform Tth polymerase, or a genetically engineered Tth polymerase. A wild-type Tth polymerase has both FEN activity and polymerase activity. A Tth polymerase for use in the methods herein can have reduced or negligible polymerase activity.

[0035] The engineered Tth polymerase may include one or more amino acid substitutions, insertions or deletions. The term "deletion," when used in reference to an amino acid, means that the amino acid has been removed or is absent. The term "insertion" means that one or more amino acids have been added. A "substitution" means that an amino acid residue is replaced by another amino acid residue. An amino acid residue may be replaced with another amino acid residue selected from the 20 naturally occurring standard amino acid residues, naturally occurring rare amino acid residues (e.g., hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methylysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline), as well as non-naturally occurring amino acid residues that are often synthetically produced (e.g., cyclohexyl-alanine). Preferably, the term "substitution" refers to the replacement of an amino acid residue by another amino acid residue selected from the 20 standard naturally occurring amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S and T).

[0036] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and can generally be subclassified as follows: a) Amino acid subclassification

[0037] [Table 1]

[0038] Conservative amino acid substitutions also include groupings based on side chains. For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic hydroxyl side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. For example, it is reasonable to expect that the replacement of leucine with isoleucine or valine, the replacement of aspartic acid with glutamic acid, the replacement of threonine with serine, or similar replacement of amino acids with structurally related amino acids will not have a significant effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional polypeptide can be easily determined by assaying its activity. Conservative substitutions are shown under the heading of exemplary substitutions in the following table. Amino acid substitutions that fall within the scope of the invention are generally achieved by selecting substitutions that do not significantly alter (a) the structure of the peptide backbone in the area of ​​substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the effect of maintaining side chain bulk. After the substitutions are introduced, the variants are screened for biological activity. b) Exemplary Amino Acid Substitutions

[0039] [Table 2]

[0040] In some embodiments, the enzyme having FEN activity is a Thermus thermophiles DNA polymerase comprising one or more amino acid substitutions. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of A599T, A604V, W606R, A607Q, A610V, I616M, I616T and E617G. In some embodiments, the Thermus thermophiles DNA polymerase comprises the following amino acid substitutions: A604V, A610V, I616M and E617G. In some embodiments, the Thermus thermophiles DNA polymerase comprises the following amino acid substitutions: A599T, W606R, A607Q and I616T.

[0041] An engineered Tth polymerase that recognizes both DNA and RNA substrates has been previously described in WO2001090337A2. The inventors further engineered this Tth polymerase variant by introducing mutations at positions 604, 610, 616 and 617 (i.e., A604V, A610V, I616M and E617G) to obtain an enzyme with the amino acid sequence shown in SEQ ID NO:1. The inventors also introduced mutations at positions 599, 606, 607 and 616 (i.e., A599T, W606R, A607Q and I616T) to obtain an enzyme with the amino acid sequence shown in SEQ ID NO:2. These enzymes have higher FEN activity on a given substrate (i.e., a given cleavage structure) and lower non-specific activity on oligonucleotides that do not form a cleavage structure.

[0042] In some embodiments, the enzyme comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the enzyme comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the enzyme comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2.

[0043] In some embodiments, step (a) of the methods herein is performed isothermally, i.e., the temperature does not change during step (a). Step (a) may be performed at a temperature of 40° C. or greater. In some embodiments, step (a) is performed at a temperature between about 40° C. and about 90° C., between about 50° C. and about 80° C., between about 55° C. and about 75° C., or between about 60° C. and about 70° C. In one embodiment, step (a) is performed isothermally at a temperature of about 65° C.

[0044] The term "adapter" refers to a nucleic acid molecule that is configured to be complementary to both the cleavage product of a structure-specific cleavage agent (e.g., a 5' flap) and the guide RNA of a type V CRISPR / Cas effector protein (e.g., Cas12). The "adapter" may include a 3' overhang that is complementary to the 5' flap. The "adapter" is beneficial in the method because it bridges step (a) and step (c) by allowing the Cas nuclease to recognize the cleavage product of step (a) in step (c).

[0045] In some embodiments, the nucleic acid adapter comprises a first adapter oligonucleotide that comprises a protospacer-adjacent motif (PAM) of a CRISPR / Cas effector protein of type V, and a second adapter oligonucleotide that is complementary to and hybridizes to the first adapter oligonucleotide. The first adapter oligonucleotide can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleobases. The second adapter oligonucleotide can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or more nucleobases.

[0046] In some embodiments, the first adaptor oligonucleotide comprises a PAM sequence of a Cas12 nuclease. In some embodiments, the PAM sequence is 5'-TTTN-3', where N can be any one of A, T, C, or G.

[0047] In some embodiments, the second adaptor oligonucleotide comprises a 5' modification, such as a modification to the nucleobase or sugar-phosphate backbone. The 5' modification may allow for the action of a subsequent enzyme in the methods herein. In one embodiment, the second adaptor oligonucleotide is phosphorylated at the 5' end to prime for subsequent ligation by a nucleic acid ligase.

[0048] In some embodiments, step (c) is carried out in the presence of a nucleic acid ligase. The 5' cleavage product of the cleavage agent can be ligated to an adaptor by the ligase following hybridization with the 3' overhang of the nucleic acid adaptor. The term "adaptor ligation product" as used herein refers to a nucleic acid (e.g., 5' flap) that is ligated to an adaptor.

[0049] Non-limiting examples of enzymes that can be used for ligation in the methods disclosed herein are ATP-dependent double-stranded polynucleotide ligases, NAD+-dependent DNA or RNA ligases, and single-stranded polynucleotide ligases. Non-limiting examples of ligases are E. coli DNA ligase, Thermus filiformis DNA ligase, Tth DNA ligase, Thermus scotoductus DNA ligase (I and II), T3 DNA ligase, T4 DNA ligase, T4 RNA ligase, T7 DNA ligase, Taq ligase, VanC type ligase, 9°N DNA ligase, Tsp DNA ligase, DNA ligase I, DNA ligase III, DNA ligase IV, Sso7-T3 DNA ligase, Sso7-T4 DNA ligase, Sso7-T7 DNA ligase, Sso7-Taq DNA ligase, Sso7-E. coli DNA ligase, and Sso7-Ampligase DNA ligase. The ligases herein can be wild type variants, mutant isoform variants, or genetically engineered variants.

[0050] In some embodiments, the ligase is a thermostable ligase, for example, Taq ligase. In some embodiments, step (b) of the methods herein is carried out isothermally, i.e., the temperature does not change during step (b). Step (b) may be carried out at a temperature of 40° C. or greater. In some embodiments, step (b) is carried out at a temperature between about 40° C. and about 90° C., between about 50° C. and about 80° C., between about 55° C. and about 75° C., or between about 60° C. and about 70° C. In one embodiment, step (b) is carried out isothermally at a temperature of about 65° C.

[0051] The above method may include CRISPR / Cas effector proteins or enzymes and guide RNA. CRISPR / Cas systems include CRISPR-Cas12 and CRISPR-Cas13, which show strong collateral activity against single-stranded DNA (ssDNA) and ssRNA targets, respectively. Recognition and cleavage of specific targets by Cas effector proteins (complexed with guide RNA), followed by collateral cleavage of non-specific targets, provides the basis for a highly specific and sensitive approach to nucleic acid detection.

[0052] In some embodiments, the CRISPR / Cas effector is a DNA editing enzyme (e.g., a DNA endonuclease) with dsDNA cleavage activity and ssDNA cleavage activity. In such embodiments, the CRISPR / Cas effector can be a class II, type V CRISPR / Cas effector, such as a Cas12 effector protein. Exemplary Cas12 effector proteins include Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, C2c4, C2c8, C2c5, C2cl0, and C2c9.

[0053] In some embodiments, the type V CRISPR / Cas effector protein is a Cas12 protein. In one embodiment, the CRISPR / Cas effector protein is Cas12a.

[0054] The Cas effector protein variants may contain one or more mutations (e.g., conservative or non-conservative mutations). For example, it is contemplated that other Cas12 variants can be evolved from those disclosed herein, for example, by targeted mutation of one or more amino acid residues in specific regions of the enzyme. Such mutations can alter substrate binding, alter the conformation of the bound substrate, alter substrate accessibility to the active site, alter tolerance to suboptimal presentation of the target sequence to the active site, and / or alter target sequence specificity (recognition).

[0055] As used herein, a nucleic acid molecule (e.g., a native crRNA) that binds to a type V CRISPR / Cas effector protein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, etc.) to form a ribonucleoprotein complex (RNP) and targets the complex to a specific target sequence within a target DNA is referred to as a "guide RNA." In some cases, a hybrid DNA / RNA can be made in which the guide RNA contains DNA bases in addition to RNA bases, however, it is understood that the term "guide RNA" is still used herein to encompass such hybrid molecules.

[0056] The guide RNA herein comprises a guide sequence (also referred to as a "spacer") that hybridizes with a portion of the 5' flap and a portion of the nucleic acid adaptor. This ensures that the Cas nuclease is active only after the 5' cleavage product is generated and hybridizes with the nucleic acid adaptor. The guide RNA further comprises a constant region (e.g., a region adjacent to the guide sequence that binds to a type V CRISPR / Cas effector protein).

[0057] A guide sequence can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 or more nucleobases. A guide RNA can be 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 or more nucleobases.

[0058] In some embodiments, step (c) of the methods herein is performed isothermally, i.e., the temperature does not change during step (c). Step (c) may be performed at a temperature of 20° C. or higher. In some embodiments, step (c) is performed at a temperature between about 20° C. and about 70° C., between about 25° C. and about 60° C., between about 30° C. and about 50° C., or between about 35° C. and about 40° C. In one embodiment, step (c) is performed at a temperature between about 20° C. and about 65° C. In one embodiment, step (c) is performed isothermally at a temperature of about 37° C.

[0059] The method may include cleaving single-stranded detector DNA (ssDNA) with a type V CRISPR / Cas effector protein to generate a detectable signal.

[0060] The ssDNA described herein can be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleobases. In one embodiment, the ssDNA is AT-rich. In some embodiments, the ssDNA is labeled. The ssDNA can be labeled at the 5' end, the 3' end, or both the 3' end and the 5' end. The ssDNA can also be labeled at an internal position. In one embodiment, the ssDNA is labeled at both ends. The label pair can be, for example, FAM and biotin, DIG and biotin, FAM and DIG, or a signal-quencher pair.

[0061] In some embodiments, the labeled ssDNA comprises a signal-quencher pair. In the signal-quencher pair, the signal partner generates detectable signal, and the quencher partner quenches (i.e., reduces) the detectable signal of the signal partner when the signal-quencher partners are in close proximity to each other, for example, when the partners are present on the same ssDNA molecule before being cut by the CRISPR / Cas effector protein of type V. The detectable signal is generated when the labeled ssDNA is cut and the signal partner is no longer in close proximity to the quencher partner.

[0062] The quencher moiety may quench the signal from the signal moiety to various degrees. The quencher moiety will quench the signal from the signal moiety when the signal detected in the presence of the quencher moiety (when the signal partners are close to each other) is 95% or less of the signal detected in the absence of the quencher moiety (when the signal partners are far apart). For example, the signal detected in the presence of the quencher moiety may be 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, or 5% or less of the signal detected in the absence of the quencher moiety. In some cases, the signal (e.g., above background) is not detectable in the presence of the quencher moiety.

[0063] The signal detected in the absence of the quencher moiety (when the signal partners are separated) may be at least 1.2 times (e.g., at least 1.3 times, at least 1.5 times, at least 1.7 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 5 times, at least 7 times, at least 10 times, at least 20 times, or at least 50 times) greater than the signal detected in the presence of the quencher moiety (when the signal partners are close to each other).

[0064] In one embodiment, the single-stranded detector DNA comprises a fluorophore-quencher pair. The quencher moiety reduces the fluorescence signal from the fluorophore, for example, by absorbing the energy of the emission spectrum of the fluorophore. Any convenient fluorophore-quencher pair can be used, and many suitable pairs are known in the art.

[0065] The quencher moiety can absorb energy from the fluorophore to emit a signal (e.g., light of a different wavelength). Thus, the quencher moiety can itself be a second fluorophore (e.g., the first fluorophore can be 6-carboxyfluorescein, while the quencher or second fluorophore can be 6-carboxy-tetramethylrhodamine). The fluorophore-quencher pair can also be a FRET pair. The quencher moiety can be a dark quencher. A dark quencher can absorb excitation energy and dissipate the excitation energy in a different pathway (e.g., as heat). Thus, a dark quencher has very little to no fluorescence of its own (does not emit fluorescence).

[0066] Examples of fluorophores include Alexa Fluor® dyes, ATTO dyes (e.g., ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740), DyLight dyes, cyanine dyes (e.g., Cy2, Cy3, Cy3.5, Cy3b, Cy5, Cy5.5, Cy7, Cy7.5), FluoProbes dyes, Sulfo Cy dyes, Seta dyes, IRIS dyes, SeTau dyes, SRfluor dyes, Square dyes, fluorescein isothiocyanate (FITC), tetramethylrhodamine (TRITC), Texas Red, Oregon Green, Pacific Blue, Pacific Green, Pacific Orange, quantum dots, and tethered fluorescent proteins.

[0067] Examples of quencher moieties include, but are not limited to, dark quenchers, Black Hole Quencher® (BHQ®) (e.g., BHQ-0, BHQ-1, BHQ-2, BHQ-3), Qxl quenchers, ATTO quenchers (e.g., ATTO 540Q, ATTO 580Q, and ATTO 612Q), dimethylaminoazobenzenesulfonic acid (dabcyl), Iowa Black RQ, Iowa Black FQ, IRDye QC-1, QSY dyes (e.g., QSY 7, QSY 9, QSY 21), AbsoluteQuencher, Eclipse, and metal clusters, such as gold nanoparticles.

[0068] In one embodiment, the single-stranded detector DNA comprises a FRET pair. FRET is a process in which non-radiative transfer of energy occurs from an excited fluorophore to a second chromophore in close proximity. The range in which energy transfer can occur is limited to about 10 nanometers (100 angstroms), and the transfer efficiency is extremely sensitive to the separation distance between the fluorophores. Thus, as used herein, the term "FRET" (also known as "fluorescence resonance energy transfer"; "Förster resonance energy transfer") refers to a physical phenomenon involving a donor fluorophore and a matching acceptor fluorophore, both of which are selected such that the emission spectrum of the donor overlaps with the excitation spectrum of the acceptor, and further, when the donor and acceptor are in close proximity to each other (usually 10 nm or less), the excitation of the donor causes the excitation of the acceptor and emission from it (because a portion of the energy is transferred from the donor to the acceptor via quantum coupling effects). Thus, the FRET signal serves as a measure of the proximity of the donor and acceptor; a signal is only produced when they are in close proximity to one another. FRET donor moieties (e.g., donor fluorophores) and FRET acceptor moieties (e.g., acceptor fluorophores) are collectively referred to herein as a "FRET pair."

[0069] FRET donor and acceptor moieties (FRET pairs) are well known to those of skill in the art, and any convenient FRET pair (e.g., any convenient donor and acceptor moiety pair) can be used. Examples of FRET pairs are shown in the table below.

[0070] [Table 3]

[0071] The labeled-detector ssDNA can produce a first detectable signal (e.g., from a FRET pair) before it is cleaved, and a second detectable signal (e.g., from a quencher / fluorophore pair) when it is cleaved. Thus, the labeled-detector ssDNA can include a FRET pair and a quencher / fluorophore pair.

[0072] In some cases, the cleavage of the labeled detector ssDNA can be detected by colorimetric reading measurement. For example, the release of a fluorophore (e.g., release from a FRET pair and release from a quencher / fluorophore pair, etc.) can result in a wavelength shift (and thus a color shift) of detectable signal. Thus, in some cases, the cleavage of the labeled detector ssDNA herein can be detected by a color shift. Such a shift can be expressed as a decrease in the amount of signal of one color (wavelength), an increase in the amount of another color, and a change in the ratio of one color to another color, etc.

[0073] In some embodiments, cleavage of the single-stranded detector DNA is detected visually. Visual detection can be by direct observation (e.g., by eye or by use of a camera or microscope) or by spectroscopic or spectrophotometric measurements.

[0074] In one embodiment, cleavage of the single-stranded detector DNA is detected by measuring a change in the fluorescent signal caused by the detector DNA. The change in the fluorescent signal can be an increase or decrease in fluorescence when the detector DNA is used in the methods herein, for example, in step (c) of the methods herein.

[0075] In one embodiment, cleavage of single-stranded detector DNA is detected visually on a lateral flow system. Lateral flow systems are typically based on flow strips. The flow strips may include one or more pads for holding the sample fluid and may further include one or more detection reagents for detecting target moieties in the sample fluid. The pads may be based on a series of capillary beds, e.g., small pieces of porous paper, microstructured polymers, or sintered polymers. Each of these pads may have the ability to spontaneously transport the sample fluid, e.g., via capillary action. The sample to be analyzed may be added to the proximal end of the strip. The liquid phase elements of the sample (which may be dissolved, suspended, emulsified, or in any other fluidized form) may migrate to the pad area where the detection reagents, typically consisting of signal molecules or particles, typically gold colloids or proteins passively or covalently linked to colored, fluorescent, or paramagnetic monodisperse latex particles, are immobilized. The signal reagent may also be another reagent that includes non-particulate substances (e.g., soluble directly labeled fluorophore gels). The label can be conjugated to one of the individual biological components of the assay, either antigen or antibody, depending on the assay format of the individual flow strip. The liquid phase sample remobilizes the dried conjugate material, incorporating it into the liquid phase sample material, allowing the analyte in the sample to interact with the conjugate. The conjugate material can be a protein, such as an antibody or antigen, placed in a band or stripe in a specific area of ​​the pad, where it functions to capture the components, analytes and conjugates of the liquid phase sample as they move past, through or beyond the capture line. Excess liquid phase material (sample and reagents) can continue to move up the strip, past the capture line and be trapped in the pad near the other end of the flow strip. The test result can be developed on the reaction matrix and expressed as the presence or absence of a test indicator (typically a continuous line) of the captured conjugate, where these indicators are read visually or using a reader device. Some of the conjugated particles may not be captured at the capture line, in which case they continue to flow towards a second line of immobilized detection reagent, the control line.This control line typically contains another detection reagent specific for the conjugated antibody on the conjugate, which generates a control signal (typically a continuous line) when the conjugated particle binds to this detection reagent.

[0076] In one embodiment, the absence of a test line on a lateral flow system indicates the presence of the polynucleotide analyte in the sample. In some embodiments, steps (a) and (b) of the methods herein are carried out in separate reaction vessels, and the products of these reaction vessels are mixed before proceeding to step (c).

[0077] In some embodiments, steps (a) and (b) of the methods herein are performed in a single reaction vessel. Steps (a) and (b) may proceed simultaneously in a single reaction vessel. Alternatively, step (a) may proceed for a known period of time, and then the reagents required for step (b) (i.e., nucleic acid ligase and nucleic acid adaptor) may be added to the reaction vessel. In some embodiments, step (c) is also performed in the same reaction vessel as steps (a) and (b). Steps (a), (b) and (c) may proceed simultaneously in a single reaction vessel. Alternatively, step (b) may proceed for a known period of time, and then the Cas nuclease, guide RNA and single-stranded detector DNA may be added to the reaction vessel.

[0078] In some embodiments, the methods herein further comprise the step of amplifying the polynucleotide analyte prior to step (a). A variety of amplification methods and components are known to those skilled in the art and any convenient method can be used (see, e.g., Zanoli and Spoto, Biosensors (Basel). 2013 March;3(1):18-43; Gill and Ghaemi, Nucleosides, Nucleotides, and Nucleic Acids, 2008, 27:224-243; Craw and Balachandrana, Lab Chip, 2012, 12, 2469-2486). Nucleic acid amplification can include polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), quantitative PCR (qPCR), reverse transcription qPCR (RT-qPCR), nested PCR, multiplexed PCR, asymmetric PCR, touchdown PCR, random primer PCR, hemi-nested PCR, polymerase cycling assembly (PCA), colony PCR, ligase chain reaction (LCR), digital PCR, methylation specific-PCR (MSP), co-amplification at lower denaturation temperature-PCR (COLD-PCR), allele-specific PCR, intersequence-specific PCR (ISS-PCR), whole genome amplification (WGA), inverse PCR, and thermal asymmetric interlaced PCR (TAIL-PCR).

[0079] In some embodiments, the amplification is isothermal amplification. The term "isothermal amplification" refers to a method of nucleic acid (e.g., DNA) amplification that uses incubation at a single temperature, thereby eliminating the need for a thermal cycler (e.g., using an enzymatic chain reaction). Isothermal amplification is a form of nucleic acid amplification that does not rely on heat denaturation of the target nucleic acid during the amplification reaction, and therefore would not require multiple rapid temperature changes. Thus, isothermal nucleic acid amplification methods can be performed inside or outside a laboratory environment. These amplification methods can be used to amplify RNA isothermally by combining with a reverse transcription step.

[0080] Examples of isothermal amplification methods include, but are not limited to, loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), Ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal mediated amplification of RNA technology (SMART), self-sustained sequence replication (3SR), genome exponential amplification reaction (GEAR), and isothermal multiple displacement amplification (IMDA).

[0081] In some cases, the amplification is recombinase polymerase amplification (RPA). Recombinase polymerase amplification (RPA) uses two opposing primers (much like PCR) and three enzymes: a recombinase, a single-stranded DNA binding protein (SSB), and a strand-displacing polymerase. The recombinase pairs the oligonucleotide primer with a homologous sequence in the double-stranded DNA, the SSB binds to the displaced strand of DNA to prevent the primer from being displaced, and the strand-displacing polymerase initiates DNA synthesis at the site where the primer bound to the target DNA. The addition of reverse transcriptase to the RPA reaction can facilitate RNA detection as well as DNA detection without the need for a separate step to create cDNA.

[0082] In transcription mediated amplification (TMA), RNA polymerase is used to create RNA from a promoter engineered into the primer region, and then reverse transcriptase synthesizes cDNA from the primer. A third enzyme, such as Rnase H, can then be used to strip the RNA target from the cDNA without a heat denaturation step. This amplification technique is similar to self-sustained sequence replication (3SR) and nucleic acid sequence-based amplification (NASBA), but the enzymes used are different.

[0083] In one embodiment, polynucleotides are isothermally amplified using loop mediated amplification (LAMP). LAMP uses a thermostable polymerase with strand displacement ability and a set of four or more specifically designed primers. Each primer is designed to have a hairpin end that, upon displacement, folds into a hairpin to facilitate self-priming and further extension by the polymerase. In the LAMP reaction, the reaction proceeds under isothermal conditions, but for double-stranded targets, an initial heat denaturation step is required. Furthermore, amplification results in products of various lengths in a ladder pattern. As yet another example, strand displacement amplification (SDA) combines the ability of a restriction endonuclease to nick the unmodified strand of its target DNA with the ability of an exonuclease-deficient DNA polymerase to extend the 3' end of the nick and displace the downstream DNA strand.

[0084] In one embodiment, polynucleotides are isothermally amplified using helicase-dependent amplification (HDA), which uses a thermostable helicase (Tte-UvrD) rather than heat to unwind dsDNA to create a single strand that is then used for hybridization and primer extension by a polymerase.

[0085] Disclosed herein is a method for detecting a polynucleotide analyte in a sample, the method comprising: a) providing a sample containing a polynucleotide analyte; i) a first nucleic acid probe and a second nucleic acid probe configured to form a cleavage structure in the presence of a polynucleotide analyte; and ii) Structure-specific nucleic acid cleavage agents contacting the Upon formation of the cleavage structure, the first nucleic acid probe is cleaved with a cleavage agent to release a 5' flap from the first nucleic acid probe; b) ligating the 5' flap to a nucleic acid adapter to form an adapter ligation product, wherein the nucleic acid adapter comprises a double-stranded region and a 3' overhang extension that is complementary to the 5' flap, and wherein the 3' overhang extension of the nucleic acid adapter hybridizes to the 5' flap; c) ligating the adapter ligation product to i) Type V CRISPR / Cas effector proteins; ii) a guide RNA comprising a region that binds to a type V CRISPR / Cas effector protein and a guide sequence that is complementary to a portion of a 5' flap and a portion of a nucleic acid adapter ligated to and adjacent to the 5' flap; and iii) Single-stranded detector DNA and contacting the d) detecting the adaptor ligation product by measuring a detectable signal resulting from cleavage of the single-stranded detector DNA by the type V CRISPR / Cas effector protein, thereby detecting the polynucleotide analyte in the sample; The method includes:

[0086] This method for detecting DNA / RNA has various diagnostic applications, since it can be used to identify any gene sequence without using a sequencer. This method allows the detection and subclassification of bacterial and viral pathogens in various human, food and environmental samples. This detection method can also distinguish different variants of such pathogens, such as antibiotic-resistant or vaccine-resistant variants. In addition, this method also allows the detection of genetic mutations in the human genome associated with the development of disease and cell-free DNA / RNA in various samples.

[0087] The method of the present invention can be useful for detecting the presence or absence of one or more polynucleotide analytes in one or more samples that are known to contain or suspected to contain the polynucleotide analytes.The method can also be used to quantify the amount of polynucleotide analytes in a sample.The method is useful for detecting polynucleotide targets in a sample, such as, for example, RNA, MRNA, rRNA, plasmid DNA, viral DNA, bacterial DNA, and chromosomal DNA.

[0088] The term "polynucleotide analyte" may be any polynucleotide that can be detected or analyzed by the methods defined herein. The analyte may be naturally occurring or synthetic. The polynucleotide analyte may be present in a sample obtained using any method known in the art. In some cases, the sample may be processed prior to analyzing the polynucleotide analyte. Polynucleotides may include DNA, RNA, peptide nucleic acid, and any hybrids thereof, where the polynucleotide includes any combination of deoxyribonucleotides and / or ribonucleotides. Polynucleotides may be single-stranded or double-stranded, or may include portions of both double-stranded or single-stranded sequences. Polynucleotides may include any combination of nucleotides or bases, including, for example, uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, isoguanine, and any nucleotide derivatives thereof. As used herein, the term "nucleotide" can include nucleotides and nucleosides, nucleoside and nucleotide analogs, and modified nucleotides, including both synthetic and naturally occurring species. The polynucleotide can be any suitable polynucleotide, including, but not limited to, cDNA, mitochondrial DNA (mtDNA), messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), nuclear RNA (nRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), small Cajal body-specific RNA (scaRNA), microRNA (miRNA), double-stranded RNA (dsRNA), ribozyme, riboswitch, or viral RNA. The polynucleotide can be contained in any suitable vector, such as a plasmid, cosmid, fragment, chromosome, or genome. The polynucleotide analyte can be a nucleic acid endogenous to a cell.As another example, a polynucleotide analyte can be a nucleic acid introduced into or expressed in a cell by infecting the cell with a pathogen, such as viral or bacterial genomic RNA or DNA, a plasmid, or viral or bacterial mRNA.

[0089] In some embodiments, the polynucleotide analyte is DNA or RNA. In some embodiments, the polynucleotide analyte is single-stranded or double-stranded. In one embodiment, the polynucleotide analyte comprises a single nucleotide polymorphism (SNP).

[0090] In one embodiment, the polynucleotide analyte is a viral nucleic acid. In one embodiment, the polynucleotide analyte is a viral nucleic acid from SARS-CoV-2. The SARS-CoV-2 genome consists of approximately 30 kb plus single-stranded RNA with a 5'-cap structure and a 3' poly-A tail, which contains several genes characteristic of coronaviruses, such as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) genes. Other elements of the genome, such as ORFla and ORFlb, code for non-structural proteins, including RNA-dependent RNA polymerase (RdRp).

[0091] As used herein, the term "sample" includes tissues, cells, body fluids and their isolates, etc., isolated from a subject, as well as tissues, cells, body fluids, etc., present in a subject (i.e., the sample is in vivo). Examples of samples include whole blood, blood liquids (e.g., serum and plasma), lymphatic and cystic fluids, sputum, feces (or stool), tears, mucus, hair, skin, ascites, cystic fluid, urine, nipple exudate, nipple aspirate, tissue sections, such as biopsy and autopsy samples, frozen sections taken for histological purposes, archival samples, explants, and primary and / or transformed cell cultures derived from patient tissues, etc.

[0092] The terms "detecting," "determining," "measuring," "evaluating," "assessing," and "assaying" are used interchangeably herein to refer to any form of measurement, including determining whether an element is present. These terms include both quantitative and / or qualitative determinations. Assessment may be relative or absolute. The methods defined herein may include measuring or visualizing the levels of two or more polynucleotide analytes in a sample.

[0093] The methods can be used for any purpose in which detection of viral, bacterial, or other nucleic acids is desirable, including, for example, diagnostic and prognostic applications in laboratory and clinical settings, hi some embodiments, the methods can be used for detection of nucleic acids for genotyping.

[0094] In some embodiments, the nucleic acid detected is useful for diagnosing a disease state.The disease state can be an infectious disease, an organ disease, a blood disease, an immune system disease, a cancer, a brain and nervous system disease, an endocrine disease, a pregnancy or birth-related disease, a genetic disease, or an environmentally acquired disease, a cancer, or a fungal infection, a bacterial infection, a parasitic infection, or a viral infection.Thus, in some embodiments, the method is useful for detecting the nucleic acid (e.g., DNA or RNA) from bacteria, fungi, viruses (e.g., double-stranded RNA viruses, positive-stranded RNA viruses, negative-stranded RNA viruses, retroviruses, etc.) or parasites.

[0095] Exemplary viruses that may be detected include Myoviridae, Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae (including human herpesviruses, and varicella-zoster virus), Malocoherpesviridae, Liposthrixviridae, Rudiviridae, Adenoviridae, Ampullaviridae, Ascoviridae, Asfarviridae (including African swine fever virus), Baculoviridae, Cicaudaviridae, Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, and the like. Virus families: Hytrosaviridae, Iridoviridae, Maseilleviridae, Mimiviridae, Nudiviridae, Nimaviridae, Pandoraviridae, Papillomaviridae, Phycodnaviridae, Plasmaviridae, Polydnaviruses, Polyomaviridae (including Simian virus 40, JC virus, and BK virus), Poxviridae (including cowpox and smallpox), Sphaerolipoviridae, Tectiviridae, Turriviridae, Dinodnavirus, Salteroporvirus, virus, Rhizidovirus, Coronaviridae, Picornaviridae, Caliciviridae, Flaviviridae, Togaviridae, Bornaviridae, Filoviridae, Paramyxoviridae, Pneumoviridae, Rhabdoviridae, Arenaviridae, Bunyaviridae, Orthomyxoviridae, or Deltavirus.In some embodiments, the virus is a coronavirus (e.g., SARS-Cov-2), SARS, poliovirus, rhinovirus, hepatitis A, Norwalk virus, yellow fever virus, West Nile virus, hepatitis C virus, dengue virus, Zika virus, rubella virus, Ross River virus, Sindbis virus, chikungunya virus, Borna disease virus, Ebola virus, Marburg virus, measles virus, mumps virus, Nipah virus, Hendra virus, Newcastle disease virus, human respiratory syncytial virus, rabies virus, Lassa virus, Hantavirus, Crimean-Congo hemorrhagic fever virus, influenza virus, or hepatitis D virus.

[0096] In some embodiments, the detected nucleic acid may be associated with a pathogen, including pathogenic bacteria such as E. faecalis, E. faecium, Listeria monocytogenes, Campylobacter jejuni, Staphylococcus aureus (e.g., MRSA), Escherichia coli O157:H7, Borrelia burgdorferi, Helicobacter pylori, Ehrlichia chaffeensis, Clostridium difficile, Vibrio cholerae 0139, Salmonella enterica, Bartonella henselae, Streptococcus pyogenes, Chlamydia pneumoniae, Clostridium botulinum, Corynebacterium amycolatum, Klebsiella pneumonia, Vibrio vulnificus, and parachlamydia.

[0097] Also provided herein are methods of treating the disease following detection of the disease. "Subject" or "patient" means any single subject for which treatment is desired, including humans, cows, horses, pigs, goats, sheep, dogs, cats, guinea pigs, rabbits, chickens, insects, etc. Also intended to be included as a subject is any subject participating in a clinical research trial and not showing any clinical signs of disease, participating in an epidemiological study, or used as a control.

[0098] Disclosed herein is a kit for carrying out the method disclosed herein. The kit may include: i) a first nucleic acid probe comprising a 3' portion that is complementary to a first portion of a polynucleotide analyte and a 5' portion that is not complementary to and does not hybridize with the polynucleotide analyte; ii) a second nucleic acid probe comprising a 5' portion that is complementary to a second portion of the polynucleotide analyte and a 3' portion that is not complementary to and does not hybridize with the polynucleotide analyte, the first portion of the polynucleotide analyte being 5' to and adjacent to the second portion of the polynucleotide analyte; and iii) a structure-specific nucleic acid cleavage agent. The structure-specific nucleic acid cleavage agent may be a polypeptide comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:1. The kit may further include a nucleic acid adapter for forming an adapter ligation product, the nucleic acid adapter comprising a double-stranded region and a 3' overhang extension that is complementary to the 5' flap, the 3' overhang extension of the nucleic acid adapter being capable of hybridizing with the 5' flap. The kit may further comprise: i) a type V CRISPR / Cas effector protein; ii) a guide RNA comprising a region that binds to the type V CRISPR / Cas effector protein and a guide sequence that is complementary to a portion of the 5' flap and a portion of the nucleic acid adaptor ligated to and adjacent to the 5' flap; and iii) a single-stranded detector DNA. A detectable signal resulting from cleavage of the single-stranded detector DNA by the type V CRISPR / Cas effector protein can be measured by a detector. The kit may further comprise one or more buffer components, metabolites, and / or other reaction components required to carry out the methods defined herein. The kit may further comprise a lateral flow device or system for detecting a signal generated by cleavage of the single-stranded detector DNA. The kit may further comprise instructions for carrying out the methods defined herein.

[0099] Disclosed herein is a kit for detecting a polynucleotide analyte in a sample, the kit comprising a structure-specific nucleic acid cleavage agent, a nucleic acid ligase and a type V CRISPR / Cas effector protein. The structure-specific nucleic acid cleavage agent may be a polypeptide comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:1. The kit may further comprise a first nucleic acid probe and a second nucleic acid probe, wherein the first and second nucleic acid probes are configured to form a cleavage structure in the presence of the polynucleotide analyte. The kit may further comprise a first adaptor polynucleotide and a second adaptor polynucleotide, wherein the first and second adaptor polynucleotides are configured to form a nucleic acid adaptor upon hybridization. The kit may further comprise a guide RNA configured to bind to the type V CRISPR / Cas effector protein and the ligation product of the nucleic acid adaptor and the cleavage product of the structure-specific nucleic acid cleavage agent. The kit may further comprise a single-stranded detector DNA. The kit may further comprise one or more buffer components, metabolites, and / or other reaction components necessary to carry out the methods defined herein. The kit may further comprise a lateral flow device or system for detecting a signal generated by cleavage of single-stranded detector DNA. The kit may further comprise instructions for carrying out the methods defined herein.

[0100] In some embodiments, the methods or kits herein exhibit attomolar (aM) detection sensitivity. In some cases, the subject compositions or methods exhibit femtomolar (fM) detection sensitivity. In some cases, the subject compositions or methods exhibit picomolar (pM) detection sensitivity. In some cases, the subject compositions or methods exhibit nanomolar (nM) detection sensitivity.

[0101] In some embodiments, the detection threshold of target polynucleotide using the method defined herein is 1 nM or less. The term "detection threshold" is used herein to describe the minimum amount of target polynucleotide that must be present in a sample for detection to occur. Thus, as an illustrative example, when the detection threshold is 1 nM, a signal can be detected when the target DNA is present in a sample at a concentration of 1 nM or more. In some embodiments, the method of the present disclosure has a detection threshold of 500 pM or less, 100 pM or less, 50 pM or less, 10 pM or less, 5 pM or less, 1 pM or less, 500 fM or less, 100 fM or less, 50 fM or less, 10 fM or less, 5 fM or less, 1 fM or less, 500 aM or less, 100 aM or less, 50 aM or less, 10 aM or less, or 1 aM or less.

[0102] In some embodiments, the detection threshold is between about 100 pM and about 1 nM, between about 10 pM and about 100 pM, between about 1 pM and about 10 pM, between about 100 fM and about 1 pM, between about 10 fM and about 100 fM, between about 1 fM and about 10 fM, between about 100 aM and about 1 fM, between about 10 aM and about 100 aM, or between about 1 aM and about 10 aM.

[0103] As used herein, "and / or" refers to and includes every possible combination of one or more of the associated listed items, as well as the lack of combination when interpreted in the alternative (or).

[0104] As used in this application, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. For example, the term "an agent" includes a plurality of agents (including mixtures thereof).

[0105] Throughout this specification and the claims which follow, unless the context indicates otherwise, the word "comprise", as well as variations such as "comprises" and "comprising", are understood to mean the inclusion of a stated integer or step or group of integers or steps, but not to the exclusion of other integers or steps or groups of integers or steps.

[0106] Reference in this specification to any prior publication (or information derived therefrom) or to any known matter is not, and should not be construed as, an acknowledgement or acceptance, or any form of suggestion, that the prior publication (or information derived therefrom) or known matter forms part of the common general knowledge in the art to which this specification pertains.

[0107] Those skilled in the art will understand that the invention described herein is capable of variations and modifications other than those specifically described. It is understood that the invention includes all such variations and modifications that fall within its spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or shown in this specification, individually or collectively, as well as any combination of any two or more of said steps or features.

[0108] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Certain embodiments of the present invention will now be described with reference to the following examples, which are for illustrative purposes only and are not intended to limit the scope of the generality of the foregoing. EXAMPLES

[0109] method Bacterial strains, plasmids and oligonucleotides used in this study E. coli TOP10 was used as the cloning host and E. coli BL21(DE3) was used for protein expression. Both bacterial strains were grown in Luria-Bertani (LB) medium at 37° C. with shaking at 220 rpm unless otherwise stated.

[0110] pET28b was used as a cloning vector for protein expression in E. coli BL21(DE3). Cas12 was obtained from pMBP-LbCas12a (Addgene plasmid # 113431), a gift from Jennifer Doudna. Tth pol (GenBank accession number WP_011228405), hFEN1 (GenBank accession number NP_004102), MjaFEN (GenBank accession number WP_010870964) and T5 exo (GenBank accession number YP_006958) sequences were codon-optimized for expression in E. coli and synthesized as gblocks from Integrated DNA Technologies (IDT).

[0111] The sequences of oligos and primers for LAMP, HDA and FEN (flap and invasion primers) are shown in Tables 1-4. Protein purification All proteins were purified using a His-tag attached to their C-terminus. For purification of Cas12, E. coli BL21(DE3)(pET28b-Cas12) was grown overnight in Luria-Bertani (LB) medium supplemented with kanamycin (50 μg / mL). After overnight growth, the culture was diluted 10-fold in Terrific medium supplemented with kanamycin and grown at 37 °C until the OD600 reached approximately 0.3-0.4. Following this, 100 mM IPTG was added to the culture and grown for an additional 24 h at 23 °C. Cells were harvested by centrifugation and resuspended in lysis buffer (50 mM Tris-Cl (pH 8), 300 mM NaCl, EDTA-free protease inhibitor tablets). Cells were lysed using a homogenizer at 23,000 bar passing the cells 5-10 times. The lysate was centrifuged and the supernatant was subjected to protein purification using a HisTrap HP column connected to an FPLC instrument. Elution was achieved after a gradient run over 2 h with lysis buffer supplemented with 1 M imidazole. Different fractions were collected and run on SDS-PAGE gel to confirm protein purification. The purified protein was washed with lysis buffer without protease inhibitor tablets to remove imidazole, then concentrated and flash frozen for storage.

[0112] Purification of the FEN enzyme was performed similarly, with minor modifications. The growth medium for protein expression was changed to LB, and the protein was expressed at 37 °C for 4–5 h. Cell lysis was performed using Y-PER (Thermo Fisher), and the soluble protein was purified using Ni-NTA resin in a PD-10 column. Elution was achieved using lysis buffer supplemented with 50 mM, 100 mM, 200 mM, and 500 mM imidazole. The purified protein (final concentration 1 μM) was stored at -20 °C in a buffer containing 50% glycerol, 0.1 mM EDTA, 1 mM DTT, and 0.1% Triton-X-100. FEN activity assay The activity of various FEN enzymes was assessed in isothermal reactions incubated at 65°C for the appropriate time, which contained reaction buffer RB (50 mM Tris-Cl (pH 8), 100 mM NaCl, 10 mM MgCl2), 100 nM flap primer, 20 nM invasion primer, 0.2% Triton-X-100, 50 nM purified FEN enzyme (unless otherwise stated), and 1 nM target template (unless otherwise stated) in a 20 μL reaction. When fluorophore- and quencher-conjugated flap primers were used, fluorescence (ex: 485 nm, em: 535 nm) was measured every 5 min. Ligation assay To ligate the synthesized flap oligo with the dsDNA adapter, the latter was formed by annealing the 5'p adapter and the ds converter (final concentration 200 nM) in 1xRB. For this, the reaction mixture was heated to 98 °C in a thermocycler and cooled to room temperature at 0.1 °C / s. The ligation was carried out with 1xRB, 5 nM dsDNA adapter, 5 nM flap oligo, and the appropriate additives and cofactors depending on the ligase used. For T4 DNA ligase and T4 RNA ligase (NEB), 1 mM DTT and 1 mM ATP (NEB) were added to the reaction. For Taq DNA ligase (NEB), 1 mM NAD (Sigma Aldrich) and 10 mM DTT were added to the reaction. The amounts of ligase used were 5 U T4 DNA ligase, 10 U T4 RNA ligase, and 80 U Taq DNA ligase. Incubation temperatures were 22°C for T4 DNA ligase, 37°C for T4 RNA ligase, and 65°C for Taq ligase. Cas12-sgRNA reaction Cas12-sgRNA complexes were formed by incubating 3.75 μL of purified Cas12 (100 μM) and 0.3125 μL of 1 mM sgRNA in 2 μL of 1×RB and 20 mM DTT (RB-DTT) with nuclease-free water to a final volume of 20 μL. Incubation was carried out at 37 °C for 30 min. Following this, the Cas12-sgRNA complex was diluted to 375 μL with water to achieve a final Cas12:sgRNA concentration ratio of 1 μM:0.833 μM.

[0113] For reactions using Cas12-sgRNA complexes, 3 μL of complexes formed as above, 4 μL of fluorophore-quencher reporter (1 μM), and 0.7 μL of 1×RB were added to the reaction and incubated at 37° C. with fluorescence measurements taken every 3 min (ex: 485 nm, em: 535 nm). For data in Figures 21, 23, and 24, 1.5 μL of Cas12-sgRNA complexes and 2 μL of fluorophore-quencher reporter were used. LAMP assay To perform LAMP, NEB's Warmstart LAMP kit (DNA and RNA) was used and the assay was performed according to the manufacturer's instructions. LAMP primers were designed using either PrimerExplorer v5 or the NEB LAMP primer design tool. Primers FIP, BIP, LF, and LB were biotinylated at the 5' end in order to biotinylate the LAMP products and subsequently purify them using streptavidin agarose beads. After LAMP, 10 μL of DNA products were purified by adding an equal volume of Pierce streptavidin agarose resin resuspended in B&W buffer (10 mM Tris-Cl (pH 7.5), 1 mM EDTA, and 2 M NaCl). The samples were further diluted by adding 30 μL of 0.5× B&W buffer and the mixture was incubated at room temperature for 30 min for biotin-streptavidin conjugates. After 30 min, the beads were allowed to settle and the supernatant was removed. These beads were then used in FELICX. HDA assay HDA was performed using the IsoAmp II Universal tHDA kit from NEB. The assay was performed according to the manufacturer's instructions with primers designed using IDT's PrimerQuest tool. All HDA reactions used MgSO4 (4 mM), NaCl (40 mM) and appropriate primers (200 nM). After completion of the assay, DNA products were either used directly in FELICX or purified using Invitrogen's ChargeSwitch PCR cleanup kit before use in FELICX. When RNA was used as template, 0.25 μL Warmstart RTx (NEB) was added for every 10 μL of HDA assay mix. FELICX(Flap Endonuclease,Taq LIgase and CRISPR-Cas for diagnostics(X)) For the first step of FELICX, the amplified DNA was added to FEN+Taq mix containing 2 µL of flap primer (1 µM), 0.4 µL of invasion primer (1 µM), 0.5 µL of dsDNA adapter (200 nM, as specified in the ligation assay), 2 µL of RB-DTT buffer, 1 µL of Triton-X-100 (2%), 1 µL of NAD (20 mM), 0.5–1 µL of FEN, and 0.5 µL of Taq ligase, and brought to a final volume of 20 µL with nuclease-free water. The reaction was carried out at 65 °C for the appropriate time. Following this, the reaction mixture was added to Cas12-sgRNA mix containing 3 μL of Cas12-sgRNA complex (described in Cas12-sgRNA reaction), 4 μL of FAM-IBFQ reporter probe (1 μM), and 0.7 μL of RB-DTT, and incubated at 37 °C while measuring fluorescence (ex: 485 nm, em: 535 nm) every 3 min using a microplate reader.

[0114] For lateral flow strips, the FAM-IBQF reporter was replaced with 0.25 μL of FAM-Bio reporter (1 μM) or 0.1 μL of DIG-Bio reporter (1 μM). Following cleavage with Cas12, the reaction mix was run on Abingdon Health PCRD Flex strips according to the manufacturer's instructions. Band intensity was quantified using ImageJ. SNP detection To detect the T478K mutation in the receptor binding domain (RBD) of SARS-CoV-2, the mutation was introduced into pCDNA3-SRARS-CoV-2-S-RBD-Fc, carrying the viral RBD region (~1.5 kB), by overlap PCR using spike-T478K-F / R primers. After confirming mutagenesis by sequencing, the wild-type and mutated RBD regions were PCR amplified using spike-F / R primers and equal amounts of plasmid. After amplification, 50–150 ng of purified PCR product was used as template for FELICX, which was performed as previously described with minor modifications. The volume of the flap primers (WT and mut probe) was increased to 3 μL (final concentration 150 nM). Both templates were analyzed using WT and mut probes. For lateral flow strips, the cleavage reaction with Cas12-sgRNA used a FAM-Bio reporter in the WT probe sample and a DIG-Bio reporter in the mut probe sample. Gene detection in mammalian cells The C666-1 cell line was kindly provided by Dr Joshua Tay, Department of Otolaryngology, Yong Loo Lin School of Medicine, National University of Singapore, and the HK-1 cell line was a gift from Associate Professor Zhong Yong Liang, Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore. Both cell lines were grown in Gibco BenchStable RPMI 1640 (Life Technologies, Cat# A4192301) supplemented with 10% FBS (Biowest, Cat# S181H) and 1% penicillin-streptomycin (Gibco, Cat# 15140122) at 37°C with 5% CO2. After reaching 70-80% confluency, cells were trypsinized, washed with RPMI, and cell numbers were counted using a Luna cell counter. A fixed number of C666-1 or HK-1 cells were lysed by incubation in lysis buffer (10 mM Tris-Cl (pH 7.5), 200 μg / mL proteinase K, 0.1% SDS, 2 mM CaCl2) at 65°C for 10 min. After lysis, the genetic material was purified using the ChargeSwitch PCR cleanup kit and subjected to FELICX as described above. Fluorescence measured 30 min after the Cas12-sgRNA reaction was recorded. Detection of genes in K. pneumonia Overnight cultures of K. pneumoniae DSM 2026 grown in LB medium at 37°C under shaking conditions were diluted to the required cfu based on the formula OD600 = approximately 108 c.fu. HK-1 cells were grown in Gibco BenchStable RPMI 1640 (Life Technologies, Cat# A4192301) supplemented with 10% FBS (Biowest, Cat# S181H) and 1% penicillin-streptomycin (Gibco, Cat# 15140122) at 37°C in 5% CO2. After reaching 70-80% confluency, cells were trypsinized, washed with RPMI, and cell numbers were counted using a Luna cell counter. Mock clinical samples were prepared by mixing K. pneumoniae with HK-1 cells as required and lysing the samples in lysis buffer (10 mM Tris-Cl (pH 7.5), 200 μg / mL proteinase K, 0.1% SDS, 2 mM CaCl2) by incubation at 65 °C for 10 min. After lysis, the genetic material was purified using the ChargeSwitch PCR Cleanup Kit and subjected to FELICX as previously described. Fluorescence was then recorded after 30 min of the Cas12-sgRNA reaction. Cancer Biomarker Detection To prepare the template, overlapping primers (scgb1-F, scgb2-R, scgb3-F and scgb4-R) were designed and PCR amplification was performed using scgb2a2-T7-F and scgb2a2-R. The final construct incorporates a T7 promoter at the 5' end of SCGB2A2, allowing in vitro transcription with the Hiscrit T7 High Yield RNA Synthesis Kit (NEB). The transcript was purified with the Monarch RNA Cleanup Kit (NEB) and used for FELICX. To simulate clinical samples, RNA was quantified and spiked into fetal bovine serum (FBS) South America, heat inactivated (Biowest, Cat# S181H). Mock samples were treated with the ChargeSwitch PCR Cleanup Kit to purify the spiked RNA, followed by HDA with RTx and FELICX as previously described. Then, fluorescence was recorded 30 min after the Cas12-sgRNA reaction.

[0115] Example 1: Characterization and optimization of FEN For the development of FELICX, we first characterized and optimized the cleavage activity of FEN. FEN is a class of enzymes that is widespread in both prokaryotes and eukaryotes. FEN plays a major role in DNA replication, where it removes the RNA primer from the 5' end of Okazaki fragments that then bind to each other to form the lagging strand. In bacteria, DNA polymerase I (consisting of an N-terminal 5' nuclease and a C-terminal polymerase) fulfills the role of FEN through its N-terminal domain. In archaea and mammalian cells, there are dedicated FEN enzymes that are similar to the N-terminal domain of bacterial DNA polymerase I.

[0116] FENs are used to detect both DNA and RNA, but have some limitations, such as slow kinetics, the need for initial denaturation to assemble the oligo onto the target, and the reliance on a fluorescent reader for signal detection. DNA polymerase I from Thermus thermophilus (Tth pol) is a well-characterized FEN that can recognize the flap structures of both DNA and RNA targets. To determine whether Tth pol is the best candidate for FELICX, we expressed and purified a panel of Tth pol variants: the N-terminal domain of Tth pol (TthN), human FEN1 (hFEN), archaeal Methanococcus jannaschii FEN1 (MjaFEN), and bacteriophage T5 exonuclease (T5 exo) (Figure 18a and c) and compared their activities with a commercially available thermostable FEN (thermo FEN) (Figure 13). Previously, wild-type Tth pol was mutated (G506K, Q509K, H786A) to enable RNA recognition and inactivate the polymerase domain. This mutant was named Tth pol v1 in this study. Mutations in Taq polymerase have also been reported to broaden the substrate spectrum of this enzyme, but their effects on FEN activity have not been studied. These mutations are in the polymerase domain of this enzyme and may affect the substrate binding and thus the FEN activity of this enzyme. Due to the high similarity between Taq and Tth polymerases, the corresponding mutations in Tth pol may have similar effects on enzyme activity. Therefore, Tth pol v1 was further mutated to contain A604V, A610V, I616M, and E617GA mutations to generate Tth pol v2. Similarly, Tth pol v1 was mutated to contain A599T, W606R, A607Q, and I616T mutations to generate Tth pol v3 (Figure 18b). Based on a previous report that found that only the N-terminal domain of Taq polymerase exhibited FEN activity, TthN was also generated, which contains only the N-terminal domain (amino acids 1-307) of full-length Tth pol, and its FEN activity was tested.

[0117] To quantify FEN activity, a flap primer with a 5' fluorophore and two accompanying quenchers (3' and internal) was used. This primer generates a fluorescent signal when cleaved by FEN (Figure 13a). Enzyme activity for various configurations of flap and invasion primers was tested for all FEN enzymes: a 5'-flap flap primer and invasion primer configuration (Figure 13a), a double flap configuration in which both the flap and invasion primer have flaps (Figure 18d), and a configuration in which no target-specific invasion primer is present. Nonspecific activity of FEN for only the fluorophore-quencher flap primer was also measured. The target used was the partial orf1ab DNA of SARS-CoV-2, which has low similarity to the SARS virus genome. Reactions were performed at 65 °C, which is the melting temperature of the flap primers used in this study.

[0118] As seen in the left panel of Figure 13b, the activity of Tth pol v1, v2, and v3 was significantly reduced when a double flap substrate was used (Figure 18d) and in the absence of an invasion primer, suggesting that the configuration shown in Figure 2a was optimal for these enzymes. For DNA substrates with flap and non-flap invasion primers, among these variants of Tth pol, Tth pol v2 showed the highest activity, followed by Tth pol v3 and v1 (Figure 13b), demonstrating our hypothesis that the mutations introduced into the enzyme affect the enzyme activity. These enzymes showed high specific FEN activity and low non-specific activity, as shown by the fluorescent signal observed even in the case of flap primer only (Figure 13b). Surprisingly, TthN did not show any activity with any of the substrates tested, which was in contrast to previous studies performed using the highly similar N-terminal domain of Taq polymerase. hFEN showed no activity, possibly due to the high reaction temperature, while thermophilic MjaFEN showed low activity against substrates with flap primer and invasion primer of 5'-flap (Fig. 13b). For T5 exo, strong fluorescent signals were observed almost immediately after the start of the reaction for all substrates containing only flap primer. This indicates that the enzyme has a very high exonuclease activity, masking its FEN activity. In this enzyme, the K83 residue has been shown to be essential for exonuclease activity, but not essential for endonuclease activity, and was mutated to eliminate exonuclease activity. However, in this study, the K83A mutation completely inactivated this enzyme, eliminating both endonuclease and exonuclease activities (Fig. 13b). On the other hand, thermos FEN showed a similar trend to Tth pol in activity against various DNA substrates. However, this enzyme had low activity compared to Tth pol and its variants.These results indicate that Tth pol v2 is the best candidate with high specific FEN activity and that the ideal substrate for this enzyme is the DNA target bound to a flap primer and a non-flap invading primer.

[0119] We then selected the best FEN candidate, Tth pol v2, and compared its activity against the RNA substrate with flap-containing flap primer and invasion primer with other Tth pol variants, v1 and v3. As in the previous experiments, the SARS-CoV-2 partial orf1ab RNA was used in the assay. As seen in the right panel of Figure 13b, Tth pol v1 and v2 performed better than Tth pol v3, but weaker signals were observed for all three enzymes compared to DNA substrates. Based on the FEN activity observed on both DNA and RNA substrates, Tth pol v2 was selected as the FEN for FELICX. Further mutagenesis of Tth pol v2 did not significantly improve the FEN activity of this enzyme.

[0120] Tth pol v2 was further characterized by assessing the effect of enzyme concentration on FEN activity towards both DNA and RNA substrates and non-specific activity towards the flap primer alone (referred to as probe). Non-specific activity, as indicated by increased fluorescence for the probe alone, was observed to increase concomitantly with increasing enzyme concentration (Figure 13c). At 10 nM, high FEN activity was observed towards both DNA and RNA substrates with minimal non-specific activity. Non-specific activity increased significantly above this concentration and then became indistinguishable from FEN activity at 220 nM, suggesting that the enzyme requires an optimal concentration (10 nM) for specific FEN activity.

[0121] Although FEN can amplify the signal through cyclic annealing and denaturation of the flap primer, it may not be robust enough for highly sensitive detection of targets in short time periods because the signal amplification is linear (one flap product is formed each cycle per target). To examine this, the detection limit for both DNA and RNA was confirmed using 10 nM Tth pol v2. During a 30 min incubation, both DNA and RNA were detected up to 100 pM, but the fluorescence signal at 100 pM was slightly higher than the no target control (NTC) (Figure 13d and e). Increasing the Tth pol v2 concentration to 50 nM reduced the detection limit to 1 nM due to higher nonspecific activity resulting in larger background fluorescence (Figure 20).

[0122] Example 2: Development and optimization of FELICX Upon recognizing the DNA substrate, CRISPR-Cas12 acts as an excellent signal amplifier due to its rapid trans-cleavage activity on ssDNA. Thus, FEN was combined with CRISPR-Cas12 by converting the FEN-generated flap oligo into a substrate for Cas12 through ligation to an adapter. In FELICX, Cas12 from ND2006, a bacterium of the Lachnospiraceae family, was used. Because Cas12 prefers dsDNA substrates over ssDNA (Figure 21), a dsDNA adapter with a 3' overhang was generated by annealing two oligos: 5'p-adapter and ds-converter. The 5'p-adapter has a 5'-phosphate that is ligated to the 3'-OH of the flap oligo by a ligase enzyme, while the ds-converter has a PAM adjacent to the binding site of the sgRNA (Figure 14a). The complementary target of the sgRNA is split between the flap and the adaptor, ensuring that neither the adaptor alone nor the uncleaved flap primer gives rise to a signal.

[0123] For each new target in FELICX, only the part of the invasion primer and the flap primer that is complementary to the target needs to be changed (Figure 13a). FEN can cleave the flap primer in a sequence-independent manner, so the 5' flap of the flap primer is left unchanged. However, the cleavage site of FEN is one base pair into the part of the flap primer that is complementary to the target. Therefore, every target can result in a flap with any of the four nucleotides at the 3' end of the flap depending on the target sequence. To address this, it becomes necessary to change the complementary nucleotide in the ds converter (see nucleotides N and N' in Figure 3a). Since this base pair is within the region recognized by the sgRNA, the recognition of the dsDNA adapter by the Cas12-sgRNA complex was tested with any of the four nucleotides at position N without changing the sgRNA sequence. It is expected that this test will simplify the use of Cas12-sgRNA and eliminate the need to change the sgRNA sequence for each new target. For this, we used a Cas12-sgRNA complex with an sgRNA complementary to a dsDNA adapter with a thymine at position N (Figure 23a). As shown in Figure 23c, Cas12-sgRNA detected all possible targets with similar efficiency, regardless of the nucleotide at the end of the flap. This is expected, since previous reports have shown that LbCas12a can tolerate mismatches at 8 to 18 nucleotides from the PAM, the region where the variable base pair (N-N') is located. Thus, for any nucleic acid target, the remainder of the dsDNA adapter, except for one base pair, and the sgRNA remain unchanged regardless of the target sequence, which facilitates reconfiguration of FELICX to detect different targets.

[0124] For flap-to-adapter ligation, Taq ligase was chosen because it has the same temperature optimum (65°C) as FEN. This allows for a one-pot reaction in which both FEN-mediated flap primer cleavage and adapter ligation occur simultaneously. The ligation efficiency of Taq ligase at 65°C was measured and compared with that of other ligases by performing reactions at the enzyme's reported optimum temperature (21°C for T4 DNA ligase and 37°C for T4 RNA ligase). Synthetic flap oligos and dsDNA adapters were used as substrates, and ligation was performed for 30 min. The ligation products were detected by Cas12-sgRNA complexes, and the fluorescent signal was measured after 10 or 30 min of incubation at 37°C (Figure 14b). Among the three ligases tested, Taq DNA ligase showed the highest fluorescent signal within 10 min of incubation with Cas12-sgRNA, which was comparable to that of the pre-ligated product (Figure 14c). Therefore, Taq ligase was used as the ligase for FELICX.

[0125] Next, the one-pot FEN+Taq ligase reaction step was optimized by varying the concentration of FEN and the reaction incubation time at 65°C. Figure 14d shows that the optimal FEN concentration was 10 nM, as it produced the strongest fluorescent signal. Meanwhile, lower and higher FEN concentrations resulted in weaker signals. Lower FEN concentrations resulted in fewer flap products being formed, resulting in weaker fluorescent signals. However, higher FEN concentrations may result in shorter flap products due to the nonspecific activity of FEN, which may not anneal to the dsDNA adapter due to the high reaction temperature, or may not be recognized by the Cas12-sgRNA complex after ligation due to incomplete hybridization of the sgRNA to the ligation product. Furthermore, the fluorescent signal improved with longer incubation times, with the strongest signal obtained by 4 h incubation. To further increase the intensity of the fluorescent signal, the concentrations of both the Cas12-sgRNA complex and the DNA reporter were increased, which resulted in a significant increase in the specific signal (Figure 22). A modified sgRNA extended with ssDNA at the 3' end was previously reported to increase the trans-cleavage activity of LbCas12a and was also evaluated. However, the activity was observed to be not improved compared to that of the unmodified sgRNA (Figure 23). Using these optimized conditions, the detection limits of DNA and RNA by FELICX were evaluated to determine whether the signal amplification by FEN and Cas12 was sufficient for sensitive detection of nucleic acids. To this end, partial orf1ab DNA or RNA substrates of SARS-CoV-2 were spiked into total DNA or RNA purified from HEK293T cells, respectively, to mimic clinical samples. When both substrates were tested, only up to 100 pM of target was detected by FELICX despite a prolonged incubation of 4 h at 65 °C (Figures 14e and f).This detection limit is similar to that observed with FEN alone (Figure 13d and e), but the fluorescent signal observed with FELICX was significantly higher than that with FEN alone, indicating that the signal was amplified by Cas12.

[0126] Previously reported nucleic acid detection methods have incorporated isothermal nucleic acid amplification techniques, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), to improve detection sensitivity. Target nucleic acids were amplified using LAMP, a commonly used nucleic acid amplification method, performed at 65°C, followed by FELICX to detect partial orf1ab DNA of SARS-CoV-2 spiked into total DNA purified from HEK293T cells. However, orf1ab DNA was not detected despite extensive optimization of the assay.

[0127] Because nonspecific amplification was observed using LAMP with six primers, alternative amplification methods that require fewer primers, such as RPA and helicase-dependent amplification (HDA), may eliminate the formation of nonspecific products and improve the sensitivity of target detection. HDA was chosen because the operating temperature of HDA (65°C) is the same as that of the FEN+Taq ligation step, simplifying the operation of HDA+FELICX. When FELICX was coupled with HDA, six copies / μL (10 aM) of orf1ab and two copies / μL (3.5 aM) of E. coli genome were detected in two of the three biological samples (Figure 14g and h), showing a notable improvement in the detection of orf1ab in particular compared to LAMP. Thus, the nucleic acid amplification step, by reducing the number of primers present, can prevent the formation of nonspecific products and increase the yield of the target, significantly improving the subsequent detection. Integration of a lateral flow detection system into FELICX FELICX was integrated with a lateral flow system for signal detection to eliminate the need for a fluorescent reader. We used the PCRD Flex lateral flow strip, which can detect two different oligos conjugated with biotin and either FAM or digoxigenin (Figure 15a). In the final step of FELICX, the Cas12-sgRNA complex cleaves a DNA reporter in the presence of its substrate. To exploit this cleavage activity in the lateral flow strip, the DNA fluorescent reporter was replaced with a probe for PCRD Flex. In the presence of the substrate, the Cas12-sgRNA complex cleaves the probe, resulting in the disappearance of the corresponding band on the lateral flow strip (Figure 15b). Thus, a positive signal for the target nucleic acid is represented by the absence of a band on the lateral flow strip. Partial orf1ab DNA was used as a target to validate the functionality of the lateral flow strip and FELICX. Because the sensitivity of the lateral flow strips is lower than that of the fluorescent reader, the HDA reaction volume was scaled up from 10 μL to 25 μL. Because FELICX observed high variability and weak signal when using unpurified HDA products (Figure 14g and h), a nucleic acid purification step was included after HDA. The ChargeSwitch PCR Cleanup Kit was used for purification. When coupled with HDA and subsequent purification, orf1ab DNA spiked into purified HEK293T DNA was detected down to 0.6 copies / μL (1 aM) as indicated by disappearance of the T1 band on the lateral flow strips using FELICX (Figure 15c). The total time of detection was 2.5 h, including 1 h HDA, 1 h FEN+Taq ligation, and 30 min Cas12-sgRNA reaction. Reducing the HDA+FELICX time to 60 min (20 min each for HDA, FEN+Taq ligation, and Cas12-sgRNA) did not reduce sensitivity (Figure 15d), demonstrating the robustness of HDA+FELICX.

[0128] Having confirmed that the method is applicable for rapid and sensitive detection of DNA, HDA+FELICX was used to detect RNA targets. Since the HDA reaction mixture lacks a dedicated reverse transcriptase, WarmStart RTx reverse transcriptase was used due to its temperature optimum of 65°C and similar buffer requirements as HDA. Since RTx has not been used with HDA before, we evaluated the function of RTx+HDA in a one-step (one-pot RTx+HDA) or two-step (sequential RTx and HDA) reaction to determine if RTx interferes with the HDA reaction. No difference was observed between the reactions (Figure 26), and the one-step RTx+HAD was chosen because it is less complex. After performing RTx+HDA, FELICX was performed to detect orf1ab RNA spiked into purified HEK293T RNA. 0.6 copies / μL RNA was detected in this process (Figure 15e). It took a total of about 90 min to detect RNA (45 min for RTx+HDA, 20 min for FEN+Taq ligation, and 20 min for Cas12-sgRNA). These results demonstrated that HDA+FELICX can detect both DNA and RNA with similar sensitivity in 60 and 90 min, respectively, using lateral flow strips.

[0129] Example 3: Detection of polynucleotide analytes using FELICX Detection of SNPs is important for disease diagnosis and treatment as well as identification of pathogen variants. FEN can be used to discriminate SNPs by designing flap and invasion primers such that a one base pair overlap between the primers is the location of the SNP (Figure 16a). If the SNP is present in the target, the absence of overlap between the flap and invasion primers abolishes the activity of FEN. Thus, the presence or absence of SNPs in a target sample can be confirmed by probing the sample with flap primers specific for the wild-type and variant target sequences (Figure 16b). For this purpose, FELICX was used to distinguish the receptor binding domain in the spike protein of wild-type SARS CoV-2 from its variants carrying the T478K mutation found in delta and omicron variants of the virus. Flap primers, i.e., WT and mut probes specific for the wild-type (WT) sequence and variant, respectively, were designed and used to probe both targets. For the WT target, after FELICX, the fluorescent signal was about 2.5-fold higher with the WT probe compared to the mut probe (Figure 16c). In contrast, the variant target with the T478K mutation showed that the fluorescent signal was about 2.5-fold higher with the mut probe than with the WT probe. Despite the difference between the WT and the T478K variant being only one base pair (ACG codon changed to AAG), FELICX readily detected the SNP as indicated by the opposite trends in the fluorescent signal observed for the WT and variant sequences.

[0130] Detection of SNPs was also demonstrated using PCRD Flex strips, with each test line corresponding to either the WT or mut probe. For this, a FAM-biotin reporter was used for the WT probe and a DIG-biotin reporter for the mut probe (Figure 16b). Similar to the results shown in Figure 16c, the WT sample showed a positive signal with the WT probe, as indicated by the disappearance of the band corresponding to this probe (Figures 16d and e). For the T478K variant, the disappearance of the band corresponding to the mut probe indicated a positive signal with this probe.

[0131] FELICX can also detect other pathogens of interest in more complex samples, such as EBV, which causes infectious mononucleosis and is associated with autoimmune diseases, nasopharyngeal carcinoma (NPC), and other neoplasms. C666-1, an NPC cell line and natural host of EBV, was used. The EBV-negative HK-1 NPC cell line was also included in the analysis, and the housekeeping gene gapdh was used as an internal control for assay functionality. After cell lysis, RTx+HDA and FELICX were performed, and both EBV and gapdh were detected at levels as low as 0.6 cells / μL of C666-1 (Figures 17a and 27, upper panel), demonstrating highly sensitive detection of EBV by HDA+FELICX. Sensitive detection of targets in whole cells using HDA+FELICX demonstrated the robustness of the method.

[0132] FELICX was also able to detect K. pneumoniae, the most common nosocomial pathogen and a major cause of patient complications worldwide. K. pneumoniae is known to be a causative agent of neonatal sepsis, pneumonia, surgical wound infections, and cystitis. K. pneumoniae is not only widespread but also a major source of antibiotic resistance genes, especially genes encoding carbapenemases. Therefore, detecting the pathogen in clinical samples and unambiguously identifying its antibiotic resistance status is essential to guide infection treatment. HDA+FELICX was used to detect carbapenemase resistance genes (blaKPC) in virulent K. pneumoniae DSM 2026 bacterial cells spiked into HK-1 whole cells. The hemolysin gene (khe), present in all K. pneumoniae isolates, was used as an internal control. Using conditions similar to those described for EBV detection, blaKPC and khe were detected in K. pneumoniae DSM2026 down to 0.6 cfu / μL (Figures 17b and 27, lower panels). No signal was observed for either gene in HK-1 cells alone. As RTx was not used in this assay, only copies of the genes in the K. pneumonia DSM2026 genome were detected, highlighting the high sensitivity of the assay. Tth-pol v2 array (query number 1) Tth-pol v3 sequence (SEQ ID NO:2) Cas12 gRNA 5'-TAATTTCTACTAAGTGTAGATCAACGTCGTGACTGGGAAAACCCT-3' (SEQ ID NO: 3)

[0133]

Table 4-1

[0134]

Table 4-2

[0135]

Table 4-3

[0136]

Table 5-1

[0137]

Table 5-2

[0138]

Table 6

[0139]

Table 7

Claims

1. A method for detecting polynucleotide analytes in a sample, a) Samples containing polynucleotide analytes, i) A first nucleic acid probe comprising a 3' portion complementary to the first portion of the polynucleotide analyte and a 5' portion that is not complementary to the polynucleotide analyte and does not hybridize with it; ii) A second nucleic acid probe comprising a 5' portion complementary to the second portion of the polynucleotide analyte and a 3' portion not complementary to the polynucleotide analyte and not hybridizing with it, wherein the first portion of the polynucleotide analyte is 5' with respect to the second portion of the polynucleotide analyte and adjacent thereto; and iii) Structure-specific nucleic acid cleavage agents The step of bringing it into contact with Hybridization of the first nucleic acid probe into the first portion of the polynucleotide analyte and hybridization of the second nucleic acid probe into the second portion of the polynucleotide analyte form a cleavage structure; The formation of a cleavage structure occurs when the first nucleic acid probe is cleaved by the cleavage agent, releasing a 5' flap containing the 5' portion of the first nucleic acid probe that is not complementary to the polynucleotide analyte and does not hybridize with it; b) A step of ligating a 5' flap with a nucleic acid adapter to form an adapter ligation product, wherein the nucleic acid adapter includes a double-stranded region and a 3' overhang extension complementary to the 5' flap, and the 3' overhang extension hybridizes with the 5' flap; c) Adapter ligation products, i) Type V CRISPR / Cas effector proteins; ii) A guide RNA comprising a region that binds to a type V CRISPR / Cas effector protein, a portion of the 5' flap, and a guide sequence that ligates to the 5' flap and is complementary to a portion of the nucleic acid adapter adjacent to it; and iii) Single-stranded detector DNA The step of making contact with it; d) The step of detecting the adapter ligation product by measuring the detectable signal produced by the cleavage of single-stranded detector DNA by the type V CRISPR / Cas effector protein, thereby detecting the polynucleotide analyte in the sample. Methods that include...

2. The method according to claim 1, wherein the polynucleotide analyte is DNA or RNA.

3. The method according to claim 1, wherein the polynucleotide analyte is single-stranded or double-stranded.

4. The method according to claim 1, further comprising the step of amplifying a polynucleotide analyte before step (a).

5. The method according to claim 1, wherein the polynucleotide analyte contains a single nucleotide polymorphism (SNP).

6. The method according to claim 1, wherein the polynucleotide analyte is viral nucleic acid.

7. The method according to claim 1, wherein the cleaving agent in step a) is an enzyme having flap endonuclease activity.

8. The method according to claim 7, wherein the enzyme having flap endonuclease activity is a DNA polymerase derived from Thermus thermophilus.

9. The method according to claim 7, wherein the enzyme having flap endonuclease activity comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence described in SEQ ID NO:

1.

10. The method according to claim 1, wherein step c) is carried out in the presence of ligase.

11. The method according to claim 10, wherein the ligase is a heat-stable ligase.

12. The method according to claim 1, wherein the type V CRISPR / Cas effector protein is the Cas12 protein.

13. The method according to claim 1, wherein the single-stranded detector DNA includes a fluoroforequencher pair.

14. The method according to claim 1, for visually detecting breaks in single-stranded detector DNA.

15. A method for detecting single nucleotide polymorphisms (SNPs) in polynucleotide analytes in a sample, a) Samples containing polynucleotide analytes, i) A first nucleic acid probe comprising a 3' portion complementary to the first portion of the polynucleotide analyte and a 5' portion that is not complementary to the polynucleotide analyte and does not hybridize with it; ii) A second nucleic acid probe comprising a 5' portion complementary to the second portion of the polynucleotide analyte and a 3' portion not complementary to the polynucleotide analyte and not hybridizing with it, wherein the first portion of the polynucleotide analyte is 5' with respect to the second portion of the polynucleotide analyte and adjacent thereto; and iii) Structure-specific nucleic acid cleavage agents The step of bringing it into contact with Hybridization of the first nucleic acid probe into the first portion of the polynucleotide analyte and hybridization of the second nucleic acid probe into the second portion of the polynucleotide analyte form a cleavage structure; The formation of a cleavage structure occurs when the first nucleic acid probe is cleaved by the cleavage agent, releasing a 5' flap containing the 5' portion of the first nucleic acid probe that is not complementary to the polynucleotide analyte and does not hybridize with it; b) A step of ligating a 5' flap with a nucleic acid adapter to form an adapter ligation product, wherein the nucleic acid adapter includes a double-stranded region and a 3' overhang extension complementary to the 5' flap, and the 3' overhang extension hybridizes with the 5' flap; c) Adapter ligation products, i) Type V CRISPR / Cas effector proteins; ii) A guide RNA comprising a region that binds to a type V CRISPR / Cas effector protein, a portion of the 5' flap, and a guide sequence that ligates to the 5' flap and is complementary to a portion of the nucleic acid adapter adjacent to it; and iii) Single-stranded detector DNA The step of making contact with it; d) The step of detecting adapter ligation products by measuring the detectable signal generated by the cleavage of single-stranded detector DNA by type V CRISPR / Cas effector protein, thereby detecting SNPs in the polynucleotide analyte in the sample. Methods that include...

16. A method for detecting polynucleotide analytes in a sample, a) Samples containing polynucleotide analytes, i) A first nucleic acid probe and a second nucleic acid probe configured to form a cleavage structure in the presence of a polynucleotide analyte; and ii) Structure-specific nucleic acid cleavage agents The step of bringing it into contact with The first nucleic acid probe is cleaved by the cleavage agent, and a 5' flap is released from the first nucleic acid probe; b) A step of ligating a 5' flap with a nucleic acid adapter to form an adapter ligation product, wherein the nucleic acid adapter includes a double-stranded region and a 3' overhang extension complementary to the 5' flap, and the 3' overhang extension of the nucleic acid adapter hybridizes with the 5' flap; c) Adapter ligation products, i) Type V CRISPR / Cas effector proteins; ii) A guide RNA comprising a region that binds to a type V CRISPR / Cas effector protein, a portion of the 5' flap, and a guide sequence that ligates to the 5' flap and is complementary to a portion of the nucleic acid adapter adjacent to it; and iii) Single-stranded detector DNA The step of making contact with it; d) The step of detecting the adapter ligation product by measuring the detectable signal produced by the cleavage of single-stranded detector DNA by the type V CRISPR / Cas effector protein, thereby detecting the polynucleotide analyte in the sample. Methods that include...

17. A kit for detecting polynucleotide analytes in a sample, comprising a structure-specific nucleic acid cleavage agent, a nucleic acid ligase, and a type V CRISPR / Cas effector protein.

18. The kit according to claim 17, wherein the structure-specific nucleic acid cleavage agent is a polypeptide comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence described in SEQ ID NO:

1.

19. The kit according to claim 17, further comprising a first nucleic acid probe and a second nucleic acid probe, wherein the first and second nucleic acid probes are configured to form cleavage structures in the presence of a polynucleotide analyte.

20. The kit according to claim 17, further comprising a first adapter polynucleotide and a second adapter polynucleotide, wherein the first and second adapter polynucleotides are configured to form a nucleic acid adapter during hybridization.

21. The kit according to claim 20, further comprising a type V CRISPR / Cas effector protein and a guide RNA configured to bind to the ligation product of the cleavage product of a nucleic acid adapter and a structure-specific nucleic acid cleavage agent.

22. The kit according to claim 17, further comprising single-stranded detector DNA.

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