Method of detecting polynucleotide analytes

Abstract

The present invention relates generally to the field of nucleic acid detection. In particular, the present specification teaches a method of detecting a polynucleotide analyte in a sample. In one aspect, a method includes the use of a cleavage agent having endonuclease activity and a V-type CRISPR / Cas effector protein. In another aspect, the V-type CRISPR / Cas effector protein is a Cast 2 protein.

Description

Technical Field

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

[0002] In vitro amplification and detection of nucleic acids has a variety of clinical applications, especially in the diagnosis of acute and chronic conditions, 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 instrumentation and are costly and time-consuming. New generation detection assays, including invader assays and CRISPR-based methods, strive to combine ease of use and cost-effectiveness without compromising assay specificity and sensitivity.

[0003] The invasion assay is a method for detecting and quantitatively analyzing DNA or RNA. It does not amplify the target of interest, but rather generates and amplifies an unrelated signal only in the presence of the correct target sequence. The assay involves generating an invasive cleavage structure in a target-dependent manner, so that a structure-specific enzyme cuts the invasive cleavage structure to release a signal that can be further detected. The assay involves the use of two primers, namely, 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 generally unrelated to the target sequence. The 5' portion of the flap primer that does not hybridize with the target forms a 5' flap. The invasion primer anneals to the 5' portion of the target annealed to the 5' portion of the flap primer, and the flap primer and the invasion primer overlap to form a forked overlapping structure, which is considered to be similar to the structure generated during strand displacement DNA synthesis. The invasion primer and the flap primer usually overlap by one nucleotide, but longer overlaps can also be used. The forked structure is cut to release the 5' flap of the 5' primer, and the released 5' flap is then used as a detectable signal. Disadvantages of invasion assays include slow detection, the need for an initial denaturation step, and the need for a fluorescent detection system.

[0004] CRISPR-based methods include DETECTR and SHERLOCK assays. The DETECTR assay relies on the Cas12 nuclease to detect DNA targets, and the SHERLOCK assay relies on the Cas13 nuclease to detect RNA targets. In both methods, recognition of the nucleic acid target results in off-target cleavage, thereby generating a signal. The disadvantage of Cas12-based assays is that a PAM sequence close to the target sequence is required to be recognized by the Cas nuclease, so the sequences that can be detected are limited. Cas13-based assays require in vitro transcription of the DNA target into RNA for detection, which makes the detection more complicated. In both cases, the guide RNA activity must also be optimized to detect each new target.

[0005] It is 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 for detecting a polynucleotide analyte in a sample, the method comprising: a) contacting a sample containing the polynucleotide analyte with: i) a first nucleic acid probe, the first nucleic acid probe comprising a 3' portion complementary to a first portion of the polynucleotide analyte and a 5' portion that is not complementary to the polynucleotide analyte and does not hybridize with the polynucleotide analyte; ii) a second nucleic acid probe, the second nucleic acid probe comprising a 5' portion complementary to a second portion of the polynucleotide analyte and a 3' portion that is not complementary to the polynucleotide analyte and does not hybridize with the polynucleotide analyte, wherein the first portion of the polynucleotide analyte is complementary to the polynucleotide analyte; the second portion of the polynucleotide analyte being adjacent to the first 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 forms a cleavage structure; wherein formation of the cleavage structure causes the cleavage agent to cleave the first nucleic acid probe to release a 5' flap, wherein the 5' flap comprises a 5' portion of the first nucleic acid probe that is not complementary to the polynucleotide analyte and is not hybridized to the polynucleotide analyte; b) ligating the 5' flap to a nucleic acid adaptor to form an adaptor ligation product, wherein the nucleic acid adaptor comprises a double-stranded region and a 3' overhang extension complementary to the 5' flap. extension), wherein the 3' overhang extension hybridizes to the 5' flap; c) contacting the adaptor 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 adaptor that is attached to and adjacent to the 5' flap; and iii) a single-stranded detection DNA; and d) measuring a detectable signal generated by cleavage of the single-stranded detection DNA by the type V CRISPR / Cas effector protein to detect the adaptor ligation product, thereby detecting a polynucleotide analyte in the sample.

[0007] Disclosed herein is a method for detecting a single nucleotide polymorphism (SNP) of a polynucleotide analyte in a sample, the method comprising: a) contacting a sample comprising a polynucleotide analyte with: i) a first nucleic acid probe, the first nucleic acid probe comprising a 3' portion complementary to a first portion of the polynucleotide analyte and a 5' portion that is not complementary to the polynucleotide analyte and does not hybridize with the polynucleotide analyte; ii) a second nucleic acid probe, the second nucleic acid probe comprising a 5' portion complementary to a second portion of the polynucleotide analyte and a 3' portion that is not complementary to the polynucleotide analyte and does not hybridize with the polynucleotide analyte, wherein the first portion of the polynucleotide analyte is adjacent to the second portion of the polynucleotide analyte at 5'; and iii) a structure-specific nucleic acid cleavage agent; wherein hybridization of the first nucleic acid probe with the first portion of the polynucleotide analyte and hybridization of the second nucleic acid probe with the second portion of the polynucleotide analyte form a cleavage structure; wherein formation of the cleavage structure causes The invention relates to a method for detecting SNPs of a polynucleotide analyte in a sample by providing a cleavage agent to cleave the first nucleic acid probe to release a 5' flap, wherein the 5' flap comprises a 5' portion of the first nucleic acid probe that is not complementary to the polynucleotide analyte and does not hybridize to the polynucleotide analyte; b) ligating the 5' flap to a nucleic acid adaptor to form an adaptor ligation product, wherein the nucleic acid adaptor comprises a double-stranded region and a 3' protruding extension that is complementary to the 5' flap, wherein the 3' protruding extension hybridizes to the 5' flap; c) contacting the adaptor ligation product with: i) a type V CRISPR / Cas effector protein; ii) a guide RNA, wherein the guide RNA comprises 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 that is attached to and adjacent to the 5' flap; and iii) a single-stranded detection DNA; and d) measuring a detectable signal generated by cleavage of the single-stranded detection DNA by the type V CRISPR / Cas effector protein to detect the adaptor ligation product, thereby detecting SNPs of the polynucleotide analyte in the sample.

[0008] Disclosed herein is a method for detecting a polynucleotide analyte in a sample, the method comprising:

[0009] a) i) contacting a sample comprising a polynucleotide analyte with a first nucleic acid probe and a second nucleic acid probe, the first nucleic acid probe and the second nucleic acid probe being configured to form a cleavage structure in the presence of the polynucleotide analyte; and

[0010] ii) structure-specific nucleic acid cleavage agents;

[0011] wherein the formation of the cleavage structure allows the cleavage agent to cleave the first nucleic acid probe to release the 5' flap from the first nucleic acid probe;

[0012] b) ligating the 5' flap to a nucleic acid adaptor to form an adaptor ligation product, wherein the nucleic acid adaptor comprises a double-stranded region and a 3' overhang extension complementary to the 5' flap, and wherein the 3' overhang extension hybridizes to the 5' flap;

[0013] c) contacting the adaptor ligation products:

[0014] i) Type V CRISPR / Cas effector proteins;

[0015] 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 the 5' flap and a portion of a nucleic acid adaptor that is attached to and adjacent to the 5' flap; and

[0016] iii) single-stranded detection DNA; and

[0017] d) measuring a detectable signal generated by cleavage of the single-stranded detection DNA by a type V CRISPR / Cas effector protein to detect the adaptor ligation product, thereby detecting the polynucleotide analyte in the sample.

[0018] Disclosed herein is a kit for detecting a polynucleotide analyte in a sample, comprising a structure-specific nucleic acid cleavage agent, a nucleic acid ligase, and a type V CRISPR / Cas effector protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Embodiments of the present invention will now be described by way of non-limiting examples with reference to the accompanying drawings, in which:

[0020] Figure 1 Schematic diagram of the overall working of flap endonuclease, Taq ligase and CRISPR-Cas12 in the disclosed method.

[0021] Figure 2 Use FELICX (flap endonuclease, Taq ligase and CRISPR- C as fordiagnostics( X ))Description of the workflow for polynucleotide detection. (a) Genetic material is extracted from clinical samples and amplified isothermally. (b) Detection of target nucleotide sequence and generation of signal by sequential activity of FEN, ligase and Cas12-sgRNA. The output of FEN is an oligonucleotide, which is connected to the adaptor to form a substrate for Cas12-sgRNA, the binding of which to Cas12 leads to the cleavage of ssDNA reporter. (c) The output can be a fluorescent signal or a readout on a lateral flow test strip.

[0022] Figure 3 Binding of the invasion primer and flap probe to the DNA target. The underlined sequence is the flap that remains constant for any target. The red arrow indicates the site where FEN cleaves the flap probe.

[0023] Figure 4The flap endonuclease activity of the commonly used DNA-targeting Tth pol and Tth pol v2. The activity is determined using flap probes labeled with a fluorophore and a quencher. After cleavage by the purified enzyme, a fluorescent signal is observed. The reaction is carried out in a solution containing Tris-Cl, NaCl, MgCl 2 , Triton-X-100 and DTT buffer.

[0024] Figure 5 The flap is ligated to a dsDNA adapter containing a 5'p adapter strand and a ds converter strand. The flap product is shaded. The box on the upper strand of the ligated product is the binding region of the gRNA, and the box on the lower strand is the PAM sequence for CRISPR-Cas12.

[0025] Figure 6 Detection of (a) orf1ab copies amplified by LAMP; (b) E. coli genome copies amplified by LAMP; (c) orf1ab copies amplified by HAD; (d) E. coli genome copies amplified by HDA. NTC - no template control. All samples were prepared by spiking target DNA into HEK293t total DNA to simulate clinical samples.

[0026] Figure 7 Lateral flow readout of orf1ab (mock sample) after HDA and detection using the methods described herein. (a) Readout after 1 hour HDA, 1 hour FEN+Taq reaction, and 30 minutes cas12 reaction; (b) Readout after 20 minutes HDA, 20 minutes FEN+Taq reaction, and 20 minutes cas12 reaction. 5'6-FAM-TTATTATTAT-3'Bio ssDNA was used as the primer corresponding to T 1 orf1ab gene reporter. C-control, T 2 - A second test lead not used in this experiment.

[0027] Figure 8 (a) Amplification of orf1ab RNA using RTx and HDA in two-step or one-step. Blue triangles indicate increasing volumes of RTx mix added to HDA. (b) Lateral flow readout of orf1ab RNA (mock sample) over 90 minutes (45 minutes HDA, 20 minutes FEN+Taq reaction, and 20 minutes 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.

[0028] Fig. 9Detection of EBV and gapdh in C666-1 and HK-1 cells. NTC - no template control. The figure shows the fluorescence readout of the Cas12 step of the detection method.

[0029] Fig.10 Detection of blaKPC and khe in Klebseilla pneumoniae spiked into HK-1 cells. NTC - No template control. The figure shows the fluorescence readout of the Cas12 step of the detection method.

[0030] Fig.11 Detection of scgb2a2 inserted into FBS. The figure shows the fluorescence readout of the Cas12 step of the detection method.

[0031] Fig.12 Detection of WT or mut DNA corresponding to the spike protein of SARS-CoV-2 by (a) wild-type flap probe (b) mut flap probe. The figure shows the fluorescence readout of the Cas12 step of the detection method.

[0032] Figure 13 shows a comparison of FEN activities of different enzymes and their engineered variants. (a) Schematic diagram of the fluorescence assay used to quantify FEN activity. (b) Activity of different FEN enzymes on 1 nM orf1ab DNA / RNA substrates with different configurations of flaps and invader primers, including the configuration shown in (a) (first column), a dual flap configuration with flaps on both the invader primer and the flap primer (second column), a configuration without an invader primer (third column), and a configuration with only a flap primer (fourth column). No FEN refers to reactions without enzymes (fifth column). The data on the left are DNA substrates and the data on the right are RNA substrates. Data represent mean values ​​(n=3 biological replicates). Thermo FEN was from NEB (Catalog No. M0645S). (c) FEN activities of Tthpol v2 against probes (flap primer only), DNA substrates (1 nM), and RNA substrates (1 nM) at different enzyme concentrations after incubation at 65°C for 60 minutes. The horizontal black bars represent mean values ​​(n=3 biological replicates). p values ​​< 0.05 calculated using a two-sample t-test are provided. (d) and (e) Limits of detection of orf1ab DNA (left) and RNA (right) after incubation with 10 nM Tthpol v2 for 30 min at 65°C. Data represent mean ± SD (n = 3 biological replicates). p values ​​< 0.05 compared to NTC are provided, calculated using a two-sample t-test. NTC: no target control.

[0033] Figure 14 shows the optimization of adapter ligation and nucleic acid detection limits using FELICX. (a) Sequence of flap product connected to dsDNA adapter. PAM is underlined and variable base pairs are shaded. (b) Schematic diagram showing that an oligonucleotide similar to a flap product formed by FEN is connected to a dsDNA adapter by a ligase. The ligation product forms a substrate for Cas12-sgRNA, which cuts the ssDNA reporter after activation to produce a fluorescent signal. (c) Synthesized flap oligonucleotides were connected to dsDNA adapters using different ligases for 30 minutes, followed by Cas12-sgRNA detection (cutting for 10 minutes or 30 minutes), and then the fluorescent signal observed. The connection volume was 15 μL, to which 5 μL Cas12-sgRNA was subsequently added. The data represent mean ± SD (n = 3 biological replicates). NTC: no target control. Connection: pre-connected substrate. (d) Optimization of FEN concentration and incubation time for FEN+Taq ligase reaction (15 μL) using orf1ab DNA (1 nM). After ligation, a 30-min Cas12-sgRNA reaction (20 μL) was performed. Data represent mean ± SD (n = 2 biological replicates). (e) and (f) Detection limits of orf1ab DNA or RNA spiked into purified HEK293T total DNA or RNA, respectively, using this detection system. FEN+Taq ligase reaction (20 μL) was performed for 4 hours and Cas12-sgRNA reaction (another 7 μL) was performed for 30 minutes. Data represent mean ± SD (n = 3 biological replicates). p-values ​​compared to NTC are provided < 0.05, 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 in 10 μL reactions for 1 hour, FEN+Taq ligase reactions were performed for 1 hour (total volume 20 μL), and Cas12-sgRNA reactions were performed for 30 minutes (total volume 27 μL). Data represent mean ± SD (n = 3 biological replicates). P values ​​compared to NTC are provided < 0.05, calculated using a two-sample t-test.

[0034] Fig.15FELICX is shown in combination with lateral flow test strips for nucleic acid detection. (a) Schematic diagram of the lateral flow test strips used in this study. Test line T1 corresponds to a reporter with FAM and biotin, and T2 corresponds to a reporter with digoxigenin (DIG) and biotin. C is a lateral flow control. (b) Schematic diagram of nucleic acid detection using HDA+FELICX and lateral flow test 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 in a 25 μL reaction for 1 hour, followed by rapid purification, FEN+Taq reaction for 1 hour (total volume 20 μL), and Cas12-sgRNA reaction for 30 minutes (total volume 27 μL). In this experiment, only T1 was used. The numbers below the test strips show the fold change in T1 band intensity compared to NTC. (d) Same as (c), HDA, FEN+Taq, and Cas12-sgRNA reactions were each performed for 20 minutes. The numbers below the test strips show the 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 in a 25 μL reaction followed by rapid purification, 20 min FEN+Taq reaction (total volume 20 μL), and 20 min Cas12-sgRNA reaction (total volume 27 μL). Only T1 was used in the experiment. The numbers below the test strips show the fold change in T1 band intensity compared to NTC.

[0035] Figure 16 shows SNP detection using FELICX. (a) Schematic diagram showing FEN cleavage of flap primers in the case of wild-type (WT) targets and no flap primer cleavage in the case of SNPs. The cleavage site of the flap primer is marked with an arrow. The last nucleotide of the invading primer does not match the target, preventing FEN cleavage. (b) Detection of WT target, target with SNP / mutation, or no target using FELICX, and the corresponding lateral flow test strip patterns observed in each case. (c)-(e) Detection of WT receptor binding domain sequences of SARS-CoV-2 or its variant (T478K) by FELICX using WT and mut probes. Panel (c) shows the fluorescence signal, and (d) shows the lateral flow results after FELICX of the pre-amplification substrate using two probes. Pre-amplification was performed by PCR, followed by DNA purification to ensure that equal amounts of WT and mutant DNA were used for accurate comparison. The FEN+Taq reaction was performed for 60 minutes (total volume 20 μL) and the Cas12-sgRNA reaction was performed for 30 minutes (total volume 27 μL). In (c), data represent mean ± SD (n = 3 biological replicates). p-values ​​< 0.05 compared to NTC are provided, calculated using a two-sample t-test. (e) Percent change in the intensity of the bands corresponding to the WT and mut probes compared to NTC in (d). NTC: no target control.

[0036] Fig.17 The versatility of FELICX is demonstrated. FELICX was used to detect targets in more complex samples, such as whole bacteria and mammalian cells as well as serum. The fluorescence signal fold changes of (a) EBV and gapdh in whole C666-1 and HK-1 cells, (b) bla in K. pneumoniae (Kp) cells spiked into HK-1 whole cells were detected. KPC and khe, and (c) SCGB2A2 RNA spiked with FBS. Samples were lysed and rapidly purified as described in the methods. HDA was performed for 1 hour in a 10 μL reaction, FEN+Taq ligase reaction was performed for 1 hour (total volume 20 μL), and Cas12-sgRNA reaction was performed for 30 minutes (total volume 27 μL). NTC: no target control.

[0037] Fig.18.(a) Schematic representation of the various FEN enzymes used in this study. Boxes represent the domains of the enzymes and lines represent the positions of the point mutations introduced in the enzymes. (b) Table showing the point mutations introduced in Tth pol v1, v2 and v3. (c) SDS-PAGE showing the purification of the different FEN enzymes. The target proteins are marked with arrows. (d) Schematic representation of the fluorescence assay used to quantify FEN activity. This configuration shows the 5'-flap of the flap primer and the 3'-flap of the invasion primer.

[0038] Fig.19 (a) Structure of T5 exonuclease (PDB ID: 1EXN) with helical arches labeled. (b) Predicted structure of Tth pol with disordered arches labeled. The structure was predicted using SWISS-MODEL5. (c) FEN activities of different enzymes with 1 nM DNA (left) and 1 nM RNA (right) as substrates. Data represent mean ± SD (n = 3 biological replicates).

[0039] Fig. 20 (a) and (b) Detection limits of orf1ab DNA and RNA, respectively, after incubation with 50 nM Tth pol v2 at 65°C for 30 min. Data represent mean ± SD (n = 3 biological replicates). p values ​​compared to NTC < 0.05 are provided, calculated using a two-sample t-test. NTC: no target control.

[0040] Fig.21 Cas12-sgRNA activity with ssDNA (1 nM) or dsDNA (1 nM) as substrates was shown, determined by co-incubation in 20 μL reactions at 37°C and measuring the fluorescent signal generated by reporter cleavage. Data represent mean values ​​(n=3 biological replicates).

[0041] Fig. 22 The fluorescence signal observed after incubation of Cas12-sgRNA complex (RNP) and its DNA reporter (rep) at 37°C for 30 minutes using dsDNA substrate (1 nM) is shown. 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 mean ± SD (n = 2 biological replicates).

[0042] Fig.23(a) and (b) Schematic diagrams of pre-ligated dsDNA adapters, where N = A, C, G, or T (shaded boxes) hybridizes to sgRNA and sgRNA-7 DNA, respectively. Both gRNAs are complementary to dsDNA adapters with N = T. The PAM sequence is underlined. (c) The activity of Cas12-sgRNA and Cas12-sgRNA-7 DNA complexes on four possible pre-ligated dsDNA adapters (5nM) was determined by co-incubating at 37°C in a 20 μL reaction and measuring the fluorescent signal generated by reporter cleavage. Data represent mean values ​​(n = 3 biological replicates). NTC: no target control.

[0043] Fig.24 (a) and (b) Detection limits 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 reactions, FEN+Taq reactions were performed for 1 h (total volume 20 μL), and Cas12-sgRNA reactions were performed for 30 min (total volume 27 μL). Data represent mean ± SD (n = 3 biological replicates). p-values ​​compared to NTC are provided < 0.05, calculated using a two-sample t-test. NTC: no target control.

[0044] Fig.25 Cq values ​​of LAMP performed at 65°C using different primer sets according to the manufacturer's instructions are shown. DNA: 60,000 copies of orf1ab DNA. NTC: no target control. For group 1, biological replicates of NTC did not show any amplification, so Cq values ​​were not determined. Horizontal black bars represent mean values ​​(n=3 biological replicates).

[0045] Fig.26 (a) Agarose gel showing 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 incubation at 65°C for 60 min. No amplification was observed for the NTC (no target control) sample. (b) Quantification of the band intensities corresponding to the amplification products shown in (a). Quantification was performed using ImageJ. Data represent mean ± SD (n = 3 biological replicates).

[0046] Fig. 27 Shown is the detection of EBV and gapdh in C666-1 and HK-1 cell lines (upper panel) and bla spiked into Klebsiella pneumoniae (Kp) in HK-1 cells KPCand khe (lower side) fluorescence signal fold change. Samples were lysed and rapidly purified as described in the methods. HDA was performed for 1 hour in a 10 μL reaction, FEN+Taq ligase reaction was performed for 1 hour (total volume 20 μL), and Cas12-sgRNA reaction was performed for 30 minutes (total volume 27 μL). NTC: no target control. DETAILED DESCRIPTION

[0047] The present specification teaches a method for detecting a polynucleotide analyte in a sample.

[0048] Methods may 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. Methods may include a) contacting a sample comprising a polynucleotide analyte with: i) a first nucleic acid probe comprising a 3' portion complementary to a first portion of the 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 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, wherein the first portion of the polynucleotide analyte is adjacent to the second portion of the polynucleotide analyte at 5'; 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; wherein formation of the cleavage structure causes the cleavage agent to cleave the first nucleic acid probe to release a 5' flap, wherein the 5' flap comprises a 5' portion of the first nucleic acid probe that is not complementary to and does not hybridize with the polynucleotide analyte. The method may further include b) ligating the 5' flap to a nucleic acid adaptor to form an adaptor ligation product, wherein the nucleic acid adaptor comprises a double-stranded region and a 3' overhang extension complementary to the 5' flap, wherein the 3' overhang extension hybridizes with the 5' flap to the nucleic acid adaptor. The method may further include c) contacting the adaptor 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 adaptor that is attached to and adjacent to the 5' flap; and iii) a single-stranded detector DNA; and d) measuring a detectable signal generated by cleavage of the single-stranded detection DNA by the type V CRISPR / Cas effector protein to detect the adaptor ligation product, thereby detecting a polynucleotide analyte in the sample.

[0049] Without being bound by theory, the present invention includes the use of structure-specific nucleic acid cleavage agents (e.g., flap endonucleases), nucleic acid ligases, and type V CRISPR-Cas nucleases to rapidly detect nucleic acids (DNA or RNA) with high sensitivity and specificity. First, the sample is incubated with a DNA oligonucleotide pair that binds to the target DNA / RNA to produce a flap structure that is recognized by the structure-specific nucleic acid cleavage agent and subsequently cleaved ( Figure 1 ). The cleaved flap product is ligated to a double-stranded DNA (dsDNA) adapter by a nucleic acid ligase to form a substrate for the CRISPR-Cas nuclease. In the presence of the substrate, the Cas nuclease bound to the guide RNA (gRNA) cleaves the single-stranded DNA (ssDNA) reporter to produce a detectable signal, which can be, for example, a visible band or a fluorescent signal on a lateral flow detection system. By using DNA oligonucleotides and specific adapters for each nucleic acid, different nucleic acids can be distinguished, even those that differ by a single nucleotide. The sensitivity of the method can be improved by adding an isothermal amplification system (e.g., loop-mediated isothermal amplification (LAMP) or helicase-dependent amplification (HDA)) prior to DNA oligonucleotide binding and using the amplified product as a substrate for a series of binding and enzymatic reactions disclosed herein.

[0050] 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), which contain purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. Nucleic acids can be double-stranded or single-stranded. References to single-stranded nucleic acids include references to sense strands or antisense strands. The backbone of a polynucleotide can contain sugar and phosphate groups (as commonly found in RNA or DNA), or modified or substituted sugar or phosphate groups. A polynucleotide can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. A nucleotide sequence can 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 their analogs.

[0051] As used herein, "oligonucleotide" refers to a single-stranded molecule that can be used in hybridization or amplification techniques. Generally speaking, the length of an oligonucleotide can be any integer number of nucleotides from about 15 to about 100, but can also be longer.

[0052] The term "complementary" refers to base pairing between nucleotides or nucleic acids, such as base pairing between the two strands of a double-stranded DNA molecule or base pairing between an oligonucleotide primer and a primer binding site on a single-stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are typically A and T (or A and U), or C and G. Two single-stranded RNA or DNA molecules are said to be complementary when the nucleotides of one strand are optimally aligned and compared, and appropriate nucleotide insertions or deletions are made, and the nucleotides of that strand are paired with at least about 80% of the nucleotides of the other strand, typically at least about 90% to 95%, and more preferably about 98% to 100%.

[0053] The term "non-complementary" or "non-complementary" can refer to the lack of base pairing between nucleotides or nucleic acids, for example, the lack of base pairing between the two strands of a double-stranded DNA molecule or the lack of base pairing 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 can be considered non-complementary when the nucleotides of one strand are less than 50%, optionally less than 40%, 30%, 20% or 10% paired with the nucleotides of the other strand.

[0054] As used herein, the term "hybridization" or "hybridize" refers to the process by which two single-stranded polynucleotides non-covalently bind to form a stable double-stranded polynucleotide, i.e., a double strand. The term "hybridization" may also refer to triple-stranded hybridization. The resulting (usually) double-stranded polynucleotide is a "hybrid." The ratio of the number of polynucleotides that form a stable hybrid is referred to herein as the "degree of hybridization."

[0055] Hybridization conditions typically include a salt concentration of less than about 1 M, more typically less than about 500 mM and less than about 200 mM. The hybridization temperature is typically higher than 22 ° C, more typically higher than about 30 ° C, and preferably greater than about 37 ° C. In one embodiment, hybridization occurs at about 65 ° C. Hybridization is typically performed under stringent conditions, i.e., the conditions under which the probe hybridizes to its target. Stringent conditions may depend on the sequence and are different in different situations. Longer fragments may require higher hybridization temperatures for specific hybridization. Since other factors may affect the stringency of hybridization, including the base composition and length of the complementary strands, the presence of organic solvents, and the degree of base mismatching, the combination of parameters is more important than the absolute measured value of any single parameter. Typically, stringent conditions are selected to be higher than the thermal melting point (T ) of the specific sequence at a defined ionic strength and pH. m ) is about 5℃ lower. m It 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.

[0056] The term "probe" refers to any molecule that can selectively bind to a specific target nucleic acid (eg, genomic DNA, polynucleotide transcripts, viral DNA or RNA). The probe can be synthesized by a person skilled in the art or derived from a suitable biological preparation.

[0057] As used herein, the term "cleavage structure" refers to a structure formed by the interaction of at least one nucleic acid probe with a target polynucleotide, the structure comprising a duplex with adjacent single-stranded nucleic acid flaps, which can be cleaved by a structure-specific nucleic acid cleavage agent. The secondary structure of the cleavage structure makes it a suitable substrate for specific cleavage by a structure-specific nucleic acid cleavage agent.

[0058] As used herein, the term "flap probe" or "flap oligonucleotide" refers to an oligonucleotide that can interact with a target polynucleotide to form a cleavage structure regardless of the presence of an invading oligonucleotide. When the flap probe hybridizes to the target polynucleotide, the flap probe and the target form a cleavage structure, and cleavage occurs within the flap probe.

[0059] As used herein, the term "invader probe" or "invader oligonucleotide" refers to an oligonucleotide that hybridizes to a target polynucleotide at a position near the hybridization region between the flap probe and the target nucleic acid, wherein the invader probe comprises a portion (e.g., a chemical moiety or nucleotide, whether or not complementary to the target) that overlaps the hybridization region between the flap probe and the target.

[0060] The first nucleic acid probe (or flap probe) disclosed herein may comprise a 3' portion that is complementary to the first portion of a polynucleotide analyte. The 3' portion may comprise 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 comprise a 5' portion that is not complementary to a polynucleotide analyte and does not hybridize with a 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.

[0061] The second nucleic acid probe (or invasion probe) disclosed herein may comprise a 5' portion complementary to the second portion of the polynucleotide analyte and a 3' portion that is not complementary to the polynucleotide analyte and does not hybridize with the polynucleotide analyte. The 5' portion may comprise or consist of about 20 to 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.

[0062] In one embodiment, when hybridized to a polynucleotide analyte, the first nucleic acid probe and the second nucleic acid probe overlap each other by at least one nucleobase. The first nucleic acid probe and the second nucleic acid probe 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.

[0063] In one embodiment, the cleavage structure is formed by hybridization of the first and second nucleic acid probes with the target polynucleotide. The cleavage structure may include a duplex formed between the first nucleic acid probe and the polynucleotide and a 5' flap, wherein the 5' flap includes a 5' portion of the first nucleic acid probe that is not hybridized with the polynucleotide. The cleavage structure may further include a 3' flap, wherein the 3' flap includes a 3' portion of the second nucleic acid probe that is not hybridized with the polynucleotide. The length of 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. The length of the 3' flap may be 1, 2, 3, 4 or 5 nucleotides.

[0064] As used herein, the term "structure-specific nucleic acid cleavage agent" or "cleavage agent" refers to any agent capable of cleaving a cleavage structure, including but not limited to enzymes. "Structure-specific nucleases" or "structure-specific enzymes" are enzymes that recognize specific secondary structures in nucleic acid molecules and cleave these structures. The structure-specific nucleic acid cleavage agents of the present disclosure cleave nucleic acid molecules in response to the formation of cleavage structures; the cleavage agent does not have to cleave the cleavage structure at any specific position within the cleavage structure. The cleavage agent can include nuclease activity from a variety of sources, including flap endonucleases (FENs) from prokaryotes and eukaryotes, DNA polymerases (e.g., Taq DNA polymerase, DNA polymerases from Thermus sp. and Escherichia coli DNA polymerase I) and exonucleases (e.g., bacteriophage T5 exonucleases). The cleavage agent can include an enzyme with 5' nuclease activity, such as Taq DNA polymerase, Escherichia coli DNA polymerase I, bacteriophage T5 exonucleases, Thermus sp. DNA polymerases. The nicking agent may also include a modified or engineered DNA polymerase that has 5' nuclease activity but lacks synthetic activity or exhibits reduced synthetic activity.

[0065] As used herein, the term "cleavage product" or "5' cleavage product" refers to the product generated by the action of a structure-specific nucleic acid cleavage agent on a cleavage structure, such as a 5' flap cleaved from a flap probe.

[0066] In one embodiment, formation of the cleavage structure allows the cleavage agent to cleave the first nucleic acid probe to release a 5' flap comprising a 5' portion of the first nucleic acid probe that is not complementary to and does not hybridize to the polynucleotide analyte.

[0067] The cleavage agent can cleave the first nucleic acid probe at the position of one 3' nucleobase of the portion where the first nucleic acid probe overlaps with the second nucleic acid probe.

[0068] In some embodiments, the structure-specific nucleic acid cleaving agent is an enzyme with flap endonuclease activity.

[0069] As used herein, the term "flap endonuclease (FEN)" refers to an enzyme having 5' exonuclease and structure-specific endonuclease activity, which recognizes a cleavage structure and performs cleavage to produce a 5' cleavage product. The enzyme having FEN activity herein can be a wild-type or engineered enzyme from a prokaryotic or eukaryotic organism, such as 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 enzymes may also contain one or more additional enzyme activities, such as a nucleic acid polymerase activity, in addition to the FEN activity. The enzyme may be engineered to reduce or remove non-FEN activity.

[0070] In some embodiments, the enzyme having FEN activity is a thermostable enzyme.

[0071] When the term "thermostable" is used to refer to an enzyme (e.g., an enzyme with FEN activity or a nucleic acid ligase), it means that the enzyme is functional or active (i.e., can catalyze) at high temperatures (e.g., 45°C or higher). In some embodiments, the thermostable enzymes herein are functional at temperatures of about 45°C to about 80°C, about 50°C to about 75°C, about 55°C to about 70°C, or about 60°C to about 70°C. In some embodiments, the thermostable enzymes are functional 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 are functional at temperatures of about 45°C to about 80°C.

[0072] In some embodiments, the enzyme is a DNA polymerase from Thermus thermophiles (ie, Tth polymerase).

[0073] The enzyme can be a wild-type, mutant isomer or genetically engineered Tth polymerase. The wild-type Tth polymerase has both FEN activity and polymerase activity. The Tth polymerase used in the method herein can have reduced or negligible polymerase activity.

[0074] The engineered Tth polymerase may comprise one or more amino acid substitutions, insertions or deletions. The term "deletion" when used in relation to an amino acid refers to an amino acid that has been removed or does not exist. The term "insertion" refers to the addition of one or more amino acids. "Replacement" refers to the replacement of an amino acid residue by another amino acid residue. An amino acid residue may be replaced by another amino acid residue, and the other amino acid residue may be selected from naturally occurring standard 20 amino acid residues, rare naturally occurring amino acid residues (e.g., hydroxyproline, hydroxylysine, isohydroxylysine, 6-N-methyllysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, isoleucine isomers (allo-isoleucine), N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline) and non-naturally occurring amino acid residues (usually synthetic, such as cyclohexylalanine). Preferably, the term "substitution" refers to replacing an amino acid residue with another amino acid residue selected from the standard 20 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).

[0075] "Conservative amino acid substitution" refers to the replacement of an amino acid residue by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and can generally be subdivided into the following categories:

[0076] a) Amino Acid Subclass

[0077]

[0078] Conservative amino acid substitutions also include grouping 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-hydroxy 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; the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. For example, it is reasonable to expect that replacing leucine with isoleucine or valine, replacing aspartic acid with glutamic acid, replacing threonine with serine, or similarly replacing 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 measuring its activity. Conservative substitutions are shown in the table below with exemplary substitutions as headings. Amino acid substitutions falling within the scope of the invention are generally achieved by selecting substitutions that do not differ significantly in maintaining (a) the peptide backbone structure in the region of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. After the substitutions are introduced, the variants are screened for biological activity.

[0079] b) Exemplary amino acid substitutions

[0080]

[0081]

[0082] In some embodiments, the enzyme having FEN activity is a Thermus thermophilus DNA polymerase comprising one or more amino acid substitutions. In some embodiments, the one or more amino acid substitutions are selected from: A599T, A604V, W606R, A607Q, A610V, I616M, I616T and E617G. In some embodiments, the Thermus thermophilus DNA polymerase comprises the following amino acid substitutions: A604V, A610V, I616M and E617G. In some embodiments, the Thermus thermophilus DNA polymerase comprises the following amino acid substitutions: A599T, W606R, A607Q and I616T.

[0083] Previously described a kind of engineered Tth polymerase that can recognize DNA and RNA substrate in WO2001090337A2.The inventor further engineered this Tth polymerase variant by introducing mutation (that is, A604V, A610V, I616M and E617G) at position 604,610,616 and 617, to produce the enzyme with the amino acid sequence given in SEQ ID NO:1.The inventor also introduced mutation (that is, A599T, W606R, A607Q and I616T) at position 599,606,607 and 616, to produce the enzyme with the amino acid sequence given in SEQ ID NO:2.These enzymes show higher FEN activity to given substrate (that is, given cleavage structure), and show lower non-specific activity to the oligonucleotide that does not form cleavage structure.

[0084] In some embodiments, the enzyme comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence shown 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 shown 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 shown in SEQ ID NO: 2.

[0085] In some embodiments, step (a) of the method herein is carried out in an isothermal manner, i.e., the temperature remains unchanged during the duration of step (a). Step (a) may be carried out at a temperature of 40°C or higher. In some embodiments, step (a) is carried out at a temperature of about 40°C to about 90°C, about 50°C to about 80°C, about 55°C to about 75°C, or about 60°C to about 70°C. In one embodiment, step (a) is carried out isothermally at a temperature of about 65°C.

[0086] The term "adapter" refers to a nucleic acid molecule that is configured to be complementary to both the cleavage product (e.g., 5' flap) of a structure-specific cleavage agent and the guide RNA of a type V CRISPR / Cas effector protein (e.g., Cas12). An "adapter" may comprise a 3' overhang that is complementary to the 5' flap. An "adapter" is advantageous in the present method because it connects steps (a) and (c) by allowing the Cas nuclease in step (c) to recognize the cleavage product of step (a).

[0087] In some embodiments, the nucleic acid adapter comprises a first adapter oligonucleotide and a second adapter oligonucleotide, wherein the first adapter oligonucleotide comprises a protospacer-adjacent motif (PAM) of a type V CRISPR / Cas effector protein, and the second adapter oligonucleotide is complementary to and hybridizes with 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 adaptor 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.

[0088] In some embodiments, the first adapter oligonucleotide comprises a PAM sequence of a Cas12 nuclease. In some embodiments, the PAM sequence is 5'-TTTN-3', wherein N can be any one of A, T, C or G.

[0089] In some embodiments, the second adaptor oligonucleotide comprises a 5' modification, such as a modification to a nucleobase or a sugar-phosphate backbone. The 5' modification can allow subsequent enzymes in the methods herein to function. In one embodiment, the second adaptor oligonucleotide is phosphorylated at its 5' end to prepare it for subsequent ligation by a nucleic acid ligase.

[0090] In some embodiments, step (c) is performed in the presence of a nucleic acid ligase. The 5' cleavage product of the cleavage agent can be ligated to the adaptor by a ligase after hybridization with the 3' overhang of the nucleic acid adaptor. As used herein, the term "adaptor ligation product" refers to a nucleic acid (e.g., a 5' flap) that has been ligated to the adaptor.

[0091] Non-limiting examples of ligated enzymes that can be used 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 Escherichia 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-Escherichia coli DNA ligase, and Sso7-Ampligase DNA ligase. The ligase herein may be a wild type, a mutant isomer or a genetically engineered variant.

[0092] In some embodiments, the ligase is a thermostable ligase, such as Taq ligase.

[0093] In some embodiments, step (b) of the method herein is carried out in an isothermal manner, i.e., the temperature remains unchanged during the duration of step (b). Step (b) may be carried out at a temperature of 40°C or higher. In some embodiments, step (b) is carried out at a temperature of about 40°C to about 90°C, about 50°C to about 80°C, about 55°C to about 75°C, or about 60°C to about 70°C. In one embodiment, step (b) is carried out isothermally at a temperature of about 65°C.

[0094] The method can include CRISPR / Cas effector proteins or enzymes and guide RNA. CRISPR / Cas systems (including CRISPR-Cas12 and CRISPR-Cas13) show strong collateral activity against single-stranded DNA (ssDNA) and ssRNA targets, respectively. After the Cas effector protein (complexed with the guide RNA) recognizes and cleaves the specific target, the collateral cleavage of non-specific targets provides the basis for a highly specific and sensitive nucleic acid detection method.

[0095] In some embodiments, the CRISPR / Cas effector is a DNA editing enzyme (e.g., DNA endonuclease) with dsDNA cleavage activity and ssDNA cleavage activity. In such embodiments, the CRISPR / Cas effector can be a Class II V-type 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.

[0096] In some embodiments, the type V CRISPR / Cas effector protein is a Cas12 protein. In one embodiment, the CRISPR / Cas effector protein is Cas12a.

[0097] Cas effector protein variants may comprise one or more mutations (e.g., conservative or non-conservative mutations). For example, it is also contemplated that other Cas12 variants may be evolved from the variants disclosed herein, such as by targeted mutations of one or more amino acid residues in a specific region of the enzyme. Such mutations may change substrate binding, change the conformation of the bound substrate, change the accessibility of the substrate to the active site, change the tolerance of the target sequence to the non-optimal presentation of the active site, and / or change the target sequence specificity (recognition).

[0098] A nucleic acid molecule (e.g., natural crRNA) that binds to a V-type CRISPR / Cas effector protein (e.g., Cas12 protein, e.g., Cas12a, Cas12b, Cas12c, Cas12d, Cas12e) to form a ribonucleoprotein complex (RNP), and targets the complex to a specific target sequence within a target DNA is referred to herein as a "guide RNA". It should be understood that in some cases, hybrid DNA / RNA can be prepared so that the guide RNA contains DNA bases in addition to RNA bases, but the term "guide RNA" is still used herein to cover such hybrid molecules.

[0099] The guide RNA herein comprises a guide sequence (also referred to as a "spacer") that hybridizes to a portion of the 5' flap and a portion of the nucleic acid adaptor. This ensures that the Cas nuclease is active only after a 5' cleavage product is generated and hybridized to the nucleic acid adaptor. The guide RNA also comprises a constant region (e.g., a region adjacent to the guide sequence and bound to a V-type CRISPR / Cas effector protein).

[0100] The guide sequence may 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 or more nucleobases.

[0101] In some embodiments, step (c) of the method herein is carried out in an isothermal manner, that is, the temperature remains unchanged during the duration of step (c). Step (c) can be carried out at a temperature of 20°C or higher. In some embodiments, step (c) is carried out at a temperature of about 20°C to about 70°C, about 25°C to about 60°C, about 30°C to about 50°C, or about 35°C to about 40°C. In one embodiment, step (c) is carried out at a temperature of about 20°C to about 65°C. In one embodiment, step (c) is carried out isothermally at a temperature of about 37°C.

[0102] The method may include cleavage of single-stranded detection DNA (ssDNA) by a type V CRISPR / Cas effector protein to produce a detectable signal.

[0103] 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 rich in AT. In some embodiments, the ssDNA is labeled. The ssDNA can be labeled at the 5' end, at the 3' end, or at 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.

[0104] In some embodiments, the labeled ssDNA comprises a signal-quencher pair. The signal partner of the signal-quencher pair generates a detectable signal, and when the signal-quencher partners are close to each other (e.g., when the partners are present on the same ssDNA molecule before being cut by a type V CRISPR / Cas effector protein), the quencher partner quenches (i.e., reduces) the detectable signal of the signal partner. When the labeled ssDNA is cut and the signal partner is no longer close to the quencher partner, a detectable signal will be generated.

[0105] The quencher moiety can quench the signal from the signal moiety to varying degrees. The quencher moiety can quench the signal from the signal moiety, wherein 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 when the quencher moiety is not present (when the signal partners are separated). For example, the signal detected in the presence of the quencher moiety can 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 when the quencher moiety is not present. No signal may be detected in the presence of the quencher moiety (e.g., above background noise).

[0106] The signal detected in the absence of the quencher moiety (when the signal partners are separated) can be at least 1.2 times greater (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).

[0107] In one embodiment, the single-stranded detection DNA comprises a fluorophore-quencher pair. The quencher portion reduces the fluorescent signal from the fluorophore, for example, by absorbing energy in the fluorophore emission spectrum. Any convenient fluorophore-quencher pair may be used, and many suitable pairs are known in the art.

[0108] The quencher portion can absorb energy from the fluorophore and then emit a signal (e.g., light of a different wavelength). Thus, the quencher portion itself can be a second fluorophore (e.g., the first fluorophore can be 6-carboxyfluorescein and the quencher or second fluorophore can be 6-carboxytetramethylrhodamine). The fluorophore-quencher pair can also be a FRET pair. The quencher portion can be a dark quencher. The dark quencher can absorb the excitation energy and dissipate the energy in a different way (e.g., as heat). Therefore, the dark quencher itself has minimal or even no fluorescence (does not emit fluorescence).

[0109] Examples of fluorophores include, but are not limited to: Alexa Dyes, ATTO dyes (such as ATTO 390, ATTO425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTORho101, 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 protein.

[0110] Examples of quencher moieties include, but are not limited to: dark quenchers, Black Hole (e.g., BHQ-0, BHQ-1, BHQ-2, BHQ-3), Qxl quencher, ATTO quencher (e.g., ATTO 540Q, ATTO 580Q and ATTO 612Q), dimethylaminoazobenzenesulfonic acid (Dabsyl), Iowa Black RQ, Iowa Black FQ, IRDye QC-1, QSY dyes (e.g., QSY 7, QSY 9, QSY 21), AbsoluteQuencher, Eclipse and metal clusters (e.g., gold nanoparticles), etc.

[0111] In one embodiment, the single-stranded detection DNA comprises a FRET pair. FRET is a process by which energy is transferred radiationlessly from an excited state fluorophore to a second chromophore in close proximity. The range of energy transfer is limited to about 10 nanometers (100 angstroms), and the efficiency of the transfer is extremely sensitive to the separation distance between the fluorophores. Therefore, as used herein, the term "FRET" ("fluorescence resonance energy transfer"; also known as " "FRET" refers to a physical phenomenon involving a donor fluorophore and a matched acceptor fluorophore, which are selected so that the emission spectrum of the donor overlaps the excitation spectrum of the acceptor, and are further selected so that when the donor and acceptor are in close proximity to each other (usually 10 nm or less), excitation of the donor will result in excitation and emission of the acceptor because part of the energy is transferred from the donor to the acceptor via quantum coupling effects. Therefore, the FRET signal acts as a criterion for the proximity between the donor and the acceptor; a signal is generated only when they are in close proximity to each other. The FRET donor moiety (e.g., donor fluorophore) and the FRET acceptor moiety (e.g., acceptor fluorophore) are collectively referred to herein as a "FRET pair."

[0112] FRET donor and acceptor moieties (FRET pairs) are known to those skilled in the art, and any convenient FRET pair (eg, any convenient pair of donor and acceptor moieties) may be used. Examples of FRET pairs are given in the table below.

[0113]

[0114]

[0115] The labeled detection ssDNA can generate a first detectable signal (e.g., from a FRET pair) before being cleaved, and generate a second detectable signal (e.g., from a quencher / fluorophore pair) when the labeled detection ssDNA is cleaved. Thus, the labeled detection ssDNA can include a FRET pair and a quencher / fluorophore pair.

[0116] In some cases, the cutting of the labeled detection ssDNA can be detected by measuring the colorimetric reading. For example, the release of the fluorophore (e.g., release from a FRET pair, release from a quencher / fluorophore pair, etc.) can cause a wavelength shift (thereby causing a color shift) of the detectable signal. Therefore, in some cases, the cutting of the labeled detection ssDNA herein can be detected by a color shift. This shift can be expressed as a loss of the amount of a color (wavelength) signal, an increase in the amount of another color, a change in the ratio of one color to another color, etc.

[0117] In some embodiments, cleavage of the single-stranded detection DNA is performed by visual detection. Visual detection can be performed by direct observation (e.g., by eye or using a camera or microscope) or by spectral or spectrophotometric measurement.

[0118] In one embodiment, the cleavage of the single-stranded detection DNA is detected by measuring the change in the fluorescent signal generated by the detection DNA. When the detection DNA is used in the method herein (eg, in step (c) of the method herein), the change in the fluorescent signal can be an increase or decrease in fluorescence.

[0119] In one embodiment, cleavage of the single-stranded detection DNA is performed by visual detection on a lateral flow system.

[0120] Lateral flow systems are usually based on mobile test strips. Mobile test strips can include one or more pads for accommodating sample fluids, and can also include one or more detection reagents for detecting target parts in sample fluids. The pad can be based on a series of capillary beds, such as porous paper, microstructured polymers or sintered polymers. Each of these pads has the ability to spontaneously transport sample fluids, such as via capillary action. The sample to be analyzed can be added to the proximal end of the test strip. The liquid phase elements of the sample (which can be dissolved, suspended, emulsified or any other liquefied form) can migrate to the region of the detection reagent fixed in the pad, and the detection reagent is usually composed of proteins that are passively or covalently connected to signal molecules or particles (usually colloidal gold or colored, fluorescent or paramagnetic monodisperse latex particles). Signal reagents can also be another reagent, including non-particulate matter (e.g., soluble, directly labeled fluorophore gel). The label can be combined with one of the specific biological components (antigens or antibodies) of the assay, depending on the assay format of the specific mobile test strip. The liquid sample re-moves the dried conjugate material so that it is incorporated into the liquid sample material, and the analyte in the sample interacts with the conjugate. The conjugate material can be a protein, such as an antibody or an antigen, which is deposited in a specific area of ​​the pad in the form of a strip or stripe, and when the components, analyte and conjugate in the liquid sample pass through, pass through or cross the capture line, the conjugate material can capture these components. Excessive liquid materials (samples and reagents) continue to migrate along the test paper, cross the capture line, and can be trapped in the pad near the other end of the mobile test paper. The test result can be manifested on the reaction matrix, and can be expressed as the presence or absence of the test mark (usually a continuous line) of the captured conjugate, which can be read by the naked eye or using a reader. Some conjugate particles may not be captured on the capture line, but will continue to flow to the second line of the fixed detection reagent, i.e. the control line. The control line usually contains another detection reagent that is specific to the binding antibody on the conjugate. The combination of the conjugate particles and the detection reagent will produce a control signal (usually a continuous line).

[0121] In one embodiment, the absence of the test line on the lateral flow system indicates the presence of the polynucleotide analyte in the sample.

[0122] In some embodiments, steps (a) and (b) of the methods herein are performed in separate reaction vessels, and the products of these reaction vessels are combined prior to performing step (c).

[0123] In some embodiments, steps (a) and (b) of the method herein are carried out in a single reaction vessel. Steps (a) and (b) can be carried out simultaneously in a single reaction vessel. Alternatively, the reagents required for step (b) (i.e., nucleic acid ligase and nucleic acid adapter) can be added to the reaction vessel after step (a) has been carried out for a known duration. In some embodiments, step (c) is also carried out in the same reaction vessel as steps (a) and (b). Steps (a), (b) and (c) can be carried out simultaneously in a single reaction vessel. Alternatively, Cas nuclease, guide RNA and single-stranded detection DNA can be added to the reaction vessel after step (b) has been carried out for a known duration.

[0124] In some embodiments, the methods herein further comprise amplifying the polynucleotide analyte prior to step (a).

[0125] Those skilled in the art will be aware of a variety of amplification methods and components, and any convenient method may be used (see, e.g., Zanoli and Spoto, Biosensors (Basel). 2013 Mar;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, multiplex PCR, asymmetric PCR, touchdown PCR, random primer PCR, semi-nested PCR, polymerase cycle assembly (PCA), colony PCR, ligase chain reaction (LCR), digital PCR, methylation-specific PCR (MSP), co-amplification PCR at low denaturing temperature (COLD-PCR), allele-specific PCR, inter-sequence-specific PCR (ISS-PCR), whole genome amplification (WGA), inverse PCR and thermal asymmetric interleaved PCR (TAIL-PCR).

[0126] In some embodiments, amplification is isothermal amplification.Term " isothermal amplification " represents a kind of nucleic acid (for example, DNA) amplification method (for example, using enzyme chain reaction), and the method uses single temperature incubation, thereby eliminates the demand for thermal cycler.Isothermal amplification is a kind of nucleic acid amplification form, and it does not depend on the thermal denaturation of target nucleic acid during amplification reaction, and therefore can not need multiple rapid temperature changes.Therefore, isothermal nucleic acid amplification method can be carried out in or outside laboratory environment.By being combined with reverse transcription step, these amplification methods can be used for isothermal amplification RNA.

[0127] 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), nickase amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), branch amplification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal-mediated RNA amplification technology (SMART), self-directed sequence replication (3SR), genomic exponential amplification reaction (GEAR), and isothermal multiple displacement amplification (IMDA).

[0128] In some cases, the amplification is recombinase polymerase amplification (RPA). Recombinase polymerase amplification (RPA) uses two opposing primers (very similar to PCR) and uses three enzymes, namely, a recombinase, a single-stranded DNA binding protein (SSB), and a strand displacement polymerase. The recombinase pairs the oligonucleotide primer with a homologous sequence in the duplex DNA, the SSB binds to the displaced DNA strand to prevent the primer from being displaced, and the strand displacement polymerase begins DNA synthesis at the point where the primer binds to the target DNA. The addition of a reverse transcriptase to the RPA reaction can facilitate the detection of RNA and DNA without the need for a separate step to generate cDNA.

[0129] In transcription-mediated amplification (TMA), RNA is prepared from an engineered promoter in the primer region using RNA polymerase, and then reverse transcriptase synthesizes cDNA from the primer. A third enzyme, such as RNase H, can then be used to degrade the RNA target from the cDNA without the need for a heat denaturation step. This amplification technique is similar to self-directed sequence replication (3SR) and nucleic acid sequence-based amplification (NASBA), but the enzymes used differ.

[0130] In one embodiment, loop-mediated amplification (LAMP) isothermally amplified polynucleotides are used. LAMP uses a thermostable polymerase with chain displacement ability and a set of four or more specifically designed primers. Each primer is designed to have a hairpin end, and once displaced (displace), it will snap into a hairpin shape to promote the further extension of self-priming (self-priming) and polymerase. In the LAMP reaction, although the reaction is carried out under isothermal conditions, the double-stranded target requires an initial thermal denaturation step. In addition, the ladder-like pattern of various length products is amplified. For another example, chain displacement amplification (SDA) combines the ability of restriction endonucleases to cut on the unmodified strand of their target DNA and the ability of DNA polymerases lacking exonucleases to extend 3' ends at the cut and displace downstream DNA chains.

[0131] In one embodiment, the polynucleotides are isothermally amplified using helicase-dependent amplification (HDA). HDA utilizes a thermostable helicase (Tte-UvrD) rather than heat to unwind dsDNA to generate single strands, which are then available for hybridization and primer extension by a polymerase.

[0132] Disclosed herein is a method for detecting a polynucleotide analyte in a sample, the method comprising:

[0133] a) i) contacting a sample comprising a polynucleotide analyte with a first nucleic acid probe and a second nucleic acid probe, the first nucleic acid probe and the second nucleic acid probe being configured to form a cleavage structure in the presence of the polynucleotide analyte; and

[0134] ii) structure-specific nucleic acid cleavage agents;

[0135] wherein the formation of the cleavage structure allows the cleavage agent to cleave the first nucleic acid probe to release the 5' flap from the first nucleic acid probe;

[0136] b) ligating the 5' flap to a nucleic acid adaptor to form an adaptor ligation product, wherein the nucleic acid adaptor comprises a double-stranded region and a 3' overhang extension complementary to the 5' flap, and wherein the 3' overhang extension hybridizes with the 5' flap to the nucleic acid adaptor;

[0137] c) contacting the adaptor ligation products:

[0138] i) Type V CRISPR / Cas effector proteins;

[0139] 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 the 5' flap and a portion of a nucleic acid adaptor that is attached to and adjacent to the 5' flap; and

[0140] iii) single-stranded detection DNA; and d) measuring a detectable signal generated by cleavage of the single-stranded detection DNA by a type V CRISPR / Cas effector protein to detect the adaptor ligation product, thereby detecting a polynucleotide analyte in the sample.

[0141] The DNA / RNA detection method has a variety of diagnostic applications because it can be used to identify any gene sequence without the need for a sequencer. The method is able to detect and type bacterial and viral pathogens in a variety of human, food, and environmental samples. The detection method is also able to distinguish between different variants of these pathogens, such as antibiotic-resistant or vaccine-resistant variants. In addition, the method can detect gene mutations in the human genome associated with disease development and cell-free DNA / RNA in different samples.

[0142] The method of the present invention can be used to detect whether one or more polynucleotide analytes are present in one or more samples known to contain or suspected to contain polynucleotide analytes. The method can also be used to quantify the amount of polynucleotide analytes in the sample. The method can be used to detect polynucleotide targets in the sample, such as RNA, mRNA, rRNA, plasmid DNA, viral DNA, bacterial DNA and chromosomal DNA.

[0143] The term "polynucleotide analyte" can be any polynucleotide that can be detected or analyzed by the method defined herein. The analyte can be naturally occurring or synthetic. The polynucleotide analyte can be present in a sample obtained using any method known in the art. In some cases, the sample can be processed before the polynucleotide analyte of the analytical sample. The polynucleotide can include DNA, RNA, peptide nucleic acid and any hybrid thereof, wherein the polynucleotide includes any combination of deoxyribonucleotides and / or ribonucleotides. The polynucleotide can be single-stranded or double-stranded, or include a portion of a double-stranded or single-stranded sequence. The polynucleotide can 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, as well as nucleosides and nucleotide analogs and modified nucleotides, including 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 (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 the cell. As another example, the polynucleotide analyte can be a nucleic acid introduced into the cell or expressed in the cell by a pathogen-infected cell, such as a viral or bacterial genomic RNA or DNA, a plasmid, a viral or bacterial mRNA, etc.

[0144] In some embodiments, the polynucleotide analyte is DNA or RNA.In some embodiments, the polynucleotide analyte is single-stranded or double-stranded.

[0145] In one embodiment, the polynucleotide analyte comprises a single nucleotide polymorphism (SNP).

[0146] 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 a ~30kb positive single-stranded RNA with a 5'-cap structure and a 3'poly-A tail, comprising multiple characteristic genes of coronaviruses, such as S (spike), E (envelope), M (membrane) and N (nucleocapsid) genes. Other elements of the genome (such as ORF1a and ORF1b) encode non-structural proteins, including RNA-dependent RNA polymerase (RdRp).

[0147] As used herein, the term "sample" includes tissues, cells, body fluids, and isolates thereof, etc., isolated from a subject, as well as tissues, cells, and body fluids, etc., present in the subject (i.e., the sample is in vivo). Examples of samples include: whole blood, blood (e.g., serum and plasma), lymph and cystic fluid, sputum, feces (or excrement), tears, mucus, hair, skin, ascites, cystic fluid, urine, nipple exudate, nipple aspirate, tissue sections (e.g., biopsy and autopsy samples), frozen sections for histological purposes, archival samples, explants, and primary and / or transformed cell cultures derived from patient tissues, etc.

[0148] The terms "detect," "determine," "measure," "evaluate," "assess," and "assay" are used interchangeably herein to refer to any form of measurement, including determining whether an element is present or not. These terms include quantitative and / or qualitative determinations. Determinations 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.

[0149] The methods can be used for any purpose requiring detection of viral, bacterial or other nucleic acids, including diagnostic and prognostic applications, such as in laboratory and clinical settings. In some embodiments, the methods can be used to detect nucleic acids for genotyping.

[0150] In some embodiments, the nucleic acid to be detected is used to diagnose a disease state. The disease state can be infection, organ disease, blood disease, immune system disease, cancer, brain and nervous system disease, endocrine disease, pregnancy or childbirth related disease, hereditary disease or environmental acquired disease, cancer or fungal infection, bacterial infection, parasitic infection or viral infection. Therefore, in some embodiments, the method can be used to detect nucleic acids (e.g., DNA or RNA) from bacteria, fungi, viruses (e.g., caused by double-stranded RNA viruses, positive RNA viruses, negative sense RNA viruses, retroviruses, etc.) or parasites.

[0151] Exemplary viruses that can be detected include, but are not limited to, Myoviridae, Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae (including human herpes virus and varicella zoster virus), Zoztervirus), Malocoherpesviridae, Lipothrixviridae, Rudiviridae, Adenoviridae, Ampullaviridae, Ascoviridae, Asfarviridae (including African swine fever virus), Baculoviridae, Cicaudaviridae, Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae, Iridoviridae, Maseilleviridae, Mimiviridae, Nudiv iridae, Nimaviridae, Pandoraviridae, Papillomaviridae, Phycodnaviridae, Plasmaviridae, Polydnaviruses, Polyomaviridae (including simian virus 40, JC virus, BK virus), Poxviridae (including vaccinia and smallpox) , Sphaerolipoviridae, Tectiviridae, Turriviridae, Dinodnavirus, Salterpro virus, Rhizidovirus, Coronaviridae virus, Picornaviridae virus, Caliciviridae virus, Flaviviridae virus,Togaviridae, Bornaviridae, Filoviridae, Paramyxoviridae, Pneumoviridae, Rhabdoviridae, Arenaviridae, Bunyaviridae, Orthomyxoviridae, or Delta virus. In some embodiments, the virus is a coronavirus (e.g., SARS-Cov-2), SARS, polio virus, 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, Hanta virus, Crimean-Congo hemorrhagic fever virus, influenza, or hepatitis D virus.

[0152] In some embodiments, the nucleic acid to be detected can be associated with a pathogen, including pathogenic bacteria, such as Enterococcus faecalis, Enterococcus 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 pneumoniae), Clostridium botulinum, Corynebacterium amycolatum, Klebsiella pneumoniae, Vibrio vulnificus, and Parachlamydia.

[0153] Also provided herein are methods of treating a disease after detecting the disease.

[0154] "Subject" or "patient" refers to any single subject in need of treatment, including humans, cattle, horses, pigs, goats, sheep, dogs, cats, guinea pigs, rabbits, chickens, insects, etc. Subjects are also intended to include any subject participating in a clinical research trial but not showing any clinical symptoms of disease, subjects participating in epidemiological studies, or subjects serving as controls.

[0155] Disclosed herein is a kit for performing the methods disclosed herein. The kit may include i) a first nucleic acid probe comprising a 3' portion complementary to a first portion of a polynucleotide analyte and a 5' portion that is not complementary to the polynucleotide analyte and does not hybridize with the polynucleotide analyte; ii) a second nucleic acid probe comprising a 5' portion complementary to a second portion of the polynucleotide analyte and a 3' portion that is not complementary to the polynucleotide analyte and does not hybridize with the polynucleotide analyte, wherein the first portion of the polynucleotide analyte is adjacent to the second portion of the polynucleotide analyte at 5'; 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 shown in SEQ ID NO: 1. The kit may further include a nucleic acid adapter for forming an adapter ligation product, wherein the nucleic acid adapter comprises a double-stranded region and a 3' protruding extension complementary to the 5' flap, wherein the 3' protruding extension is capable of hybridizing to the nucleic acid adapter with the 5' flap. The kit may further comprise i) a V-type CRISPR / Cas effector protein; ii) a guide RNA comprising a region that binds to a V-type CRISPR / Cas effector protein and a guide sequence that is complementary to a portion of the 5' flap and a portion of a nucleic acid adapter that is connected to and adjacent to the 5' flap; and iii) a single-stranded detection DNA. The detectable signal generated by the V-type CRISPR / Cas effector protein cleaving the single-stranded detection DNA can be measured by a detector. The kit may further comprise one or more buffer components, metabolites, and / or other reaction components required to perform the methods defined herein. The kit may further comprise a lateral flow device or system for detecting a signal generated by cleaving the single-stranded detection DNA. The kit may further comprise instructions for performing the methods defined herein.

[0156] The present invention discloses a kit for detecting a polynucleotide analyte in a sample, comprising a structure-specific nucleic acid cleavage agent, a nucleic acid ligase, and a V-type 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 shown 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 a polynucleotide analyte. The kit may further comprise a first adapter polynucleotide and a second adapter polynucleotide, wherein the first and second adapter polynucleotides are configured to form a nucleic acid adapter upon hybridization. The kit may further comprise a guide RNA configured to bind to a V-type CRISPR / Cas effector protein and a connection product of a nucleic acid adapter and a cleavage product of a structure-specific nucleic acid cleavage agent. The kit may further comprise a single-stranded detection DNA. The kit may further comprise one or more buffer components, metabolites, and / or other reaction components required for carrying out the method defined herein. The kit may further comprise a lateral flow device or system for detecting a signal generated by cleavage of a single-stranded detection DNA. The kit may further comprise instructions for carrying out the method defined herein.

[0157] In some embodiments, the method or kit herein show attomole (aM) detection sensitivity. In some cases, the subject composition or method show femtomolar (fM) detection sensitivity. In some cases, the subject composition or method show picomolar (pM) detection sensitivity. In some cases, the subject composition or method show nanomolar (nM) detection sensitivity.

[0158] In some embodiments, using the method defined herein, the detection threshold of target polynucleotide is 1nM or lower.Term " detection threshold " is used in this article to describe the minimum amount of target polynucleotide that must be present in order to detect in sample.Therefore, as illustrative example, when the detection threshold is 1nM, then when there is concentration in sample and it is 1nM or higher target DNA, signal can be detected.In some embodiments, the detection threshold of the method disclosed herein is 500pM or lower, 100pM or lower, 50pM or lower, 10pM or lower, 5pM or lower, 1pM or lower, 500fM or lower, 100fM or lower, 50fM or lower, 10fM or lower, 5fM or lower, 1fM or lower, 500aM or lower, 100aM or lower, 50aM or lower, 10aM or lower or 1aM or lower.

[0159] In some embodiments, the detection threshold is about 100 pM to about 1 nM, about 10 pM to about 100 pM, about 1 pM to about 10 pM, about 100 fM to about 1 pM, about 10 fM to about 100 fM, about 1 fM to about 10 fM, about 100 aM to about 1 fM, about 10 aM to about 100 aM, or about 1 aM to about 10 aM.

[0160] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted as an alternative (or).

[0161] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an agent" includes a plurality of agents, including mixtures thereof.

[0162] Throughout the specification and the appended claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or steps or groups of integers or steps but not the exclusion of any other integer or steps or groups of integers or steps.

[0163] Reference in this specification to any previous publication (or information derived therefrom) or any known matter is not and should not be taken as an admission or recognition or any form of implication that the previous publication (or information derived therefrom) or known matter constitutes part of the common general knowledge in the field to which this specification relates.

[0164] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It should be understood that the present invention includes all such variations and modifications as long as they fall within the spirit and scope of the present invention. The present invention also includes all steps, features, compositions and compounds mentioned or indicated in this specification, either individually or collectively, and any and all combinations of any two or more of the steps or features.

[0165] Unless defined otherwise, 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.

[0166] Certain embodiments of the invention will now be described with reference to the following examples, which are provided for illustrative purposes only and are not intended to limit the general scope of the foregoing.

[0167] Example

[0168] method

[0169] Bacterial strains, plasmids, and oligonucleotides used in the study

[0170] E. coli TOP10 was used as cloning host and E. coli BL21 (DE3) was used for protein expression. Unless otherwise stated, both bacterial strains were grown in Luria-Bertani (LB) broth at 37°C with shaking at 220 rpm.

[0171] pET28b was used as a cloning vector to express proteins in E. coli BL21 (DE3). Cas12 was from pMBP-LbCas12a, a gift from Jennifer Doudna (Addgene plasmid #113431). Tth pol (GenBank Accession No. WP_011228405), hFEN1 (GenBank Accession No. NP_004102), MjaFEN (GenBank Accession No. WP_010870964), and T5 exo (GenBank Accession No. YP_006958) sequences were codon optimized for expression in E. coli and synthesized as gblocks from Integrated DNA Technologies (IDT).

[0172] Tables 1 to 4 provide the sequences of oligonucleotides and primers for LAMP, HDA and FEN (flap and invasion primers).

[0173] Protein purification

[0174] All proteins were purified by using a His tag attached to its C-terminus. For Cas12 purification, Escherichia coli BL21 (DE3) (pET28b-Cas12) was grown overnight in Luria-Bertani (LB) broth supplemented with kanamycin (50 μg / mL). After overnight growth, the culture was diluted 10 times in Terrific broth supplemented with kanamycin and grown at 37 ° C until OD600 reached ~ 0.3-0.4. Then, 100mM IPTG was added to the culture and further grown at 23 ° C for 24 hours. The cells were collected by centrifugation and resuspended in lysis buffer (50mM Tris-Cl (pH 8), 300mM NaCl, EDTA-free protease inhibitor tablets). The cells were lysed by passing the homogenizer 5 to 10 times at 23,000 bar using a homogenizer. The lysate was centrifuged, and the supernatant was subjected to protein purification using a HisTrap HP column connected to an FPLC instrument. Elution was performed using lysis buffer supplemented with 1 M imidazole, with a gradient run of 2 hours. Different fractions were collected and tested 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.

[0175] FEN enzyme purification was performed in the same manner with slight modifications. The growth medium used for protein expression was changed to LB, and the protein was expressed at 37°C for 4 to 5 hours. Cell lysis was performed using Y-PER (Thermo Fisher), and soluble protein was purified using Ni-NTA resin in a PD-10 column. Elution was performed using a lysis buffer supplemented with 50mM, 100mM, 200mM, and 500mM imidazole. The purified protein (final concentration was 1 μM) was stored at -20°C in a buffer containing 50% glycerol, 0.1mM EDTA, 1mM DTT, and 0.1% Triton-X-100.

[0176] FEN activity assay

[0177] The activity of each FEN enzyme was evaluated in an isothermal reaction containing 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 incubated at 65° C. for the appropriate time. When flap primers with a bound fluorophore and quencher were used, fluorescence was measured every 5 minutes (ex: 485 nm, em: 535 nm).

[0178] Ligation assay

[0179] To ligate the synthesized flap oligonucleotide to the dsDNA adapter, the dsDNA adapter was formed by annealing the 5'p adapter and the ds converter in 1×RB (final concentration of 200 nM). To this end, the reaction mixture was heated to 98°C in a thermal cycler and cooled to room temperature at a rate of 0.1°C / s. Ligation was performed by adding 1×RB, 5nM dsDNA adapter, 5nM flap oligonucleotide, and appropriate additives and cofactors depending on the ligase used. For T4 DNA ligase and T4 RNA ligase (NEB), 1mM DTT and 1mM ATP (NEB) were added to the reaction. For Taq DNA ligase (NEB), 1mNAD (Sigma Aldrich) and 10mM DTT were added to the reaction. The amount of ligase used was 5U of T4 DNA ligase, 10U of T4 RNA ligase, and 80U of Taq DNA ligase. The incubation temperature was 22°C for T4 DNA ligase, 37°C for T4 RNA ligase, and 65°C for Taq ligase.

[0180] Cas12-sgRNA reaction

[0181] The Cas12-sgRNA complex was 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), and the final volume was set to 20 μL with nuclease-free water. The incubation was performed at 37°C for 30 minutes. The Cas12-sgRNA complex was then diluted to 375 μL with water to achieve a final Cas12:sgRNA concentration ratio of 1 μM:0.833 μM.

[0182] For the reaction of Cas12-sgRNA complex, 3 μL of the complex formed as described 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, and fluorescence measurements were taken every 3 minutes (ex: 485 nm, em: 535 nm). Fig.21 , Fig.23 and Fig.24 For the data in , 1.5 μL of Cas12-sgRNA complex and 2 μL of fluorophore-quencher reporter were used.

[0183] LAMP assay

[0184] For LAMP, the Warmstart LAMP kit (DNA and RNA) from NEB was used and assayed according to the manufacturer's instructions. LAMP primers were designed using 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 product for subsequent purification with streptavidin agarose beads. After LAMP, 10 μL of DNA product was 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 sample was further diluted by adding 30 μL 0.5× B&W buffer and the mixture was incubated at room temperature for 30 minutes for biotin-streptavidin binding. After 30 minutes, the supernatant was removed after the beads settled. These beads were then used for FELICX.

[0185] HDA assay

[0186] HDA was performed using the IsoAmp II Universal tHDA kit from NEB. The assay was performed according to the manufacturer's instructions using primers designed using the PrimerQuest tool from IDT. For all HDA reactions, MgSO 4(4 mM), NaCl (40 mM) and appropriate primers (200 nM). After the assay was completed, the DNA product was used directly for FELICX or purified using the ChargeSwitch PCR Clean-Up kit from Invitrogen and then subjected to FELICX. When RNA was used as a template, 0.25 μL Warmstart RTx (NEB) was added per 10 μL HDA assay mixture.

[0187] FELICX( F lap E ndonuclease,Taq LI gas and C RISPR-Cas for diagnostics X ))

[0188] In the first step of FELICX, the amplified DNA was added to a FEN+Taq mixture containing: 2 μL flap primer (1 μM), 0.4 μL invasion primer (1 μM), 0.5 μL dsDNA adapter (200 nM, as described in the ligation assay), 2 μL RB-DTT buffer, 1 μL Triton-X-100 (2%), 1 μL NAD (20 mM), 0.5-1 μL FEN and 0.5 μL Taq ligase, made up to a final volume of 20 μL with nuclease-free water. The reaction was carried out at 65°C for an appropriate duration. Subsequently, the reaction mixture was added to the Cas12-sgRNA mixture containing 3 μL Cas12-sgRNA complex (as described in Cas12-sgRNA reaction), 4 μL FAM-IBFQ reporter probe (1 μM) and 0.7 μL RB-DTT and incubated at 37°C, and fluorescence was measured every 3 minutes in a microplate reader (ex: 485 nm, em: 535 nm).

[0189] For lateral flow test strips, the FAM-IBQF reporter was replaced with 0.25 μL FAM-Bio reporter (1 μM) or 0.1 μL DIG-Bio reporter (1 μM). After Cas12 cleavage, the reaction mixture was detected on a PCRD Flex test strip from Abingdon Health according to the manufacturer's instructions. Band intensity was quantified using ImageJ.

[0190] SNP Detection

[0191] To detect the T478K mutation in the SARS-CoV-2 receptor binding domain (RBD), the mutation was introduced into pCDNA3-SRARS-CoV-2-S-RBD-Fc carrying the viral RBD region (~1.5 kB) by overlapping PCR using primers spike-T478K–F / R. After confirmation of mutagenesis by sequencing, wild-type and mutated RBD regions were PCR amplified using primers spike-F / R and equal amounts of plasmid. After amplification, 50–150 ng of purified PCR product was used as template for FELICX, which was performed as described previously with slight modifications. The volume of flap primers (WT and mut probes) was increased to 3 μL (final concentration 150 nM). Both templates were analyzed using WT and mut probes. For lateral flow test strips, the FAM-Bio reporter was used in the Cas12-sgRNA cleavage reaction for the WT probe sample and the DIG-Bio reporter was used for the mut probe sample.

[0192] Detection of genes in mammalian cells

[0193] The C666-1 cell line was kindly provided by Dr. Joshua Tay from the Department of Otolaryngology, Yong Loo Lin School of Medicine, National University of Singapore, and the HK-1 cell line was kindly provided by Associate Professor Zhong Yong Liang from the Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore. Both cell lines were cultured in Gibco BenchStable RPMI 1640 (Life Technologies, catalog number A4192301) at 37°C and 5% CO. 2 , supplemented with 10% FBS (Biowest, catalog number S181H) and 1% penicillin-streptomycin (Gibco, catalog number 15140122). After reaching 70-80% confluence, cells were trypsinized and washed with RPMI, and the number of cells was counted using a Luna cell counter. The cells were cultured in lysis buffer (10 mM Tris-Cl (pH 7.5), 200 μg / mL proteinase K, 0.1% SDS, 2 mM CaCl 2) and incubated at 65°C for 10 minutes to lyse a fixed number of C666-1 or HK-1 cells. After lysis, the genetic material was purified using the ChargeSwitch PCR Clean-Up Kit and FELICX was performed as described above. The fluorescence measured after 30 minutes of the Cas12-sgRNA reaction was then reported.

[0194] Detection of genes in Klebsiella pneumoniae

[0195] An overnight culture of Klebsiella pneumoniae DSM 2026 grown in LB broth at 37°C with shaking was diluted to obtain the required cfu according to the formula OD600 = ~108 c.fu. HK-1 cells were cultured at 37°C and 5% CO 2 The cells were grown in Gibco BenchStable RPMI 1640 (Life Technologies, catalog number A4192301) supplemented with 10% FBS (Biowest, catalog number S181H) and 1% penicillin-streptomycin (Gibco, catalog number 15140122) at 4 °C. After reaching 70-80% confluence, the cells were trypsinized and washed with RPMI, and the number of cells was counted using a Luna cell counter. Klebsiella pneumoniae was mixed with HK-1 cells as needed and lysed in lysis buffer (10 mM Tris-Cl (pH 7.5), 200 μg / mL proteinase K, 0.1% SDS, 2 mM CaCl 2 ) and incubated at 65°C for 10 minutes to lyse the sample to prepare a simulated clinical sample. After lysis, the genetic material was purified using the ChargeSwitch PCR Clean-Up Kit and FELICX was performed as described previously. Then, the fluorescence after 30 minutes of Cas12-sgRNA reaction was reported.

[0196] Detection of cancer biomarkers

[0197] To prepare the template, overlapping primers (scgb1-F, scgb2-R, scgb3-F, and scgb4-R) were designed and PCR amplification was performed with scgb2a2-T7-F and scgb2a2-R. The final construct contained a T7 promoter at the 5' end of SCGB2A2, which can be transcribed in vitro by the HiscribeT7 high-yield RNA synthesis kit (NEB). The transcripts were purified by the Monarch RNAClean-Up kit (NEB) and used for FELICX. To simulate clinical samples, RNA was quantified and added to heat-inactivated South American fetal bovine serum (FBS) (Biowest, catalog number S181H). The simulated samples were treated with the ChargeSwitch PCR Clean-Up kit to purify the spiked RNA, and then HDA was performed using RTx and FELICX as described above. Then, the fluorescence after 30 minutes of the Cas12-sgRNA reaction was reported.

[0198] Example 1: Characterization and optimization of FEN

[0199] To develop FELICX, the cleavage activity of FEN was first characterized and optimized. FEN is a class of enzymes that are ubiquitous in prokaryotes and eukaryotes. The main role of FEN is in DNA replication, where it removes the RNA primer from the 5' end of the Okazaki fragment, which is then linked together to form the lagging strand. In bacteria, DNA polymerase I (composed of an N-terminal 5' nuclease and a C-terminal polymerase) plays the role of FEN via its N-terminal domain. In archaea and mammalian cells, there is a dedicated FEN enzyme that resembles the N-terminal domain of bacterial DNA polymerase I.

[0200] FENs have been used to detect DNA and RNA, although they have several limitations, such as slow speed, the need for initial denaturation to assemble oligonucleotides on the target, and reliance on fluorescent readers for signal detection. DNA polymerase I from Thermus thermophilus (Tth pol) is a well-characterized FEN that can recognize flap structures on DNA and RNA targets. To determine whether Tthpol is the best candidate for FELICX, a panel of Tth pol variants were expressed and purified: the N-terminal domain of Tth pol (TthN), human FEN1 (hFEN), archaeal Methanococcus jannaschii FEN1 (MjaFEN), bacteriophage T5 exonuclease (T5 exo) ( Fig.18a and c), and compared their activities with those of a commercially available thermostable FEN (thermoFEN) (Figure 13). Previously, wild-type Tth pol was mutated (G506K, Q509K, H786A) to enable it to recognize RNA and inactivate the polymerase domain. In this study, this mutant was named Tth pol v1. Mutations in Taq polymerase have been reported to expand its substrate spectrum, although their effects on the FEN activity of the enzyme have not been studied. These mutations are located in the polymerase domain of the enzyme and may affect its substrate binding and thus FEN activity. 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 polv1 was further mutated to include A604V, A610V, I616M and E617GA mutations to produce Tth pol v2. Similarly, Tth pol v1 was mutated to contain A599T, W606R, A607Q, and I616T mutations, thereby generating Tth pol v3 ( Fig.18 b). TthN was also generated, consisting of only the N-terminal domain (amino acids 1-307) of full-length Tth pol, and tested for FEN activity based on previous reports showing that the N-terminal domain of Taq polymerase alone exhibits FEN activity.

[0201] To quantify FEN activity, a flap primer with a 5' fluorophore and two linked quenchers (3' and internal) was used. This primer generates a fluorescent signal upon cleavage by FEN (Figure 13a). For all FEN enzymes, the enzyme activity of different configurations of flap primer and invasion primer was tested: flap primer with a 5' flap and invasion primer (Figure 13a), double flap with both flap primer and invasion primer having flap (Figure 13b). Fig.18 d), and in the absence of a target-specific invader primer. The nonspecific activity of FEN alone against a fluorophore-quencher flap primer was also measured. For the target, a partial orf1ab DNA of SARS-CoV-2 with low similarity to the SARS virus genome was used. The reaction was performed at 65°C, which is the melting temperature of the flap primer used in this study.

[0202] As shown in the left side of Figure 13b, when using a double-flap substrate ( Fig.18 d) and when the invasion primer was missing, the activities of Tth pol v1, v2, and v3 were greatly reduced, indicating that Figure 2The configuration shown in a is optimal for these enzymes. Among these Tth pol variants, Tth pol v2 showed the highest activity on flap primers and invasion primers without flap-bound DNA substrates, followed by Tth pol v3 and v1 (Figure 13b), which verified our hypothesis that the introduced enzyme mutations affect enzyme activity. Fluorescence signals were also observed in the case of flap primers alone (Figure 13b), indicating that these enzymes have low nonspecific activity as well as high specific FEN activity. Unexpectedly, in contrast to previous studies using the highly similar N-terminal domain of Taq polymerase, TthN did not show any activity towards any of the substrates tested. hFEN did not show any activity, probably due to the high reaction temperature, while the thermophilic bacterium MjaFEN had poor activity towards substrates with flap primers and invasion primers with a 5'-flap (Figure 13b). For T5 exo, strong fluorescence signals were observed for all substrates (including flap primers alone) almost immediately after initiating the reaction. This indicates that the enzyme has very high exonuclease activity, which masks its FEN activity. In order to eliminate its exonuclease activity, the K83 residue was mutated, and the K83 residue has been shown to be essential for exonuclease activity but insignificant for endonuclease activity. However, in this study, the K83A mutation completely inactivated the enzyme, inhibiting both endonuclease and exonuclease activities (Figure 13b). At the same time, thermophilic bacteria FEN showed activity trends similar to Tth pol on various DNA substrates. However, compared with Tth pol and its variants, thermophilic bacteria FEN has lower activity. These results indicate that Tth pol v2 is the best candidate with high specific FEN activity, and the ideal substrate for the enzyme is a DNA target bound to a flap primer and a flapless invasion primer.

[0203] Next, the best FEN candidate, Tth pol v2, was selected and its activity was compared with that of other Tth pol variants v1 and v3 on RNA substrates combined with a flap primer and an invasion primer with a flap. Similar to previous experiments, the assay used a partial orf1ab RNA from SARS-CoV-2. As shown on the right side of Figure 13b, Tth pol v1 and v2 performed better than Tth pol v3, although weaker signals were observed for all three enzymes compared to the DNA substrate. Based on the FEN activity observed on 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 the enzyme.

[0204] We further characterized Tth pol v2 by evaluating the effect of enzyme concentration on FEN activity on DNA and RNA substrates and on nonspecific activity with a flap primer alone (labeled as a probe). We observed that as the enzyme concentration increased, the nonspecific activity also increased, as indicated by the increase in fluorescence when the probe was used alone (Figure 13c). At 10 nM, the nonspecific activity was minimal, and high FEN activity was observed for both DNA and RNA substrates. Above this concentration, the nonspecific activity increased significantly and then became indistinguishable from FEN activity at 220 nM. This suggests that an optimal concentration (10 nM) of enzyme is required for specific FEN activity.

[0205] Although FEN can amplify the signal by cyclically annealing and denaturing the flap primers, it may not be sufficient to achieve high-sensitivity detection of targets in a short period of time due to the linear amplification of the signal (one flap product is formed per target per cycle). To investigate this, the detection limits of DNA and RNA were determined by using 10nM Tth pol v2. Within 30 minutes of incubation, up to 100pM of DNA and RNA were detected, although the fluorescence signal of 100pM was slightly higher than that of the no-target control (NTC) (Figures 13d and 13e). Increasing the Tth pol v2 concentration to 50nM reduced the detection limit to 1nM due to higher background fluorescence caused by higher nonspecific activity ( Fig. 20 ).

[0206] Example 2: Development and optimization of FELICX

[0207] After recognizing the DNA substrate, CRISPR-Cas12 acts as an excellent signal amplifier through its rapid trans-cleavage activity on ssDNA. Therefore, FEN is combined with CRISPR-Cas12 to convert the flap oligonucleotide generated by FEN into a substrate for Cas12 by connecting it to the adapter. For FELICX, Cas12 from Lachnospiraceae bacterium ND2006 was used. Since Cas12 prefers dsDNA substrates to ssDNA ( Fig.21 ), we created a dsDNA adapter with a 3' overhang by annealing two oligonucleotides (5'p-adaptor and ds-converter). The 5'p-adaptor carries a 5'-phosphate that is ligated to the 3'-OH of the flap oligonucleotide by ligase, and the ds-converter carries a PAM adjacent to the sgRNA binding site (Figure 14a). The complementary target of the sgRNA is split between the flap and the adapter, ensuring that neither the adapter itself nor the uncleaved flap primer will generate a signal.

[0208] For each new target in FELICX, only the invader primer and the part of the flap primer complementary to the target need to be changed (Figure 13a). The 5' flap of the flap primer remains unchanged, because FEN can cleave the flap primer in a sequence-independent manner. However, the cleavage site of FEN is one base pair away from the complementary part of the flap primer to the target. Therefore, each target can produce a flap with one of four nucleotides at the 3' end of the flap, depending on the target sequence. To account for this, the complementary nucleotide in the ds converter must be changed (see Figure 3 Since this base pair is located within the region recognized by the sgRNA, the recognition of the dsDNA adapter by the Cas12-sgRNA complex is tested using one of the four nucleotides at position N without changing the sgRNA sequence. This will simplify the use of Cas12-sgRNA, without the need to change the sgRNA sequence for each new target. To this end, a Cas12-sgRNA complex is used in which the sgRNA is complementary to the dsDNA adapter with a thymine at position N ( Fig.23 a). Fig.23 As shown in c, Cas12-sgRNA detects all possible targets with similar efficiency, regardless of the nucleotides at the end of the flap. This is expected, as previous reports have shown that LbCas12a can tolerate 8-18 nucleotide mismatches from the PAM, which is the region where the variable base pair (N-N') is located. Therefore, for any nucleic acid target, the rest of the dsDNA adapter except for one base pair and sgRNA remains unchanged, regardless of the target sequence, making it easier to reprogram FELICX to detect different targets.

[0209] For the connection of the flap to the adapter, Taq ligase was selected because its optimal temperature (65°C) is the same as that of FEN. This will enable FEN to cut the flap primer and connect the adapter simultaneously in one reaction system (one-pot). The connection efficiency of Taq ligase at 65°C was measured and compared with the connection efficiency of other ligases, where the reaction was carried out at the optimal temperature of the reported enzyme: T4 DNA ligase at 21°C and T4 RNA ligase at 37°C. Using synthetic flap oligonucleotides and dsDNA adapters as substrates, the connection lasted for 30 minutes. The connection product was detected by Cas12-sgRNA complex, where the fluorescence signal was measured after incubation at 37°C for 10 minutes or 30 minutes (Figure 14b). Among the three ligases tested, Taq DNA ligase had the strongest fluorescence signal after incubation with Cas12-sgRNA for 10 minutes, which was comparable to the signal of the pre-connected product (Figure 14c). Therefore, Taq ligase was used as the ligase for FELICX.

[0210] Next, the FEN+Taq ligase reaction step in a reaction system was optimized by changing the FEN concentration and the reaction incubation time at 65°C. Figure 14d shows that the optimal FEN concentration is 10nM because it produces the strongest fluorescence signal. At the same time, lower and higher FEN concentrations produce weaker signals. At low concentrations of FEN, fewer flap products are formed, resulting in weaker fluorescence signals. However, at high FEN concentrations, the nonspecific activity of FEN may produce flap products of shorter lengths, which will not anneal to the dsDNA adapter due to the high reaction temperature, or cannot be recognized by the Cas12-sgRNA complex after connection due to incomplete hybridization of the sgRNA with the connection product. In addition, the fluorescence signal increased with increasing incubation time, with the strongest signal produced by incubation for 4 hours. To further enhance the fluorescence signal, the concentrations of the Cas12-sgRNA complex and the DNA reporter were increased, which resulted in a significant increase in the specific signal ( Fig. 22 ). Modified sgRNAs were also evaluated, with their 3' ends extended with ssDNA, which has been previously reported to increase the trans-cleavage activity of LbCas12a. However, no improvement in activity was observed compared to unmodified sgRNAs ( Fig.23 ). Using these optimized conditions, the detection limits of FELICX for DNA and RNA were evaluated to determine whether the signal amplification of 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 purified total DNA or RNA from HEK293T cells, respectively, to simulate clinical samples. Despite continuous incubation at 65°C for 4 hours, only up to 100 pM of target was detected by FELICX when both substrates were tested (Figures 14e and f). Although this detection limit is similar to that observed using FEN alone (Figures 13d and e), the fluorescence signal observed using FELICX was significantly higher than that using FEN alone, indicating that the signal was amplified by Cas12.

[0211] Previously reported nucleic acid detection methods combined isothermal nucleic acid amplification techniques, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), to improve detection sensitivity. The target nucleic acid was amplified using LAMP, a commonly used nucleic acid amplification method performed at 65°C, and then partial orf1ab DNA of SARS-CoV-2 spiked into purified total DNA from HEK293T cells was detected by FELICX. However, despite extensive optimization of the assay, orf1ab DNA was not detected.

[0212] Since 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 thus improve the sensitivity of target detection. HDA was chosen because its operating temperature (65°C) is the same as that of the FEN+Taq ligation step, simplifying the operation of HDA+FELICX. When FELICX was coupled to HDA, six copies / μL (10 aM) of orf1ab and two copies / μL (3.5 aM) of the E. coli genome were detected in two of the three biological samples (Figures 14g and h), which is a significant improvement compared to LAMP, especially for orf1ab detection. Therefore, the presence of fewer primers in the nucleic acid amplification step can prevent the formation of nonspecific products and increase the target yield, greatly improving its subsequent detection.

[0213] Integrate lateral flow assays into FELICX

[0214] Integrate FELICX with lateral flow systems for signal detection, eliminating the need for a fluorescence reader. Using PCRD Flex lateral flow test strips, it is able to detect two different oligonucleotides conjugated with biotin and FAM or digoxigenin ( Fig.15 a). In the last step of FELICX, the Cas12-sgRNA complex cleaves the DNA reporter in the presence of its substrate. To exploit this cleavage activity of the lateral flow test 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 test strip ( Fig.15 b). Therefore, a positive signal for the target nucleic acid is indicated by the absence of a band on the lateral flow test strip. The functionality of the lateral flow test strip was validated using FELICX using partial orf1ab DNA as a target. Since the sensitivity of the lateral flow test strip is poorer than that of the fluorescence reader, the HDA reaction volume was increased from 10 μL to 25 μL. Since high variability and weak signals were observed when using unpurified HDA product in FELICX (Figures 14g and h), a nucleic acid purification step was added after HDA. Purification was performed using the ChargeSwitch PCRClean-Up Kit. When combined with HDA and subsequent purification, up to 0.6 copies / μL (1aM) of orf1ab DNA incorporated into purified HEK293T DNA was detected using FELICX, as indicated by the disappearance of the T1 band in the lateral flow test strip ( Fig.15c). The total detection time was 2.5 hours, including 1 hour HDA, 1 hour FEN+Taq ligation, and 30 minutes Cas12-sgRNA reaction. When the HDA+FELICX time was shortened to 60 minutes (20 minutes each for HDA, FEN+Taq ligation, and Cas12-sgRNA), there was no loss in sensitivity ( Fig.15 d), demonstrating the stability of HDA+FELICX.

[0215] After confirming the suitability of the method 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 as it has an optimal temperature of 65°C and similar buffer requirements as HDA. Since RTx has not been used previously with HDA, we evaluated the functionality of RTx+HDA in either a one-step (RTx+HDA in one reaction) or two-step (continuous RTx and HDA) reaction to determine if RTx interfered with the HDA reaction. No differences were observed between the reactions ( Fig.26 ), the one-step RTx+HAD was chosen due to its lower complexity. RTx+HDA was performed followed by FELICX to detect orf1ab RNA spiked into purified HEK293T RNA. 0.6 copies / μL RNA was detected in this process ( Fig.15 e). Detection of RNA required a total of ~90 minutes (RTx+HDA for 45 minutes, FEN+Taq ligation for 20 minutes, and Cas12-sgRNA for 20 minutes). These results indicate that HDA+FELICX can detect DNA and RNA with similar sensitivity using lateral flow test strips within 60 minutes and 90 minutes, respectively.

[0216] Example 3: Detection of polynucleotide analytes using FELICX

[0217] Detecting SNPs is very important for disease diagnosis and treatment, as well as for identifying pathogen variants. By designing flap and invasion primers so that one base pair overlap between the primers falls at the SNP position, FEN can be used to distinguish SNPs (Figure 16a). If there is a SNP in the target, the absence of overlap between the flap and invasion primers will eliminate the activity of FEN. Therefore, the presence of SNPs in the target sample can be confirmed by probing the sample with flap primers specific for wild-type and variant target sequences (Figure 16b). To this end, FELICX was used to distinguish the receptor binding domain in the spike protein of wild-type SARS CoV-2 and its variants carrying the T478K mutation, which is present in the Delta and Omicron variants of the virus. Flap primers (i.e., WT and mut probes) specific for the wild-type (WT) sequence and variants, respectively, were designed for detecting the two targets. For the WT target, after FELICX, the fluorescence signal of the WT probe was about 2.5 times higher than that of the mut probe (Figure 16c). In contrast, for the variant target with T478K mutation, the fluorescence signal of the mut probe was about 2.5 times higher than that of the WT probe. Although there was only one base pair difference between the WT and T478K variants (ACG codon changed to AAG), FELICX easily detected the SNP because opposite trends in the fluorescence signals were observed for the WT and variant sequences.

[0218] The PCRD Flex test strips also demonstrate SNP detection, where each test line corresponds to a WT or mut probe. To this end, the WT probe uses a FAM-biotin reporter and the mut probe uses a DIG-biotin reporter (Figure 16b). Similar to the results shown in Figure 16c, the WT sample shows a positive signal using the WT probe, as shown by the disappearance of the band corresponding to the probe (Figures 16d and e). For the T478K variant, the band corresponding to the mut probe disappears, indicating that a positive signal was obtained using this probe.

[0219] FELICX can also detect other target pathogens in more complex samples, such as EBV, which causes infectious mononucleosis and is associated with autoimmune diseases, nasopharyngeal carcinoma (NPC), and other tumors. C666-1, an NPC cell line that is also the natural host of EBV, was used. The EBV-negative HK-1 NPC cell line was also included in the analysis, where the housekeeping gene gapdh was used as an internal control for the functional assay. Cell lysis followed by RTx+HDA and FELICX detected EBV and gapdh at levels as low as 0.6 cells / μL of C666-1 ( Fig.17 a and Fig. 27, top), indicating that HDA+FELICX has high sensitivity for EBV detection. The robustness of this method was verified by sensitive detection of targets in intact cells using HDA+FELICX.

[0220] FELICX can also detect Klebsiella pneumoniae, which is the most common nosocomial pathogen worldwide and a major source of patient complications. Klebsiella pneumoniae is the causative agent of neonatal sepsis, pneumonia, surgical wound infections, and cystitis. In addition to its widespread prevalence, Klebsiella pneumoniae is a major source of antibiotic resistance genes, especially genes encoding carbapenemases. Therefore, detecting pathogens in clinical samples and clearly determining their antibiotic resistance status is critical to guiding infection treatment. HDA+FELICX was used to detect carbapenemase resistance genes (blaKPC) in pathogenic Klebsiella pneumoniae DSM 2026 bacterial cells spiked into whole HK-1 cells. The hemolysin gene (khe), which is present in all Klebsiella pneumoniae isolates, was used as an internal control. Using conditions similar to those for EBV detection, blaKPC and khe ( Fig.17 b and Fig. 27 , lower part). Only HK-1 cells did not observe signals for any gene. Since RTx was not used in this assay, only gene copies in the K. pneumoniae DSM2026 genome were detected, emphasizing the high detection sensitivity of this assay.

[0221] Tth-pol v2 sequence

[0222]

[0223] Tth-pol v3 sequence

[0224]

[0225] Cas12 gRNA

[0226] 5'-TAATTTCTACTAAGTGTAGATCAACGTCGTGACTGGGAAAACCCT-3'(SEQ ID NO:3)

[0227] Table 1: Oligonucleotides used in this study

[0228]

[0229]

[0230]

[0231] Table 2: LAMP primers used in this study

[0232]

[0233]

[0234] Table 3: HDA primers used in this study

[0235]

[0236]

[0237] Table 4: Flap and invasion primers used in this study. The constant portion of the flap primers is underlined.

[0238] The box is the non-complementary part of the invasion primer.

[0239]

[0240]

Claims

1. A method for detecting a polynucleotide analyte in a sample, the method comprising: a) contacting a sample comprising the polynucleotide analyte with: i) a first nucleic acid probe comprising a 3' portion complementary to a first portion of the polynucleotide analyte and a 5' portion that is not complementary to and does not hybridize to the polynucleotide analyte; ii) a second nucleic acid probe comprising a 5' portion complementary to the second portion of the polynucleotide analyte and a 3' portion that is not complementary to and does not hybridize to the polynucleotide analyte, wherein the first portion of the polynucleotide analyte is 5' adjacent to the second portion of the polynucleotide analyte; and iii) structure-specific nucleic acid cleavage agents; 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 forms a cleavage structure; wherein the formation of the cleavage structure allows the cleavage agent to cleave the first nucleic acid probe to release a 5' flap, the 5' flap comprising a 5' portion of the first nucleic acid probe that is not complementary to the polynucleotide analyte and does not hybridize to the polynucleotide analyte; b) ligating the 5' flap to a nucleic acid adaptor to form an adaptor ligation product, wherein the nucleic acid adaptor comprises a double-stranded region and a 3' overhang extension complementary to the 5' flap, wherein the 3' overhang extension hybridizes to the 5' flap; c) contacting the adaptor 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 and a guide sequence that is complementary to a portion of the 5' flap and to a portion of a nucleic acid adaptor that is attached to and adjacent to the 5' flap; and iii) Single-stranded detection DNA; and d) measuring a detectable signal generated by cleavage of the single-stranded detection DNA by a type V CRISPR / Cas effector protein to detect the adaptor ligation product, thereby detecting the polynucleotide analyte in the sample.

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

3. The method according to claim 1 or 2, wherein the polynucleotide analyte is single-stranded or double-stranded.

4. The method according to any one of claims 1 to 3, wherein the method further comprises amplifying the polynucleotide analyte prior to step (a).

5. The method according to any one of claims 1 to 4, wherein the polynucleotide analyte comprises a single nucleotide polymorphism (SNP).

6. The method according to any one of claims 1 to 5, wherein the polynucleotide analyte is a viral nucleic acid.

7. The method according to any one of claims 1 to 6, wherein the cleavage agent of 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 from Thermus thermophilus.

9. The method according to claim 7 or 8, wherein the enzyme having flap endonuclease activity comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

10. The method according to any one of claims 1 to 9, wherein step c) is performed in the presence of a ligase. The method of claim 10 , wherein the ligase is a thermostable ligase.

12. The method according to any one of claims 1 to 11, wherein the V-type CRISPR / Cas effector protein is a Cas12 protein.

13. The method according to any one of claims 1 to 12, wherein the single-stranded detection DNA comprises a fluorophore-quencher pair.

14. The method according to any one of claims 1 to 13, wherein cleavage of the single stranded detection DNA is performed by visual detection.

15. A method for detecting a single nucleotide polymorphism (SNP) of a polynucleotide analyte in a sample, the method comprising: a) contacting a sample comprising said polynucleotide analyte with: i) a first nucleic acid probe comprising a 3' portion complementary to a first portion of the polynucleotide analyte and a 5' portion that is not complementary to and does not hybridize to the polynucleotide analyte; ii) a second nucleic acid probe comprising a 5' portion complementary to the second portion of the polynucleotide analyte and a 3' portion that is not complementary to and does not hybridize to the polynucleotide analyte, wherein the first portion of the polynucleotide analyte is 5' adjacent to the second portion of the polynucleotide analyte; and iii) structure-specific nucleic acid cleavage agents; 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 forms a cleavage structure; wherein the formation of the cleavage structure allows the cleavage agent to cleave the first nucleic acid probe to release a 5' flap, the 5' flap comprising a 5' portion of the first nucleic acid probe that is not complementary to the polynucleotide analyte and does not hybridize to the polynucleotide analyte; b) ligating the 5' flap to a nucleic acid adaptor to form an adaptor ligation product, wherein the nucleic acid adaptor comprises a double-stranded region and a 3' overhang extension complementary to the 5' flap, wherein the 3' overhang extension hybridizes to the 5' flap; c) contacting the adaptor ligation products: i) Type V CRISPR / Cas effector proteins; 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 a nucleic acid adaptor that is attached to and adjacent to the 5' flap; and iii) Single-stranded detection DNA; and d) measuring a detectable signal generated by cleavage of the single-stranded detection DNA by the type V CRISPR / Cas effector protein to detect the adaptor ligation product, thereby detecting the SNP of the polynucleotide analyte in the sample.

16. A method for detecting a polynucleotide analyte in a sample, comprising a) i) contacting a sample comprising the polynucleotide analyte with a first nucleic acid probe and a second nucleic acid probe, wherein the first nucleic acid probe and the second nucleic acid probe are configured to form a cleavage structure in the presence of the polynucleotide analyte; and ii) structure-specific nucleic acid cleavage agents; wherein the formation of the cleavage structure allows the cleavage agent to cleave the first nucleic acid probe to release the 5' flap from the first nucleic acid probe; b) ligating the 5' flap to a nucleic acid adaptor to form an adaptor ligation product, wherein the nucleic acid adaptor comprises a double-stranded region and a 3' overhang extension complementary to the 5' flap, and wherein the 3' overhang extension hybridizes with the 5' flap to the nucleic acid adaptor; c) contacting the adaptor ligation products with: i) Type V CRISPR / Cas effector proteins; 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 a nucleic acid adaptor that is attached to and adjacent to the 5' flap; and iii) Single-stranded detection DNA; and d) measuring a detectable signal generated by cleavage of the single-stranded detection DNA by the type V CRISPR / Cas effector protein to detect the adaptor ligation product, thereby detecting a polynucleotide analyte in the sample.

17. A kit for detecting a polynucleotide analyte 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 of 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 shown in SEQ ID NO:

1.

19. The kit according to claim 17 or 18, further comprising a first nucleic acid probe and a second nucleic acid probe, wherein the first nucleic acid probe and the second nucleic acid probe are configured to form a cleavage structure in the presence of the polynucleotide analyte.

20. The kit of any one of claims 17 to 19, further comprising a first adaptor polynucleotide and a second adaptor polynucleotide, wherein the first adaptor polynucleotide and the second adaptor polynucleotide are configured to form a nucleic acid adaptor upon hybridization.

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

22. The kit according to any one of claims 17 to 21, further comprising a single-stranded detection DNA.

Images