Use of a high temperature resistant Cas protein and a detection method and kit for target nucleic acid molecules

Abstract

The present invention provides a purpose of a thermostable Cas protein and a detection system and a kit for a target nucleic acid molecule. Specifically, the present invention provides a reaction system for detecting a target nucleic acid molecule, wherein the reaction system includes: a guide RNA, Cas12b (formerly known as C2c1), and a nucleic acid probe, which is detected after the reaction is completed. In addition, by combining with nucleic acid amplification techniques (such as LAMP, etc.), the sensitivity of the detection method can be greatly improved. The detection system provided by the present invention can be used for rapid detection of pathogenic microorganisms, gene mutations, single nucleotide polymorphisms, and specific target DNA, etc., and nucleic acid samples can be quantified.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular, relates to the use of a thermostable Cas protein and a method and a kit for detecting a target nucleic acid molecule. Background Art

[0002] Rapid nucleic acid molecular detection is a critical technology in the fields of public health, environmental testing, criminal investigation, etc. Nucleic acid molecular detection can not only be used to detect whether humans, animals, plants, etc. are infected with pathogens, but also can be used to detect genetic diseases or cancer risks, provide auxiliary references for personal medication, and detect whether water bodies are contaminated by microorganisms. At present, many nucleic acid detection methods have been developed, such as Realtime PCR, FISH hybridization technology (Fluorescence in situ hybridization), etc. However, there is still a need to develop nucleic acid detection technologies that are both fast, cheap, and sensitive.

[0003] In recent years, CRISPR technology has shown great application value in the field of genome editing. The main task is to complete this task with DNA (or RNA) targeted nucleases such as Cas9. By simply designing a guide RNA, people can target proteins such as Cas9 to any nucleic acid target sequence containing PAM (such as Cas9's PAM sequence is NGG) and cause double-stranded DNA breaks (or RNA cuts).

[0004] In addition to genome editing, CRISPR can also be used for gene regulation, epigenetic editing, functional gene screening, genome imaging, etc. Recently, this rich toolbox of CRISPR has begun to appear and attract attention in the field of nucleic acid detection.

[0005] The first CRISPR-related nucleic acid detection technology was based on the dCas9 (dead Cas9) protein. dCas9 mutates the two catalytic cleavage active sites of Cas9, thereby losing the activity of cutting double strands, but dCas9 still retains the ability to be guided by guide RNA and bind to specific DNA. Chen et al. used dCas9 to fuse with EGFP fluorescent protein, which can be guided by specific guide RNA, and the fluorescence at the target sequence can be observed under a microscope. In 2017, Guk et al. reported a DNA-FISH system based on dCas9 / sgRNA-SG I, which can selectively detect methicillin-resistant Staphylococcus aureus.

[0006] In 2013, Collins' team established a method for detecting Zika virus on paper. The method involves RNA extraction, amplification and toehold reaction detection. By design, if one virus strain happens to be different from another in the PAM site sequence (i.e. one is NGG and the other is different), Cas9 can be used to target and cut the strain containing PAM, thereby affecting the amplification process and toehold detection.

[0007] Recently, another Cas9-based nucleic acid detection method is based on the principle of CRISPR / Cas9-triggered isothermal amplification, which can also be used for site-specific nucleic acid detection. This technology involves CRISPR / Cas9 cleavage, nickase and DNA polymerase. This method can not only detect a target concentration of 0.82amol, but also distinguish the difference between single bases of nucleic acid molecules. The target differentiation of this method is more generally applicable than the previous method.

[0008] Another principle of nucleic acid detection is based on the "trans-cleavage" (or "bypass cleavage") effect of some Cas proteins. In 2016, Zhang Feng et al. discovered that Cas13a (formerly known as C2c2) has bypass cleavage activity, that is, when Cas13a binds to the target RNA sequence, it will show the characteristics of randomly cutting other RNAs, which can be used for specific nucleic acid detection, called SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) technology.

[0009] Although there are already many CRISPR-related nucleic acid detection methods, this field still needs to develop faster, simpler and cheaper nucleic acid detection methods. Summary of the invention

[0010] The purpose of the present invention is to develop a CRISPR-related faster, simpler and cheaper nucleic acid detection method.

[0011] In a first aspect of the present invention, a detection system for detecting a target nucleic acid molecule is provided, the system comprising:

[0012] (a) a Cas12b protein, wherein the Cas12b protein is Cas12b or a Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b;

[0013] (b) a guide RNA, wherein the guide RNA guides the Cas12b protein to specifically bind to the target nucleic acid molecule; and

[0014] (c) a nucleic acid probe, wherein the nucleic acid probe is a single-stranded DNA;

[0015] Wherein, the target nucleic acid molecule is target DNA.

[0016] In another preferred embodiment, the detection includes: qualitative detection or quantitative detection.

[0017] In another preferred embodiment, the detection system further contains (d) a buffer solution.

[0018] In another preferred embodiment, the detection system further contains a target nucleic acid molecule to be detected.

[0019] In another preferred embodiment, the detection system further comprises:

[0020] (e1) a polymerase for amplifying target DNA;

[0021] (e2) optionally a reverse transcriptase for reverse transcription;

[0022] (e3) dNTPs used for amplification reaction and / or reverse transcription reaction.

[0023] In another preferred embodiment, the detection system further contains reagents for LAMP reaction.

[0024] In another preferred embodiment, the concentration of the target nucleic acid molecule to be detected in the detection system is 1×10 -9 nM-1×10 3 nM; preferably 1×10 -8 nM-1×10 2 nM.

[0025] In another preferred embodiment, the concentration of the target nucleic acid molecule to be detected in the detection system is 1-100 copies / μl or 1-1×10 15 copies / μl, preferably 1-10 copies / μl, more preferably 1-5 copies / μl.

[0026] In another preferred embodiment, in the detection system, the molar ratio of the nucleic acid probe to the target nucleic acid molecule is 10 3 : 1 to 10 14 ∶1, preferably 10 4 : 1 to 10 7 ∶1.

[0027] In another preferred embodiment, the length of the guide RNA is 16-25 nt, preferably 16-22 nt, and more preferably 16-20 nt.

[0028] In another preferred embodiment, the length of the guide RNA is 18-25 nt, preferably 18-22 nt, and more preferably 18-20 nt.

[0029] In another preferred embodiment, when the target nucleic acid molecule is ssDNA, the detection site of the target nucleic acid molecule is located at position 9 or position 10-16 downstream of the PAM sequence of the guide RNA, preferably position 10-14, and more preferably position 10-12.

[0030] In another preferred embodiment, when the detection site is located at the 9th position downstream of the PAM sequence of the guide RNA, the detection site is G.

[0031] In another preferred embodiment, the detection site is located at positions 9-20 downstream of the PAM sequence of the guide RNA, preferably positions 10-18, and more preferably positions 10-16.

[0032] In another preferred embodiment, the detection site of the target nucleic acid molecule is located at positions 1-12 downstream of the PAM sequence of the guide RNA, and more preferably at positions 1, 3, and 10.

[0033] In another preferred embodiment, the length of the guide RNA is 15-30 nt, preferably 15-18 nt.

[0034] In another preferred embodiment, the target DNA includes DNA formed based on RNA reverse transcription.

[0035] In another preferred embodiment, the target DNA includes cDNA.

[0036] In another preferred embodiment, the target DNA is selected from the following group: single-stranded DNA, double-stranded DNA, or a combination thereof.

[0037] In another preferred embodiment, the nucleic acid probe carries a fluorescent group and a quenching group.

[0038] In another preferred embodiment, the fluorescent group and the quencher group are independently located at the 5' end, the 3' end and the middle of the nucleic acid probe.

[0039] In another preferred embodiment, the length of the nucleic acid probe is 3-300 nt, preferably 5-100 nt, more preferably 6-50 nt, and most preferably 8-20 nt.

[0040] In another preferred embodiment, the target nucleic acid molecule includes a target nucleic acid molecule derived from a group consisting of plants, animals, insects, microorganisms, viruses, or a combination thereof.

[0041] In another preferred embodiment, the target DNA is artificially synthesized or naturally occurring DNA.

[0042] In another preferred embodiment, the target DNA includes wild-type or mutant DNA.

[0043] In another preferred embodiment, the target DNA includes DNA obtained by reverse transcription or amplification of RNA, such as cDNA.

[0044] In another preferred embodiment, the Cas12b protein is selected from the following group: AacCas12b (Alicyclobacillus acidoterrestris), Aac2Cas12b (Alicyclobacillus acidiphilus), AkaCas12b (Alicyclobacillus kakegawensis), AmaCas12b (Alicyclobacillus macrosporangiidus), AheCas12b (Alicyclobacillus herbarius), and AcoCas12b (Alicyclobacillus contaminans).

[0045] In another preferred embodiment, the nucleic acid probe comprises a single-stranded DNA with a detectable label.

[0046] In another preferred embodiment, the single-stranded DNA is fluorescent and biotin-labeled single-stranded DNA.

[0047] In another preferred embodiment, the single-stranded DNA is fluorescently labeled single-stranded DNA.

[0048] In another preferred embodiment, the single-stranded DNA is a fluorescent probe labeled with a fluorescent group HEX at the 5' end and a quenching group BHQ1 at the 3' end.

[0049] In a second aspect of the present invention, there is provided a detection system for detecting SNP (single nucleotide polymorphism) or nucleotide mutation (including single base or multi-base mutation), the system comprising a first detection system and a second detection system;

[0050] The first detection system includes:

[0051] (a1) Cas12b protein, wherein the Cas12b protein is Cas12b or a Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b;

[0052] (b1) a first guide RNA, wherein the first guide RNA guides the Cas12b protein to specifically bind to the target nucleic acid molecule; and

[0053] (c1) a nucleic acid probe, wherein the nucleic acid probe is a single-stranded DNA;

[0054] The second detection system includes:

[0055] (a2) Cas12b protein, wherein the Cas12b protein is Cas12b or a Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b;

[0056] (b2) a second guide RNA, wherein the second guide RNA guides the Cas12b protein to specifically bind to the target nucleic acid molecule; and

[0057] (c2) a nucleic acid probe, wherein the nucleic acid probe is a single-stranded DNA;

[0058] Wherein, the target nucleic acid molecule is a target DNA;

[0059] Furthermore, the first guide RNA and the second guide RNA target the same nucleic acid sequence region containing the SNP site, and the first guide RNA targets the wild-type (or non-mutated) nucleic acid sequence of the SNP site, while the second guide RNA targets the mutant nucleic acid sequence of the SNP site.

[0060] In another preferred embodiment, the system further includes a blank control system.

[0061] In another preferred embodiment, the blank control system comprises:

[0062] (a3) a Cas12b protein, which is Cas12b or a Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b; and

[0063] (c3) A nucleic acid probe, wherein the nucleic acid probe is a single-stranded DNA.

[0064] In another preferred embodiment, the SNP site is located at position 9 or position 10-16 downstream of the PAM sequence of the guide RNA, preferably position 10-14, and more preferably position 10-12.

[0065] In another preferred example, the SNP site is located at the 9th position downstream of the PAM sequence of the guide RNA, and the detection site is G.

[0066] In another preferred embodiment, the SNP site is located at positions 9-20 downstream of the PAM sequence of the guide RNA, preferably positions 10-18, and more preferably positions 10-16.

[0067] In another preferred embodiment, the length of the first or second guide RNA is 16-25 nt, preferably 16-22 nt, and more preferably 16-20 nt.

[0068] In another preferred embodiment, the length of the first or second guide RNA is 18-25 nt, preferably 18-22 nt, and more preferably 18-20 nt.

[0069] In a third aspect of the present invention, a kit for detecting a target nucleic acid molecule is provided, the kit comprising:

[0070] i) a first container and a Cas12b protein in the first container, wherein the Cas12b protein is Cas12b or a Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b;

[0071] ii) an optional second container and a guide RNA located in the second container, wherein the guide RNA guides the Cas protein to specifically bind to a target nucleic acid molecule;

[0072] iii) a third container and a nucleic acid probe located in the third container;

[0073] iv) an optional fourth container and a buffer within the fourth container;

[0074] Wherein, the target nucleic acid molecule is target DNA.

[0075] In another preferred embodiment, any two, three, or four (or all) of the first container, the second container, the third container, and the fourth container may be the same or different containers.

[0076] In another preferred embodiment, the nucleic acid probe carries a fluorescent group and a quenching group.

[0077] In another preferred embodiment, the kit further comprises a buffer.

[0078] In another preferred embodiment, the kit further comprises:

[0079] v) a fifth container and a polymerase for amplifying the target DNA located in the fifth container;

[0080] vi) an optional sixth container and a reverse transcriptase for reverse transcription located in the sixth container;

[0081] vii) a seventh container and dNTPs in the seventh container for an amplification reaction and / or a reverse transcription reaction.

[0082] In another preferred embodiment, the detection system further contains reagents for LAMP reaction.

[0083] In another preferred example, the fifth container, the sixth container and the seventh container may be the same container or different containers.

[0084] In another preferred embodiment, two, more or all of the first to seventh containers may be the same container or different containers.

[0085] In a fourth aspect of the present invention, a method for detecting whether a target nucleic acid molecule is present in a sample is provided, comprising the following steps:

[0086] (i) providing the detection system for detecting a target nucleic acid molecule according to the first aspect of the present invention, wherein the detection system further comprises a sample to be detected; and

[0087] (ii) detecting whether the nucleic acid probe in the detection system is cut by the Cas12b protein, wherein the cutting is a trans cutting that bypasses the single-stranded DNA;

[0088] Among them, if the nucleic acid probe is cut by the Cas12b protein, it means that the target nucleic acid molecule exists in the sample; if the nucleic acid probe is not cut by the Cas12b protein, it means that the target nucleic acid molecule does not exist in the sample.

[0089] In another preferred embodiment, the Cas12b protein is Cas12b or a Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b.

[0090] In another preferred embodiment, the sample to be detected includes a non-amplified sample and an amplified (or nucleic acid amplified) sample.

[0091] In another preferred embodiment, the sample to be detected is a sample obtained by amplification.

[0092] In another preferred embodiment, the method of nucleic acid amplification is selected from the following group: PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification, rolling circle amplification, HDA, SPIA, NEAR, TMA and SMAP2.

[0093] In another preferred embodiment, the PCR includes high temperature PCR, normal temperature PCR, or low temperature PCR.

[0094] In another preferred embodiment, the method is used to detect whether the nucleic acid at the target site has a SNP, a point mutation, a deletion, and / or an insertion.

[0095] In another preferred embodiment, when there is a lack of PAM sequence upstream and downstream of the target site (within the range of -20nt to +20nt, more preferably within the range of -16nt to +16nt), a primer introducing PAM is used for nucleic acid amplification.

[0096] In another preferred embodiment, the primer introducing PAM has a structure of Formula I from 5' to 3':

[0097] P1-P2-P3 (I)

[0098] In the formula,

[0099] P1 is a 5' segment sequence located at the 5' end that is complementary or non-complementary to the sequence of the target nucleic acid molecule;

[0100] P2 is the PAM sequence;

[0101] P3 is a 3' segment sequence located at the 3' end and complementary to the sequence of the target nucleic acid molecule.

[0102] In another preferred embodiment, the PAM primer specifically binds to the upstream or downstream of the target nucleic acid molecule.

[0103] In another preferred embodiment, the length of P1 is 0-20 nt.

[0104] In another preferred embodiment, the length of P3 is 5-20 nt.

[0105] In another preferred embodiment, the length of the PAM primer is 16-50 nt, preferably 20-35 nt.

[0106] In another preferred embodiment, the complementarity includes complete complementarity and partial complementarity.

[0107] In another preferred embodiment, at least one primer used in the nucleic acid amplification contains a PAM sequence.

[0108] In another preferred embodiment, when the upstream and downstream of the target site (in the range of -20nt to +20nt, preferably in the range of -15nt to +15nt, and more preferably in the range of -10nt to +10nt) contain a PAM sequence, primers containing or not containing a PAM sequence can be used, and the amplified product contains the PAM sequence.

[0109] In another preferred embodiment, the detection in step (ii) comprises fluorescence detection.

[0110] In another preferred embodiment, the fluorescence detection method is performed using an enzyme reader or a fluorescence spectrophotometer.

[0111] In a fifth aspect of the present invention, a method for detecting whether a target nucleic acid molecule is present in a sample is provided, comprising the following steps:

[0112] (i) providing a detection system, the detection system comprising:

[0113] (a) a Cas12b protein, wherein the Cas12b protein is Cas12b or a Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b;

[0114] (b) a guide RNA, wherein the guide RNA guides the Cas12b protein to specifically bind to the target nucleic acid molecule;

[0115] (c) a nucleic acid probe, wherein the nucleic acid probe is a single-stranded DNA;

[0116] Wherein, the target nucleic acid molecule is a target DNA;

[0117] (d) Buffer

[0118] (e1) a polymerase for amplifying target DNA;

[0119] (e2) optionally a reverse transcriptase for reverse transcription;

[0120] (e3) dNTPs for amplification reaction and / or reverse transcription reaction; and

[0121] (f) test samples to be tested;

[0122] (ii) performing a reverse transcription and / or amplification reaction on the detection system to obtain a reverse transcribed and / or amplified detection system;

[0123] (iii) detecting whether the nucleic acid probe in the detection system in the previous step is cut by the Cas12b protein, wherein the cutting is a trans cutting that bypasses the single-stranded DNA;

[0124] Among them, if the nucleic acid probe is cut by the Cas12b protein, it means that the target nucleic acid molecule exists in the sample; if the nucleic acid probe is not cut by the Cas12b protein, it means that the target nucleic acid molecule does not exist in the sample.

[0125] In another preferred embodiment, in the step (ii), the temperature of the detection system is maintained at 50-70°C, preferably 50-65°C, and more preferably 55-65°C.

[0126] In another preferred embodiment, in the step (iii), the temperature of the detection system is maintained at 25-70°C, preferably 48-65°C.

[0127] In another preferred embodiment, the method of nucleic acid amplification is selected from the following group: PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification, rolling circle amplification, HDA, SPIA, NEAR, TMA and SMAP2.

[0128] In another preferred embodiment, the nucleic acid amplification includes: LAMP amplification.

[0129] In another preferred embodiment, in the detection system, the nucleic acid concentration of the test sample to be detected is 1×10 -11 -1×10 -5 nM, preferably 1×10 -9 -1×10 -6 nM, preferably 1×10 -8 -1×10 -7 nM.

[0130] In another preferred embodiment, the detection includes: qualitative detection or quantitative detection.

[0131] In another preferred embodiment, the quantitative detection is an absolute quantitative detection (for example, quantitative detection combined with digital PCR technology).

[0132] In another preferred embodiment, the total time of steps (ii) and (iii) is ≤2 hours, preferably ≤1.5 hours, and more preferably ≤1 hour (eg, 30-60 minutes).

[0133] In another preferred embodiment, the target nucleic acid molecule is a methylated nucleic acid sequence, or the methylated nucleic acid sequence is a nucleic acid sequence obtained by converting non-methylated C to uracil.

[0134] In another preferred embodiment, the methylated nucleic acid sequence is treated with bisulfite to convert the unmethylated C into uracil.

[0135] In another preferred embodiment, the target nucleic acid molecule is a DNA molecule formed by reverse transcription of a linear or circular RNA molecule, or a DNA molecule formed by RT-PCR.

[0136] In another preferred embodiment, the RT-PCR includes RT-LAMP.

[0137] In a sixth aspect of the present invention, a use of a Cas12b protein is provided, wherein the Cas12b protein is used to prepare a detection reagent or kit for detecting a target nucleic acid molecule based on bypass single-stranded DNA cleavage, wherein the Cas12b protein is Cas12b or a Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b.

[0138] In another preferred embodiment, the Cas12b protein is selected from the following group: AacCas12b (Alicyclobacillus acidoterrestris), Aac2Cas12b (Alicyclobacilius acidiphilus), AkaCas12b (Alicyclobacillus kakegawensis), AmaCas12b (Alicyclobacillus macrosporangiidus), AheCas12b (Alicyclobacillus herbarius), and AcoCas12b (Alicyclobacillus contaminans).

[0139] In a seventh aspect of the present invention, a device for detecting whether a target nucleic acid molecule is present in a sample is provided, the device comprising:

[0140] (a) an amplification reaction-bypass cleavage reaction module, the module is used to perform a nucleic acid amplification reaction and a bypass cleavage reaction on a digitized reaction system, wherein the bypass cleavage reaction is a bypass cleavage mediated by Cas12b protein; and (b) a signal detection module, the module is used to detect whether Cas12b protein-mediated bypass cleavage occurs in each digitized reaction system.

[0141] In another preferred embodiment, the nucleic acid amplification reaction and the bypass cleavage reaction are carried out simultaneously.

[0142] In another preferred embodiment, the amplification reaction-bypass cutting reaction module further includes a temperature control unit, and the temperature control unit is used to amplify the temperature of the digital reaction system located in the device to a predetermined temperature.

[0143] In another preferred embodiment, the predetermined temperature is 50-70°C, preferably 50-65°C, and more preferably 55-65°C.

[0144] In another preferred embodiment, the predetermined temperature is 25-70°C, preferably 48-65°C.

[0145] In another preferred embodiment, the predetermined temperature during the entire amplification reaction and the bypass cleavage reaction is substantially the same.

[0146] In another preferred embodiment, the fluctuation range of the predetermined temperature is within ±5°C, preferably within ±3°C, and more preferably within ±1°C.

[0147] In another preferred embodiment, the nucleic acid amplification is isothermal amplification, or amplification in which the difference in denaturation-annealing-extension temperature is ≤10°C (preferably ≤5°C, more preferably ≤3°C).

[0148] In another preferred embodiment, the nucleic acid amplification includes: LAMP amplification.

[0149] In another preferred embodiment, the digital reaction system includes a droplet digital PCR (ddPCR) reaction system or a chip digital PCR (cdPCR) reaction system.

[0150] In another preferred embodiment, the digital reaction system comprises a plurality of independent reaction system units, each of which comprises:

[0151] (a) a Cas12b protein, wherein the Cas12b protein is Cas12b or a Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b;

[0152] (b) a guide RNA, wherein the guide RNA guides the Cas12b protein to specifically bind to the target nucleic acid molecule;

[0153] (c) a nucleic acid probe, wherein the nucleic acid probe is a single-stranded DNA;

[0154] Wherein, the target nucleic acid molecule is a target DNA;

[0155] (d) Buffer

[0156] (e1) a polymerase for amplifying target DNA;

[0157] (e2) optionally a reverse transcriptase for reverse transcription;

[0158] (e3) dNTPs for amplification reaction and / or reverse transcription reaction; and

[0159] (f) the test sample to be tested,

[0160] Due to the dilution process (ie, digital processing), each independent reaction system contains either 1 copy of the nucleic acid from the test sample or 0 copies of the nucleic acid from the test sample.

[0161] In another preferred embodiment, each reaction system unit is identical except that each reaction system unit contains 1 or 0 copies of nucleic acid from the test sample.

[0162] In another preferred embodiment, the reaction system unit is a droplet.

[0163] In another preferred embodiment, the reaction system unit is a micro-reaction system located in a micro-well of a chip.

[0164] In another preferred example, the device further includes a sample adding module and / or a control module.

[0165] In another preferred example, the signal detection module includes an imaging module.

[0166] In another preferred embodiment, the imaging module includes a fluorescence detection unit.

[0167] In another preferred example, the fluorescence detection unit illuminates the digital reaction system to excite the reaction system to generate a fluorescence signal, converts the fluorescence signal into a digital signal, and preferably sends it to the control module.

[0168] In another preferred embodiment, the control module performs calculation analysis based on the number of the fluorescent signal (preferably, the fluorescent signal has been converted into a digital signal) and the total number of the reaction system units to obtain a quantitative detection result (such as concentration or copy number) of the target nucleic acid.

[0169] In another preferred embodiment, the device is a digital PCR detection device.

[0170] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0171] Figure 1 The fluorescence intensity generated by trans-cutting probes with different concentrations of Cas12b and sgRNA when dsDNA and ssDNA are used as target DNA, respectively, is shown.

[0172] Figure 2 The figure shows the effect of different bases at the PAM sequence on the trans-cutting fluorescence detection intensity when dsDNA and ssDNA are used as target DNA respectively.

[0173] Figure 3 The trans cutting rate of Cas12b when ssDNA or dsDNA is used as the target is shown.

[0174] Figure 4 The trans-cutting fluorescence intensity of Cas12b targeting dsDNA or ssDNA under different temperature conditions is shown.

[0175] Figure 5 The detection sensitivity (i.e., fluorescence intensity of trans cleavage) of Cas12b targeting dsDNA or ssDNA is shown by gradient dilution of the target sequence.

[0176] Figure 6The detection sensitivity of Cas12b combined with LAMP amplification reaction (i.e., HOLMES v2.0 method) to the target is shown.

[0177] Figure 7 Using sgRNAs with different guide sequence lengths, the effects of single-base mutations in positions 1-12 of the ssDNA target sequence on the fluorescence intensity of Cas12b trans cutting were determined.

[0178] Figure 8 Using a 16nt sgRNA as the guide sequence, the effects of single-base mutations in positions 1-16 of the ssDNA target sequence on the fluorescence intensity of Cas12b trans cutting were measured.

[0179] Fig. 9 Using a 16nt sgRNA as the guide sequence, the effects of single-base mutations in positions 1-16 of the ssDNA target sequence to any other three bases on the fluorescence intensity of Cas12b trans cutting were measured.

[0180] Fig.10 Using a 16nt sgRNA as the guide sequence, the effects of single-base mutations at positions 8-16 of three different ssDNA target sequences (rs5082, rs1467558, and rs2952768) on the fluorescence intensity of Cas12b trans cutting were measured.

[0181] Fig.11 Using sgRNAs with different guide sequence lengths, the effect of any single base mutation in positions 1-16 of the dsDNA target sequence on the fluorescence intensity of Cas12b trans cutting was determined.

[0182] Fig.12 It shows that LAMP and Cas12b detection can be achieved in a one-step reaction under the conditions of 50°C, 55°C, 60°C, and 65°C.

[0183] Fig.13 HOLMES v2.0 (LAMP combined with Cas12b) tests the gyrB site of E. coli, identifies the presence of E. coli, and tests the sensitivity of detection by gradient dilution. (After the plate test, 0.005 OD of E. coli is about 7,000)

[0184] Fig.14 HOLMES v2.0 (LAMP combined with Cas12b) tests the Y chromosome sry site and uses saliva samples to identify gender (DNMT1-3 sites are gene sites on the chromosome and serve as positive controls).

[0185] Fig.15Asymmetric or symmetric PCR amplification combined with Cas12b to detect SNP sites (rs5082). It can be seen that since there is no PAM sequence near the rs5082 site, asymmetric PCR that produces single-stranded DNA can more clearly distinguish SNP sites.

[0186] Fig.16 By designing primers, after introducing the PAM sequence through LAMP amplification, the SNP site (rs5082) is detected in combination with Cas12b. It can be seen that since there is no PAM sequence near the rs5082 site, a LAMP amplification method with PAM added (with PAM sequence in the primer) is generated, which can better distinguish the SNP site.

[0187] Fig.17 Detection of RNA virus JEV (Japanese encephalitis virus) by HOLMES v2.0 (LAMP combined with Cas12b).

[0188] Fig.18 HOLMES v2.0 (LAMP combined with Cas12b) one-step method for quantifying trace amounts of DNA. By diluting template DNA of different concentrations, the fluorescence value is detected in real time in a fluorescence quantifier using the LAMP-Cas12b one-step method (55°C reaction).

[0189] Fig.19 The time point when the fluorescence value reaches 600,000 is used as the y-axis (using Fig.18 The data in the table are plotted with the absolute value of the concentration as the X-axis to obtain a trend line that approximates a straight line. This equation can be used to quantify the target sequence.

[0190] Fig. 20 Digital HOLMES (LAMP combined with Cas12b and clarity chip) method for absolute quantitative detection of nucleic acid samples.

[0191] Fig.21 This is the schematic diagram of HOLMESv2 detecting target DNA methylation. After bisulfite conversion and PCR amplification, the C base in the unmethylated CpG site will be converted to T; by designing an sgRNA guide sequence that completely matches the methylated target site, and using the HOLMESv2 reaction (i.e., adding Cas12b, sgRNA, and a single-stranded DNA probe labeled with a fluorescent group at one end and a quenching group at the other end), the content of methylated sites in the system is detected. Similarly, the content of unmethylated sites in the system can be detected by designing an sgRNA guide sequence that completely matches the unmethylated target site, and using the HOLMESv2 reaction to detect the content of unmethylated sites in the system, and then the ratio of methylated sites can be calculated.

[0192] Fig. 22 It is the experimental result of detecting target DNA methylation by sgRNA of different sites and lengths. target-C, the test result of this site is C (representing methylation); target-T, the test result of this site is T (representing the base before bisulfite conversion treatment is not methylated). The signal value of the same sgRNA sequence targeting target-C is high, while the signal value targeting target-T is low, which can be used to distinguish whether the site is modified by methylation.

[0193] Fig.23 This is a schematic diagram of the principle of using m3-C12-17 site sgRNA to detect target DNA methylation. The left picture shows that the sgRNA sequence and the target sequence are completely matched; the right picture shows that after the non-methylated site is treated with bisulfite and PCR amplified, the M3 site becomes T, resulting in the sgRNA sequence and the target sequence not being completely matched.

[0194] Fig.24 The results are the test results of the methylation level of the M3 CpG site (COL1A2) in 4 cell lines. The results of 0%, 10%, 30% and 50% are the test results for making the standard curve, and 293T, SW480, NCI-N87 and MCF-7 are the actual test values ​​of this site in the 4 cell lines; from left to right, it represents 3 independent repeated experiments, and each experiment was repeated 3 times.

[0195] Fig.25 is the standard curve of the methylation level of the M3 CpG site (COL1A2). Figure 5 Draw a standard curve graph based on the results of the standard curve test.

[0196] Fig.26 These are the test results of the methylation rate of the M3 CpG site (COL1A2) in four cell lines (293T, SW480, NCI-N87, MCF-7). As can be seen from the figure, the test results of the HOLMESv2 system are closest to the NGS sequencing results (High-throughput sequencing).

[0197] Fig. 27The results of circular RNA detection using HOLMESv2 technology are shown. (Top) Schematic diagram of HOLMESv2 detection of RNA and target site selection; (Lower left) Result diagram of HOLMESv2 detection of target GAPDH; (Lower right) Result diagram of HOLMESv2 detection of target CDR1as. target site, target site; NTC, is the experimental control group, that is, the sample is sterile water; target is the experimental group, that is, the sample is an RNA sample; the fluorescence values ​​in the lower left and lower right figures are the fluorescence values ​​after deducting the experimental background fluorescence. Both the NTC and target reaction groups were amplified by LAMP reaction, and their amplified products were used for Cas12b reaction testing; and the background fluorescence is the fluorescence value of the Cas12b reaction system directly added with sterile water for testing. DETAILED DESCRIPTION

[0198] The inventors have conducted extensive and in-depth research and have developed a technical solution for target nucleic acid detection for the first time through research on the cutting characteristics of Cas12b. The experimental results show that the technical solution of the present invention can detect trace nucleic acids quickly, with high sensitivity and high accuracy, and can be applied to different fields such as rapid identification of gender in saliva samples, detection of Escherichia coli contamination, rapid identification of SNP genotypes, RNA virus detection, and concentration determination of trace DNA samples. The present invention was completed on this basis.

[0199] the term

[0200] The term "guide RNA" or "gRNA" or "sgRNA" refers to an RNA that guides a Cas protein (such as a Cas12b protein) to specifically bind to a target DNA sequence.

[0201] The term "CRISPR" refers to clustered regularly interspaced short palindromic repeats, which are the immune system of many prokaryotes.

[0202] The term "Cas protein" refers to CRISPR-associated protein, which is the associated protein in the CRISPR system.

[0203] The term "Cas12a" (formerly known as "Cpf1") refers to a crRNA-dependent endonuclease, which is a type VA enzyme in the CRISPR system classification.

[0204] The terms "Cas12b" and "C2c1" are used interchangeably to refer to the sgRNA-dependent endonuclease, which is a type VB enzyme in the CRISPR system classification.

[0205] The term "LAMP" stands for loop-mediated isothermal amplification, which is a constant temperature nucleic acid amplification technology suitable for genetic diagnosis.

[0206] The term "PAM" refers to the protospacer-adjacent motif, which is necessary for Cas12b to cut double-stranded DNA. The PAM of AacCas12b is the TTN sequence.

[0207] The term "PCR" refers to "polymerase chain reaction", a method used to amplify a target DNA fragment in large quantities.

[0208] HOLMES v2.0 refers to one-HOur Low-cost Multipurpose highly Efficient System version 2.0, which is a nucleic acid detection method based on Cas12b.

[0209] Detection Methods

[0210] The present invention discloses a method for detecting a target nucleic acid molecule. A guide RNA, a Cas protein, a nucleic acid probe and a buffer are placed in a reaction system containing the target nucleic acid molecule to be detected, and then fluorescence detection is performed on the target nucleic acid molecule.

[0211] The present invention provides a detection method for rapidly detecting target nucleic acid molecules with high specificity. Once the target DNA (single-stranded or double-stranded), sgRNA and Cas12b protein form a ternary complex, the complex will cut other single-stranded DNA molecules in the system.

[0212] By designing sgRNA to target the target DNA (a DNA sequence to be detected); adding sgRNA and Cas12b protein to the detection system; when the target DNA exists (single-stranded DNA or double-stranded DNA), Cas12b forms a ternary complex with sgRNA and target DNA, and the complex simultaneously performs its bypass cutting activity and cuts the nucleic acid probe labeled with a fluorescent signal, thereby emitting fluorescence. .

[0213] A typical nucleic acid probe is a single-stranded DNA with a luminescent group and a quencher group attached to both ends, so that once the probe is cut, the luminescent group can emit light.

[0214] In the present invention, whether the system to be detected contains target DNA molecules can be known by detecting fluorescence.

[0215] In the present invention, the suitable Cas protein is Cas12b, and the preferred Cas12b is AacCas12b (Alicyclobacillus acidoterrestris), Aac2Cas12b (Alicyclobacillus acidiphilus), AkaCas12b (Alicyclobacillus kakegawensis), AmaCas12b (Alicyclobacillus macrosporangiidus), AheCas12b (Alicyclobacillus herbarius), and AcoCas12b (Alicyclobacillus contaminans).

[0216] The target nucleic acid molecule to be detected in the reaction system of the target nucleic acid molecule to be detected may be unamplified, or obtained after amplification, and / or after reverse transcription amplification.

[0217] The detection method of the present invention can detect nucleic acid molecules of different species, such as nucleic acid molecules of mammals, plants, or microorganisms and viruses. The method of the present invention is particularly suitable for detecting pathogenic microorganisms, gene mutations or specific target DNA or RNA.

[0218] The method of the present invention can be used to quickly detect whether a sample contains a specific DNA sequence. In addition, by combining with amplification techniques (such as LAMP, PCR, asymmetric PCR, RPA, etc.), the sensitivity of the detection method can be greatly improved.

[0219] Preferably, the bypass cleavage of Cas12b can be combined with nucleic acid amplification (such as isothermal amplification, LAMP amplification) and other techniques (such as digital PCR).

[0220] In a preferred embodiment of the present invention, when the detection of bypass cleavage based on Cas12b is combined with nucleic acid amplification, the detection sensitivity can be increased to 10 -8 nM or lower concentrations.

[0221] In addition, when the detection of Cas12b-based bypass cleavage is combined with digital PCR, the detection sensitivity can be increased to 1 copy / 1 reaction system (such as a microdroplet), which can almost meet all detection sensitivity needs.

[0222] Cas12b-based nucleic acid detection method and HOLMES v2.0 (one-HOur Low-cost Multipurpose highly Efficient System version 2.0)

[0223] Establishment of nucleic acid detection method

[0224] Using the characteristics of Cas12b, the present invention has developed a method for specific detection of nucleic acid molecules, called HOLMESv2.0 (one-HOur Low-cost Multipurpose highly Efficient System version 2.0). As the name of the technology suggests, it is characterized by a one-hour, low-cost, multi-purpose, high-efficiency, and very simple test method.

[0225] In the entire reaction system, it can be divided into two major steps, one is the amplification of the template nucleic acid, and the other is the specific nucleic acid detection of the Cas12b protein; or these two steps can be combined into one step.

[0226] In the present invention, in a preferred example, the LAMP method or the asymmetric PCR method is used for nucleic acid amplification, but in fact, any amplification method can be combined with the second step of nucleic acid detection, such as the isothermal amplification method RPA.

[0227] The initial nucleic acid is not limited to double-stranded DNA, but can also be single-stranded DNA or RNA, so the method is applicable to various types of nucleic acid molecules.

[0228] For the nucleic acid detection stage, three components are the key to the experiment, namely Cas12b, sgRNA and nucleic acid probe. In addition to the AacCas12b mentioned in the embodiment, other Cas12b proteins are also applicable to this method. In addition, other types of Cas proteins (such as Cas12c proteins) are also applicable to the scope of protection of the present invention.

[0229] For the sgRNA that acts as a guide, it will be more stable in the system after artificial modification. In the selection of nucleic acid probes, the present invention uses short single-stranded DNA labeled with HEX and BHQ1 (FAM and Eclipse are also used for labeling). Any other detectable labeling method is theoretically applicable as long as the nucleic acid probe produces a detectable difference after being cut. Alternatively, the nucleic acid probe can also be designed to fluoresce after binding to the compound, thereby detecting whether the probe is cut.

[0230] For ease of understanding, various different characteristics of the nucleic acid detection method based on Cas12b of the present invention (including the preferred "HOLMES" v2.0 method) are further described.

[0231] Identification of Cas12b trans-cutting activity: Although Cas12a and Cas12b differ greatly in sequence, the experimental results of the inventors show that Cas12b also has trans-cutting activity.

[0232] First, the bypass DNA is designed into a fluorescent probe, which is composed of a 12nt random sequence, and is labeled with a fluorescent group HEX at the 5' end and a quenching group BHQ1 (HEX-N12-BHQ1) at the 3' end. When the system contains a target DNA fragment (dsDNA or ssDNA), a ternary complex of target DNA, sgRNA and Cas12b protein is formed. At this time, the probe is cut, and the HEX fluorescent group will emit fluorescence (excitation light 535nm, emission light 556nm) through the detection of the fluorescence detector.

[0233] like Figure 1 As shown, when the concentration of Cas12b and sgRNA is 250nM or 500nM, both dsDNA and ssDNA as targets have higher fluorescence intensity. When the concentration of Cas12b and sgRNA is reduced to 50nM and 100nM, ssDNA as the target sequence still has higher fluorescence intensity.

[0234] PAM properties of Cas12b trans cutting: Studies have shown that when Cas12b cis cuts double-stranded DNA (cis cutting), a sequence called PAM is required, that is, 5'-TTN-3' is immediately followed by the 5' end to be cut by Cas12b. In order to test whether the target sequence of Cas12b requires PAM during trans cutting, the inventors designed different sequences (including TTC, TAC, ATC, AAC, GGC and CCC) at the PAM position to test whether PAM is required during trans cutting.

[0235] like Figure 2 As shown, when double-stranded DNA is used as the target, the trans cutting of Cas12b is more sensitive to the PAM sequence, that is, TTC can have a higher trans cutting fluorescence intensity (the fluorescence intensity produced after becoming TAC is slightly lower than TTC), while when single-stranded DNA is used as the target, it is insensitive to the PAM sequence and always shows a higher trans cutting fluorescence intensity.

[0236] Cas12b trans cutting rate: When ssDNA and dsDNA are used as target DNA, the trans cutting rate of Cas12b is measured.

[0237] like Figure 3As shown, it can be seen that when ssDNA is used as a substrate, the trans cutting rate of Cas12b is very fast, and it is close to the maximum value in only 6 minutes. When dsDNA is used as a substrate, the cutting speed is relatively slow, and there is a gradual increase process. This suggests that for some occasions that require rapid detection (such as ≤10 minutes), ssDNA should be used as a substrate for Cas12 bypass cutting.

[0238] Applicable temperature range of Cas12b trans cutting: Cas12b has a very wide temperature range for trans cutting. Whether it is ssDNA or dsDNA as the target sequence, the temperature range is 45-65℃, and the temperature range of ssDNA target is wider, 25-70℃ (see Figure 4 ).

[0239] Cas12b detection sensitivity: The inventors tested the detection sensitivity of Cas12b by diluting the concentration of target dsDNA or ssDNA. In a preferred embodiment, the target ssDNA (DNMT1-3 (TTC PAM) -R) or dsDNA (DNMT1-3 (TTCPAM) -F, DNMT1-3 (TTC PAM) -R oligonucleotides produced by annealing) was diluted to a concentration of 100nM, 10nM, 1nM, ... to 10 -4 nM.

[0240] The results showed that when the DNA concentration was 1 nM, the target molecule could still be detected by direct detection with Cas12b ( Figure 5 ).

[0241] HOLMES v2.0 sensitivity test: In one embodiment, when Cas12b and LAMP reactions are combined (i.e., HOLMES v2.0), the sensitivity of the detection can reach 10 -8 nM, greatly improving the sensitivity of target nucleic acid sequence detection ( Figure 6 ).

[0242] SNP testing for ssDNA targets: The methods of the present invention are well suited for detecting nucleic acid mutations, including SNPs, especially SNPs in ssDNA targets.

[0243] The SNP detection of the target single-stranded DNA showed that when the sgRNA guide sequence was 18nt or 20nt, the mutation of bases 1-12 had little effect on the fluorescence signal generated by trans cutting. When the sgRNA guide sequence was 14nt or 15nt, the fluorescence values, including the wild-type control, were very low. When the sgRNA guide sequence was 16nt, the fluorescence value of bases 10-12 was significantly reduced compared to the control ( Figure 7 ).

[0244] Further studies have found that when the guide sequence is 16nt sgRNA, the effect of base mutations at positions 10-16 on the fluorescence value is very obvious, especially at positions 10-14 where the fluorescence value is almost undetectable ( Figure 8 ).

[0245] In addition, when the target single-stranded DNA mutates into different types of bases at different positions, there is no essential effect on the fluorescence value. Only the fluorescence value changes greatly when the 9th position mutates into G ( Fig. 9 ).

[0246] In addition, the inventors also tested three other target sequences. Although the fluorescence change values ​​of different sequences for a certain site mutation were different, they could all find sites with obvious changes at positions 8-16, such as positions 8, 10, 11, and 12 of rs5082, position 11 of rs1467558, and positions 8-15 of rs2952768 ( Fig.10 ).

[0247] SNP testing for dsDNA targets: The methods of the present invention are suitable for detecting nucleic acid mutations, including SNPs in dsDNA targets.

[0248] The SNP detection of the target double-stranded DNA shows that the fluorescence signal generated by single base mutations on trans cutting is different from that of ssDNA. For example, sgRNA with a guide sequence of 18-20nt has different degrees of sensitivity to base mutations at positions 10-16 (although the fluorescence signal also decreases to varying degrees after mutations at positions 1 and 3) ( Fig.11 ). Generally, these sgRNAs are most sensitive to positions 10 and 16.

[0249] One-step detection at different temperatures: The present invention also provides a one-step detection combining nucleic acid amplification with Cas12b-based detection.

[0250] Preferably, LAMP amplification and Cas12b detection can be combined to achieve one-step detection. In the present invention, the trans cutting of Cas12b was tested at different temperatures (50°C, 55°C, 60°C, and 65°C). Fig.12 As shown, at 50 and 55°C, one-step detection can be achieved, especially at 55°C, the fluorescence value rises faster.

[0251] RNA target testing: The method of the present invention is not only suitable for the detection of target DNA, but also can be used for the detection of target RNA. In the present invention, RNA can be reverse transcribed to form DNA first, and then the target sequence can be detected.

[0252] A preferred method is to add reverse transcriptase to the reaction system to achieve reverse transcription of RNA; then amplify with, for example, Bst 2.0 DNA polymerase, and achieve trans cleavage with Cas12b. Another method is to directly use Bst3.0 DNA polymerase (this enzyme can amplify RNA templates to produce DNA) to directly achieve reverse transcription and amplification of RNA. ( Fig.17 )

[0253] Quantitative testing of trace amounts of DNA templates: The method of the present invention can be used for quantitative testing.

[0254] In one example, a one-step method based on LAMP amplification and Cas12b was used at 55°C to quantitatively test trace amounts of DNA. By diluting templates of different concentrations, the fluorescence value of trans cutting was detected in real time, and a standard curve was prepared. After calculation, the concentration of the target template sequence can be obtained ( Fig.18 and 19 ).

[0255] Another quantitative detection is to combine the Cas12b-based detection method of the present invention with digital PCR technology (see "Digital HOLMES Method" below).

[0256] Primer design and sgRNA design

[0257] Based on the teachings of the present invention, those skilled in the art can synthesize corresponding primers and / or sgRNA according to actual needs to perform nucleic acid detection based on Cas12b of the present invention. In the present invention, some preferred primers and sgRNA designs can refer to the following suggestions.

[0258] For primer design, please refer to the following:

[0259] Case 1. Pre-amplification reaction, if the LAMP method is selected, the conventional method can be used for primer design (http: / / primerexplorer.jp / elamp4.0.0 / index.html). It is recommended to add LoopF ​​and LoopB primers between primer regions F2 / F3 and B2 / B3, that is, 6 pairs of primers.

[0260] Case 2. Preliminary amplification reaction: If an asymmetric PCR method is selected, several primers need to be selected to test the most suitable combination for producing single-stranded DNA.

[0261] Case 3. When testing SNP, if there is a suitable PAM site near the site, use case 1; if there is no suitable PAM site, a PAM site can be introduced into the primer so that the product produces a PAM site. Usually, the SNP site is 8-16 bases away from the PAM, but the specific target sequence still needs to be tested specifically.

[0262] Case 4. When it is difficult to find suitable LAMP amplification primers near the SNP site or it is impossible to introduce a suitable PAM, asymmetric PCR amplification and other methods can be used to amplify single-stranded DNA.

[0263] For sgRNA design, you can refer to the following situations:

[0264] Case 1. For the detection of target genes, it is generally advisable to select sgRNA with 20bp complementary pairing, and the target sequence has a PAM sequence.

[0265] Scenario 2. For SNP detection, if double-strand amplification such as LAMP is used as the amplification step, it is advisable to first test the position of the SNP in the target sequence. Prioritize the sgRNA with a guide sequence of 18 nt in length, and test at positions 10-16.

[0266] Case 3. For SNP detection, if single-strand amplification such as asymmetric PCR is used as the amplification step, it is advisable to first test the position of the SNP in the target sequence. Priority should be given to testing sgRNAs with a 16nt guide sequence, and testing at positions 10-16, especially 10-12.

[0267] Digital HOLMES method

[0268] In the present invention, a technique combining the HOLMES technique of the present invention with digital PCR (dPCR) is also provided, which is referred to as the "digital HOLMES method"

[0269] Digital PCR is an absolute quantitative technology for nucleic acid molecules that has developed rapidly in recent years. Currently, there are two main types of digital PCR: droplet digital PCR (ddPCR) and chip digital PCR (cdPCR). That is, before traditional PCR amplification, the sample is processed into droplets or added to the chip to divide the reaction system containing nucleic acid molecules into thousands of nanoliter-scale reaction systems. Compared with ordinary PCR, digital PCR has an additional pre-treatment step and a later fluorescence detection step. At present, digital PCR detection still requires the use of an ordinary PCR instrument to perform the amplification step, and the conditions of the PCR reaction are relatively strict and take a long time (about 3-4 hours).

[0270] By using the HOLMES method in combination with the current fluorescence reading instrument for chip digital PCR (ie, the digital HOLMES method), absolute quantitative detection can be performed by simply maintaining a constant temperature.

[0271] In addition, when digital HOLMES is amplified using methods such as LAMP, it can be amplified in a wide temperature range (50-70°C, or around 50-55°C), without the need for precise temperature control like conventional PCR instruments. Since Cas12b also has bypass cleavage activity within this temperature range, it can be effectively integrated and amplified and bypassed at the same time.

[0272] The present invention also provides a device for digital HOLMES detection, in particular, the device as described in the seventh aspect of the present invention.

[0273] Therefore, using digital HOLMES technology to integrate pre-treatment, amplification and detection into one instrument can not only reduce human operating errors, but also speed up detection and reduce instrument costs.

[0274] Reagent test kit

[0275] The present invention also provides a kit, including guide RNA, Cas12b protein, and nucleic acid probe. In addition, the kit of the present invention may also include other reagents, such as buffer, reagents required for amplification, reagents required for reverse transcription, or a combination thereof.

[0276] Typically, the kit of the invention comprises:

[0277] i) a first container and a Cas12b protein in the first container, wherein the Cas12b protein is Cas12b or a Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b;

[0278] ii) an optional second container and a guide RNA located in the second container, wherein the guide RNA guides the Cas protein to specifically bind to a target nucleic acid molecule;

[0279] iii) a third container and a nucleic acid probe (preferably a fluorescent probe) located in the third container;

[0280] iv) an optional fourth container and a buffer within the fourth container;

[0281] Wherein, the target nucleic acid molecule is target DNA.

[0282] In addition, the kit of the present invention may further include:

[0283] v) a fifth container and a polymerase for amplifying the target DNA located in the fifth container;

[0284] vi) an optional sixth container and a reverse transcriptase for reverse transcription located in the sixth container;

[0285] vii) a seventh container and dNTPs in the seventh container for an amplification reaction and / or a reverse transcription reaction.

[0286] In the present invention, one or more or all of the containers may be the same or different containers.

[0287] The main advantages of the present invention are:

[0288] Utilizing the characteristics of Cas12b, the inventors have developed a method for specific detection of nucleic acid molecules, called HOLMESv2.0 (one-HOur Low-cost Multipurpose highly Efficient System version 2.0). It is characterized by being fast (1 hour), low-cost, multi-channel, efficient and simple. The method can be used in the fields of rapid pathogen detection, SNP detection, etc. Compared to the first-generation HOLMES based on Cas12a, the second-generation HOLMES based on the high-temperature enzyme Cas12b has more advantages.

[0289] (1) Fast: When the test conditions are ready, it only takes about 1 hour from getting the sample to getting the test results.

[0290] (2) Low cost: There are no special materials or enzymes in the experiment, and the materials and reagents involved are relatively small, so micro-quantitative testing and analysis can be performed.

[0291] (3) High efficiency: The present invention has extremely high sensitivity and can detect DNA at a concentration of 10aM.

[0292] (4) Multi-purpose: It can detect different nucleic acid samples, including DNA samples and RNA samples.

[0293] (5) Simple: There are no special and complicated steps. Once the kit is made and the program is set, you only need to simply add the sample and perform other operations.

[0294] (6) One-step reaction: The amplification reaction and the detection reaction can be carried out simultaneously to achieve a one-step test.

[0295] (7) Isothermal: Since LAMP amplification and Cas12b detection are both performed under constant temperature conditions, the instrument requirements are lower.

[0296] (8) Wide reaction temperature range: Cas12b can be detected at 45-65°C, so the instrument requirements are relatively low.

[0297] (9) Two enzymes are needed to achieve amplification and detection of DNA or RNA: Since Bst 3.0 DNA polymerase has DNA polymerase activity using DNA or RNA as a template, only two enzymes, Bst 3.0 and Cas12b, are needed to achieve amplification and detection of DNA or RNA, saving production costs.

[0298] (10) Highly sensitive SNP detection: By rationally designing sites and sgRNA, single base differences can be distinguished with high sensitivity.

[0299] (11) Quantitative detection can be achieved: Through the one-step HOLMES method for real-time fluorescence detection, or combined with the chip and detection system of a digital PCR instrument, quantitative detection of samples can be achieved.

[0300] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0301] In this application, unless otherwise stated, the Cas12b trans-cleavage activity system is: 250 nM of sgRNA and Cas12b, and the reaction conditions are 48°C for 30 min.

[0302] Material

[0303] 1. RNase inhibitor was purchased from TaKaRa, high-fidelity DNA polymerase KOD FX was purchased from ToYoBo; primers (oligonucleotides) were synthesized by Shanghai Bioengineering; T7 RNA polymerase was purchased from Thermo; RNA purification and concentration kit (RNAClean & Concentrator TM -5) purchased from Zymo Research; SV Gel and PCR Clean-Up System were purchased from Promega; culture media (eg, Tryptone, Yeast Extract, etc.) were purchased from OXOID.

[0304] 2. Culture medium formula: Liquid LB (1% Tryptone, 0.5% Yeast extract, 1% NaCl). When preparing solid LB, just add 2% agar to the liquid LB.

[0305] Example 1 Cas12b protein detects double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) targets

[0306] Single-stranded DNA or double-stranded DNA (target T1) was selected as the target sequence, and the response values ​​of Cas12b protein and sgRNA at different concentrations were tested.

[0307] 1. Preparation of guide RNA (sgRNA)

[0308] First, the plasmid pUC18-sgRNA-T1 was constructed with pUC18 as the plasmid backbone. The plasmid had a T7 promoter and a template DNA sequence for transcribing sgRNA inserted into pUC18 (Note: the sgRNA transcribed from this template targets a sequence called T1 in this study). The method was to use the pUC18 plasmid as a template, pUC18-1-F and pUC18-1-R as primers, perform a round of PCR, connect the PCR products with T4 DNA Ligase, transform into DH10b, and obtain the correct clone after sequencing, which was called pUC18-sgRNA-T1-pre. Then, using pUC18-sgRNA-T1-pre as a template, pUC18-2-F and pUC18-2-R as primers, perform a second round of PCR, connect the PCR products and transform in the same way, and finally obtain the plasmid pUC18-sgRNA-T1 with correct sequencing.

[0309] Then, using plasmid pUC18-sgRNA-T1 as a template, T7-crRNA-F and sgRNA-T1-R as primers, PCR was used to amplify the DNA template required for in vitro transcription. Dpn I was then added to the PCR product (1 μl Dpn I (10 U / μl) was added to each 50 μl PCR system), and the plasmid DNA template was digested in a 37°C water bath for 30 min, and the PCR product was recovered using a gel and PCR product column recovery kit (Promega). Using this PCR recovery product as a template, the T7 high-yield transcription kit (Thermo) was used to synthesize sgRNA (named sgRNA-T1), and the reaction was carried out at 37°C overnight (12-16h).

[0310] Finally, DNase I was added to the transcription system (2 μl DNase I (5 U / μl) for every 50 μl transcription system), and the plasmid DNA template was eliminated by water bath at 37°C for 30 min. RNA was purified using an RNA purification and concentration kit, quantified using NanoDrop 2000C, and stored in a -80°C refrigerator for later use.

[0311] 2. Preparation of target DNA

[0312] (1) If the target DNA is single-stranded, directly synthesize an oligonucleotide target DNA (T1-R) containing the 20 bp target sequence (T1) recognized by the sgRNA.

[0313] (2) If the target DNA is double-stranded, directly synthesize two complementary oligonucleotides (T1-F; T1-R), which contain the 20bp target sequence (T1) recognized by sgRNA. Anneal the two oligonucleotides to obtain double-stranded target DNA. Specifically, the paired oligonucleotides (2μM) were mixed in 1× PCR buffer (Transgen Biotech) with a total volume of 20μL, and then annealed: initially denatured at 95℃ for 5 minutes, then cooled from 95℃ to 20℃, and reduced by 1℃ per minute using a thermal cycler.

[0314] 3. Cas12b reaction

[0315] (1) sgRNA annealing: dilute sgRNA to an appropriate concentration and anneal in a PCR instrument. Annealing procedure: denature at 75°C for 5 min, then cool from 75°C to 20°C, decreasing by 1°C per minute.

[0316] (2) Incubation of sgRNA and Cas12b: Mix the annealed sgRNA with Cas12b of equal molar concentration and place at 30°C for 20-30 minutes.

[0317] (3) Cas12b reaction: In a 20 μl reaction system, add the mixture of sgRNA and Cas12b incubated in step (2) (final concentrations of both are 50, 100, 250 or 500 nM), fluorescent quenching probe (HEX-N12-BHQ1, final concentration 500 nM), 2 μl of 10×NEB Buffer 3.1 and 0.5 μl of RNase inhibitor (40 U / μl). After mixing, react at 48°C for 30 min. Then, heat at 98°C for 5 min to inactivate.

[0318] 4. Detection of bypass single-stranded DNA cleavage activity of Cas12b by fluorescence microplate reader

[0319] The inactivated 20 μL reaction solution was added to a 96-well plate and detected using an ELISA reader (excitation light 535 nm, emission light 556 nm).

[0320] The results are as follows Figure 2 As shown, when the concentration of Cas12b and sgRNA is 250nM or 500nM, both dsDNA and ssDNA as targets have higher fluorescence intensity. When the concentration of Cas12b and sgRNA is reduced to 50nM and 100nM, ssDNA as the target sequence still has higher fluorescence intensity.

[0321] Example 2 HOLMES v2.0 (LAMP combined with Cas12b) test response sensitivity

[0322] By detecting the fluorescence intensity of the excited fluorescent probe (HEX-N12-BHQ1), the target DNA concentration required for Cas12b to exercise the bypass single-stranded DNA cleavage activity, that is, the sensitivity of the Cas12b bypass single-stranded DNA cleavage reaction, is determined.

[0323] 1. Preparation of sgRNA

[0324] First, using the previously constructed plasmid pUC18-sgRNA-T1 as a template, primers named sgRNA-DNMT1-3-F and sgRNA-DNMT1-3-R were designed. Through PCR, the 20 bases of the target DNA T1 in the sgRNA were replaced with the sgRNA targeting DNMT1-3 to obtain another plasmid pUC18-sgRNA-DNMT1-3.

[0325] Then, the DNA template required for in vitro transcription was amplified by PCR using plasmid pUC18-sgRNA-DNMT1-3 as a template and T7-crRNA-F and ZLsgRNA-DNMT1-3-R as primers. Dpn I was then added to the PCR product (1 μl Dpn I (10 U / μl) was added to each 50 μl PCR system), and the plasmid DNA template was digested in a 37°C water bath for 30 min, and the PCR product was recovered using a gel and PCR product column recovery kit (Promega). Using this PCR recovery product as a template, the sgRNA (named sgRNA-DNMT1-3) was synthesized using a T7 high-yield transcription kit (Thermo), and the reaction was carried out overnight at 37°C (12-16 h).

[0326] Finally, DNase I was added to the transcription system (2 μl DNase I (5 U / μl) for every 50 μl transcription system), and the plasmid DNA was eliminated by water bath at 37°C for 30 min. RNA was purified using an RNA purification and concentration kit, quantified using NanoDrop 2000C, and stored in a -80°C refrigerator for later use.

[0327] 2. Preparation of target DNA

[0328] For target DNA, the first method is to directly add Cas12b to the reaction system without amplification. The method is as follows:

[0329] (1) If the target DNA is single-stranded, directly synthesize a 50 bp oligonucleotide as the target DNA (DNMT1-3(TTC PAM)-R), which contains the 20 bp target sequence (DNMT1-3) recognized by the sgRNA.

[0330] (2) If the target DNA is double-stranded, two complementary 50-bp oligonucleotides (DNMT1-3(TTCPAM)-F; DNMT1-3(TTC PAM)-R) are directly synthesized, which contain the 20-bp target sequence (DNMT1-3) recognized by the sgRNA. The two oligonucleotides are annealed to obtain double-stranded target DNA. Specifically, the paired oligonucleotides (2 μM) are annealed in 1× PCR buffer (Transgen Biotech) with a total volume of 20 μL, and then the annealing procedure is performed: initial denaturation at 95°C for 5 minutes, followed by cooling from 95°C to 20°C, and decreasing by 1°C per minute using a thermal cycler.

[0331] (3) Dilute the single-stranded or double-stranded target DNA in a gradient manner to 2 μM, 0.2 μM, 0.02 μM, 0.002 μM, and 0.0002 μM for later use.

[0332] The second method is to insert a fragment containing the target sequence (DNMT1-3) into a plasmid vector and amplify it through LAMP reaction.

[0333] (1) Using the pEasy-Blunt Zero Cloning Kit from Transgen, the fragment containing the target sequence (DNMT1-3) was inserted into the pEasy-Blunt Zero Cloning Vector, and the correct clone was obtained after sequencing verification.

[0334] (2) LAMP expansion reaction

[0335] The above plasmid was used as a template for LAMP amplification reaction. The template was added at 0, 1 nM, 0.1 nM, and then diluted 10 times to 10 -11 nM. The total volume of each reaction system was 25 μL, and the primers were 1.6 μM LAMP-DNM-FIP and LAMP-DNM-BIP, 0.2 μM LAMP-DNM-F3 and LAMP-DNM-B3, 0.4 μM LAMP-DNM-LoopF ​​and LAMP-DNM-LoopB. The kit used for LAMP reaction was LAMP Kit (NEB). The LAMP reaction procedure is 65℃ for 30min. After LAMP is completed, inactivate at 85℃ for 10min and then directly use it for Cas12b reaction.

[0336] 3. Cas12b reaction

[0337] (1) sgRNA annealing: dilute sgRNA to an appropriate concentration (5 μM) and anneal in a PCR instrument. Annealing procedure: denature at 75°C for 5 min, then cool from 75°C to 20°C, decreasing by 1°C per minute.

[0338] (2) Incubation of sgRNA and Cas12b: Mix the annealed sgRNA with Cas12b of equal molar concentration and place at 30°C for 20-30 minutes.

[0339] (3) Cas12b reaction: In a 20 μl reaction system, add the mixture of sgRNA and Cas12b incubated in step (2) (both final concentrations are 250 nM), 1 μl of target DNA or 1 μl of LAMP product, fluorescent probe (HEX-N12-BHQ1) (final concentration 500 nM), and 2 μl of 10×NEB Buffer 3.1 and 0.5 μl of RNase inhibitor (40 U / μl). After mixing, react at 48°C for 30 min. Then, heat at 98°C for 5 min to inactivate.

[0340] 4. Detection of bypass single-stranded DNA cleavage activity of Cas12b by fluorescence microplate reader

[0341] The inactivated 20 μL reaction solution was added to a 96-well plate and detected using an ELISA reader (excitation light 535 nm, emission light 556 nm).

[0342] like Figure 5 As shown in the figure, it can be seen that Cas12b can detect single-stranded or double-stranded DNA at a concentration of 1 nM; after combining with LAMP amplification, Cas12b (i.e., HOLMES v2.0 method) can detect 10 -8 nM concentration of DNA ( Figure 6 ).

[0343] Example 3 Cas12b testing single base mutation target

[0344] Single-stranded DNA or double-stranded DNA is selected as the target sequence to test the change in the Cas12btrans cutting fluorescence signal when there is a single base mutation in the target DNA, thereby detecting the single base mutation.

[0345] 1. Preparation of guide RNA (sgRNA)

[0346] The DNA template required for in vitro transcription was amplified by PCR using plasmid pUC18-sgRNA-DNMT1-3 as a template, T7-crRNA-F as an upstream primer, and oligonucleotides containing guide sequences complementary to different targets as downstream primers. Dpn I was then added to the PCR product (1 μl Dpn I (10 U / μl) was added to each 50 μl PCR system), and the plasmid DNA template was digested in a 37°C water bath for 30 min, and the PCR product was recovered using a gel and PCR product column recovery kit (Promega). Using this PCR recovery product as a template, a T7 high-yield transcription kit (Thermo) was used to synthesize full-length sgRNA (where the guide sequence length is 20 nt) or truncated sgRNA (where the guide sequence length is less than 20 nt), and the reaction was carried out overnight at 37°C (12-16 h).

[0347] Afterwards, DNase I was added to the transcription system (2 μl DNase I (5 U / μl) was added to every 50 μl transcription system), and the DNA template was eliminated by water bath at 37°C for 30 min. RNA was purified using an RNA purification and concentration kit, and then quantified using NanoDrop 2000C and stored in a -80°C refrigerator for later use.

[0348] 2. Preparation of target DNA

[0349] (1) If the target DNA is single-stranded, directly synthesize a 50 bp oligonucleotide as the target DNA, which contains the target sequence recognized by the sgRNA.

[0350] (2) If the target DNA is double-stranded, directly synthesize two complementary 50 bp oligonucleotides containing the target sequence recognized by the sgRNA. Anneal the two oligonucleotides to obtain double-stranded target DNA. Specifically, the paired oligonucleotides (1 μM) were annealed in 1× PCR buffer (Transgen Biotech) with a total volume of 20 μL, and then the annealing procedure was performed: initial denaturation at 95°C for 5 minutes, followed by cooling from 95°C to 20°C, using a thermal cycler to reduce 1°C per minute.

[0351] 3. Cas12b reaction

[0352] (1) sgRNA annealing: dilute sgRNA to an appropriate concentration (5 μM) and anneal in a PCR instrument. Annealing procedure: denature at 75°C for 5 min, then cool from 75°C to 20°C, decreasing by 1°C per minute.

[0353] (2) Incubation of sgRNA and Cas12b: Mix the annealed sgRNA with Cas12b of equal molar concentration and place at 30°C for 20-30 minutes.

[0354] (3) Cas12b reaction: In a 20 μl reaction system, add the mixture of sgRNA and Cas12b incubated in step (2) (both final concentrations are 250 nM), target DNA (final concentration 50 nM), fluorescent quenching probe (HEX-N12-BHQ1, final concentration 500 nM), 2 μl of 10×NEB Buffer 3.1 and 0.5 μl of RNase inhibitor (40 U / μl). After mixing, react at 48°C for 30 min. Then, heat at 98°C for 5 min to inactivate.

[0355] 4. Detection of bypass single-stranded DNA cleavage activity of Cas12b by fluorescence microplate reader

[0356] The inactivated 20 μL reaction solution was added to a 96-well plate and then detected using an ELISA reader (excitation light 535 nm, emission light 556 nm).

[0357] First, with regard to the target sequence of DNMT1-3, for the target single-stranded DNA, such as Figure 7 As shown in the figure, when the sgRNA guide sequence is 20nt, the mutation of bases 1-12 has little effect on the fluorescence signal generated by trans cutting. When the sgRNA guide sequence is 14nt or 15nt, the fluorescence values ​​including the control are very low. When the sgRNA guide sequence is 16nt, the fluorescence value is significantly reduced after the mutation of bases 10-12.

[0358] Further research found that ( Figure 8 ), the effect of base mutations at positions 10-16 on the fluorescence value is very obvious, especially after mutations at positions 10-14, the fluorescence value is almost undetectable. Then, the target single-stranded DNA is mutated into different types of bases at different positions, which has no essential effect on the fluorescence value. Only the fluorescence value changes greatly when the 9th position is mutated into G ( Fig. 9 ).

[0359] Next, we tested three other target sequences. Although the fluorescence change values ​​of different sequences for a certain site mutation were different, we could find sites with obvious changes at positions 8-16, such as positions 8, 10, 11, and 12 of rs5082, position 11 of rs1467558, and positions 8-15 of rs2952768 ( Fig.10 ).

[0360] For target double-stranded DNA (such as Fig.11As shown in Figure 2, the effect of single base mutation on the fluorescence signal generated by trans cutting is different from that of ssDNA. Taking the target sequence of DNMT1-3 as an example, the sgRNA with a guide sequence of 18-20nt is sensitive to base mutations at positions 10-16. In addition, in this example, several sgRNAs are most sensitive to positions 10 and 16.

[0361] In addition, at 50°C, 55°C, 60°C, and 65°C, LAMP and Cas12b detection achieved a one-step reaction ( Fig.12 ).

[0362] Example 4 Testing of Escherichia coli and other microorganisms in environmental water

[0363] The Escherichia coli gyrB gene was selected as the detection target to indirectly test Escherichia coli and other microorganisms in water, and the detection limit was determined.

[0364] 1. Preparation of guide RNA (sgRNA)

[0365] Using plasmid pUC18-sgRNA-DNMT1-3 as a template, T7-crRNA-F as an upstream primer, and ZL-gyrB-crRNA2-R as a downstream primer, PCR was used to amplify the DNA template required for in vitro transcription. Dpn I was then added to the PCR product (1 μl Dpn I (10 U / μl) was added to each 50 μl PCR system), and the plasmid DNA template was digested in a 37°C water bath for 30 min, and the PCR product was recovered using a gel and PCR product column recovery kit (Promega). Using this PCR recovery product as a template, sgRNA was synthesized using a T7 high-yield transcription kit (Thermo), and the reaction was carried out overnight at 37°C (12-16 h).

[0366] Afterwards, DNase I was added to the transcription system (2 μl DNase I (5 U / μl) was added to every 50 μl transcription system), and the DNA template was eliminated by water bath at 37°C for 30 min. RNA was purified using an RNA purification and concentration kit, and then quantified using NanoDrop 2000C and stored in a -80°C refrigerator for later use.

[0367] 2. LAMP amplification reaction

[0368] E. coli MG1655 was cultured to OD 600 When the OD value is 1.0, a portion of the bacterial solution is placed at 100°C for 15 min. Then, LAMP amplification reaction is performed, and the template is added to the bacterial solution of different dilutions. 600 Diluted to 0, 5×10 -3 , 5×10 -4 , to 5×10-9 .

[0369] After coating test, 5×10 -3 The total volume of the reaction system was 25 μL, and the primers were 1.6 μM LAMP-gyrB-FIP and LAMP-gyrB-BIP, 0.2 μM LAMP-gyrB-F3 and LAMP-gyrB-B3, 0.4 μM LAMP-gyrB-LoopF ​​and LAMP-gyrB-LoopB. The kit used for the LAMP reaction was LAMP Kit (NEB). The LAMP reaction procedure is 65℃ for 30min. After LAMP is completed, inactivate at 85℃ for 10min and then directly use it for Cas12b reaction.

[0370] 3. Cas12b reaction

[0371] (1) sgRNA annealing: dilute sgRNA to an appropriate concentration (5 μM) and anneal in a PCR instrument. Annealing procedure: denature at 75°C for 5 min, then cool from 75°C to 20°C, decreasing by 1°C per minute.

[0372] (2) Incubation of sgRNA and Cas12b: Mix the annealed sgRNA with Cas12b of equal molar concentration and place at 30°C for 20-30 minutes.

[0373] (3) Cas12b reaction: In a 20 μl reaction system, add the mixture of sgRNA and Cas12b incubated in step (2) (both final concentrations are 250 nM), 1 μl of LAMP product, fluorescent probe (HEX-N12-BHQ1) (final concentration 500 nM), 2 μl of 10×NEB Buffer 3.1 and 0.5 μl of RNase inhibitor (40 U / μl). After mixing, react at 48°C for 30 min. Then, heat at 98°C for 5 min to inactivate.

[0374] 4. Detection of bypass single-stranded DNA cleavage activity of Cas12b by fluorescence microplate reader

[0375] The inactivated 20 μL reaction solution was added to a 96-well plate and detected using an ELISA reader (excitation light 535 nm, emission light 556 nm).

[0376] like Fig.13 As shown in the figure, after combining with LAMP amplification, the sensitivity of Cas12b (i.e., HOLMES v2.0 method) can reach the level of detecting several Escherichia coli (after plate test, OD 600 =5×10 -6 About 7 E. coli).

[0377] Example 5 Saliva Gender Test

[0378] In this example, the sry gene locus unique to the Y chromosome was selected as the test target, and the gender of the saliva-derived sample was identified by HOLMES v2.0.

[0379] 1. Preparation of guide RNA (sgRNA)

[0380] Using plasmid pUC18-sgRNA-DNMT1-3 as a template, T7-crRNA-F as an upstream primer, ZL-sry-crRNA3-R and ZLsgRNA-DNMT1-3-R as downstream primers, two DNA templates required for in vitro transcription were amplified by PCR. Then, Dpn I was added to the PCR product (1 μl Dpn I (10U / μl) was added to each 50 μl PCR system), and the plasmid DNA template was digested in a 37°C water bath for 30 min, and the PCR product was recovered using a gel and PCR product column recovery kit (Promega). Using this PCR recovery product as a template, sgRNA was synthesized using a T7 high-yield transcription kit (Thermo), and the reaction was carried out overnight at 37°C (12-16h).

[0381] Afterwards, DNase I was added to the transcription system (2 μl DNase I (5 U / μl) was added to every 50 μl transcription system), and the DNA template was eliminated by water bath at 37°C for 30 min. RNA was purified using an RNA purification and concentration kit, and then quantified using NanoDrop 2000C and stored in a -80°C refrigerator for later use.

[0382] 2. LAMP amplification reaction

[0383] The saliva of males and females was used as template for LAMP amplification reaction. The total volume of each reaction system was 25 μL, and the primers were 1.6 μM LAMP-sry-FIP and LAMP-sry-BIP, 0.2 μM LAMP-sry-F3 and LAMP-sry-B3, and 0.4 μM LAMP-sry-LoopF ​​and LAMP-sry-LoopB. The kit used for LAMP reaction was LAMPKit (NEB). The LAMP reaction procedure is 65℃ for 30min. After LAMP is completed, inactivate at 85℃ for 10min and then directly use it for Cas12b reaction.

[0384] 3. Cas12b reaction

[0385] (1) sgRNA annealing: dilute sgRNA to an appropriate concentration (5 μM) and anneal in a PCR instrument. Annealing procedure: denature at 75°C for 5 min, then cool from 75°C to 20°C, decreasing by 1°C per minute.

[0386] (2) Incubation of sgRNA and Cas12b: Mix the annealed sgRNA with Cas12b of equal molar concentration and place at 30°C for 20-30 minutes.

[0387] (3) Cas12b reaction: In a 20 μl reaction system, add the mixture of sgRNA and Cas12b incubated in step (2) (both final concentrations are 250 nM), 1 μl of LAMP product, fluorescent probe (HEX-N12-BHQ1) (final concentration 500 nM), 2 μl of 10×NEB Buffer 3.1 and 0.5 μl of RNase inhibitor (40 U / μl). After mixing, react at 48°C for 30 min. Then, heat at 98°C for 5 min to inactivate.

[0388] 4. Detection of bypass single-stranded DNA cleavage activity of Cas12b by fluorescence microplate reader

[0389] The inactivated 20 μL reaction solution was added to a 96-well plate and detected using an ELISA reader (excitation light 535 nm, emission light 556 nm).

[0390] like Fig.14 As shown, after combined with LAMP amplification, Cas12b (i.e., HOLMES v2.0 method) can efficiently detect human gender in saliva samples.

[0391] Example 6 Human SNP Testing

[0392] In this example, the rs5082 locus of human was detected. The target DNA was prepared by asymmetric PCR or LAMP isothermal amplification method to identify the SNP type of the saliva-derived sample.

[0393] 1. Preparation of guide RNA (sgRNA)

[0394] Using plasmid pUC18-sgRNA-DNMT1-3 as a template, T7-crRNA-F as an upstream primer, and oligonucleotides of different lengths containing a guide sequence complementary to the rs5082 site as a downstream primer, the DNA template required for in vitro transcription was amplified by PCR. Dpn I was then added to the PCR product (1 μl Dpn I (10U / μl) was added to each 50 μl PCR system), and the plasmid DNA template was digested in a 37°C water bath for 30 min, and the PCR product was recovered using a gel and PCR product column recovery kit (Promega). Using this PCR recovery product as a template, a truncated sgRNA was synthesized using a T7 high-yield transcription kit (Thermo), and the reaction was performed overnight at 37°C (12-16h). For target DNA generated by asymmetric PCR, a truncated sgRNA with a guide sequence of 16nt was used in the Cas12b reaction; for target DNA generated by LAMP amplification, a truncated sgRNA with a guide sequence of 18nt was used in the Cas12b reaction.

[0395] Afterwards, DNase I was added to the transcription system (2 μl DNase I (5 U / μl) was added to every 50 μl transcription system), and the plasmid DNA template was eliminated by water bath at 37°C for 30 min. RNA was purified using an RNA purification and concentration kit, and then quantified using NanoDrop 2000C and stored in a -80°C refrigerator for later use.

[0396] 2. Amplification reaction (asymmetric PCR or LAMP)

[0397] Asymmetric PCR amplification reaction: human cell genome HEK293T was used as a template, primers ASP-primer, ASP-rs5082-F, and ASP-rs5082-R were used for asymmetric PCR amplification (while ordinary PCR did not add ASP-primer), and KOD FX enzyme was used for amplification reaction.

[0398] LAMP amplification reaction: The human cell genome HEK293T was used as a template for LAMP amplification reaction. The total volume of each reaction system was 25 μL, and primers 1.6 μM LAMP-rs5082-FIP-10PAM (control: LAMP-rs5082-FIP) and LAMP-rs5082-BIP, 0.2 μM LAMP-rs5082-F3 and LAMP-rs5082-B3, 0.4 μM LAMP-rs5082-LoopF ​​and LAMP-rs5082-LoopB were used. The kit used for LAMP reaction was LAMP Kit (NEB). The LAMP reaction procedure is 65℃30min. After LAMP is completed, it is inactivated at 85℃10min and then directly used for Cas12b reaction. Since the primer LAMP-rs5082-FIP-10PAM adds the PAM sequence, the amplified DNA contains the PAM sequence.

[0399] 3. Cas12b reaction

[0400] (1) sgRNA annealing: dilute sgRNA to an appropriate concentration (5 μM) and anneal in a PCR instrument. Annealing procedure: denature at 75°C for 5 min, then cool from 75°C to 20°C, decreasing by 1°C per minute.

[0401] (2) Incubation of sgRNA and Cas12b: Mix the annealed sgRNA with Cas12b of equal molar concentration and place at 30°C for 20-30 minutes.

[0402] (3) Cas12b reaction: In a 20 μl reaction system, add the mixture of sgRNA and Cas12b incubated in step (2) (both final concentrations are 500 nM, Cas12b is 250 nM in the LAMP amplification test), 1 μl of target DNA or 1 μl of LAMP product, fluorescent probe (HEX-N12-BHQ1) (final concentration 500 nM), and 2 μl of 10×NEB Buffer 3.1 and 0.5 μl of RNase inhibitor (40 U / μl). After mixing, react at 48°C for 60 min. Afterwards, heat at 98°C for 5 min in a PCR instrument to inactivate.

[0403] 4. Detection of bypass single-stranded DNA cleavage activity of Cas12b by fluorescence microplate reader

[0404] The inactivated 20 μL reaction solution was added to a 96-well plate and detected using an ELISA reader (excitation light 535 nm, emission light 556 nm).

[0405] like Fig.15 As shown, since there is no PAM sequence near the rs5082 site, asymmetric PCR that produces single-stranded DNA can more clearly distinguish the SNP site.

[0406] If combined with LAMP amplification, since there is no PAM sequence near the rs5082 site, the detection site needs to be introduced into the PAM sequence, which can better distinguish the SNP site ( Fig.16 ).

[0407] Example 7 RNA virus testing

[0408] In this example, RNA virus JEV (Japanese encephalitis virus) was selected, and HOLMES v2.0 was used to identify whether the sample was infected with the virus. The sites selected for testing were E453 and NS170.

[0409] 1. Preparation of guide RNA (sgRNA)

[0410] The DNA template required for in vitro transcription was amplified by PCR using plasmid pUC18-sgRNA-DNMT1-3 as a template, T7-crRNA-F as an upstream primer, and an oligonucleotide containing a guide sequence complementary to the corresponding target as a downstream primer. Dpn I was then added to the PCR product (1 μl Dpn I (10 U / μl) was added to each 50 μl PCR system), and the plasmid DNA template was digested in a 37°C water bath for 30 min, and the PCR product was recovered using a gel and PCR product column recovery kit (Promega). Using this PCR recovery product as a template, sgRNA was synthesized using a T7 high-yield transcription kit (Thermo), and the reaction was performed overnight at 37°C (12-16 h).

[0411] Afterwards, DNase I was added to the transcription system (2 μl DNase I (5 U / μl) was added to every 50 μl transcription system), and the plasmid DNA template was eliminated by water bath at 37°C for 30 min. RNA was purified using an RNA purification and concentration kit, and then quantified using NanoDrop 2000C and stored in a -80°C refrigerator for later use.

[0412] 2. LAMP amplification reaction

[0413] The cell fluid infected with JEV virus was used as a template for LAMP amplification reaction. The total volume of each reaction system was 25 μL, and the primers were 1.6 μM LAMP-E453-FIP and LAMP-E453-BIP, 0.2 μM LAMP-E453-F3 and LAMP-E453-B3, 0.4 μM LAMP-E453-LoopF ​​and LAMP-E453-LoopB, (or 1.6 μM LAMP-NS170-FIP and LAMP-NS170-BIP, 0.2 μM LAMP-NS170-F3 and LAMP-NS170-B3, 0.4 μM LAMP-NS170-LoopF ​​and LAMP-NS170-LoopB) The kit used for LAMP reaction was LAMP Kit (NEB) or Bst 3.0 DNA polymerase (NEB). The LAMP reaction procedure is 65℃30min. After LAMP is completed, inactivate at 85℃10min and then directly use it for Cas12b reaction.

[0414] 3. Cas12b reaction

[0415] (1) sgRNA annealing: dilute sgRNA to an appropriate concentration (5 μM) and anneal in a PCR instrument. Annealing procedure: denature at 75°C for 5 min, then cool from 75°C to 20°C, decreasing by 1°C per minute.

[0416] (2) Incubation of sgRNA and Cas12b: Mix the annealed sgRNA with Cas12b of equal molar concentration and place at 30°C for 20-30 minutes.

[0417] (3) Cas12b reaction: In a 20 μl reaction system, add the mixture of sgRNA and Cas12b incubated in step (2) (both final concentrations are 250 nM), 1 μl of LAMP product, fluorescent probe (HEX-N12-BHQ1) (final concentration 500 nM), and 2 μl of 10×NEB Buffer 3.1 and 0.5 μl of RNase inhibitor (40 U / μl). After mixing, react at 48°C for 30 min. Afterwards, heat at 98°C for 5 min in a PCR instrument to inactivate.

[0418] 4. Detection of bypass single-stranded DNA cleavage activity of Cas12b by fluorescence microplate reader

[0419] The inactivated 20 μL reaction solution was added to a 96-well plate and detected using an ELISA reader (excitation light 535 nm, emission light 556 nm).

[0420] like Fig.17 As shown, after adding LAMP amplification, Cas12b (i.e., HOLMES v2.0 method) can very sensitively detect whether JEV virus is present.

[0421] Example 8 Relative quantitative detection of trace DNA (real-time HOLMES method)

[0422] 1. Preparation of guide RNA (sgRNA)

[0423] The DNA template required for in vitro transcription was amplified by PCR using plasmid pUC18-sgRNA-DNMT1-3 as a template, T7-crRNA-F as an upstream primer, and an oligonucleotide containing a guide sequence complementary to the target as a downstream primer. Dpn I was then added to the PCR product (1 μl Dpn I (10 U / μl) was added to each 50 μl PCR system), and the plasmid DNA template was digested in a 37°C water bath for 30 min, and the PCR product was recovered using a gel and PCR product column recovery kit (Promega). Using this PCR recovery product as a template, sgRNA was synthesized using a T7 high-yield transcription kit (Thermo), and the reaction was performed overnight at 37°C (12-16 h).

[0424] Afterwards, DNase I was added to the transcription system (2 μl DNase I (5 U / μl) was added to every 50 μl transcription system), and the plasmid DNA template was eliminated by water bath at 37°C for 30 min. RNA was purified using an RNA purification and concentration kit, and then quantified using NanoDrop 2000C and stored in a -80°C refrigerator for later use.

[0425] 2. One-step LAMP amplification-fluorescence detection reaction (using real-time PCR instrument)

[0426] The LAMP amplification reaction was performed using DNMT1-3 plasmids of different dilution concentrations as templates. The total volume of each reaction system was 20 μL, and the primers were 1.6 μM LAMP-DNM-FIP and LAMP-DNM-BIP, 0.2 μM LAMP-DNM-F3 and LAMP-DNM-B3, and 0.4 μM LAMP-DNM-LoopF ​​and LAMP-DNM-LoopB. The kit used for the LAMP reaction was LAMP Kit (NEB). Add a mixture of sgRNA and Cas12b (final concentration of both is 500nM), a fluorescent probe (FAM-N12-Eclipse) (final concentration 500nM), and 0.5μl of RNase inhibitor (40U / μl). After mixing, perform real-time fluorescence detection on a fluorescence quantifier, and the reaction conditions are 55℃ for 120min.

[0427] 3. Results Analysis

[0428] like Fig.18 Shown are real-time fluorescence measurements of target DNA at different dilution concentrations.

[0429] By plotting the time point when the fluorescence value reaches 600,000 as the y-axis and the absolute value of the concentration lg as the x-axis, a trend line that approximates a straight line can be obtained ( Fig.19 ).

[0430] The above results show that the method of the present invention can be used to treat 10 -1 -10 -6 Accurately quantify nM of DNA.

[0431] Example 9 Absolute quantitative detection of trace DNA (digital HOLMES method)

[0432] In this embodiment, the HOLMES method is combined with chip digital PCR to perform absolute quantitative detection through a reaction temperature.

[0433] 1. Preparation of guide RNA (sgRNA)

[0434] The DNA template required for in vitro transcription was amplified by PCR using plasmid pUC18-sgRNA-DNMT1-3 as a template, T7-crRNA-F as an upstream primer, and oligonucleotides of different lengths containing a guide sequence complementary to the rs5082 site as downstream primers. Dpn I was then added to the PCR product (1 μl Dpn I (10 U / μl) was added to each 50 μl PCR system), and the plasmid DNA template was digested in a 37°C water bath for 30 min, and the PCR product was recovered using a gel and PCR product column recovery kit (Promega). Using this PCR recovery product as a template, sgRNA was synthesized using a T7 high-yield transcription kit (Thermo), and the reaction was performed overnight at 37°C (12-16 h).

[0435] Afterwards, DNase I was added to the transcription system (2 μl DNase I (5 U / μl) was added to every 50 μl transcription system), and the plasmid DNA template was eliminated by water bath at 37°C for 30 min. RNA was purified using an RNA purification and concentration kit, and then quantified using NanoDrop 2000C and stored in a -80°C refrigerator for later use.

[0436] 2. One-step LAMP amplification-fluorescence detection reaction (using jnmedsys's ddPCR instrument)

[0437] The human 293T cell genome at different dilution concentrations was used as a template for absolute quantitative detection. The total volume of each reaction system was 15 μL, and the primers were 1.6 μM LAMP-rs5082-FIP-10PAM, LAMP-rs5082-BIP, 0.2 μM LAMP-rs5082-F3 and LAMP-rs5082-B3, 0.4 μM LAMP-rs5082-LoopF ​​and LAMP-rs5082-LoopB. The kit used for the LAMP reaction was LAMP Kit (NEB). Add a mixture of sgRNA and Cas12b (both at a final concentration of 500 nM), fluorescent probes (HEX-N12-BHQ1 and FAM-N12-Eclipse) (both at a final concentration of 500 nM), 0.375 μl of RNase inhibitor (40 U / μl), and 0.75 μl of JN solution.

[0438] After mixing, the sample is delivered to the chip (the chip is in a PCR tube) using a slider to partition the system. Then it is transferred to the Sealing Enhancer instrument for processing.

[0439] Next, 235ul of blocking solution was added and the reaction was carried out in a metal bath at 55°C for 120min. At 0, 60, 90, and 120min, the cells were placed in a View Jig instrument to detect fluorescence.

[0440] 3. Results Analysis

[0441] like Fig. 20 As shown, after 120 minutes of reaction, 51.75 copies were detected by calculation of template 1, and 6.3 copies were calculated for template 2 after 10-fold dilution, while no copy number was detected for the control without template.

[0442] The results show that the digital HOLMES method of the present invention can not only perform quantitative detection, but also can accurately detect a single copy of a template in a micro-detection system with extreme sensitivity, and the time consumption is greatly shortened.

[0443] Example 10 Methylation site detection

[0444] In this example, a promoter region (250 bp) of the collagen α2 (I) gene (COL1A2) containing 13 CpG sites (M1 to M13) was selected through NCBI search query as the target gene detection sequence of this example (NCBI sequence number: AF004877.1). The collagen α2 (I) gene (COL1A2) encodes collagen and is related to tumorigenesis.

[0445] In COL1A2(BSP)-C, all CpG sites are methylated. After bisulfite conversion (converting unmethylated C to uracil) and PCR amplification, the methylated C base in CpG remains C.

[0446] In COL1A2(BSP)-T, none of the CpG sites were methylated. After bisulfite conversion and PCR amplification, the C base in the CpG was changed to T.

[0447] The principle of the methylation site detection method of the present invention is described in Fig.21 .

[0448] Experimental methods:

[0449] 1. Extract the genomes of four cancer cell lines (293T, SW480, NCI-N87, MCF-7).

[0450] 2. The genome was treated with bisulfite, the promoter region of the COL1A2 gene containing the M3 site was selected, and the corresponding BSP primers (primer COL1A2(BSP)-F / R) were designed. PCR amplification was performed using the cell genome after bisulfite conversion as a template, and the product was recovered and purified by gel recovery and used as target DNA for subsequent detection.

[0451] 3. Preparation of target nucleic acid for standard curve

[0452] (1) The gene fragment COL1A2 was amplified from SW480-gDNA using primers (primer COL1A2-F / R) and assembled into the pClone007S T vector (TSINGKE, TSV-007S). DH10b was then transformed and cultured at 37°C in LB medium supplemented with appropriate antibiotics. The plasmid was extracted and sequenced to obtain the plasmid pClone007-COL1A2.

[0453] (2) Plasmid pClone007-COL1A2 was treated with CpG methyltransferase (MSss I) (M0226V, NEB) to methylate all cytosine residues in its 5′-CG-3′ sequence. Plasmid pClone007-COL1A2 and pClone007-COL1A2 treated with CpG methyltransferase were then subjected to bisulfite conversion to convert unmethylated cytosine into uracil. A BSP primer pair (primer COL1A2(BSP)-F / R) was designed for PCR amplification. The amplified fragments were purified by gel recovery and assembled into the pClone007ST vector to construct plasmids pClone007-COL1A2(BSP)-C and pClone007-COL1A2(BSP)-T, thereby obtaining fragments COL1A2(BSP)-C (Target C) and COL1A2(BSP)-T (Target T).

[0454] 4. Methylation detection

[0455] gDNA of four cancer cell lines (293T, SW480, NCI-N87, MCF-7) were subjected to EZ DNA methylation-direct TM After bisulfite conversion using a PCR amplification kit (D5021, Zymo Research), the COL1A2 (BSP) (250 bp) gene fragments of the four cancer cell lines were amplified using a BSP primer pair (primer COL1A2 (BSP) -F / R) and purified by gel recovery.

[0456] Bisulfite cloning sequencing

[0457] The above four COL1A2 (BSP) gene fragments were connected to the pClone007S T vector, and then transformed into DH10b. More than 10 positive monoclonal clones were picked from the plate for sequencing analysis, and the methylation degree of 13 CpG sites in the COL1A2 gene was calculated by the methylation rate calculation tool QUantification tool for Methylation Analysis (QUMA).

[0458] Direct bisulfite sequencing

[0459] The above four COL1A2 (BSP) gene fragments were analyzed by Sanger sequencing, and the methylation rate was obtained by calculating the peak ratio C / (C+T) at 13 CpG sites in the COL1A2 gene.

[0460] Bisulfite NGS high-throughput sequencing

[0461] The above four COL1A2 (BSP) gene fragments were subjected to NGS high-throughput sequencing library construction and NGS high-throughput sequencing and analysis (completed by Sino-Sci Biotech Co., Ltd.), and the methylation levels of 13 CpG sites in the COL1A2 gene were analyzed.

[0462] HOLMESv2 system methylation detection

[0463] (1) Extraction of CRISPR-Cas12b protein

[0464] The codon-optimized AacCas12b full-length gene was synthesized by Shanghai Tolo Biotechnology Co., Ltd. and cloned into the vector pET28a. The recombinant Cas12b protein with a His tag at the N-terminus was expressed in Escherichia coli BL21 (DE3). 600When it reaches 0.6, 0.25mM isopropylthiogalactoside (IPTG) is added to induce culture at 16°C for 14-18h and then centrifuged to collect the bacteria. The bacterial solution was suspended with buffer A (50mM Tris-HCl (pH 7.6), 150mM NaCl, 20mM imidazole and 1 / 500 phenylmethylsulfonyl fluoride (v / v)), lysed with a high-pressure homogenizer (Avestin), centrifuged at 15,000rpm for 30min, pre-equilibrated with buffer A Ni column (GE Healthcare), the supernatant was transferred to the Ni column, and then eluted with buffer B (50mM Tris-HCl (pH 7.6), 200mM NaCl and 30mM imidazole), and the recombinant protein was eluted with buffer B (where the imidazole concentration was adjusted to 300mM). The eluted fraction containing AacCas12b protein was collected by ultrafiltration and concentrated to about 5mL. Then it was loaded onto a HiLoad 16 / 600 Superdex 200 pg column (GE Healthcare) equilibrated with buffer C (40 mM Tris-HCl (pH 7.6), 200 mM NaCl, 2 mM DTT, and 5% glycerol (v / v)), and the eluted fractions containing AacCas12b protein were collected and concentrated to 4 mg / mL, and 50% glycerol was added and stored at -20°C.

[0465] (2) Preparation of sgRNA

[0466] Using plasmid pUC18-sgRNA-DNMT1-3 as a template, T7-sgRNA-F as an upstream primer, and a target-specific downstream primer, the template required for in vitro transcription of sgRNA was amplified by PCR. Then, Dpn I was added to the PCR product (1 μL Dpn I was added to each 50 μL PCR system), and the plasmid DNA template was digested in a 37°C water bath for 30 min, and the PCR product was recovered using a gel and PCR product column recovery kit (Promega). Using this PCR recovery product as a template, sgRNA was synthesized using a T7 high-yield transcription kit (Thermo), and the reaction was carried out at 37°C overnight (12-16h). Finally, DNase I was added to the transcription system (2 μL DNase I was added to each 50 μL transcription system), and the DNA template was eliminated by a 37°C water bath for 30 min, and RNA was purified using an RNA purification and concentration kit (Zymo Research). The resulting sgRNA was named sgRNA-DNMT1-3. The samples were quantified using NanoDrop 2000C and stored in a -80°C refrigerator for future use.

[0467] (3) Screening of sgRNA

[0468] In COL1A2, various sgRNAs of different lengths and CpG sites at different positions were designed to match the upstream and downstream of each CpG site. The sgRNA (final concentration of 10 μM) was annealed on a PCR instrument, and the annealing program was: 75°C, 5min; then decreased by 1°C every 1min until 20°C. After annealing, sgRNA and AacCas12b protein (final concentration of 5 μM) were mixed in equal volumes and incubated at 30°C for 15-20min. Finally, a 20 μL Cas12b reaction system was prepared (operated on ice):

[0469]

[0470] After reacting in a 48°C water bath for 1 h, 20 μL of the system was transferred to a 96-well plate. The excitation wavelength was set to 535 nm and the emission wavelength was set to 556 nm on the microplate reader. The fluorescence value was measured and the sgRNA used in the group of reactions with the largest fluorescence value difference of the target DNA (COL1A2(BSP)-C / T) was selected as the most suitable sgRNA.

[0471] (4) Methylation detection based on the HOLMESv2 system

[0472] The fragments COL1A2(BSP)-C and COL1A2(BSP)-T were mixed evenly according to the proportion of COL1A2(BSP)-C of 0%, 10%, 30%, and 50%, respectively, as the target DNA for the methylation detection standard curve. The sample fragments COL1A2(BSP) treated with bisulfite from four cancer cell lines (293T, SW480, NCI-N87, and MCF-7) were mixed with the fragment COL1A2(BSP)-T at equal molar concentrations, as the target DNA for detecting the methylation ratio of the real sample. Then, sgRNA-COL1A2m3-C12-17 was used in the HOLMESv2 system to detect the methylation standard curve of the M3 site in the COL1A2 promoter region and the methylation ratio of the M3 site in the four cancer cell lines (293T, SW480, NCI-N87, and MCF-7).

[0473] Anneal the sgRNA (final concentration of 10 μM) on a PCR instrument. The annealing program is: 75°C, 5 min; then decrease 1°C every 1 min until 20°C. After annealing, mix sgRNA and AacCas12b protein (final concentration of 5 μM) in equal volumes and incubate at 30°C for 15-20 min. Finally, prepare a 20 μL Cas12b reaction system (operate on ice):

[0474]

[0475]

[0476] 20 μL of the system was transferred into a 96-well plate and placed in a real-time PCR instrument. The program was set to react at 48°C for 1 hour, and the fluorescence value was measured every 2 minutes.

[0477] The methylation standard curve of the M3 site uses the fluorescence value at 30min and 10min of reaction as the ordinate Y, and the proportion of COL1A2(BSP)-C at 0%, 10%, 30%, and 50% as the abscissa X. The equation model is: Y = aX + b. The concentration of COL1A2(BSP) in the four cancer cell lines is 1 / 2 of the target DNA in the standard curve. Therefore, the methylation ratio calculation model is: X unknown =2(Y unknown -b) / a.

[0478] Experimental results:

[0479] 1. Detection mode diagram

[0480] After the gene containing methylated CpG sites is treated with bisulfite, all non-methylated sites CG in the gene are converted to UG, and all methylated sites CG remain unchanged. The converted gene is used as the target DNA to form a ternary complex with AacCas12b protein and sgRNA. In a single-stranded DNA probe system with a fluorescent group and a quenching group (i.e., the HOLMESv2 system), the reaction is carried out at 48°C for a certain time, and its fluorescence value is detected. If the detection site is methylated, the DNA probe emits light and the fluorescence value is high; if the detection site is unmethylated, the DNA probe does not emit light, and the fluorescence value is very low or zero.

[0481] 2. Screening of sgRNA

[0482] like Fig. 22 As shown, according to the comparison of the fluorescence values ​​of each sμRNA and two targets (target-C, target-T, representing the COL1A2 fragments after bisulfite treatment with 100% and 0% methylation degree of all CpG sites) in the HOLMESv2-Cas12b reaction system, the most suitable sgRNA can be selected. Among them, 9 sgRNAs including m3-C12-17, m4-C1-18, m4-C1-19, m4-C1-20, m5-C14-18, m6m7m8-C7-18, m6m7m8-19, m6m7m8-20, and m9m10-C1-18 have obvious differences in fluorescence values ​​in the two targets (target-C, target-T), and can all be used for methylation detection. In this study, a single-site sgRNA (m3-C12-17) was selected for subsequent methylation detection. Its sgRNA is as follows Fig.23 shown.

[0483] 3. Quantitative detection of methylation of M3 CpG site (COL1A2)

[0484] The fragments COL1A2(BSP)-C and COL1A2(BSP)-T were mixed at a ratio of 0%, 10%, 30%, and 50% of the COL1A2(BSP)-C content, and the fluorescence value of the reaction was detected in the HOLMESv2 system using sgRNA-COL1A2m3-C12-17 at 30 minutes. After data analysis, the fluorescence value of 0% was used as the zero point for standardization, and a quantitative standard curve of M3 CpG site (COL1A2) methylation was drawn. A standard curve was drawn correspondingly for each detection. The inventors detected three times, with three replicates each time, and the equations of the standard curves were: y=758939x+9320.9(R 2 =0.9975);y=671675x+12415(R 2 =0.9943);y=645621x+8601.7(R 2 =0.997), the linear correlation of the three standard curves was very good, R 2 Reaching above 0.99, the fluorescence value of the gene locus in the real sample can be detected by the HOLMESv2 system to calculate the corresponding methylation degree ( Fig.25 ).

[0485] COL1A2 exhibits different methylation levels in different cancers. To verify the practicality of the HOLMESv2-Cas12b methylation detection method, the fluorescence values ​​of the M3 CpG site (COL1A2) in four cancer cells (293T, SW480, NCI-N87, MCF-7) at 30 min were detected using sgRNA-COL1A2m3-C12-17 in the HOLMESv2 system. Fig.24 ), where the target gene concentration of cancer cells is 1 / 2 of the target gene concentration of the standard curve. According to the above standard curve equation, their methylation levels are calculated to be 63.3%, 81.3%, 54.8% and 77.1%, respectively.

[0486] In addition, the methylation detection method currently used widely was used to detect the methylation level of the COL1A2 gene in the above four cancer cells, and compared with the method of the present invention to verify the feasibility of the HOLMESv2-Cas12b methylation detection method ( Fig.26 ).

[0487] The bisulfite NGS high-throughput sequencing method can perform quantitative detection of methylation, and the methylation rates of the M3 CpG site (COL1A2) in four cancer cells (293T, SW480, NCI-N87, and MCF-7) were detected to be 65.26%, 81.06%, 48.27%, and 67.3%, respectively, which are consistent with the HOLMESv2-Cas12b detection results.

[0488] Bisulfite direct sequencing and bisulfite cloning sequencing can currently only perform semi-quantitative detection of methylation; the methylation detection results of these two methods for the M3 CpG site (COL1A2) in the above four cancer cells (293T, SW480, NCI-N87, MCF-7) were 63.2%, 63.6%, 30.4%, 20% and 92.3% (12 / 13), 100% (15 / 15), 78.6% (11 / 14), 71.4% (10 / 14), respectively. Compared with the results of bisulfite NGS high-throughput sequencing, the former is lower and the latter is higher.

[0489] The above results show that the quantitative results of the HOLMESv2-Cas12b detection method of the present invention are very accurate, fast and simple.

[0490] Example 11 Detection of alternatively spliced ​​RNA and circular RNA

[0491] Pre-mRNA alternative splicing is a key link in post-transcriptional regulation in higher eukaryotes and plays an important role in human diseases such as tumor progression. Some pre-mRNAs are processed to form circular RNAs ().

[0492] In this example, HOLMESv2 was used to specifically target the splicing site to detect mRNA splicing, and combined with RT-LAMP amplification, to detect the mature spliced ​​mRNA of the GAPDH gene ( Fig. 27 In addition, a similar strategy was adopted in crRNA design, and the circular RNA of Cdr1as in HEK293T was successfully detected in this example ( Fig. 27 ).

[0493] 1. Preparation of guide RNA (sgRNA)

[0494] The DNA template required for in vitro transcription was amplified by PCR using plasmid pUC18-sgRNA-DNMT1-3 as a template, T7-sgRNA-F as an upstream primer, and an oligonucleotide containing a guide sequence complementary to the corresponding target as a downstream primer (see Table 2). Dpn I was then added to the PCR product (1 μL Dpn I (10 U / μL) was added to each 50 μL PCR system), and the plasmid DNA template was digested in a 37°C water bath for 30 min, and the PCR product was recovered using a gel and PCR product column recovery kit (Promega). The sgRNA was synthesized using the T7 high-yield transcription kit (Thermo) using the PCR recovery product as a template, and the reaction was carried out overnight at 37°C (12-16 h).

[0495] Afterwards, DNase I was added to the transcription system (2 μL DNase I (5 U / μL) was added to every 50 μL transcription system), and the plasmid DNA template was eliminated by water bath at 37°C for 30 min. RNA was purified using an RNA purification and concentration kit, and then quantified using NanoDrop 2000C and stored in a -80°C refrigerator for later use.

[0496] 2. LAMP amplification reaction

[0497] The LAMP amplification reaction was performed using the 293T cell genome. The total volume of each reaction system was 25 μL, and the primers were 1.6 μM LAMP-FIP and LAMP-BIP, 0.2 μM LAMP-F3 and LAMP-B3, and 0.4 μM LAMP-LoopF ​​and LAMP-LoopB. The kit used for the LAMP reaction was LAMP Kit (NEB) or Bst 3.0 DNA polymerase (NEB). The LAMP reaction procedure is 65℃30min. After LAMP is completed, inactivate at 85℃10min and then directly use it for Cas12b reaction.

[0498] 3. Cas12b reaction

[0499] (1) sgRNA annealing: dilute sgRNA to an appropriate concentration (5 μM) and anneal in a PCR instrument. Annealing procedure: denature at 75°C for 5 min, then cool from 75°C to 20°C, decreasing by 1°C per minute.

[0500] (2) Incubation of sgRNA and Cas12b: Mix the annealed sgRNA with Cas12b of equal molar concentration and place at 30°C for 20-30 minutes.

[0501] (3) Cas12b reaction: In a 20 μL reaction system, add the mixture of sgRNA and Cas12b incubated in step (2) (both final concentrations are 250 nM), 1 μL of LAMP product, fluorescent probe (HEX-N12-BHQ1) (final concentration 500 nM), and 2 μL of 10×NEB Buffer 3.1 and 0.5 μL of RNase inhibitor (40 U / μL). After mixing, react at 48°C for 30 minutes. Afterwards, heat at 98°C for 5 minutes in a PCR instrument to inactivate.

[0502] 4. Detection of bypass single-stranded DNA cleavage activity of Cas12b by fluorescence microplate reader

[0503] Add 20 μL of the inactivated reaction solution to a 96-well plate and detect using an ELISA reader (excitation light 535 nm, emission light 556 nm). Fig. 27 As shown, it can be seen that after LAMP amplification, Cas12b (i.e., HOLMES v2.0 method) can detect and identify whether the RNA conforms to the expected RNA splicing situation or whether it is a circular RNA.

[0504] Table 1 Oligonucleotides used as substrates

[0505]

[0506]

[0507]

[0508] Table 2 Template sequences used to transcribe sgRNA

[0509]

[0510]

[0511] Table 3 Oligonucleotides used as amplification primers

[0512]

[0513]

[0514] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application. Sequence Listing <110> Shanghai Tolo Biotechnology Co., Ltd. <120> Use of a high temperature resistant Cas protein and a detection method and kit for target nucleic acid molecules <130> P2019-0684 <150> CN201810560284.8 <151> 2018-06-03 <160> 217 <170> SIPOSequenceListing 1.0 <210> 1 <211> 66 <212> DNA <213> Artificial Sequence <400> 1 tttctgtttg ttatcgcaac tttctactga attcaagctt tactctagaa agaggagaaa 60 ggatcc 66 <210> 2 <211> 66 <212> DNA <213> Artificial Sequence <400> 2 ggatcctttc tcctctttct agagtaaagc ttgaattcag tagaaagttg cgataacaaa 60 cagaaa 66 <210> 3 <211> 66 <212> DNA <213> Artificial Sequence <400> 3 ggatcctttc tcctctttct agagtaaagc ttgaattcag tagaaagttg cgataacaaa 60 cagaaa 66 <210> 4 <211> 66 <212> DNA <213> Artificial Sequence <400> 4 ggatcctttc tcctctttct agagtaaagc ttgaattcag tagaaagttg cgataacaaa 60 cagaaa 66 <210> 5 <211> 50 <212> DNA <213> Artificial Sequence <400> 5 aatgttccct gatggtccat gtctgttat cgcctgtcaa gtggcgtgac 50 <210> 6 <211> 50 <212> DNA <213> Artificial Sequence <400> 6 gtcacgccac ttgacaggcg agtaacagac atggaccatc agggaacatt 50 <210> 7 <211> 50 <212> DNA <213> Artificial Sequence <400> 7 aatgaaccct gatggtccat gtctgttat cgcctgtcaa gtggcgtgac 50 <210> 8 <211> 50 <212> DNA <213> Artificial Sequence <400> 8 gtcacgccac ttgacaggcg agtaacagac atggaccatc agggttcatt 50 <210> 9 <211> 50 <212> DNA <213> Artificial Sequence <400> 9 aatgatccct gatggtccat gtctgttat cgcctgtcaa gtggcgtgac 50 <210> 10 <211> 50 <212> DNA <213> Artificial Sequence <400> 10 gtcacgccac ttgacaggcg agtaacagac atggaccatc agggatcatt 50 <210> 11 <211> 50 <212> DNA <213> Artificial Sequence <400> 11 aatgtaccct gatggtccat gtctgttat cgcctgtcaa gtggcgtgac 50 <210> 12 <211> 50 <212> DNA <213> Artificial Sequence <400> 12 gtcacgccac ttgacaggcg agtaacagac atggaccatc agggtacatt 50 <210> 13 <211> 50 <212> DNA <213> Artificial Sequence <400> 13 gtcacgccac ttgacaggcg agtaacagac atggaccatc agggcccatt 50 <210> 14 <211> 50 <212> DNA <213> Artificial Sequence <400> 14 gtcacgccac ttgacaggcg agtaacagac atggaccatc agggggcatt 50 <210> 15 <211> 50 <212> DNA <213> Artificial Sequence <400> 15 aatgttcgct gatggtccat gtctgttat cgcctgtcaa gtggcgtgac 50 <210> 16 <211> 50 <212> DNA <213> Artificial Sequence <400> 16 gtcacgccac ttgacaggcg agtaacagac atggaccatc agcgaacatt 50 <210> 17 <211> 50 <212> DNA <213> Artificial Sequence <400> 17 aatgttccgt gatggtccat gtctgttat cgcctgtcaa gtggcgtgac 50 <210> 18 <211> 50 <212> DNA <213> Artificial Sequence <400> 18 gtcacgccac ttgacaggcg agtaacagac atggaccatc acggaacatt 50 <210> 19 <211> 50 <212> DNA <213> Artificial Sequence <400> 19 aatgttccca gatggtccat gtctgttat cgcctgtcaa gtggcgtgac 50 <210> 20 <211> 50 <212> DNA <213> Artificial Sequence <400> 20 gtcacgccac ttgacaggcg agtaacagac atggaccatc tgggaacatt 50 <210> twenty one <211> 50 <212> DNA <213> Artificial Sequence <400> twenty one aatgttccct catggtccat gtctgttat cgcctgtcaa gtggcgtgac 50 <210> twenty two <211> 50 <212> DNA <213> Artificial Sequence <400> twenty two gtcacgccac ttgacaggcg agtaacagac atggaccatg agggaacatt 50 <210> twenty three <211> 50 <212> DNA <213> Artificial Sequence <400> twenty three aatgttccct gttggtccat gtctgttat cgcctgtcaa gtggcgtgac 50 <210> twenty four <211> 50 <212> DNA <213> Artificial Sequence <400> twenty four gtcacgccac ttgacaggcg agtaacagac atggaccaac agggaacatt 50 <210> 25 <211> 50 <212> DNA <213> Artificial Sequence <400> 25 aatgttccct gaaggtccat gtctgttat cgcctgtcaa gtggcgtgac 50 <210> 26 <211> 50 <212> DNA <213> Artificial Sequence <400> 26 gtcacgccac ttgacaggcg agtaacagac atggaccttc agggaacatt 50 <210> 27 <211> 50 <212> DNA <213> Artificial Sequence <400> 27 aatgttccct gatcgtccat gtctgttat cgcctgtcaa gtggcgtgac 50 <210> 28 <211> 50 <212> DNA <213> Artificial Sequence <400> 28 gtcacgccac ttgacaggcg agtaacagac atggacgatc agggaacatt 50 <210> 29 <211> 50 <212> DNA <213> Artificial Sequence <400> 29 aatgttccct gatgctccat gtctgttat cgcctgtcaa gtggcgtgac 50 <210> 30 <211> 50 <212> DNA <213> Artificial Sequence <400> 30 gtcacgccac ttgacaggcg agtaacagac atggagcatc agggaacatt 50 <210> 31 <211> 50 <212> DNA <213> Artificial Sequence <400> 31 aatgttccct gatggaccat gtctgttat cgcctgtcaa gtggcgtgac 50 <210> 32 <211> 50 <212> DNA <213> Artificial Sequence <400> 32 gtcacgccac ttgacaggcg agtaacagac atggtccatc agggaacatt 50 <210> 33 <211> 50 <212> DNA <213> Artificial Sequence <400> 33 aatgttccct gatggtgcat gtctgttat cgcctgtcaa gtggcgtgac 50 <210> 34 <211> 50 <212> DNA <213> Artificial Sequence <400> 34 gtcacgccac ttgacaggcg agtaacagac atgcaccatc agggaacatt 50 <210> 35 <211> 50 <212> DNA <213> Artificial Sequence <400> 35 aatgttccct gatggtcgat gtctgttat cgcctgtcaa gtggcgtgac 50 <210> 36 <211> 50 <212> DNA <213> Artificial Sequence <400> 36 gtcacgccac ttgacaggcg agtaacagac atcgaccatc agggaacatt 50 <210> 37 <211> 50 <212> DNA <213> Artificial Sequence <400> 37 aatgttccct gatggtcctt gtctgttat cgcctgtcaa gtggcgtgac 50 <210> 38 <211> 50 <212> DNA <213> Artificial Sequence <400> 38 gtcacgccac ttgacaggcg agtaacagac aaggaccatc agggaacatt 50 <210> 39 <211> 50 <212> DNA <213> Artificial Sequence <400> 39 aatgttccct gatggtccaa gtctgttat cgcctgtcaa gtggcgtgac 50 <210> 40 <211> 50 <212> DNA <213> Artificial Sequence <400> 40 gtcacgccac ttgacaggcg agtaacagac ttggaccatc agggaacatt 50 <210> 41 <211> 50 <212> DNA <213> Artificial Sequence <400> 41 aatgttccct gatggtccat ctctgttat cgcctgtcaa gtggcgtgac 50 <210> 42 <211> 50 <212> DNA <213> Artificial Sequence <400> 42 gtcacgccac ttgacaggcg agtaacagag atggaccatc agggaacatt 50 <210> 43 <211> 50 <212> DNA <213> Artificial Sequence <400> 43 aatgttccct gatggtccat gactgttat cgcctgtcaa gtggcgtgac 50 <210> 44 <211> 50 <212> DNA <213> Artificial Sequence <400> 44 gtcacgccac ttgacaggcg agtaacagtc atggaccatc agggaacatt 50 <210> 45 <211> 50 <212> DNA <213> Artificial Sequence <400> 45 aatgttccct gatggtccat gtgtgttatact cgcctgtcaa gtggcgtgac 50 <210> 46 <211> 50 <212> DNA <213> Artificial Sequence <400> 46 gtcacgccac ttgacaggcg agtaacacac atggaccatc agggaacatt 50 <210> 47 <211> 50 <212> DNA <213> Artificial Sequence <400> 47 aatgttccct gatggtccat gtcagttat cgcctgtcaa gtggcgtgac 50 <210> 48 <211> 50 <212> DNA <213> Artificial Sequence <400> 48 gtcacgccac ttgacaggcg agtaactgac atggaccatc agggaacatt 50 <210> 49 <211> 50 <212> DNA <213> Artificial Sequence <400> 49 agacttagat ctgagccctc cctcttccca gcacaggcag gggtagaagc 50 <210> 50 <211> 50 <212> DNA <213> Artificial Sequence <400> 50 agacttagat ctgagccctc cctcttccca gaacaggcag gggtagaagc 50 <210> 51 <211> 50 <212> DNA <213> Artificial Sequence <400> 51 tgtgtttgga tttgcagtag gctgaagcgt tatactatga ctggagtcca 50 <210> 52 <211> 50 <212> DNA <213> Artificial Sequence <400> 52 tgtgtttgga tttgcagtag gctgaagcgt tgtactatga ctggagtcca 50 <210> 53 <211> 50 <212> DNA <213> Artificial Sequence <400> 53 aaaatagtgc tttttacttt tatctgaatg attgaaatgt ccttttccca 50 <210> 54 <211> 50 <212> DNA <213> Artificial Sequence <400> 54 aaaatagtgc tttttacttt tatctgaatg actgaaatgt ccttttccca 50 <210> 55 <211> 50 <212> DNA <213> Artificial Sequence <400> 55 gtcacgccac ttgacaggcg agtaacagac atggaccatc agtgaacatt 50 <210> 56 <211> 50 <212> DNA <213> Artificial Sequence <400> 56 gtcacgccac ttgacaggcg agtaacagac atggaccatc agagaacatt 50 <210> 57 <211> 50 <212> DNA <213> Artificial Sequence <400> 57 gtcacgccac ttgacaggcg agtaacagac atggaccatc aaggaacatt 50 <210> 58 <211> 50 <212> DNA <213> Artificial Sequence <400> 58 gtcacgccac ttgacaggcg agtaacagac atggaccatc atggaacatt 50 <210> 59 <211> 50 <212> DNA <213> Artificial Sequence <400> 59 gtcacgccac ttgacaggcg agtaacagac atggaccatc cgggaacatt 50 <210> 60 <211> 50 <212> DNA <213> Artificial Sequence <400> 60 gtcacgccac ttgacaggcg agtaacagac atggaccatc ggggaacatt 50 <210> 61 <211> 50 <212> DNA <213> Artificial Sequence <400> 61 gtcacgccac ttgacaggcg agtaacagac atggaccata agggaacatt 50 <210> 62 <211> 50 <212> DNA <213> Artificial Sequence <400> 62 gtcacgccac ttgacaggcg agtaacagac atggaccatt agggaacatt 50 <210> 63 <211> 50 <212> DNA <213> Artificial Sequence <400> 63 gtcacgccac ttgacaggcg agtaacagac atggaccacc agggaacatt 50 <210> 64 <211> 50 <212> DNA <213> Artificial Sequence <400> 64 gtcacgccac ttgacaggcg agtaacagac atggaccagc agggaacatt 50 <210> 65 <211> 50 <212> DNA <213> Artificial Sequence <400> 65 gtcacgccac ttgacaggcg agtaacagac atggaccctc agggaacatt 50 <210> 66 <211> 50 <212> DNA <213> Artificial Sequence <400> 66 gtcacgccac ttgacaggcg agtaacagac atggaccgtc agggaacatt 50 <210> 67 <211> 50 <212> DNA <213> Artificial Sequence <400> 67 gtcacgccac ttgacaggcg agtaacagac atggacaatc agggaacatt 50 <210> 68 <211> 50 <212> DNA <213> Artificial Sequence <400> 68 gtcacgccac ttgacaggcg agtaacagac atggactatc agggaacatt 50 <210> 69 <211> 50 <212> DNA <213> Artificial Sequence <400> 69 gtcacgccac ttgacaggcg agtaacagac atggaacatc agggaacatt 50 <210> 70 <211> 50 <212> DNA <213> Artificial Sequence <400> 70 gtcacgccac ttgacaggcg agtaacagac atggatcatc agggaacatt 50 <210> 71 <211> 50 <212> DNA <213> Artificial Sequence <400> 71 gtcacgccac ttgacaggcg agtaacagac atggcccatc agggaacatt 50 <210> 72 <211> 50 <212> DNA <213> Artificial Sequence <400> 72 gtcacgccac ttgacaggcg agtaacagac atgggccatc agggaacatt 50 <210> 73 <211> 50 <212> DNA <213> Artificial Sequence <400> 73 gtcacgccac ttgacaggcg agtaacagac atgaaccatc agggaacatt 50 <210> 74 <211> 50 <212> DNA <213> Artificial Sequence <400> 74 gtcacgccac ttgacaggcg agtaacagac atgtaccatc agggaacatt 50 <210> 75 <211> 50 <212> DNA <213> Artificial Sequence <400> 75 gtcacgccac ttgacaggcg agtaacagac atagaccatc agggaacatt 50 <210> 76 <211> 50 <212> DNA <213> Artificial Sequence <400> 76 gtcacgccac ttgacaggcg agtaacagac attgaccatc agggaacatt 50 <210> 77 <211> 50 <212> DNA <213> Artificial Sequence <400> 77 gtcacgccac ttgacaggcg agtaacagac acggaccatc agggaacatt 50 <210> 78 <211> 50 <212> DNA <213> Artificial Sequence <400> 78 gtcacgccac ttgacaggcg agtaacagac agggaccatc agggaacatt 50 <210> 79 <211> 50 <212> DNA <213> Artificial Sequence <400> 79 gtcacgccac ttgacaggcg agtaacagac ctggaccatc agggaacatt 50 <210> 80 <211> 50 <212> DNA <213> Artificial Sequence <400> 80 gtcacgccac ttgacaggcg agtaacagac gtggaccatc agggaacatt 50 <210> 81 <211> 50 <212> DNA <213> Artificial Sequence <400> 81 gtcacgccac ttgacaggcg agtaacagaa atggaccatc agggaacatt 50 <210> 82 <211> 50 <212> DNA <213> Artificial Sequence <400> 82 gtcacgccac ttgacaggcg agtaacagat atggaccatc agggaacatt 50 <210> 83 <211> 50 <212> DNA <213> Artificial Sequence <400> 83 gtcacgccac ttgacaggcg agtaacagcc atggaccatc agggaacatt 50 <210> 84 <211> 50 <212> DNA <213> Artificial Sequence <400> 84 gtcacgccac ttgacaggcg agtaacaggc atggaccatc agggaacatt 50 <210> 85 <211> 50 <212> DNA <213> Artificial Sequence <400> 85 gtcacgccac ttgacaggcg agtaacaaac atggaccatc agggaacatt 50 <210> 86 <211> 50 <212> DNA <213> Artificial Sequence <400> 86 gtcacgccac ttgacaggcg agtaacatac atggaccatc agggaacatt 50 <210> 87 <211> 50 <212> DNA <213> Artificial Sequence <400> 87 agacttagat ctgagccctc cctcttccca gcacacgcag gggtagaagc 50 <210> 88 <211> 50 <212> DNA <213> Artificial Sequence <400> 88 agacttagat ctgagccctc cctcttccca gcactggcag gggtagaagc 50 <210> 89 <211> 50 <212> DNA <213> Artificial Sequence <400> 89 agacttagat ctgagccctc cctcttccca gcagaggcag gggtagaagc 50 <210> 90 <211> 50 <212> DNA <213> Artificial Sequence <400> 90 agacttagat ctgagccctc cctcttccca gctcaggcag gggtagaagc 50 <210> 91 <211> 50 <212> DNA <213> Artificial Sequence <400> 91 agacttagat ctgagccctc cctcttccca ggacaggcag gggtagaagc 50 <210> 92 <211> 50 <212> DNA <213> Artificial Sequence <400> 92 agacttagat ctgagccctc cctcttccca ccacaggcag gggtagaagc 50 <210> 93 <211> 50 <212> DNA <213> Artificial Sequence <400> 93 agacttagat ctgagccctc cctcttccct gcacaggcag gggtagaagc 50 <210> 94 <211> 50 <212> DNA <213> Artificial Sequence <400> 94 agacttagat ctgagccctc cctcttccga gcacaggcag gggtagc <210> 95 <211> 50 <212> DNA <213> Artificial Sequence <400> 95 agacttagat ctgagccctc cctcttcgca gcacaggcag gggtagc <210> 96 <211> 50 <212> DNA <213> Artificial Sequence <400> 96 tgtgtttgga tttgcagtag gctgaagcgt tatacaatga ctggagtcca <210> 97 <211> 50 <212> DNA <213> Artificial Sequence <400> 97 tgtgtttgga tttgcagtag gctgaagcgt tatagtatga ctggagtcca <210> 98 <211> 50 <212> DNA <213> Artificial Sequence <400> 98 tgtgtttgga tttgcagtag gctgaagcgt tattctatga ctggagtcca <210> 99 <211> 50 <212> DNA <213> Artificial Sequence <400> 99 tgtgtttgga tttgcagtag gctgaagcgt taaactatga ctggagtcca <210> 100 <211> 50 <212> DNA <213> Artificial Sequence <400> 100 tgtgtttgga tttgcagtag gctgaagcgt tttactatga ctggagtcca <210> 101 <211> 50 <212> DNA <213> Artificial Sequence <400> 101 tgtgtttgga tttgcagtag gctgaagcgt aatactatga ctggagtcca <210> 102 <211> 50 <212> DNA <213> Artificial Sequence <400> 102 tgtgtttgga tttgcagtag gctgaagcga tatactatga ctggagtcca <210> 103 <211> 50 <212> DNA <213> Artificial Sequence <400> 103 tgtgtttgga tttgcagtag gctgaagcct tatactatga ctggagtcca <210> 104 <211> 50 <212> DNA <213> Artificial Sequence <400> 104 tgtgtttgga tttgcagtag gctgaagggt tatactatga ctggagtcca 50 <210> 105 <211> 50 <212> DNA <213> Artificial Sequence <400> 105 aaaatagtgc tttttacttt tatctgaatg attgatatgt ccttttccca 50 <210> 106 <211> 50 <212> DNA <213> Artificial Sequence <400> 106 aaaatagtgc tttttacttt tatctgaatg attgtaatgt ccttttccca 50 <210> 107 <211> 50 <212> DNA <213> Artificial Sequence <400> 107 aaaatagtgc tttttacttt tatctgaatg attcaaatgt ccttttccca 50 <210> 108 <211> 50 <212> DNA <213> Artificial Sequence <400> 108 aaaatagtgc tttttacttt tatctgaatg atagaaatgt ccttttccca 50 <210> 109 <211> 50 <212> DNA <213> Artificial Sequence <400> 109 aaaatagtgc tttttacttt tatctgaatg aatgaaatgt ccttttccca 50 <210> 110 <211> 50 <212> DNA <213> Artificial Sequence <400> 110 aaaatagtgc tttttacttt tatctgaatg tttgaaatgt ccttttccca 50 <210> 111 <211> 50 <212> DNA <213> Artificial Sequence <400> 111 aaaatagtgc tttttacttt tatctgaatc attgaaatgt ccttttccca 50 <210> 112 <211> 50 <212> DNA <213> Artificial Sequence <400> 112 aaaatagtgc tttttacttt tatctgaaag attgaaatgt ccttttccca 50 <210> 113 <211> 50 <212> DNA <213> Artificial Sequence <400> 113 aaaatagtgc tttttacttt tatctgattg attgaaatgt ccttttccca 50 <210> 114 <211> 50 <212> DNA <213> Artificial Sequence <400> 114 ggatctcctg gcggaggtgg tggtagaagg tccaggagca ggggtagccg <210> 115 <211> 50 <212> DNA <213> Artificial Sequence <400> 115 ggatctcctg gcggaggtgg tggtagaagg tctaggagca ggggtagccg <210> 116 <211> 50 <212> DNA <213> Artificial Sequence <400> 116 agcacatgtt tgttgaatga gtcagtggat gacgtagtc agtatcttgt <210> 117 <211> 50 <212> DNA <213> Artificial Sequence <400> 117 agcacatgtt tgttgaatga gtcagtggat gatgtagtc agtatcttgt <210> 118 <211> 50 <212> DNA <213> Artificial Sequence <400> 118 gcttctttcc ctgcctgtgc tgggaagagg gagggctcag atctaagtct <210> 119 <211> 50 <212> DNA <213> Artificial Sequence <400> 119 agacttagat ctgagccctc cctcttccca gcacaggcag ggaaagaagc 50 <210> 120 <211> 50 <212> DNA <213> Artificial Sequence <400> 120 gcttctttcc ctgcctgttc tgggaagagg gagggctcag atctaagtct 50 <210> 121 <211> 50 <212> DNA <213> Artificial Sequence <400> 121 agacttagat ctgagccctc cctcttccca gaacaggcag ggaaagaagc 50 <210> 122 <211> 50 <212> DNA <213> Artificial Sequence <400> 122 gcttttaccc ctgcctgtgc tgggaagagg gagggctcag atctaagtct 50 <210> 123 <211> 50 <212> DNA <213> Artificial Sequence <400> 123 agacttagat ctgagccctc cctcttccca gcacaggcag gggtaaaagc 50 <210> 124 <211> 50 <212> DNA <213> Artificial Sequence <400> 124 gcttttaccc ctgcctgttc tgggaagagg gagggctcag atctaagtct 50 <210> 125 <211> 50 <212> DNA <213> Artificial Sequence <400> 125 agacttagat ctgagccctc cctcttccca gaacaggcag gggtaaaagc 50 <210> 126 <211> 12 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 126 nnnnnnnnnn nn 12 <210> 127 <211> 41 <212> DNA <213> Artificial Sequence <400> 127 ctaccgtaat actaaaagtg ccacttctca gatttgagaa g 41 <210> 128 <211> 25 <212> DNA <213> Artificial Sequence <400> 128 gaaattaata cgactcacta taggg 25 <210> 129 <211> 68 <212> DNA <213> Artificial Sequence <400> 129 tcgcgcttgt cgcgcagacg aatgatctac aacagtagaa attccctata gtgagtcgta 60 ttaatttc 68 <210> 130 <211> twenty three <212> DNA <213> Artificial Sequence <400> 130 aacagacatg gaccatcagg gtg 23 <210> 131 <211> twenty one <212> DNA <213> Artificial Sequence <400> 131 catggaccat cagggtgcca c 21 <210> 132 <211> twenty two <212> DNA <213> Artificial Sequence <400> 132 acatggacca tcagggtgcc ac 22 <210> 133 <211> twenty one <212> DNA <213> Artificial Sequence <400> 133 gacatggacc atcagggtgc c 21 <210> 134 <211> twenty one <212> DNA <213> Artificial Sequence <400> 134 agacatggac catcagggtg c 21 <210> 135 <211> twenty one <212> DNA <213> Artificial Sequence <400> 135 cagacatgga ccatcagggt g 21 <210> 136 <211> twenty two <212> DNA <213> Artificial Sequence <400> 136 acagacatgg accatcaggg tg 22 <210> 137 <211> 40 <212> DNA <213> Artificial Sequence <400> 137 ccagcacagg caggggggtgc cacttctcag atttgagaag 40 <210> 138 <211> 40 <212> DNA <213> Artificial Sequence <400> 138 cgttatacta tgactggtgc cacttctcag atttgagaag 40 <210> 139 <211> 40 <212> DNA <213> Artificial Sequence <400> 139 atgattgaaa tgtcctgtgc cacttctcag atttgagaag 40 <210> 140 <211> 40 <212> DNA <213> Artificial Sequence <400> 140 ccagaacagg caggggggtgc cacttctcag atttgagaag 40 <210> 141 <211> 40 <212> DNA <213> Artificial Sequence <400> 141 cgttgtacta tgactggtgc cacttctcag atttgagaag 40 <210> 142 <211> 40 <212> DNA <213> Artificial Sequence <400> 142 atgactgaaa tgtcctgtgc cacttctcag atttgagaag 40 <210> 143 <211> 40 <212> DNA <213> Artificial Sequence <400> 143 aggtccagga gcaggggtgc cacttctcag atttgagaag 40 <210> 144 <211> 40 <212> DNA <213> Artificial Sequence <400> 144 aggtctagga gcaggggtgc cacttctcag atttgagaag 40 <210> 145 <211> 40 <212> DNA <213> Artificial Sequence <400> 145 gatgacgtag atcagtgtgc cacttctcag atttgagaag 40 <210> 146 <211> 40 <212> DNA <213> Artificial Sequence <400> 146 gatgatgtag atcagtgtgc cacttctcag atttgagaag 40 <210> 147 <211> 44 <212> DNA <213> Artificial Sequence <400> 147 atatcttcta cttctccact gtgccacttc tcagatttga gaag 44 <210> 148 <211> 44 <212> DNA <213> Artificial Sequence <400> 148 tctagagaat cccagaatgc gtgccacttc tcagatttga gaag 44 <210> 149 <211> 40 <212> DNA <213> Artificial Sequence <400> 149 gcgttatact atgactgtgc cacttctcag atttgagaag 40 <210> 150 <211> 40 <212> DNA <213> Artificial Sequence <400> 150 gcgttgtact atgactgtgc cacttctcag atttgagaag 40 <210> 151 <211> 42 <212> DNA <213> Artificial Sequence <400> 151 cttcccagca caggcagggt gccacttctc agatttgaga ag 42 <210> 152 <211> 42 <212> DNA <213> Artificial Sequence <400> 152 cttcccagaa caggcagggt gccacttctc agatttgaga ag 42 <210> 153 <211> 42 <212> DNA <213> Artificial Sequence <400> 153 tcccagcaca ggcagggggt gccacttctc agatttgaga ag 42 <210> 154 <211> 42 <212> DNA <213> Artificial Sequence <400> 154 tcccagaaca ggcagggggt gccacttctc agatttgaga ag 42 <210> 155 <211> 44 <212> DNA <213> Artificial Sequence <400> 155 tgtgatccaa gacattcccc gtgccacttc tcagatttga gaag 44 <210> 156 <211> 44 <212> DNA <213> Artificial Sequence <400> 156 ctcgtcagtg ctctcctctc gtgccacttc tcagatttga gaag 44 <210> 157 <211> 43 <212> DNA <213> Artificial Sequence <400> 157 tcttcccagc acaggcaggg tgccacttct cagatttgag aag 43 <210> 158 <211> 43 <212> DNA <213> Artificial Sequence <400> 158 tcttcccaga acaggcaggg tgccacttct cagatttgag aag 43 <210> 159 <211> 43 <212> DNA <213> Artificial Sequence <400> 159 ttcccagcac aggcaggggg tgccacttct cagatttgag aag 43 <210> 160 <211> 43 <212> DNA <213> Artificial Sequence <400> 160 ttcccagaac aggcaggggg tgccacttct cagatttgag aag 43 <210> 161 <211> 20 <212> DNA <213> Artificial Sequence <400> 161 gtgaacgttc ccttagcact 20 <210> 162 <211> 19 <212> DNA <213> Artificial Sequence <400> 162 gggagggcag aactagtcc 19 <210> 163 <211> 41 <212> DNA <213> Artificial Sequence <400> 163 cgccacttga caggcgagta actgccactt attgggtcag c 41 <210> 164 <211> 39 <212> DNA <213> Artificial Sequence <400> 164 gcgtgttccc cagagtgact tagcagcttc ctcctcctt 39 <210> 165 <211> twenty two <212> DNA <213> Artificial Sequence <400> 165 aggaaacatt aacgtactga tg 22 <210> 166 <211> twenty one <212> DNA <213> Artificial Sequence <400> 166 ttccttttat ttcccttcag c 21 <210> 167 <211> 18 <212> DNA <213> Artificial Sequence <400> 167 cgacggcaaa gaagacca 18 <210> 168 <211> 18 <212> DNA <213> Artificial Sequence <400> 168 agcctgccag gtgagtac 18 <210> 169 <211> 40 <212> DNA <213> Artificial Sequence <400> 169 cgggtggatc ggcgttttgt tcactatgaa ggcggcatca 40 <210> 170 <211> 40 <212> DNA <213> Artificial Sequence <400> 170 gtattggcgt cgaagtggcg ttcgctgcgg aatgttgttg 40 <210> 171 <211> twenty two <212> DNA <213> Artificial Sequence <400> 171 ttgttcagat attcaacgaa cg 22 <210> 172 <211> 20 <212> DNA <213> Artificial Sequence <400> 172 gtggaacgat ggcttccagg 20 <210> 173 <211> 19 <212> DNA <213> Artificial Sequence <400> 173 tctctgtgca tggcctgta 19 <210> 174 <211> 20 <212> DNA <213> Artificial Sequence <400> 174 aacagtaaag gcaacgtcca 20 <210> 175 <211> 40 <212> DNA <213> Artificial Sequence <400> 175 gcagctggga taccagtgga agtgcctcct ggaagaatgg 40 <210> 176 <211> 40 <212> DNA <213> Artificial Sequence <400> 176 tctctagagc catcttgcgc ctgaagcgac ccatgaacgc 40 <210> 177 <211> twenty two <212> DNA <213> Artificial Sequence <400> 177 tgcttactga agccgaaaaa tg 22 <210> 178 <211> twenty one <212> DNA <213> Artificial Sequence <400> 178 tgatcgcgag accacacgat g 21 <210> 179 <211> 20 <212> DNA <213> Artificial Sequence <400> 179 ggtttcggat gttacagcgt 20 <210> 180 <211> 35 <212> DNA <213> Artificial Sequence <400> 180 caagcacccc acccgctcac ccacctcctc ctttg 35 <210> 181 <211> 43 <212> DNA <213> Artificial Sequence <400> 181 ggtttcggat gttacagcgt gtgctggaag acttagatct gag 43 <210> 182 <211> 19 <212> DNA <213> Artificial Sequence <400> 182 gctggaaagg tcaagggac 19 <210> 183 <211> 19 <212> DNA <213> Artificial Sequence <400> 183 ggggtttgtt gcacagtcc 19 <210> 184 <211> 40 <212> DNA <213> Artificial Sequence <400> 184 ggtagaagca aaggcaggag gtttgcccaa ggtcacacag 40 <210> 185 <211> 40 <212> DNA <213> Artificial Sequence <400> 185 gaaagaagca aaggcaggag gtttgcccaa ggtcacacag 40 <210> 186 <211> 41 <212> DNA <213> Artificial Sequence <400> 186 ctgggaagag ggagggctca gtgttgccac actttcactg g 41 <210> 187 <211> 18 <212> DNA <213> Artificial Sequence <400> 187 gtgagcgggt ggggtgct 18 <210> 188 <211> twenty three <212> DNA <213> Artificial Sequence <400> 188 tctaagtctt ccagcacggg atc 23 <210> 189 <211> 18 <212> DNA <213> Artificial Sequence <400> 189 tgacacagcc tgggactt 18 <210> 190 <211> 19 <212> DNA <213> Artificial Sequence <400> 190 cacacctcct gtggctaag 19 <210> 191 <211> 40 <212> DNA <213> Artificial Sequence <400> 191 gagtgttctg aaggcaccac cagctccatt ggaggggtct 40 <210> 192 <211> 41 <212> DNA <213> Artificial Sequence <400> 192 acacaagggc taatgggtgc cgccaaagca attgatcggt c 41 <210> 193 <211> twenty two <212> DNA <213> Artificial Sequence <400> 193 ttggtgaacg gcttttccta tg 22 <210> 194 <211> twenty one <212> DNA <213> Artificial Sequence <400> 194 tgctctggat gggcgtcaac g 21 <210> 195 <211> 20 <212> DNA <213> Artificial Sequence <400> 195 gagacaaagg aatgccctga 20 <210> 196 <211> 20 <212> DNA <213> Artificial Sequence <400> 196 gccctctcaa gtttccatgt 20 <210> 197 <211> 41 <212> DNA <213> Artificial Sequence <400> 197 cgggttgatg tgatgccaaa gcgagcacag agcttggaac a 41 <210> 198 <211> 42 <212> DNA <213> Artificial Sequence <400> 198 ggagcgatca taggtacggc tggcgactct caatccagta cg 42 <210> 199 <211> 19 <212> DNA <213> Artificial Sequence <400> 199 gaagtcttcg atttgcatg 19 <210> 200 <211> twenty two <212> DNA <213> Artificial Sequence <400> 200 acatgtggca gtccatagtg ac 22 <210> 201 <211> 41 <212> DNA <213> Artificial Sequence <400> 201 agggatctcg ctcctggaag atcaccgtca aggctgagaa c 41 <210> 202 <211> 38 <212> DNA <213> Artificial Sequence <400> 202 cgatgctggc gctgagtacg aatgagcccc agccttct 38 <210> 203 <211> 19 <212> DNA <213> Artificial Sequence <400> 203 tccacccatg gcaaattcc 19 <210> 204 <211> 19 <212> DNA <213> Artificial Sequence <400> 204 agggggcaga gatgatgac 19 <210> 205 <211> twenty one <212> DNA <213> Artificial Sequence <400> 205 tccattgatg acaagcttcc c 21 <210> 206 <211> 20 <212> DNA <213> Artificial Sequence <400> 206 tcgtggagtc cactggcgtc 20 <210> 207 <211> 46 <212> DNA <213> Artificial Sequence <400> 207 ccagatcttc caggaaaatc cacatctgta tttgatggaa gacctt 46 <210> 208 <211> 41 <212> DNA <213> Artificial Sequence <400> 208 agaccagtaa ttgctggaag acttgtcttc caagaagctc c 41 <210> 209 <211> twenty one <212> DNA <213> Artificial Sequence <400> 209 agatttttct ggaagacatg g 21 <210> 210 <211> 18 <212> DNA <213> Artificial Sequence <400> 210 atgtcttccg gacaatcc 18 <210> 211 <211> twenty three <212> DNA <213> Artificial Sequence <400> 211 tgctggaaga cttgatttac tgg 23 <210> 212 <211> twenty four <212> DNA <213> Artificial Sequence <400> 212 ggaggcaccc tagggccagg gaaa 24 <210> 213 <211> 25 <212> DNA <213> Artificial Sequence <400> 213 gttactgcaa gcagcaacaa agtcc 25 <210> 214 <211> twenty four <212> DNA <213> Artificial Sequence <400> 214 ggaggtattt tagggttagg gaaa 24 <210> 215 <211> 25 <212> DNA <213> Artificial Sequence <400> 215 attactacaa acaacaacaa aatcc 25 <210> 216 <211> 44 <212> DNA <213> Artificial Sequence <400> 216 ggcgtcttca ccaccatgga gtgccacttc tcagatttga gaag 44 <210> 217 <211> 44 <212> DNA <213> Artificial Sequence <400> 217 ctccaagtct tccagtaaat gtgccacttc tcagatttga gaag 44

Claims

1. A detection system for detecting a target nucleic acid molecule, characterized in that: The system comprises: (a) Cas12b protein, wherein the Cas12b protein is Cas12b or a Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b, and the Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b is Cas12c; (b) a guide RNA, wherein the guide RNA guides the Cas12b protein to specifically bind to the target nucleic acid molecule; and (c) a nucleic acid probe, wherein the nucleic acid probe is a single-stranded DNA; Wherein, the target nucleic acid molecule is a target DNA, the detection system is a system for performing a nucleic acid amplification reaction and a bypass cleavage reaction, and the nucleic acid amplification reaction and the bypass cleavage reaction are performed simultaneously, and the detection system is a one-step detection system; The target DNA includes DNA obtained by reverse transcription or amplification of RNA.

2. The detection system according to claim 1, characterized in that When the target nucleic acid molecule is ssDNA, the detection site of the target nucleic acid molecule is located at the 9th position or the 10th to 16th position downstream of the PAM sequence of the guide RNA.

3. The detection system according to claim 2, characterized in that When the target nucleic acid molecule is ssDNA, the detection site of the target nucleic acid molecule is located at positions 10-14 downstream of the PAM sequence of the guide RNA.

4. The detection system according to claim 2, characterized in that When the target nucleic acid molecule is ssDNA, the detection site of the target nucleic acid molecule is located at the 10th to 12th position downstream of the PAM sequence of the guide RNA.

5. The detection system according to claim 2, characterized in that When the detection site is located at the 9th position downstream of the PAM sequence of the guide RNA, the detection site is G.

6. The detection system according to claim 1, characterized in that The detection site of the target nucleic acid molecule is located at positions 1-12 downstream of the PAM sequence of the guide RNA.

7. The detection system according to claim 6, characterized in that The detection site of the target nucleic acid molecule is located at the first position downstream of the PAM sequence of the guide RNA.

8. The detection system according to claim 6, characterized in that The detection site of the target nucleic acid molecule is located at the third position downstream of the PAM sequence of the guide RNA.

9. The detection system according to claim 6, characterized in that The detection site of the target nucleic acid molecule is located at the 10th position downstream of the PAM sequence of the guide RNA.

10. The detection system according to claim 1, characterized in that The target DNA includes cDNA obtained by reverse transcription or amplification of RNA; The cDNA is obtained by adding reverse transcriptase to the detection system to achieve reverse transcription of RNA, and then amplifying it with Bst2.0 DNA polymerase; Alternatively, the cDNA is obtained by directly using Bst3.0 DNA polymerase to directly achieve reverse transcription and amplification of RNA.

11. The detection system according to claim 1, characterized in that The Cas12b protein is selected from the group consisting of AacCas12b, Aac2Cas12b, AkaCas12b, AmaCas12b, AheCas12b, and AcoCas12b.

12. The detection system according to claim 1, characterized in that The nucleic acid probe is a single-stranded DNA with a detectable label.

13. The detection system according to claim 1, characterized in that In the detection system, the molar ratio of the nucleic acid probe to the target nucleic acid molecule is 10 3 : 1 to 10 14 :

1.

14. The detection system according to claim 1, characterized in that The length of the guide RNA is 15-30nt.

15. The detection system according to claim 1, characterized in that The target DNA is selected from the following group: single-stranded DNA, double-stranded DNA, or a combination thereof.

16. The detection system according to claim 1, characterized in that The length of the nucleic acid probe is 3-300 nt.

17. A detection system for detecting SNP, characterized in that: The system comprises a first detection system and a second detection system; The first detection system includes: (a1) Cas12b protein, wherein the Cas12b protein is Cas12b or a Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b, and the Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b is Cas12c; (b1) a first guide RNA, wherein the first guide RNA guides the Cas12b protein to specifically bind to the target nucleic acid molecule; and (c1) a nucleic acid probe, wherein the nucleic acid probe is a single-stranded DNA; The second detection system includes: (a2) Cas12b protein, wherein the Cas12b protein is Cas12b or a Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b, and the Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b is Cas12c; (b2) a second guide RNA, wherein the second guide RNA guides the Cas12b protein to specifically bind to the target nucleic acid molecule; and (c2) a nucleic acid probe, wherein the nucleic acid probe is a single-stranded DNA; Wherein, the target nucleic acid molecule is a target DNA, and the target DNA includes DNA obtained by reverse transcription or amplification of RNA; Furthermore, the first guide RNA and the second guide RNA are directed to the same nucleic acid sequence region including the SNP site, and the first guide RNA is directed to the non-mutated nucleic acid sequence of the SNP site, while the second guide RNA is directed to the mutant nucleic acid sequence of the SNP site; The detection system is a system for performing nucleic acid amplification reaction and bypass cleavage reaction, and the nucleic acid amplification reaction and bypass cleavage reaction are performed simultaneously. The detection system is a one-step detection system.

18. The detection system according to claim 17, characterized in that The system also includes a blank control system, which includes: (a3) Cas12b protein, wherein the Cas12b protein is Cas12b or a Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b, and the Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b is Cas12c; and (c3) A nucleic acid probe, wherein the nucleic acid probe is a single-stranded DNA.

19. The detection system according to claim 17, characterized in that The SNP site is located at the 9th position or the 10th to 16th position downstream of the PAM sequence of the guide RNA.

20. The detection system according to claim 19, characterized in that The SNP site is located at positions 10-14 downstream of the PAM sequence of the guide RNA.

21. The detection system according to claim 19, characterized in that The SNP site is located at positions 10-12 downstream of the PAM sequence of the guide RNA.

22. A kit for detecting a target nucleic acid molecule, characterized in that: The kit comprises: i) a first container and a Cas12b protein in the first container, wherein the Cas12b protein is Cas12b or a Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b, and the Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b is Cas12c; ii) an optional second container and a guide RNA located in the second container, wherein the guide RNA guides the Cas protein to specifically bind to a target nucleic acid molecule; iii) a third container and a nucleic acid probe located in the third container; iv) an optional fourth container and a buffer within the fourth container; Wherein, the target nucleic acid molecule is a target DNA; The kit performs nucleic acid amplification reaction and bypass cleavage reaction, and the nucleic acid amplification reaction and bypass cleavage reaction are performed simultaneously, which is a one-step detection; The kit also includes: v) a fifth container and a polymerase for amplifying the target DNA located in the fifth container, wherein the polymerase for amplifying the target DNA comprises Bst2.0 DNA polymerase and / or Bst3.0 DNA polymerase; vi) an optional sixth container and a reverse transcriptase for reverse transcription located in the sixth container; vii) a seventh container and dNTPs in the seventh container for an amplification reaction and / or a reverse transcription reaction; All of the first to seventh containers are identical containers.

23. A method for detecting the presence of a target nucleic acid molecule in a sample for non-disease diagnosis purposes, characterized in that: The following steps are involved: (i) providing a detection system for detecting a target nucleic acid molecule according to any one of claims 1 to 16, wherein the detection system further comprises a sample to be detected; and (ii) detecting whether the nucleic acid probe in the detection system is cut by the Cas12b protein, wherein the cutting is a trans cutting that bypasses the single-stranded DNA; Among them, if the nucleic acid probe is cut by the Cas12b protein, it means that the target nucleic acid molecule exists in the sample; if the nucleic acid probe is not cut by the Cas12b protein, it means that the target nucleic acid molecule does not exist in the sample.

24. A method for detecting the presence of a target nucleic acid molecule in a sample for non-disease diagnosis purposes, characterized in that: The following steps are involved: (i) providing a detection system, the detection system comprising: (a) Cas12b protein, wherein the Cas12b protein is Cas12b or a Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b, and the Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b is Cas12c; (b) a guide RNA, wherein the guide RNA guides the Cas12b protein to specifically bind to the target nucleic acid molecule; (c) a nucleic acid probe, wherein the nucleic acid probe is a single-stranded DNA; Wherein, the target nucleic acid molecule is a target DNA; (d) buffer; (e1) a polymerase for amplifying the target DNA, wherein the polymerase for amplifying the target DNA comprises Bst2.0 DNA polymerase and / or Bst3.0 DNA polymerase; (e2) optionally a reverse transcriptase for reverse transcription; (e3) dNTPs for amplification reaction and / or reverse transcription reaction; and (f) test samples to be tested; The detection system is a system for performing nucleic acid amplification reaction and bypass cleavage reaction, and the nucleic acid amplification reaction and bypass cleavage reaction are performed simultaneously, and the detection system is a one-step detection system; (ii) performing a reverse transcription and / or amplification reaction on the detection system to obtain a reverse transcribed and / or amplified detection system; (iii) detecting whether the nucleic acid probe in the detection system in the previous step is cut by the Cas12b protein, wherein the cutting is a trans cutting that bypasses the single-stranded DNA; Among them, if the nucleic acid probe is cut by the Cas12b protein, it means that the target nucleic acid molecule exists in the sample; if the nucleic acid probe is not cut by the Cas12b protein, it means that the target nucleic acid molecule does not exist in the sample.

25. The method of claim 24, wherein: The detection includes: qualitative detection or quantitative detection.

26. The method of claim 25, wherein: The quantitative detection is an absolute quantitative detection.

27. The method of claim 25, wherein: The quantitative detection is a quantitative detection combined with digital PCR technology.

28. The method of claim 24, wherein: The amplification method is selected from the following group: PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification, rolling circle amplification, HDA, SPIA, NEAR, TMA and SMAP2.

29. The method of claim 24, wherein: The target nucleic acid molecule is a methylated nucleic acid sequence.

30. The method of claim 29, wherein: The methylated nucleic acid sequence is a nucleic acid sequence obtained after conversion of non-methylated C to uracil.

31. The method of claim 30, wherein: The methylated nucleic acid sequence is treated with bisulfite to convert the unmethylated C into uracil.

32. The method of claim 24, wherein: In the step (ii), the temperature of the detection system is maintained at 50-70°C.

33. The method of claim 24, wherein: In the step (iii), the temperature of the detection system is maintained at 25-70°C.

34. The method of claim 24, wherein: In the detection system, the nucleic acid concentration of the test sample to be detected is 1×10 -11 -1×10 -5 nM.

35. The method of claim 24, wherein: The total time of steps (ii) and (iii) is ≤ 2 hours.

36. The method of claim 24, wherein: The total time of steps (ii) and (iii) is ≤ 1 hour.

37. A use of Cas12b protein, characterized in that The Cas12b protein is used to prepare a detection reagent or kit for detecting a target nucleic acid molecule based on bypass single-stranded DNA cleavage, wherein the Cas12b protein is Cas12b or a Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b, and the Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b is Cas12c; The detection reagent or kit performs a nucleic acid amplification reaction and a bypass cleavage reaction, and the nucleic acid amplification reaction and the bypass cleavage reaction are performed simultaneously, which is a one-step detection; The detection reagent or kit comprises: i) a first container and a Cas12b protein in the first container, wherein the Cas12b protein is Cas12b or a Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b, and the Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b is Cas12c; ii) an optional second container and a guide RNA located in the second container, wherein the guide RNA guides the Cas protein to specifically bind to a target nucleic acid molecule; iii) a third container and a nucleic acid probe located in the third container; iv) an optional fourth container and a buffer within the fourth container; v) a fifth container and a polymerase for amplifying the target DNA located in the fifth container, wherein the polymerase for amplifying the target DNA comprises Bst2.0 DNA polymerase and / or Bst3.0 DNA polymerase; vi) an optional sixth container and a reverse transcriptase for reverse transcription located in the sixth container; vii) a seventh container and dNTPs in the seventh container for an amplification reaction and / or a reverse transcription reaction; All of the first to seventh containers are identical containers.

38. A device for detecting the presence of a target nucleic acid molecule in a sample, characterized in that: The device comprises: (a) an amplification reaction-bypass cleavage reaction module, the module being used to perform a nucleic acid amplification reaction and a bypass cleavage reaction on a digital reaction system, wherein the bypass cleavage reaction is a bypass cleavage mediated by a Cas12b protein; and (b) a signal detection module, which is used to detect whether Cas12b protein-mediated bypass cleavage occurs in each digitized reaction system; The nucleic acid amplification reaction and the bypass cleavage reaction are carried out simultaneously, and the digital reaction system is a one-step reaction system; And the digital reaction system includes a plurality of independent reaction system units, each of which includes: (a) Cas12b protein, wherein the Cas12b protein is Cas12b or a Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b, and the Cas protein having a bypass single-stranded DNA cleavage activity similar to that of Cas12b is Cas12c; (b) a guide RNA, wherein the guide RNA guides the Cas12b protein to specifically bind to the target nucleic acid molecule; (c) a nucleic acid probe, wherein the nucleic acid probe is a single-stranded DNA; Wherein, the target nucleic acid molecule is a target DNA; (d) Buffer (e1) a polymerase for amplifying the target DNA, wherein the polymerase for amplifying the target DNA comprises Bst2.0 DNA polymerase and / or Bst3.0 DNA polymerase; (e2) optionally a reverse transcriptase for reverse transcription; (e3) dNTPs for amplification reaction and / or reverse transcription reaction; and (f) Test samples to be tested.

39. The device according to claim 38, characterized in that The amplification reaction-bypass cutting reaction module also includes a temperature control unit, which is used to amplify the temperature of the digital reaction system in the device to a predetermined temperature.

40. The apparatus of claim 38, wherein: The digital reaction system includes a droplet digital PCR (ddPCR) reaction system or a chip digital PCR (cdPCR) reaction system.

41. The apparatus of claim 38, wherein: The signal detection module includes an imaging module, and the imaging module includes a fluorescence detection unit.

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