Method for detecting nucleic acid markers based on ligase chain reaction and gene editing technology

Abstract

The present invention provides a method for detecting nucleic acid markers based on ligase chain reaction and gene editing technology, comprising the following steps: (a) designing two pairs of DNA probes for ligase chain reaction according to the sequence of the target nucleic acid marker; wherein the first pair of DNA probes is complementary to the target nucleic acid marker, the second pair of DNA probes is complementary to the sequence of the first pair of DNA probes, and a PAM sequence is designed on the DNA probes; (b) mixing the nucleic acid to be detected with the two pairs of DNA probes, and adding ligase to carry out ligase chain reaction; (c) adding gRNA, CRISPR / Cas12 system and reporter probe to the ligation product, and the CRISPR / Cas12 system is activated to cut the reporter probe and release a fluorescent signal. The present invention innovatively combines LCR reaction with CRISPR / Cas12a to achieve homogeneous and ultrasensitive detection of nucleic acid markers.

Description

Technical Field

[0001] The present invention relates to the field of molecular diagnostic detection technology, and specifically to a method for detecting nucleic acid markers based on ligase chain reaction and gene editing technology. Background Art

[0002] With the development and intersection of disciplines such as chemistry, molecular biology, and biomedicine, the study and detection of nucleic acid molecular markers has important application value in the diagnosis and treatment of major diseases at the genetic level and the study of drug responses between different individuals. Studies have shown that the occurrence and development of many diseases such as new coronavirus infection, genetic diseases, and cancer are closely related to the abnormal expression of nucleic acid molecular markers (such as gene mutations, RNA, and mircoRNA (miRNA)). Therefore, the detection of nucleic acid markers such as gene mutations or RNA in the initial stage of changes in the life system is of great significance for the early diagnosis of the disease, individualized treatment, and efficacy judgment. The development and establishment of new methods for simple, homogeneous, ultra-sensitive and accurate detection of nucleic acid markers will surely provide strong support for the early diagnosis and treatment of the disease.

[0003] A variety of methods have been reported for the detection of nucleic acid molecular markers. Due to the small amount of biological samples and the low content of nucleic acid molecular markers therein, high-sensitivity nucleic acid detection generally requires pre-amplification of the target sequence. At present, the most widely used nucleic acid amplification technologies are polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), etc. Among them, PCR is the most widely used amplification technology. PCR amplification has high sensitivity, but its recognition ability for single bases is poor. In the presence of a large number of other nucleic acid molecules, amplification of low-abundance target nucleic acid molecules is prone to erroneous results. In addition, the commonly used real-time quantitative PCR technology needs to be combined with TaqMan probes for detection. Each mutation site needs to design a TaqMan probe related to the sequence, which is expensive. As the latest generation of PCR technology, digital PCR (ddPCR) has high sensitivity and good specificity, but its instruments and equipment are still expensive and cumbersome to operate, which limits the further development of PCR technology.

[0004] LCR is a technology based on ligation reaction and relying on template cyclic amplification. LCR uses two pairs of oligonucleotide probes, each pair contains two DNA probes, and adjacent probes complementary to the target nucleic acid are connected by specific DNA ligase. The connected probes are used as templates in subsequent thermal cycles to produce exponential amplification. Since the recognition ability of enzyme ligation reaction for single-base mismatch is much stronger than that of primer extension amplification reaction (such as PCR and LAMP), LCR has better specificity for the detection of single-base gene mutations than amplification technology relying on primer extension. Moreover, LCR only requires one ligase, which is simple and fast, and its sensitivity is comparable to PCR. However, due to the unique reaction mechanism of LCR, that is, double-stranded DNA before and after the reaction, there is only a difference in length. LCR cannot use TaqMan probes or molecular beacon probes to achieve homogeneous fluorescence detection like PCR. Therefore, LCR detection generally needs to be combined with magnetic bead separation or electrophoresis separation, which is cumbersome to operate and its application is limited. Although in previous studies, our group and other groups have achieved homogeneous detection of LCR based on cationic conjugated polymer (CCP) fluorescence resonance energy transfer technology (FRET) and fluorescent dyes, the sensitivity of the method is low (1fmol / L). Therefore, further innovation and development of new methods for homogeneous and highly sensitive detection of LCR faces major challenges. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detecting nucleic acid markers based on ligase chain reaction and gene editing technology, so as to solve the problems of cumbersome operation and low sensitivity of the existing methods.

[0006] The object of the present invention is achieved as follows: A method for detecting nucleic acid markers based on ligase chain reaction and gene editing technology, comprising the following steps:

[0007] (a) According to the sequence of the target nucleic acid marker, two pairs of DNA probes for ligase chain reaction are designed. The first pair of DNA probes are complementary to the target nucleic acid marker, and the 5′ end of the first pair of DNA probes at the connection point is phosphorylated; the second pair of DNA probes are complementary to the first pair of DNA probes or the 3′ end of the second pair of DNA probes at the connection point protrudes one base, and the 5′ end is phosphorylated; a PAM sequence is designed at the 5′ end of the DNA probe away from the connection point;

[0008] (b) mixing the nucleic acid to be detected with two pairs of DNA probes, and adding ligase to carry out ligase chain reaction. If the target nucleic acid marker is present, the reaction generates a double-stranded DNA ligation product containing a PAM sequence;

[0009] (c) adding gRNA, CRISPR / Cas12 system and reporter probe to the ligation product obtained in step (b), wherein one end of the reporter probe is modified with a fluorescent group and the other end is modified with a quenching group; the gRNA can bind to the double-stranded DNA ligation product and activate the CRISPR / Cas12 system, and after the CRISPR / Cas12 system is activated, it cuts the reporter probe and releases a fluorescent signal;

[0010] (d) Detecting the fluorescence signal of the reaction system to obtain the content of the target nucleic acid marker in the nucleic acid to be detected.

[0011] In step (a), the PAM sequence and the connection point are separated by 10-16 bases.

[0012] In step (a), the target nucleic acid marker is DNA or RNA. When the target nucleic acid marker is a single-base mutant DNA, the 3′-end protruding base at the connection point of the second pair of DNA probes is at the position of the mutant base.

[0013] In step (a), the RNA target nucleic acid markers include microRNA, messenger RNA, circular RNA, long non-coding RNA, and viral RNA.

[0014] In step (b), the ligase used in the ligase chain reaction includes Amp DNA ligase, Taq DNA ligase, and 9°N DNA ligase; when performing the ligase chain reaction, the ligation temperature is 45-65°C, the number of thermal cycles is 20-50, and the amount of ligase used is 0.2-20U.

[0015] In step (b), when the target nucleic acid marker is RNA, the first pair of DNA probes are first connected using RNA as a template under the action of a ligase, wherein the ligase is SplintR ligase or T4 RNA ligase 2.

[0016] In step (c), the gRNA comprises a detection and recognition region designed according to the target nucleic acid marker.

[0017] In step (c), the Cas12 enzyme of the CRISPR / Cas12 system may be at least one of AsCas12a, LbCas12a, FnCas12a, ArCas12a, LpCas12a, BsCas12a, HkCas12a, PrCas12a, and PxCas12a, and at this time, the PAM sequence is 5′-TTTN or 5′-TTN, wherein N represents any base; the Cas12 enzyme of the CRISPR / Cas12 system may also be HkCas12a, and at this time, the PAM sequence is 5′-YYN, wherein Y represents C or T base, and N represents any base; the Cas12 enzyme of the CRISPR / Cas12 system may also be Cas12b, and at this time, the PAM sequence is 5′-TTN, wherein N represents any base.

[0018] Before performing step (c), the PAM-containing probe and its complementary probe, gRNA and CRISPR / Cas12 system can be mixed, and the fluorescence of the system can be detected. If there is a fluorescent signal, the ligation product obtained in step (b) is digested with Lambda exonuclease before performing the reaction in step (c).

[0019] In step (c), the fluorescent group of the reporter probe is FAM, FITC, HEX, TET, TAMRA, ROX or Cy5, and the quencher group is BHQ1, TAMRA, BHQ2, BHQ3 or Dabcyl.

[0020] In step (d), fluorescence detection can be performed using a fluorescence detection device, including a fluorescence photometer, a real-time fluorescence PCR instrument, an enzyme-labeled instrument, or a fluorescence detector. It can also be directly observed with the naked eye under blue light, ultraviolet light, or a fluorescence microscope.

[0021] The present invention combines the specific and efficient amplification ability of LCR reaction and the unique endonuclease activity of Cas12a, constructs an ultrasensitive cascade signal amplification system, and provides a novel method for homogeneous and ultrasensitive detection of a nucleic acid marker. The present invention innovatively designs the probe sequence. After the LCR amplification product is generated, the Cas12a endonuclease activity is activated, and the signal probes of the two ends respectively labeled with fluorescence and quenching groups are cyclically cut to achieve the secondary signal amplification of the target nucleic acid molecule. Direct detection of the fluorescent signal can be performed quantitative detection of nucleic acid markers. In addition, since the sequence of the reporter probe is unrelated to the target sequence, the signal probe of the above reaction system can also be extended to any target sequence, saving cost and simplifying the design.

[0022] The method of the present invention will greatly expand the application of LCR technology. It can be used for both DNA and RNA detection, provide a new homogeneous detection strategy for high-sensitivity analysis of nucleic acid markers, and provide new technology for clinical diagnosis and treatment and biomedical research. It has important significance and potential application prospects in the early diagnosis of major diseases, personalized medication, and effective prevention and control of sudden epidemics. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the principle of detecting gene mutations based on ligase chain reaction and gene editing technology.

[0024] Figure 2 It is the fluorescence spectrum of the mutant DNA amplification product detection with different concentrations.

[0025] Figure 3 It is a standard curve graph showing the change of fluorescence intensity with mutant DNA concentration.

[0026] Figure 4 It is a fluorescence trend graph after mutant DNA and wild-type DNA are mixed and amplified in different proportions.

[0027] Figure 5 This is the fluorescence signal graph corresponding to the same concentration of let-7a, miR-214, miR-141, miR-221 and miR-222 in Example 2.

[0028] Figure 6 This is a real-time fluorescence curve diagram generated by different concentrations of miRNA-221 in Example 2.

[0029] Figure 7 This is a standard curve diagram of the fluorescence intensity in Example 2 as it changes with the concentration of miRNA-221. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with examples. The following examples are only for illustration and are not intended to limit the scope of protection of the present invention in any way.

[0031] The processes and methods not described in detail in the following examples are conventional methods known in the art, and the reagents used in the examples are all analytically pure or chemically pure, and can be purchased commercially or prepared by methods well known to those of ordinary skill in the art. The following examples all achieve the purpose of the present invention.

[0032] Example 1

[0033] Based on ligase chain reaction and gene editing technology to detect gene mutations, the detection principle is as follows Figure 1 The specific detection method is as follows:

[0034] 1. Select the JAK2 gene fragment as the site to be tested. The wild-type sequence is 5′-CACAAGCATTTGGTTTTAAATTATGGAGTATGTGTCTGTGGAGACGAGAGTAAGTAA-3′, and its complementary sequence is 5′-TTACTTACTCTCGTCTCCACAGACACATACTCCATAATTTAAAACCAAATGCTTGTGTG-3′. The DNA sequence containing the JAK2V617F gene mutation site is 5′-CACAAGCATTTGGTTTTAAATTATGGAGTATGTTTCTGTGGAGACGAGAGTAAGTAA-3′, and its complementary sequence is 5′-TTACTTACTCTCGTCTCCACAGAAACATACTCCATAATTTAAAACCAAATGCTTGTGTG-3′. Four LCR probes X, XX, Y and YY were designed and synthesized, of which the base sequence of the X probe was 5′-PO4-ACATACTCCATAATTTAAAACCTTT-3′, the base sequence of the XX probe was 5′-TTAGGTTTTAAATTATGGAGTATGTT-3′, the base sequence of the Y probe was 5′-CAAGCATCTTTACTCGTCTCCACAGAA-3′, the base sequence of the YY probe was 5′-PO4-TCTGTGGAGACGAGTAAAGATGCTTGT-3′, and the base sequence of the gRNA was 5′-UAAUUUCUACUAAGUGUAGAU CUCGUCU CCACAGAAACA -3′ (the underlined part is the detection recognition region), and the base sequence of the reporter probe is 5′-FAM-CACACCTCAGCG-BHQ1-3′.

[0035] 2. Add 13.4 μL sterile water, 0.8 μL of 2 μM X, Y, XX and YY probe aqueous solutions, 2.0 μL 10×Amp DNA ligase reaction buffer (200 mM Tris-HCl, 250 mM KCl, 100 mM MgCl2, 5 mM adenine nucleoside triphosphate and 0.1% Triton 100, pH 8.3, 25°C), and 1.0 μL of mutant double-stranded DNA aqueous solutions of different concentrations to a 200 μL centrifuge tube, then place in a PCR instrument, denature at 95°C for 5 min, hybridize at 25°C for 25 min, and mix thoroughly with a pipette to obtain solution A. Add 0.4 μL 5U / μL Amp DNA ligase to a 200 μL centrifuge tube, and the entire process from the start of the experiment to the end of enzyme addition is performed on ice to maintain a low temperature environment. Preheat the PCR instrument to 95°C first, and immediately transfer Solution A to the preheated PCR instrument after adding the enzyme, and keep it at 95°C for 3 minutes to fully denature the four probes and the mutant DNA target chain. Then keep it at 95°C for 30 seconds and then cool it down to 60°C for 30 seconds. Perform 34 thermal cycles under this temperature condition, and immediately place the ligation product on ice after the amplification is completed.

[0036] 3. Add 0.3μL 5U / μL Lambda exonuclease to the LCR product in step 2, incubate at 37℃ for 1.5h, and then keep at 80℃ for 30min to inactivate the Lambda exonuclease. The enzyme can act on double-stranded DNA and gradually cut off the 5' end single nucleotide along the 5'→3' direction. The most suitable substrate is double-stranded DNA with phosphorylation at the 5' end. After adding Lambda exonuclease, the ligated probe that did not participate in the reaction in step 2 will be digested to reduce the background signal.

[0037] 4. Add 1.0 μL 10×NEBuffer 2.1 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 μg / mL bovine serum albumin, pH 7.9, 25°C) to a 200 μL centrifuge tube. Then add 1.0 μL 400 nM reporter probe, 0.5 μL 100 mM dithiothreitol, 0.5 μL 40 U / μL recombinant RNase inhibitor, 1.0 μL 2 μM gRNA solution and 0.5 μL RNase-free water as solution B. Take 5.0 μL of the amplified product in step 3 and add it to solution B, then add 0.5 μL 1 μM Cas12a nuclease, mix well and add to solution B. The whole process is all done on ice. Then transfer the centrifuge tube containing solution B to the PCR instrument, incubate at 37°C for 60min, and then keep at 80°C for 20min to inactivate the Cas12a enzyme. After the reaction, add 90 μL 1×TE buffer solution (10mM Tris-HCl, 1mM EDTA, pH 8.0, 25°C) to a 200 μL centrifuge tube to fully dilute and mix, then transfer all the diluted samples to a 200 μL microfluorescence pool, and perform spectral scanning on an F-7000 fluorescence spectrometer. The excitation wavelength is 480nm, the emission wavelength scanning range is 500nm-650nm, and the fluorescence value is recorded at an emission wavelength of 525nm.

[0038] 5. Under optimized experimental conditions, this method is used to quantitatively detect the concentration of mutant DNA. Figure 2 This is the fluorescence spectrum produced by different concentrations of mutant DNA and a blank experiment without any target. It can be seen from the figure that as the concentration of mutant DNA increases from 0.1fM to 1pM, the fluorescence value corresponding to the emission wavelength of 525nm shows a clear upward trend. Three sets of amplification experiments were carried out in parallel, and different concentrations of mutant DNA were detected, and the fluorescence values ​​of mutant DNA at each concentration at the emission wavelength of 525nm were recorded. Figure 3 It is the quantitative standard curve of three sets of parallel experiments, the linear equation is △F=135.9lg(C / fM)+177.0, and the linear correlation coefficient is R=0.9948. Figure 3 It can be seen that within the mutant DNA concentration range of 0.1fM-1pM, the fluorescence intensity and the logarithm of the mutant DNA concentration show a good linear relationship. The detection limit (3σ, n=11) is calculated to be 0.077fM. The precision is calculated by 7 parallel determinations of 100fM mutant DNA, and the relative standard deviation is 4.6%.

[0039] 6. The mutation frequency of alleles is closely related to certain diseases. Testing the mutation frequency of alleles in patients can achieve the purpose of diagnosis. Therefore, it is of great significance to measure the mutation frequency of alleles. The mutation frequency of alleles is measured using this experimental method, in which mutant DNA and wild-type DNA are mixed in different proportions (0%, 0.5%, 1%, 2%, 5%, 10%, 20%, 50%, 100%), and the sum of their concentrations is 1pM. The experimental steps are the same as above. Figure 4 The fluorescence values ​​corresponding to different mutation frequencies are shown. It can be clearly seen from the figure that as the mutation frequency (i.e. the proportion of mutant DNA) increases, the corresponding fluorescence intensity shows a clear upward trend. This experimental method can accurately detect mutation frequencies as low as 0.5% and has high specificity.

[0040] Example 2

[0041] The application of ligase chain reaction combined with gene editing technology in the detection of microRNA (miRNA) is as follows:

[0042] 1. Four LCR probes, X1, X2, Y1 and Y2, were designed and synthesized based on the RNA sequence of miR-221 5′-AGCUACAUUGUCUGCUGGGUUUC-3′. Probe X1 (base sequence 5′-CAAGCATCTTTCGAAACCCAGCA-3′) and probe Y1 (base sequence 5′-PO4-GACAATGTAGCTCTCAAC-3′) are partially complementary to the target sequence miR-221. Probe X2 (base sequence 5′-PO4-TGCTGGGTTTCGAAAGATGCTTGT-3′) is complementary to probe X1, and probe Y2 (base sequence 5′-GTTGAGAGCTACATTGTC-3′) is complementary to probe Y1. The base sequence of mir-gRNA is 5′-UAAUUUCUACUAAGUGUAGAU GAAACCCAGCAGACAAUGU -3′ (the underlined area is the detection recognition region).

[0043] 2. Add 5.3 μL RNase-free water, 1.0 μL 10×SplintR ligase reaction buffer (500 mM Tris-HCl, 100 mM MgCl2, 10 mM adenine nucleoside triphosphate, 100 mM dithiothreitol, pH 7.5, 25°C), 1.0 μL each of 100 nM probes X1 and Y1, 0.5 μL 40 U / μL recombinant ribonuclease inhibitor and 1.0 μL of different concentrations of miRNA-221 aqueous solution into a 200 μL centrifuge tube (do a blank experiment at the same time, add 1.0 μL RNase-free water in the blank experiment), mix well and use as solution A1 (the whole process is performed on ice). Place the centrifuge tube containing solution A1 in a PCR instrument and heat at 80°C for 3 minutes to denature probes X1, Y1 and miR-221, then lower the temperature to 25°C and keep it constant for 20 minutes to allow probes X1 and Y1 to fully hybridize with the target miR-221. After 20 minutes, add 0.2μL 25U / μL SplintR ligase to solution A1, incubate at 25°C for 40 minutes, then heat solution A1 to 80°C and keep it for 20 minutes to fully denature and inactivate SplintR ligase. After the reaction is completed, immediately store solution A1 on ice.

[0044] 3. Take a new 200 μL centrifuge tube and add the corresponding LCR reaction mixture into the tube, wherein the LCR reaction mixture includes 13.6 μL RNase-free water, 2.0 μL 10×Amp DNA ligase reaction buffer (200 mM Tris-HCl, 250 mM KCl, 100 mM MgCl2, 5 mM adenine nucleoside triphosphate, 0.1% Triton 100, pH 8.3, 25°C), 0.5 μL each of 1 μM four LCR probes X1, X2, Y1 and Y2, 2.0 μL of the ligation product in step 2 and 0.4 μL 5U / μL Amp DNA ligase. Mix well as solution B1. The whole process is operated on ice to maintain a low temperature environment. Preheat the PCR instrument to 95°C first. After adding the enzyme, transfer solution B1 to the preheated PCR instrument immediately and keep it at 95°C for 3 min to denature the four probes and the connection products in step 2. Then perform 38 thermal cycles, each thermal cycle at 95°C for 30 s and 58°C for 30 s. After the reaction is completed, store the amplified product on ice immediately.

[0045] 4. Add 3.5 μL RNase-free water, 1.0 uL 10×NEBuffer 2.1 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 μg / mL bovine serum albumin, pH 7.9, 25°C), 1.0 μL 400 nM reporter probe, 0.5 μL 100 mM dithiothreitol, 0.5 μL 40 U / μL recombinant ribonuclease inhibitor, 1.0 μL 2 μM mir-gRNA aqueous solution to a 200 μL centrifuge tube, and mix thoroughly as solution C. Take 2.0 μL of the amplified product in step 3 and add it to solution C, then immediately add 0.5 μL 1 μM Cas12a nuclease, mix thoroughly and transfer to a fluorescent quantitative PCR instrument, perform 40 thermal cycles at 37°C, and collect the fluorescence intensity signal every 2 minutes.

[0046] 5. For miRNA detection, since the sequence of miRNA is short and the miRNA sequences of the same family are sometimes very similar, specific detection experiments are required. Figure 5 The difference graph of fluorescence signals corresponding to the same concentration (4fM) of let-7a, miR-214, miR-141, miR-222 and miR-221 is shown. The fluorescence signal increase caused by let-7a, miR-214 and miR-141 is very low, and only the signal value generated by miR-222 is slightly higher, because it has some of the same base sequences as miR-221. Despite this, the signal values ​​between miR-221 and miR-222 are also significantly different, indicating that this method has good specificity for the detection of miR-221.

[0047] 6. Based on the optimized experiment, the sensitivity of the method was determined. Figure 6 This is the real-time fluorescence curve produced by different concentrations of miR-221 and blank experiments. It can be seen from the figure that when the concentration of the target is 0.4aM, there is still a significant difference between it and the blank. Figure 7 is the standard curve of the change of fluorescence intensity with the concentration of miR-221. Figure 6 Based on the fluorescence intensity corresponding to the reaction time of 30 min and the logarithm of the miR-221 concentration, a standard curve was obtained. Figure 7It can be seen that there is a linear relationship in the target concentration range of 0.4aM-40aM, and its linear equation is △F1=8019.9lg(C / aM)+13809.8, and the linear correlation coefficient is R1=0.997. When the target concentration range is 40aM–4fM, there is another linear relationship, and its linear equation is △F2=94226.5lg(C / aM)-124069.2, and the linear correlation coefficient is R2=0.993. It can be seen that there is a good linear relationship in both linear segments. The precision of the method was obtained by parallel determination of 200aM miR-221 7 times, and its relative standard deviation RSD was 2.9%.

[0048] 7. In order to evaluate the applicability of this method in actual samples, we measured the content of miR-221 in cervical cancer cells (Hela) and breast cancer cells (MCF-7). It was determined that 100pg of Hela and MCF-7 cell extracts contained 114 and 24.5zmol of miR-221, respectively. Then we performed spike recovery determination, adding different amounts of miR-221 to the total small RNA extracted from Hela and MCF-7 cells. The recovery rate was 93.0–104.7%, and the corresponding RSD was 4.4-8.7% (Table 1), indicating that this method can be used for the determination of samples extracted from cells.

[0049] Table 1. Recovery rate of miR-221 in HeLa and MCF-7 cell extracts (n=3).

[0050]

Claims

1. A method for detecting nucleic acid markers based on ligase chain reaction and gene editing technology, characterized in that: The following steps are involved: (a) designing two pairs of DNA probes for ligase chain reaction according to the sequence of the target nucleic acid marker; wherein the first pair of DNA probes are complementary to the target nucleic acid marker, and the 5′ end of the first pair of DNA probes at the connection point is phosphorylated, and the second pair of DNA probes are complementary to the sequence of the first pair of DNA probes or the 3′ end of the second pair of DNA probes at the connection point protrudes one base, and the 5′ end is phosphorylated; designing a PAM sequence at the 5′ end of the DNA probe away from the connection point, and the PAM sequence is separated from the connection point by 10-16 bases; The target nucleic acid marker is DNA or RNA. When the target nucleic acid marker is a single-base mutant DNA, the 3′-end protruding base at the connection point of the second pair of DNA probes is at the position of the mutant base; (b) mixing the nucleic acid to be detected with two pairs of DNA probes, and adding ligase to carry out ligase chain reaction. If the target nucleic acid marker is present, the reaction generates a double-stranded DNA connection product containing a PAM sequence; when the target nucleic acid marker is RNA, the first pair of DNA probes is first connected under the action of ligase using RNA as a template; (c) adding gRNA, CRISPR / Cas12 system and reporter probe to the ligation product obtained in step (b), wherein one end of the reporter probe is modified with a fluorescent group and the other end is modified with a quenching group; the gRNA comprises a detection recognition region designed according to the target nucleic acid marker, which can bind to the double-stranded DNA ligation product and activate the CRISPR / Cas12 system, and after the CRISPR / Cas12 system is activated, it cuts the reporter probe and releases a fluorescent signal; (d) Detecting the fluorescence signal of the reaction system to obtain the content of the target nucleic acid marker in the nucleic acid to be detected.

2. The method according to claim 1, characterized in that In step (a), the RNA target nucleic acid markers include microRNA, messenger RNA, circular RNA, long non-coding RNA, and viral RNA.

3. The method according to claim 1, characterized in that In step (b), the ligase used in the ligase chain reaction includes Amp DNA ligase, Taq DNA ligase, and 9°N DNA ligase; during the ligase chain reaction, the ligation temperature is 45-65°C, the number of thermal cycles is 20-50, and the amount of ligase used is 0.2-20U.

4. The method according to claim 1, characterized in that In step (b), the ligase is SplintR ligase or T4 RNA ligase 2.

5. The method according to claim 1, characterized in that In step (c), the Cas12 enzyme of the CRISPR / Cas12 system may be one of AsCas12a, LbCas12a, FnCas12a, ArCas12a, LpCas12a, BsCas12a, HkCas12a, PrCas12a, and PxCas12a, and at this time, the PAM sequence is 5′-TTTN or 5′-TTN, wherein N represents any base; the Cas12 enzyme of the CRISPR / Cas12 system may also be HkCas12a, and at this time, the PAM sequence is 5′-YYN, wherein Y represents C or T base, and N represents any base; the Cas12 enzyme of the CRISPR / Cas12 system may also be Cas12b, and at this time, the PAM sequence is 5′-TTN, wherein N represents any base.

6. The method according to claim 1, characterized in that Before performing step (c), the PAM-containing probe and its complementary probe, gRNA and CRISPR / Cas12 system can be mixed, and the fluorescence of the system can be detected. If there is a fluorescent signal, the ligation product obtained in step (b) is digested with Lambda exonuclease before performing the reaction in step (c).

7. The method according to claim 1, characterized in that In step (c), the fluorescent group of the reporter probe is FAM, FITC, HEX, TET, TAMRA, ROX or Cy5, and the quencher group is BHQ1, TAMRA, BHQ2, BHQ3 or Dabcyl.

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