MSRE-RPA-CRISPR / Cas12a detection system aiming at breast cancer methylation site and application of MSRE-RPA-CRISPR / Cas12a detection system

Abstract

The invention relates to the technical field of biology, in particular to an MSRE-RPA-CRISPR / Cas12a detection system aiming at a breast cancer methylation site and application of the MSRE-RPA-CRISPR / Cas12a detection system. The MSRE-RPA-CRISPR / Cas12a systems of the four breast cancer methylation sites are constructed and optimized, the visual detection method for the breast cancer methylation sites based on the MSRE-RPA-CRISPR / Cas12a systems is established, the detection method has the advantages of being high in sensitivity, high in specificity and short in detection time, does not depend on large-scale instruments and equipment, the result is visual and visual, and the method is suitable for large-scale popularization and application. The method is suitable for point-of-care testing (POCT).

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a MSRE-RPA-CRISPR / Cas12a detection system for breast cancer methylation sites and application thereof. BACKGROUND

[0002] Breast cancer (BC) is the most common malignancy in women worldwide. At present, the screening method of breast cancer mainly depends on imaging, which seriously limits the probability of finding early lesions and even precancerous lesions. DNA methylation begins in precancerous lesions, and abnormal changes in DNA methylation are considered as potential biomarkers for early diagnosis, prognosis and treatment monitoring of cancer. Through the detection of plasma free nucleic acid (cfDNA) methylation, it is possible to make up for the deficiency of imaging examination, facilitate the disease course monitoring of patients, and has important theoretical and practical significance. The genomic methylation level is affected by environment and heredity, and has regional and ethnic characteristics. The diagnostic cfDNA methylation site should be obtained by screening the differentially methylated sites of the whole genome of a specific population, and then a larger range of verification should be carried out in the same regional and ethnic population. Circulating Cell-free Genome Atlas (CCGA) is the largest study to evaluate the use of methylated cfDNA for tumor diagnosis today, initiated by GRAIL company. CCGA recruited 15,254 participants from 142 partner clinics in North America, and is a prospective, case-control, observational study. The follow-up study of CCGA enrolled patients found that methylated cfDNA can identify cancer types with poor prognosis, and can predict prognosis. The sensitivity is higher for high-invasive cancer types, such as hormone receptor-negative breast cancer, while the undetected cancers are less invasive. Compared with high-throughput sequencing, candidate gene methylation detection has low cost and simple process, and is more suitable for converting the findings of high-throughput sequencing to clinical application, and solving specific clinical problems. According to different methylation DNA site identification or acquisition methods, nucleic acid amplification methods and detection principles, there are many methylation cfDNA detection methods which can be freely combined.

[0003] Bisulfite treatment and TET enzyme conversion are complex and time-consuming processes, especially bisulfite treatment, which can significantly degrade DNA and affect the subsequent positive detection rate. The methylation-binding protein method cannot detect methylation at the single-base level and is more suitable for methylation assessment of specific DNA regions. The methylation-sensitive restriction endonuclease method (MSRE) is highly sensitive to base methylation and can perform specific cutting. If the digestion is incomplete, it may lead to false positive results, but the process is mild and time-saving, and it can detect single-base methylated DNA. Among the many amplification methods, recombinase polymerase amplification (RPA) has low reaction condition requirements (37-42°C, 15min) and does not require expensive, large-scale PCR equipment, making it particularly suitable for promotion and application at grassroots units. In addition to its important applications in the field of gene editing, CRISPR technology also has important applications in the field of in vitro diagnostics by utilizing its recognition or search functions.

[0004] The emergence of various new technologies has made point-of-care testing (POCT) of methylated cfDNA possible. The WHO once proposed the ASSURED criteria that point-of-care testing needs to meet: Affordable, Sensitivity and Specificity, User-friendly, Rapid and robust, Equipment-free, and Deliverable. Droplet digital PCR (ddPCR), represented by the Bio-rad QX200, has played an important role in cfDNA testing. However, ddPCR equipment is expensive, heavy, and complex to operate, making it unsuitable for POCT. Therefore, it is imperative to develop a method for the immediate detection of breast cancer methylation that can combine microfluidics operations and simple equipment to make up for the shortcomings of existing methods. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide an MSRE-RPA-CRISPR / Cas12a detection system for breast cancer methylation sites and its application.

[0006] The present invention provides a breast cancer methylation detection system, which includes at least one of system 1 or system 2 shown below:

[0007] The system 1 comprises: a methylation-sensitive restriction endonuclease, Cas12a (Cpf1), a probe 1, and a primer set targeting at least one of the target fragments of nucleotide sequences such as SEQ ID NO: 1 to SEQ ID NO: 4, and a crRNA combination;

[0008] The system 2 includes: a methylation-sensitive restriction endonuclease, Cas12a (Cpf1), and a primer set and a probe 2 targeting at least one of the target fragments of the nucleotide sequence such as SEQ ID NO: 1 to SEQ ID NO: 4;

[0009] The methylation-sensitive restriction endonuclease includes: HpaII, BstUI or SsiI;

[0010] The nucleotide sequence of the probe 1 is shown in SEQ ID NO:49.

[0011] Furthermore, in the system 1,

[0012] The combination of the primer set and crRNA targeting the target fragment with a nucleotide sequence as shown in SEQ ID NO: 1 includes: an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 2, a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 3, and a crRNA with a nucleotide sequence as shown in SEQ ID NO: 4;

[0013] The combination of the primer set and crRNA targeting the target fragment with a nucleotide sequence as shown in SEQ ID NO: 2 includes: an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 10, a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 11, and a crRNA with a nucleotide sequence as shown in SEQ ID NO: 12;

[0014] The combination of the primer set and crRNA targeting the target fragment with a nucleotide sequence as shown in SEQ ID NO: 3 includes: an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 18, a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 19, and a crRNA with a nucleotide sequence as shown in SEQ ID NO: 20;

[0015] The combination of the primer set and crRNA targeting the target fragment of the nucleotide sequence as shown in SEQ ID NO: 4 includes: an upstream primer of the nucleotide sequence as shown in SEQ ID NO: 26, a downstream primer of the nucleotide sequence as shown in SEQ ID NO: 27, and a crRNA of the nucleotide sequence as shown in SEQ ID NO: 28.

[0016] Meanwhile, for probe 1 in system 1, the two ends of the probe 1 have a fluorescent group (FAM) and a quenching group (BHQ) respectively.

[0017] In the system 2,

[0018] The combination of the primer set and the crRNA targeting the target fragment of the nucleotide sequence as shown in SEQ ID NO: 1 comprises an upstream primer of the nucleotide sequence as shown in SEQ ID NO: 2, a downstream primer of the nucleotide sequence as shown in SEQ ID NO: 3 and a probe of the nucleotide sequence as shown in SEQ ID NO: 5;

[0019] The combination of the primer set and the crRNA targeting the target fragment of the nucleotide sequence as shown in SEQ ID NO: 2 comprises an upstream primer of the nucleotide sequence as shown in SEQ ID NO: 10, a downstream primer of the nucleotide sequence as shown in SEQ ID NO: 11 and a probe of the nucleotide sequence as shown in SEQ ID NO: 13;

[0020] The combination of the primer set and the crRNA targeting the target fragment of the nucleotide sequence as shown in SEQ ID NO: 3 comprises an upstream primer of the nucleotide sequence as shown in SEQ ID NO: 18, a downstream primer of the nucleotide sequence as shown in SEQ ID NO: 19 and a probe of the nucleotide sequence as shown in SEQ ID NO: 21;

[0021] The combination of the primer set and the crRNA targeting the target fragment of the nucleotide sequence as shown in SEQ ID NO: 4 comprises an upstream primer of the nucleotide sequence as shown in SEQ ID NO: 26, a downstream primer of the nucleotide sequence as shown in SEQ ID NO: 27 and a probe of the nucleotide sequence as shown in SEQ ID NO: 29.

[0022] For probe 2 in system 2, the probe has a nucleotide analogue THF (tetrahydrofuran) in the middle of the probe, the two ends of the nucleotide analogue THF have a fluorescent group (FAM) and a quenching group (BHQ), and the 3' end of the probe has a 3' end Block, and suitable 3' end Block includes C3-spacer (represented by sp).

[0023] In the present application, a breast cancer methylation detection MSRE-RPA-CRISPR / Cas12a-based detection system 1 and a MSRE-RPA-EXO-based detection system 2 are provided; the difference between the detection system 1 and the detection system 2 is the difference of the probe, in addition to the detection system 1, the crRNA is used for specific recognition of the target sequence.

[0024] The system 1 and the system 2 both detect four methylation sites corresponding to four genes; specifically, the target fragment with the nucleotide sequence as shown in SEQ ID NO: 1 is a target fragment of the TLR5 gene; the target fragment with the nucleotide sequence as shown in SEQ ID NO: 2 is a target fragment of the OTP gene; the target fragment with the nucleotide sequence as shown in SEQ ID NO: 3 is a target fragment of the TSHZ3 gene; and the target fragment with the nucleotide sequence as shown in SEQ ID NO: 4 is a target fragment of the FEZF2 gene; wherein the methylation-sensitive restriction endonuclease SsiI can cut the target fragment of the TLR5 gene; the methylation-sensitive restriction endonuclease HpaII can cut the target fragment of the OTP gene; the methylation-sensitive restriction endonuclease SsiI can cut the target fragment of the TSHZ3 gene; and the methylation-sensitive restriction endonuclease BstUI can cut the target fragment of the FEZF2 gene; in the actual detection of a specific target fragment, a suitable methylation-sensitive restriction endonuclease, a primer set and a probe are selected for detection.

[0025] In the system 1 of the present application, the concentration ratio of Cas12a (Cpf1) to crRNA is (1-4):(1-7).

[0026] Specifically, the concentration ratio of Cas12a (Cpf1) to crRNA in the detection system 1 based on MSRE-RPA-CRISPR / Cas12a is optimized; under the condition of fixing the concentrations of other components (primers, Mg 2+ , FQ-ssDNA reporter molecules), by adjusting the concentrations of Cas12a (30-70 nM) and crRNA (30-70 nM) in a gradient and monitoring the relative fluorescence intensity (RFU), it is found that the TLR5 and TSHZ3 target fragment detection system reaches the maximum fluorescence signal intensity when Cas12a / crRNA = 50 nM / 50 nM (i.e., the concentration ratio of Cas12a / crRNA is 1:1), while the OTP and FEZF2 target fragments need to be adjusted to Cas12a / crRNA = 40 nM / 70 nM and 40 nM / 60 nM, respectively, due to the difference in the secondary structure of crRNA, in order to obtain the maximum fluorescence signal intensity (i.e., the concentration ratio of Cas12a / crRNA is 4:7 and 2:3, respectively).

[0027] The present application provides a kit comprising a DNA extraction reagent, an RNA extraction reagent, a purification reagent, a DNA polymerase and dNTPs, etc., and the present application does not limit this.

[0028] The present application provides at least one of the following A) to B) for use in breast cancer methylation detection:

[0029] A), the breast cancer methylation detection system of the present invention;

[0030] B) the kit of the present invention.

[0031] The present invention provides a method for detecting breast cancer methylation, which comprises detecting a sample using at least one of the following a) to b):

[0032] a), the breast cancer methylation detection system of the present invention;

[0033] b) the kit of the present invention.

[0034] Furthermore, the method of the present invention comprises the following steps:

[0035] Step 1: The sample DNA is digested with a methylation-sensitive restriction endonuclease to obtain a digestion product;

[0036] Step 2, the enzyme digestion product is reacted in the presence of an upstream primer, a downstream primer, probe 1, crRNA, Cas12a (Cpf1), dNTP and MgOAc to obtain a reaction product;

[0037] Step 3: Excite the fluorescence of the reaction product and observe it.

[0038] 9. The method according to claim 9, characterized in that in step 1,

[0039] The enzyme digestion system includes: 8-12 U of methylation-sensitive restriction endonuclease, 0.5-1.5 ng of sample DNA and enzyme digestion buffer; specifically, the methylation-sensitive restriction endonuclease is 10 U and the sample DNA is 1 ng;

[0040] The methylation-sensitive restriction endonuclease is selected from HpaII, BstUI or SsiI, and the methylation-sensitive restriction endonuclease selectively cuts unmethylated DNA; specifically, the enzyme digestion system is a 20 μL system; the 20 μL system includes: 2 μL of 10×CutSmart buffer, 1 μL of 10U MSRE, and 17 μL of template DNA;

[0041] The enzyme cleavage conditions are 37°C for 30 to 40 minutes; specifically, the fluorescence signals of the unmethylated targets TLR5 and OTP are completely quenched after 40 minutes of MSRE treatment, and the fluorescence signals of the unmethylated targets TSHZ3 and FEZF2 are completely quenched after 30 minutes of MSRE treatment; the enzyme cleavage is followed by an enzyme inactivation step, and the enzyme inactivation conditions are 60°C for 10 minutes.

[0042] In step 2 of the method of the present invention,

[0043] The concentration of the enzyme digestion product is 1 ng / 30 μL;

[0044] The concentration of the upstream primer is 0.3-0.35 μM; specifically, 0.33 μM;

[0045] The concentration of the downstream primer is 0.3-0.35 μM; specifically, 0.33 μM;

[0046] The concentration of the probe 1 (FQ-ssDNA) is 0.4-0.6 μM; specifically, 0.5 μM;

[0047] The concentration of the Cas12a (Cpf1) is 2-2.5 nM; specifically, 2.5 μM when detecting the target TLR5 and TSHZ3, and 2 μM when detecting the target FEZF2 and OTP;

[0048] The concentration of the MgOAc is 14 nM;

[0049] The concentration of the crRNA is 2.5-3.5 μM; specifically, 2.5 μM when detecting the target TLR5 and TSHZ3, 3 μM when detecting the target FEZF2, and 3.5 μM when detecting the target OTP;

[0050] The condition of the reaction is 39℃, 60 min.

[0051] In the present application, the concentration is the concentration used, and more specifically, the concentration when the reactants contact each other; in a specific reaction, the initial concentration, the size of the system, and the size of the volume added in the system are determined.

[0052] The method of the present application is a one-pot detection method established by combining RPA isothermal amplification and CRISPR / Cas12a; in the process of establishing the one-pot detection method, the efficient combination of the CRISPR detection module and the RPA amplification module is realized by optimizing the concentration and ratio of the reaction components and the reaction conditions of each step; specifically, the optimization of the following components or conditions in step 2 is included:

[0053] 1. Optimization of the temperature and time of the reaction in step 2; RPA-CRISPR / Cas12a detection was carried out under constant temperature conditions of 35℃, 37℃, 39℃ and 41℃ and time intervals of 10, 20, 30, 60, 90 and 120 min; it is shown that 39℃, 60 min is optimal;

[0054] 2. The molar concentration ratio of Cas12a protein to crRNA was optimized. The experimental results showed that the target fragment detection system of TLR5 and TSHZ3 genes reached the maximum fluorescence signal intensity when Cas12a / crRNA=50nM / 50nM; while the concentration ratio of Cas12a / crRNA in the detection system of the target fragment of OTP and FEZF2 genes needed to be adjusted to 40nM / 70nM and 40nM / 60nM, respectively, to obtain the maximum fluorescence signal intensity;

[0055] 3. The enzyme digestion time was optimized, indicating that the unmethylated sample DNA could be completely digested at 37°C for 30 to 40 minutes.

[0056] In the prior art, CRISPR / Cas systems are used for nucleic acid detection, but they primarily target direct detection of nucleic acid sequences rather than identification of specific methylation states. In particular, these methods lack the ability to efficiently and specifically identify methylation sites and cannot be directly applied to the detection of methylation modifications. Therefore, the present invention pioneered the development of a breast cancer methylation detection method using MSRE-RPA-CRISPR / Cas12a that specifically identifies methylation states.

[0057] There are methylation differences between breast cancer tissue and normal tissue, and the release of methylated circulating tumor DNA (ctDNA) by cancer tissue provides a basis for the use of methylated cfDNA for breast cancer liquid biopsy. This study combined the methylation-sensitive restriction endonuclease (MSRE) with the recombinase polymerase amplification (RPA)-assisted CRISPR / Cas12a system to construct an RPA-CRISPR / Cas12a single-tube reaction system for the detection of four breast cancer cfDNA methylation markers. In this method, the unmethylated target is cut by MSRE, while the methylated target site remains intact and serves as a template for subsequent RPA amplification. Cas12a activated by the target molecule cuts the fluorescence quenching (FQ) reporter molecule, releasing a fluorescent signal, thereby indicating the presence of the target molecule. This system can specifically identify the target molecule, with a minimum detection limit of 1×10 -8 ng / μL (approximately 70 copies / μL), of which the FEZF2 methylation site detection system can effectively distinguish methylation levels of 0.05%, with a sensitivity and specificity of 100% for clinical plasma samples. This highly sensitive, efficient, and portable detection method will significantly improve the level of liquid biopsy for early breast cancer and assist in the diagnosis and differential diagnosis of breast cancer.

[0058] The application establishes a visual detection method of breast cancer cfDNA methylation sites based on the MSRE-RPA-CRISPR / Cas12a system by constructing and optimizing the MSRE-RPA-CRISPR / Cas12a system of four breast cancer cfDNA methylation sites. The detection method has the characteristics of high sensitivity, strong specificity, short detection time, does not depend on large instruments and equipment, the results are intuitive and visual, and is suitable for instant detection (POCT). BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 MSRE-RPA-CRISPR method for detecting DNA methylation schematic diagram; wherein A is that the target fragment is recognized and cut by the CRISPR / Cas12a system to release the fluorescence signal of the FQ-ssDNA reporter molecule (reporting probe); B is that the methylation-sensitive restriction enzyme digests the unmethylated DNA in the patient's clinical sample and leaves the intact methylated DNA, and the intact methylated DNA triggers the RPA reaction to amplify the target fragment, and the subsequent CRISPR reaction releases the fluorescence signal to prompt the detection of the target molecule;

[0060] Figure 2 Synthetic four-gene target fragment length and methylation-sensitive restriction enzyme cleavage site and fragment length after enzyme digestion; wherein A is the fragment length of the TLR5 target fragment generated under the action of the methylation-sensitive restriction enzyme (SsiI); B is the fragment length of the OTP target fragment generated under the action of the methylation-sensitive restriction enzyme (HpaII); C is the fragment length of the TSHZ3 target fragment generated under the action of the methylation-sensitive restriction enzyme (SsiI); D is the fragment length of the FEZF2 target fragment generated under the action of the methylation-sensitive restriction enzyme (BstUI).

[0061] Figure 3Figure 3 shows the crRNA specificity verification of four target genes (TLR, OTP, TSHZ3 and FEZF2); in Figures A to D, from top to bottom are: methylated target fragments (TLR5-mC, OTP-mC, TSHZ3-mC and FEZF2-mC) + MSRE detection, fluorescence was detected; unmethylated target fragments (TLR5-C, OTP-C, TSHZ3-C and FEZF2-C) + MSRE detection, no fluorescence was detected; no MSRE group (TLR5-mC + TLR5-C, OTP-C, TSHZ3-C and FEZF2-C) + MSRE detection, no fluorescence was detected; -mC+OTP-C, TSHZ3-mC+TSHZ3-C, and FEZF2-mC+FEZF2-C), fluorescence was detected; negative control (NTC); target fragment + methylated target fragment + MSRE (TLR5-mC+TLR5-C+MSRE, OTP-mC+OTP-C+MSRE, TSHZ3-mC+TSHZ3-C+MSRE, and FEZF2-mC+FEZF2-C+MSRE), fluorescence was detected; mean ± SD, n = 3 technical replicates;

[0062] Figure 4 Optimize the detection time of the RPA-CRISPR / Cas12a detection system for four targets (TLR, OTP, TSHZ3 and FEZF2);

[0063] Figure 5 Figure 3 shows the optimization of the detection time of the RPA-CRISPR / Cas12a detection system after direct observation by the naked eye under blue light at 60, 90, and 120 min of reaction, respectively, and the four-target RPA-CRISPR / Cas12a detection system, where A represents TLR5, B represents OTP, C represents TSHZ3, and D represents FEZF2; + indicates the presence of methylated target fragments, and - indicates the absence of methylated target fragments;

[0064] Figure 6 Figure 2 shows the optimization of the RPA-CRISPR / Cas12a and MSRE enzyme cleavage system; A is the RPA reaction temperature optimization of four target fragments (TLR, OTP, TSHZ3 and FEZF2); B is the MSRE enzyme cleavage time optimization of four target fragments (TLR, OTP, TSHZ3 and FEZF2);

[0065] Figure 7 Figure 3 shows the optimization of Cas12a and crRNA concentrations in the four-target (TLR, OTP, TSHZ3, and FEZF2) detection system; A is TLR5; B is OTP; C is TSHZ3; D is FEZF2; mean ± SD, n = 3 technical replicates, variance calculated by t-test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, ns indicates no significant difference;

[0066] Figure 8 The sensitivity of the MSRE-RPA-Cas12a detection system is shown; AD are four methylated target fragments (A is TLR5; B is OTP; C is TSHZ3; D is FEZF2) diluted in equal proportions (the concentrations are 1×10 -3 ng / μL, 1×10 -4 ng / μL, 1×10 -5 ng / μL, 1×10 -6 ng / μL, 1×10 -7 ng / μL, 1×10 -8 ng / μL, 1×10 -9 ng / μL and NTC) fluorescence quantitative PCR instrument to collect real-time relative fluorescence signals (RFU); EH are the standard curves after linear fitting of the real-time relative fluorescence signals of the four target fragments and the target fragment concentration (target DNA concentration), mean ± standard deviation, n = 3 technical replicates;

[0067] Figure 9 Figure 3 shows the specificity of the MSRE-RPA-Cas12a detection system, which is achieved by detecting the real-time relative fluorescence signals generated by the methylated target fragment (tDNA) + gDNA1 (tDNA + gDNA1), methylated target fragment + gDNA2 (tDNA + gDNA2), gDNA1, gDNA2, and no-target negative control (NTC) as the detection objects of the detection system; A is TLR5 specificity; B is OTP specificity; C is TSHZ3 specificity; D is FEZF2 specificity; mean ± SD, n = 3 technical replicates;

[0068] Figure 10 Figure 3 shows the fluorescence signals generated by the MSRE-RPA-Cas12a detection system using the methylated target fragments (TLR5-mC, OTP-mC, TSHZ3-mC, and FEZF2-mC), target fragments (TLR5-C, OTP-C, TSHZ3-C, and FEZF2-C), and random DNA sequences (random1, random2, and random3) of the four target fragments as the detection objects of the detection system; mean ± standard deviation, n = 3 technical replicates; A is TLR5; B is OTP; C is TSHZ3; D is FEZF2;

[0069] Figure 11Figure 3 shows agarose gel electrophoresis of MSRE digestion and RPA reaction; in A, lane 1 is methylated TLR5 target fragment (TLR5-mC) + SsiI; lane 2 is unmethylated TLR5 target fragment (TLR5-C) + SsiI; lane 3 is RPA product; lane 4 is blank control without template; in B, lane 1 is methylated OTP target fragment (OTP-mC) + HpaII; lane 2 is unmethylated OTP target fragment (OTP-C) + HpaII; lane 3 is RPA product; lane 4 is blank control without template Plate blank control; in C, lane 1 is methylated TSHZ3 target fragment (TSHZ3-mC) + SsiI; lane 2 is unmethylated TSHZ3 target fragment (TSHZ3-C + SsiI; lane 3 is RPA product; lane 4 is no template blank control; in D, lane 1 is methylated FEZF2 target fragment (FEZF2-mC) + BstUI; lane 2 is unmethylated FEZF2 target fragment (FEZF2-C) + BstUI; lane 3 is RPA product; lane 4 is no template blank control;

[0070] Figure 12 Figure 2 shows the specificity of the Methylight assay system for the detection of methylated DNA-mC (TLR5-mC, OTP-mC, TSHZ3-mC, and FEZF2-mC); unmethylated DNA-C (TLR5-C, OTP-C, TSHZ3-C, and FEZF2-C), and NTC of four target fragments; A is TLR5; B is OTP; C is TSHZ3; and D is FEZF2; mean ± SD, n = 3 technical replicates;

[0071] Figure 13 Figure 3 shows the fluorescence intensity detected by the MSRE-RPA-Cas12a detection system; wherein, A is the relative real-time fluorescence signal generated by the MSRE-RPA-Cas12a detection system for detecting methylated and unmethylated four gene target sites mixed in different proportions; B is the endpoint fluorescence intensity of the four gene targets after the MSRE-RPA-Cas12a detection system reaction is completed;

[0072] Figure 14 Figure 2 shows the flow chart of the Methylight assay, in which bisulfite conversion treatment converts unmethylated cytosine (C) into uracil (U), while methylated C remains unchanged, resulting in sequence differences. The amplification process is monitored in real time by the fluorescent signal released by the specific probe.

[0073] Figure 15Detection ability of Methylight method detection system under different methylation levels, wherein A is the relative real-time amplification signal generated by Methylight method for detecting different proportions of mixed methylation and unmethylation four-gene target sites; B is the end-point signal intensity of four-gene targets after Methylight method reaction;

[0074] Figure 16 Detection ability of MSRE-RPA-EXO probe method detection system under different methylation levels, wherein A is the relative real-time fluorescence signal generated by MSRE-RPA-EXO probe method for detecting different proportions of mixed methylation and unmethylation four-gene target sites; B is the end-point fluorescence intensity of four-gene targets after MSRE-RPA-EXO probe method reaction; mean ± standard deviation, n = 3 technical replicates; variance was calculated by t test, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, **** represents P < 0.0001, ns represents no significant difference;

[0075] Figure 17 Pyrosequencing results of breast cancer tissue (Ca) and paracancerous normal breast tissue (p) in clinical sample detection are shown; wherein Meth. (%) represents the methylation degree of the site;

[0076] Figure 18 The MSRE-RPA-Cas12a detection clinical sample process schematic diagram is shown.

[0077] Figure 19 The fluorescence signal generated by MSRE-RPA-Cas12a detection of four target fragments methylation sites in 15 clinical tissue samples and the visualization results under blue light are shown; wherein A is the fluorescence signal generated by MSRE-RPA-Cas12a detection of TLR5 methylation sites in 15 clinical tissue samples and the visualization results under blue light; B is the fluorescence signal generated by MSRE-RPA-Cas12a detection of OTP methylation sites in 15 clinical tissue samples and the visualization results under blue light; C is the fluorescence signal generated by MSRE-RPA-Cas12a detection of TSHZ3 methylation sites in 15 clinical tissue samples and the visualization results under blue light; D is the fluorescence signal generated by MSRE-RPA-Cas12a detection of FEZF2 methylation sites in 15 clinical tissue samples and the visualization results under blue light; mean ± standard deviation, n = 3 technical replicates; variance was calculated by t test, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, **** represents P < 0.0001, ns represents no significant difference;

[0078] Figure 20Diagnostic performance of MSRE-RPA-Cas12a detection of clinical samples, wherein A is TLR5; B is OTP; C is TSHZ3; D is FEZF2;

[0079] Figure 21 Methylight detection of clinical tissue samples; wherein A-D are the relative amplification signals generated by Methylight detection of 4 target methylation sites (A is TLR5; B is OTP; C is TSHZ3; D is FEZF2) in 15 clinical tissue samples; E-H are the diagnostic performance of Methylight detection of clinical samples (E is TLR5; F is OTP; G is TSHZ3; H is FEZF2).

[0080] Figure 22 Detection of clinical plasma cfDNA samples; A is the fluorescence signal generated by MSRE-RPA-Cas12a detection of TLR5 methylation sites in 15 clinical plasma cfDNA samples and the visualization result under blue light; B is the fluorescence signal generated by MSRE-RPA-Cas12a detection of OTP methylation sites in 15 clinical plasma cfDNA samples and the visualization result under blue light; C is the fluorescence signal generated by MSRE-RPA-Cas12a detection of TSHZ3 methylation sites in 15 clinical plasma cfDNA samples and the visualization result under blue light; D is the fluorescence signal generated by MSRE-RPA-Cas12a detection of FEZF2 methylation sites in 15 clinical plasma cfDNA samples and the visualization result under blue light;

[0081] Figure 23 Methylight detection of 4 target methylation sites in 15 clinical plasma cfDNA samples; wherein A is TLR5; B is OTP; C is TSHZ3; D is FEZF2; mean ± standard deviation, n = 3 technical replicates; variance was calculated by t test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, ns indicates no significant difference. DETAILED DESCRIPTION

[0082] The present application provides a MSRE-RPA-CRISPR / Cas12a detection system for breast cancer methylation sites and its application. Those skilled in the art can refer to the content herein and appropriately improve the process parameters. It is particularly important to note that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0083] As Cas12a(Cpf1) was purchased from Meigao Biotech Co., Ltd. (Guangzhou, China); CpG methyltransferase (M.SssI), HpaII endonuclease, BstUI endonuclease, SsiI endonuclease, proteinase K, GeneRuler 100bp DNA Marker and DNA loading dye were purchased from Thermo Fisher Scientific (USA); TwistAmp Basic kit for recombinase polymerase amplification (RPA) was purchased from TwistDx Limited (Maidenhead, UK), which has a premix enzyme lyophilized ball (dNTP in the premix enzyme lyophilized ball) and rehydration buffer; Human methylated / non-methylated DNA standard was purchased from Tianjiansha (Beijing, China); DNA probes and primers were purchased from Shengong Bioengineering (Shanghai) Co., Ltd. (China); Cell DNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing, China); DNA template was purchased from Suzhou Hongxun Biotechnology Co., Ltd.; RPA-EXO reaction kit was purchased from Shenzhen Yizhi Biotechnology Co., Ltd. (containing E-mix (10x) and P-mix (10x)); The fluorescence signal acquisition instrument was ABI Stepone Plus fluorescence quantitative PCR instrument, and the blue shield 580 visible light electrophoresis-transmission instrument (Xiamen, China) was used to observe the fluorescence signal with naked eyes, and pyrosequencing was entrusted to Guangzhou Percy Biotechnology Co., Ltd.

[0084] Advantages of the present application:

[0085] (1) Methyl-sensitive restriction enzyme (MSRE) specific cleavage: MSRE can accurately recognize the methylation state in specific DNA sequences. This feature enables MSRE to specifically detect methylation at specific CpG sites, reducing non-specific interference.

[0086] (2) 39℃ isothermal reaction: Compared with the PCR technology required for conventional nucleic acid detection, the isothermal condition makes the RPA reaction free from dependence on heating-related instruments and equipment such as PCR instrument, and the nucleic acid amplification reaction is completed in a short time, with the characteristics of simple operation and rapidness.

[0087] (3) For RPA-CRISPR / Cas12a methylation cfDNA site detection method, small and portable fluorescence instruments can be used, and the results can be read by naked eye, with low requirements for operators, and laboratory staff can complete the operation after simple training.

[0088] (4) The MSRE-RPA-CRISPR / Cas12a breast cancer cfDNA methylation site detection system has high specificity and sensitivity, and the primers only amplify the target fragment, which can detect the methylation of the target cfDNA site.

[0089] (5) Application range is wide: without large instrument equipment, reaction can be carried out at constant temperature, the result can be judged by naked eyes, and it is suitable for large-scale screening.

[0090] (6) The breast cancer methylation cfDNA site detection system based on the MSRE-RPA-CRISPR / Cas12a constructed by the application increases the detection sensitivity, and can accurately identify 0.05% of the methylation target (FEZF2 gene) in a complex sample with a very high proportion of unmethylated DNA, which outperforms the EXO probe method (0.1%) and the Methylight detection method (0.1%) and compared with other detection methods that can easily cause false positives in the clinic and laboratory, the method of the application shows very high specificity, and provides a new technical scheme for the detection of breast cancer cfDNA methylation sites in the clinic.

[0091] The methylation target fragment can be represented by tDNA, and the tDNA can represent any one of TLR5-mC, OTP-mC, TSHZ3-mC and FEZF2-mC;

[0092] Table 1.

[0093]

[0094]

[0095]

[0096] The u in the crRNA in Table 1 is replaced by t in the sequence table;

[0097] Table 2. Differential methylation sites of 4 marker probe regions obtained by capture sequencing.

[0098]

[0099]

[0100] Table 3. Target site methylation level of pyrosequencing

[0101]

[0102]

[0103] The Cas12a protein in the application is Cas12a (cpf1), which is also replaced by Cas12a in the application; in the specific test process, other Cas12a proteins can be replaced in the art, and then appropriate primer probe combinations are designed according to the selected Cas12a action site.

[0104] The test materials used in the present application are all ordinary commercially available products and can be purchased in the market. The present application will be further described below in combination with examples: Example 1 Construction of breast cancer cfDNA methylation site detection method

[0105] 1. Construction of breast cancer cfDNA methylation site detection method based on MSRE-RPA-CRISPR / Cas12a

[0106] The working principle diagram of breast cancer cfDNA methylation site based on MSRE-RPA-CRISPR / Cas12a is shown in Figure 1

[0107] 1.1. Determining the detection target fragment

[0108] The team of the present application obtained 3288 methylation markers specific to breast cancer (including intraductal carcinoma) from TCGA database analysis, and captured the DNA fragments where the markers were located by probes, and obtained 4 cfDNA methylation markers for breast cancer diagnosis by bisulfite sequencing analysis. The 4 methylation regions (Illumina 450K methylation chip capture probe code / located gene) are cg23035715 / TLR5, cg16304215 / OTP, cg20072171 / FEZF2, and cg21501525 / TSHZ3, and the selected 4 methylation sites in each region are: hg19 chr1:223302762, chr5:76928853, chr3:62356944 and chr19:31843950; The fragment containing the above four sites is selected as the target segment, and the target segment is the TLR5 target fragment of the nucleotide sequence shown in SEQ ID NO: 1, the OTP target fragment of the nucleotide sequence shown in SEQ ID NO: 9, the TSHZ3 target fragment of the nucleotide sequence shown in SEQ ID NO: 17, and the FEZF2 target fragment of the nucleotide sequence shown in SEQ ID NO: 2, and the site information is shown in Table 2.

[0109] 1.2. Selection of methylation-sensitive restriction enzyme (MSRE) cleavage site

[0110] ​Four breast cancer diagnostic methylation markers were mapped to locations in the hg19 genome. Within the capture probe sequence coverage for each methylation marker, the site with the highest AUC value was selected, which also served as a methylation-sensitive restriction enzyme cleavage site. If no corresponding restriction enzyme was available, the site with the second highest AUC value was selected. Having two different restriction enzyme cleavage sites within the amplicon region reduces the false-positive rate but sacrifices sensitivity. Based on these criteria, the methylation-sensitive restriction enzymes (MSREs) identified in this study were: HpaII: C|CGG; BstUI: CG|CG; and SsiI: C|CGC. Figure 2 Schematic diagram of target fragment lengths for four genes, the restriction endonuclease cleavage sites, and the lengths of the fragments after cleavage. Restriction sites were determined using a database (http: / / rebase.neb.com / rebase / rebase.html).

[0111] 1.3 Design and synthesis of isothermal amplification primers

[0112] Considering the polyselectivity of primers and crRNA, all RPA primers and crRNA binding sequences within 200 bp upstream and downstream of the target methylation site (cfDNA length is generally no more than 200 bp) are listed. Homology alignment is performed on the amplified sequences to ensure specificity. RPA primers are selected that can amplify the target methylation site and whose amplicons contain the crRNA binding sequence. RPA primer design principles: The length is generally 30 to 35 nucleotides. The 3 to 5 nucleotides at the 5' end should avoid polyguanine (G). For the 3' end, guanine (G) and cytosine (C) contribute to stable polymerase binding. Special sequences, such as long stretches of polypurines (A, G) or polypyrimidines (T, C), are best avoided in primers. High (>70%) or low (<30%) GC content is not conducive to RPA amplification. When designing primers, sequences that are prone to forming secondary structures, primer-primer interactions, and hairpin structures should be avoided to minimize primer-dimer formation. According to the above principles, there is only one pair of primers that meet the conditions, and the primer sequences are shown in Table 1: among them, the upstream primer of RPA of TLR5 is shown in SEQ ID NO: 2; the downstream primer of RPA of TLR5 is shown in SEQ ID NO: 3; the upstream primer of RPA of OTP is shown in SEQ ID NO: 10; the downstream primer of RPA of OTP is shown in SEQ ID NO: 11; the upstream primer of RPA of TSHZ3 is shown in SEQ ID NO: 18; the downstream primer of RPA of TSHZ3 is shown in SEQ ID NO: 19; the upstream primer of RPA of FEZF2 is shown in SEQ ID NO: 26; and the downstream primer of RPA of FEZF2 is shown in SEQ ID NO: 27.

[0113] 1.4. Synthesis and dilution of methylation targets

[0114] (1) Methylation of target fragments

[0115] All primers and target fragments were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (China). The target fragment was then methylated to obtain a methylated target fragment; specifically, the target fragment (DNA double strand) was treated with CpG methyltransferase (M.SssI) to generate CpG methylated double-stranded DNA (methylated target fragment). The specific steps are as follows: 2 μg of target fragment (DNA double strand) was added to the mixture; the mixture included: 10 μL 10×NEBuffer 2.0 (500mM NaCl, 100mM Tris-HCl, 100mM MgCl2, 10mM DTT, pH 7.9), 1 μL S-adenosylmethionine (32mM), 3 μL M.SssI (4U·μL -1 ) and 86 μL of deionized water (ddH2O); then incubate at 37°C for 5 hours. The product was then purified using a PCR product purification kit. The final CpG methylated DNA was quantified using a NanoDrop 2000 / 2000C UV-visible spectrophotometer.

[0116] (2) Dilution of target fragment

[0117] The concentrations of methylated and unmethylated target fragments were serially diluted ten-fold until they reached a single copy per microliter.

[0118] 1.5. Design of crRNA

[0119] The crRNA seed region is located on the amplified sequence between the upstream and downstream primers of RPA, with a length of more than 15 bases. The PAM sequence in front of the seed region is TTTV (5'-3'), with V = A / G / C / T. According to the above principles, there is only one crRNA that meets the conditions. The crRNA sequence is shown in Table 1. The specific crRNA of TLR5 is shown in SEQ ID NO: 4; the crRNA of OTP is shown in SEQ ID NO: 12; the crRNA of TSHZ3 is shown in SEQ ID NO: 20; and the crRNA of FEZF2 is shown in SEQ ID NO: 28.

[0120] 1.6. Synthesis of single-stranded DNA fluorescent reporter probes

[0121] Methods: The single-stranded DNA probe was constructed by labeling the two ends of the single-stranded DNA sequence with FAM fluorescence group and BHQ fluorescence quenching group, respectively. The gene sequence of the reporter probe (FQ-ssDNA) was 5'-FAM-TTATT-BHQ1-3', which was synthesized by Sheng Wu Bioengineering (Shanghai) Co., Ltd. (China).

[0122] 1.7, Verification of CRISPR / Cas12a feasibility and crRNA specificity

[0123] To prove that crRNA can specifically recognize the methylation DNA RPA amplification product that cannot be digested by MSRE, CRISPR-Cas12a detection was performed by setting up the following four groups:

[0124] CRISPR / Cas12a detection reaction system (50 μL): crRNA 50 nmol / L, Cas12a (Magigen Reagent Co., Ltd.; Guangzhou, China) 50 nmol / L, 10x Cas buffer (Magigen Reagent Co., Ltd.; Guangzhou, China), FQ-ssDNA 160 nmol / L, 1 μL RPA product, and enzyme water to make up. After mixing, ABI Stepone Plus was used at 39°C for 30 min, and the fluorescence signal was read every 1 min.

[0125] The amplification curve rises over time, proving that crRNA can detect target nucleic acids in template DNA. Figure 3 The fluorescence results observed under the blue light transilluminator after the reaction. Among them, Figure 3 The target methylation DNA + MSRE was detected to have fluorescence, proving that MSRE did not cut the methylation target; the target fragment + MSRE was not detected to have fluorescence, and MSRE effectively digested the non-target; the target fragment + target methylation DNA was detected to have fluorescence, showing target specificity; the no target fragment negative control group (NTC) was not detected to have fluorescence, excluding reagent contamination; the target fragment + target methylation DNA + MSRE was detected to have fluorescence, confirming that the methylation target was retained after enzyme digestion. Statistically, the fluorescence intensity values of three repeated experiments were statistically different from the negative control.

[0126] 1.8, Optimization of MSRE-RPA-CRISPR / Cas12a fluorescence detection method

[0127] RPA isothermal amplification and CRISPR / Cas12a were combined to establish a one-pot detection system, and the efficient combination of CRISPR detection module and RPA amplification module was achieved by optimizing the concentration and ratio of reaction components.

[0128] Reaction temperature and reaction time are two important parameters affecting RPA amplification. To determine the appropriate reaction temperature and detection time, RPA-CRISPR / Cas12a detection was performed under constant temperature conditions at 35°C, 37°C, 39°C and 41°C, and time intervals of 10, 20, 30, 60, 90 and 120 min, respectively, as shown in Figure 4 The fluorescence signal gradually increased after the reaction started, and the difference was significant compared with the negative control. In addition, the visual results were compared under blue light after 60, 90 and 120 min, respectively, and it was found that the visual results were consistent with the fluorescence signal results (A-D in Figure 5 The fluorescence signal gradually increased after the reaction started, and the difference was significant compared with the negative control. In addition, the visual results were compared under blue light after 60, 90 and 120 min, respectively, and it was found that the visual results were consistent with the fluorescence signal results (A-D in Figure 6 A in FIG. 6 shows that the fluorescence signal reached the highest value at 39°C, so the experiment determined that 39°C and 60 min were the reaction conditions of the system.

[0129] In the CRISPR / Cas12a related experiment, the molar concentration ratio of Cas12a protein to crRNA is a key parameter affecting the transcleavage efficiency of the system. Under the condition of fixing other components (10 μM RPA forward / reverse primer 1 μL each, 280 nM MgOAc 1.5 μL, 10 μM single-stranded fluorescent reporter molecule (FQ-ssDNA) 1.5 μL, total 30 μL system; among them, when adjusting the concentration of Cas12a, the concentration of crRNA is fixed at 50 nM; when adjusting the concentration of crRNA, the concentration of Cas12a is fixed at 50 nM;), by adjusting the concentrations of Cas12a (30-70 nM) and crRNA (30-70 nM) by gradient and monitoring the relative fluorescence intensity (RFU), it was found that the TLR5 and TSHZ3 target fragment detection system reached the maximum fluorescence signal intensity when Cas12a / crRNA=50 nM / 50 nM (A and C in FIG. 6), while the OTP and FEZF2 target fragments needed to be adjusted to Cas12a / crRNA=40 nM / 70 nM and 40 nM / 60 nM, respectively, to obtain the maximum fluorescence signal intensity (B and D in FIG. 6) due to the difference in the secondary structure of crRNA. Figure 7 The fluorescence signal gradually increased after the reaction started, and the difference was significant compared with the negative control. In addition, the visual results were compared under blue light after 60, 90 and 120 min, respectively, and it was found that the visual results were consistent with the fluorescence signal results (A-D in Figure 7B and D in the figure). Under the premise that the independent sample t-test results did not reach the statistically significant level, the experimental dose was preferably lower to optimize resource allocation and reduce research costs. Therefore, the TLR5 detection system Cas12a / crRNA=40nM / 50nM; OTP detection system Cas12a / crRNA=40nM / 50nM; TSHZ3 detection system Cas12a / crRNA=50nM / 40nM; FEZF2 detection system Cas12a / crRNA=30nM / 50nM were selected. The optimization results show that there is a synergistic effect between the target binding free energy (ΔG) of crRNA and the activity of Cas12a. It is necessary to customize the concentration ratio according to the GC content and hairpin structure tendency of different genes to balance the trans-cleavage activity and background signal. This method enables the system to maintain the convenience of single-tube operation while improving the efficiency of fluorescence signal output.

[0130] To save reagents and reduce detection costs, a 30μL one-pot RPA-CRISPR / Cas12a reaction system was finally determined, as follows:

[0131] TLR5: Add 18 μL rehydration buffer, 1 μL each of 10 μM upstream / downstream primers, 3 μL of 10× CRISPR reaction buffer, 1.5 μL of 10 μM single-stranded fluorescent reporter (FQ-ssDNA), 1.5 μL of 50 nM crRNA, 1.5 μL of 50 nM Cas12a, 1 μL of 1 ng / μL target fragment, and 1.5 μL of 280 nM MgOAc to the PCR tube containing the premixed enzyme lyophilized pellet. Finally, add RNase-free water to 30 μL.

[0132] OTP: Add 18 μL rehydration buffer, 1 μL each of 10 μM upstream / downstream primers, 3 μL of 10× CRISPR reaction buffer, 1.5 μL of 10 μM single-stranded fluorescent reporter molecule (FQ-ssDNA), 1.5 μL of 70 nM crRNA, 1.5 μL of 40 nM Cas12a, 1 μL of 1 ng / μL target fragment, and 1.5 μL of 280 nM MgOAc to the PCR tube containing the premixed enzyme lyophilized pellet. Finally, add RNase-free water to 30 μL.

[0133] TSHZ3: Add 18 μL rehydration buffer, 1 μL each of 10 μM upstream / downstream primers, 3 μL of 10× CRISPR reaction buffer, 1.5 μL of 10 μM single-stranded fluorescent reporter (FQ-ssDNA), 1.5 μL of 50 nM crRNA, 1.5 μL of 50 nM Cas12a, 1 μL of 1 ng / μL target fragment, and 1.5 μL of 280 nM MgOAc to the PCR tube containing the premixed enzyme lyophilized pellet. Finally, add RNase-free water to 30 μL.

[0134] FEZF2: To the PCR tube containing the premixed enzyme lyophilized pellet, add 18 μL rehydration buffer, 1 μL each of 10 μM upstream / downstream primers, 3 μL of 10× CRISPR reaction buffer, 1.5 μL of 10 μM single-stranded fluorescent reporter (FQ-ssDNA), 1.5 μL of 60 nM crRNA, 1.5 μL of 40 nM Cas12a, 1 μL of 1 ng / μL target fragment, and 1.5 μL of 280 nM MgOAc. Finally, add RNase-free water to 30 μL.

[0135] After determining the optimal fluorescence generation efficiency conditions for RPA-CRISPR / Cas12a, the enzyme digestion time of methylation-sensitive restriction endonuclease (MSRE) was optimized under these conditions. To ensure complete digestion, 1 μL of unmethylated target fragment at a concentration of 1 ng / μL was added to the enzyme digestion system of each of the four target fragments. The results are shown in the figure. Figure 6 As shown in Figure B, the relative fluorescence signal (RFU) decreases with the digestion time from 10 min to 60 min. The fluorescence signals of the unmethylated targets TLR5 and OTP are completely quenched after 40 min of MSRE treatment, and the fluorescence signals of the unmethylated targets TSHZ3 and FEZF2 are completely quenched after 30 min of MSRE treatment. This shows that it takes at least 30 to 40 min for MSRE to completely digest the unmethylated target fragments. Therefore, 30 min and 40 min are set as the enzyme digestion time of MSRE, and different targets can be adjusted as needed.

[0136] 1.9. Performance testing of the MSRE-RPA-CRISPR / Cas12a assay

[0137] 1.9.1 Sensitivity test

[0138] The performance of the MSRE-RPA-Cas12a detection system was verified by systematic gradient dilution experiments and multidimensional specificity testing. A 10-fold serial dilution method was used to construct a concentration gradient (1×10-3 to 1×10-3) of methylated DNA templates for four target fragments (TLR5, TSHZ3, FEZF2, OTP).-9 The detection results show that Figure 8 A-D in FIG. 6, the TLR5, TSHZ3 and FEZF2 systems can detect the weak but significant fluorescence signals at 1x10 -8 The detection limit of the OTP system is slightly lower (1x10 -7 The detection limit of the OTP system is slightly lower (1x10 -7 The detection limit of the OTP system is slightly lower (1x10 -3 The detection limit of the OTP system is slightly lower (1x10 Figure 8 E-H in FIG. 6, proving that the system has reliable quantitative detection capability.

[0139] 1.9.2, Specificity experiment of MSRE-RPA-CRISPR / Cas12a detection method

[0140] To evaluate the specificity in complex sample background, five groups of controls were designed: the methylated target fragments were mixed with gDNA of two normal human white blood cells (to simulate high background interference in clinical samples), and pure gDNA and target-free negative controls were set. The detection results (FIG. 7) show that only the mixed group containing the target fragment produces a strong fluorescence signal, while the gDNA group and the negative group have no signal, confirming that MSRE pretreatment can effectively eliminate unmethylated background interference. Figure 9 Further, the methylated target fragments, non-methylated homologous sequences and other random DNA fragments (FIG. 8) were used as detection objects, and the system only responded to the methylated target, excluding the risk of non-specific binding. Figure 10 The random DNA fragments specifically include: TLR5 Random 1 (SEQ ID NO: 6), TLR5 Random 2 (SEQ ID NO: 7) and TLR5 Random 3 (SEQ ID NO: 8) for TLR5; OTP Random 1 (SEQ ID NO: 14), OTP Random 2 (SEQ ID NO: 15) and OTP Random 3 (SEQ ID NO: 16) for OTP; TSHZ3 Random 1 (SEQ ID NO: 22), TSHZ3 Random 2 (SEQ ID NO: 23) and TSHZ3 Random 3 (SEQ ID NO: 24) for TSHZ3; FEZF2 Random 1 (SEQ ID NO: 30), FEZF2 Random 2 (SEQ ID NO: 31) and FEZF2 Random 3 (SEQ ID NO: 32) for FEZF2.

[0141] 2. Breast cancer cfDNA methylation site detection method based on MSRE-RPA-EXO is constructed

[0142] 2.1. Determine the detection target fragment

[0143] The target fragment is as shown in 1.1.

[0144] 2.2. Selection of methylation-sensitive restriction enzyme (MSRE) cleavage site

[0145] The methylation-sensitive restriction enzyme (MSRE) cleavage site is as shown in 1.2.

[0146] 2.3. Design and synthesis of isothermal amplification primers

[0147] The isothermal amplification primers are as shown in 1.3.

[0148] 2.4. Synthesis and dilution of methylation targets

[0149] The synthesis and dilution of the methylation target are as shown in 1.4.

[0150] 2.5. Design of MSRE-RPA-EXO detection probe

[0151] The difference between MSRE-RPA-Cas12a and MSRE-RPA-EXO is the exo isothermal amplification probe; the probe of MSRE-RPA-EXO includes: TLR5 probe as shown in SEQ ID NO: 5; OTP probe as shown in SEQ ID NO: 13; TSHZ3 probe as shown in SEQ ID NO: 21; FEZF2 probe as shown in SEQ ID NO: 29; wherein, the middle of the probe has a nucleotide analogue THF (tetrahydrofuran), the two ends of the nucleotide analogue THF have a fluorescent group (FAM) and a quenching group (BHQ), the 3' end of the probe has a 3' end Block, suitable 3' end Block includes C3-spacer, a phosphate, a BiotinTEG or an amine; this embodiment uses C3-spacer, represented by sp).

[0152] 2.6. Cleavage and RPA reaction

[0153] Methylation sensitive restriction enzyme (MSRE, HpaII / BstUI / SsiI) was used to selectively cleave the unmethylated target fragment. The cleavage system was 20 μL, including: 10×CutSmart buffer 2 μL, 10 U MSRE 1 μL, 1 ng / μL template DNA (including unmethylated target fragment and methylated target fragment) 1 μL, and enzyme-free water to make up to 20 μL system. 37°C, reaction for 15 min; 60°C, 10 min to terminate the enzyme digestion, and the methylated target fragment was retained as the template for subsequent RPA amplification.

[0154] According to the TwistAMP Liquid Basic Kit instructions, the RPA reaction system was prepared: 2×Reaction Buffer 12.5 μL, dNTPs (2.6 mM) 4.6 μL, 10×Basic E-mix 2.5 μL, 20×Core Reaction Mix 1.25 μL, upstream and downstream primers (10 μM) 1.2 μL each, template DNA (methylated target fragment) 1 μL, MgOAc 1.25 μL. PCR reaction conditions: 39°C, 30 min.

[0155] Product recovery, Figure 11 The agarose gel electrophoresis showed that the primer could amplify the methylated target nucleic acid sequence, and the target sequence without methylation was digested. The nucleic acid amplification effect of the primer pair could be identified and detected by DNA gel electrophoresis.

[0156] 2.7 MSRE-RPA-EXO probe detection

[0157] Reaction system (20 μL): 2×Reaction Buffer (EZassay Biotechnology Ltd.) 10 μL, probe (4 μM) 0.6 μL, upstream primer (20 μM) 0.42 μL, downstream primer (20 μM) 0.42 μL, E-mix (10×) 2 μL, P-mix (10×) 2 μL, ddH2O to 20 μL, Starter (10×) 2 μL, DNA template 2 μL.

[0158] The reaction product needs to be judged by an instrument with fluorescence collection. The product after the constant temperature amplification exo reaction is generally cut by exo, so the product is generally not run out of the target band by agarose gel electrophoresis.

[0159] 3. Methylated fluorescent quantitative PCR (Methylight)

[0160] 3.1. Determine the detection target

[0161] As shown in MSRE-RPA-CRISPR / Cas12a, but the target fragments are slightly different; specifically, for four methylated regions (Illumina450K methylation chip capture probe encoding / located genes; cg23035715 / TLR5, cg16304215 / OTP, cg20072171 / FEZF2, and cg21501525 / TSHZ3), target fragments containing four methylation sites (hg19 chr1:223302762, chr5:76928853, chr3:62356944 and chr19:31843950) were selected; after screening, the target fragments used for methylation fluorescence quantitative PCR include: TLR5 target fragment as shown in SEQ ID NO:33; OTP target fragment as shown in SEQ ID NO:37; TSHZ3 target fragment shown in NO:41; FEZF2 target fragment shown in SEQ ID NO:45.

[0162] 3.2. Methylation and dilution of target fragments for methylation fluorescence quantitative PCR

[0163] The steps for methylating and diluting the target fragments for methylation fluorescence quantitative PCR are shown in MSRE-RPA-CRISPR / Cas12a.

[0164] 3.3 Bisulfite Conversion

[0165] Methods: Target fragments were converted using a DNA bisulfite conversion kit purchased from Beyotime Biotech (Shanghai, China). Unmethylated Cs were converted to Us, while 5-methylcytosine (5mC) remained unchanged. The methylated and unmethylated target fragments were used for primer and probe validation in the Methylight assay. The sequences of the four gene target fragments and primers and probes required for the Methylight assay are shown in Table 1.

[0166] 3.4. Methylation fluorescence quantitative PCR (Methylight).

[0167] Methylation fluorescence quantitative PCR technology is used as a reference method for methylation site detection. Its core principle is based on the specific recognition of methylation in DNA sequences after sulfite treatment, achieving quantitative detection of methylated alleles. Specifically:

[0168] (1) Design of primer probes for methylation fluorescence quantitative PCR (Methylight)

[0169] TaqMan probes and primer pairs were designed for the converted sequence (methylated cytosine was retained as C, and unmethylated C was converted to T), wherein the upstream primer of Methylight of TLR5 was shown in SEQ ID NO:34; the downstream primer of Methylight of TLR5 was shown in SEQ ID NO:35; the probe of Methylight of TLR5 was shown in SEQ ID NO:36; the upstream primer of Methylight of OTP was shown in SEQ ID NO:38; the downstream primer of Methylight of OTP was shown in SEQ ID NO:39; the probe of Methylight of OTP was shown in SEQ ID NO:40; the upstream primer of Methylight of TSHZ3 was shown in SEQ ID NO:42; the downstream primer of Methylight of TSHZ3 was shown in SEQ ID NO:43; the probe of Methylight of TSHZ3 was shown in SEQ ID NO:44; the upstream primer of Methylight of FEZF2 was shown in SEQ ID NO:46; the downstream primer of Methylight of FEZF2 was shown in SEQ ID NO: NO:47; the downstream primer of Methylight of FEZF2 is shown in SEQ ID NO:48.

[0170] A strategy of combining primers without methylation sites (the amplification region covers the target CpG site, the specific sequence is as above) with methylation site-specific probes is used: the primer design avoids CpG islands to ensure that amplification is not affected by methylation status, while the probe specifically binds to the conserved methylation region after sulfite treatment.

[0171] (2) Reaction system (25 μL) and reaction procedure

[0172] The reaction system was (25 μL): 12.5 μL Probe qPCR Mix MultiPlus (Takara Bio Inc.), 0.5 μL 10 μM upstream / downstream primers, 0.5 μM probe, 4 μL DNA template, and 6.5 μL enzyme-free water;

[0173] Reaction procedure: Two-step amplification was performed on the ABI 7500 system; specifically: 95°C for 20 seconds; 95°C for 1 second, 60°C for 20 seconds, 45 cycles, amplification curve Figure 12 The probe was confirmed to be able to specifically distinguish between methylated and unmethylated DNA.

[0174] 4. Comparison of MSRE-RPA-CRISPR / Cas12a with other breast cancer methylation site detection methods

[0175] In actual clinical detection, the target site will be in a state of different proportions of methylation and non-methylation coexistence, so this study evaluates the MSRE-RPA-CRISPR / Cas12a detection system by detecting the methylation level. The methylated / unmethylated target fragments are mixed at a ratio of 1%, 0.5%, 0.1%, 0.05%, and 0.01% (total DNA concentration 1 pg / μL), and the results show that the detection limit of the FEZF2 site reaches 0.05% methylation level, and the remaining three sites can stably detect 0.1% methylation DNA, and the negative control (NTC) has no cross reaction. Figure 13 Compared with CRISPR-related technology, this study innovatively integrates enzyme digestion (MSRE), recombinase polymerase amplification (RPA) and CRISPR-Cas12a one-pot reaction system to realize naked-eye readable fluorescence signal difference detection. In the same methylation level test, the detection ability of MSRE-RPA-EXO probe method and MethyLight method Figure 14 and Figure 16 ) for FEZF2 site is lower than that of MSRE-RPA-CRISPR / Cas12a detection system Figures 14 to 16 ), while the detection ability of the three methods for the methylation level of the other three target sites is the same. In the lateral comparison experiment, MSRE-RPA-EXO detection method also has the advantage of constant temperature amplification, but its dependence on the characteristics of fluorescent probes leads to two technical limitations: 1) special instruments are needed for fluorescence signal quantitative analysis; 2) the background fluorescence of the probe interferes significantly, and cannot realize visual judgment. MethyLight has high sensitivity (can detect 0.1% methylation level), but its limitations are: 1) depends on the DNA fragmentation caused by sulfite treatment (average degradation rate > 50%), which affects the detection of low concentration samples (such as serum ctDNA); 2) cannot realize naked-eye judgment; 3) the detection time is > 4 hours (including 16-20 hours of sulfite treatment).

[0176] Example 2 Detection of clinical samples

[0177] 1. Nucleic acid extraction of clinical tissue sample gDNA and plasma cfDNA sample

[0178] Ten breast cancer tissue and peripheral blood samples, five paracancerous normal tissue and peripheral blood samples from patients with benign tumors, and two peripheral blood samples from healthy volunteers were included in the study. Genomic DNA (gDNA) was extracted from the tissues using a genomic DNA extraction kit (Tiangen, Beijing, China). The tissues were first crushed into a cell suspension, then centrifuged at 10,000 rpm (~11200 x g) for 1 min, added with 200 μL buffer GA, and shaken until completely suspended. 4 μL of RNase A (100 mg / ml) (Cat: RT405-12, Tiangen, Beijing, China) was added, shaken for 15 s, and incubated at room temperature for 5 min to remove the RNA enzyme. 20 μL of Proteinase K (20 mg / ml) (Cat: RT403-02, Tiangen, Beijing, China) was added and incubated at 56°C for 2 h. 200 μL of buffer GB was added and incubated at 70°C for 10 min. Then, 200 μL of anhydrous ethanol, 500 μL of GD, and 600 μL of PW were sequentially washed at 12,000 rpm (~133400 x g) for 30 s. After drying at room temperature for 2 min, 100 μL of elution buffer TE was added for elution to obtain the breast cancer gDNA purification solution. The gDNA concentration was determined by absorbance at 260 nm using a NanoDrop One (Thermofisher Scientific, USA), and stored at -20°C.

[0179] Whole blood was collected in cfDNA preservation tubes (Ardent BioMed, Guangzhou, China) and stored at 4°C and processed within one day. First, centrifuge at 1600g for 10 min at 4°C, and the cell pellet was taken. The upper plasma was transferred to a new 2 mL centrifuge tube. Then, the plasma was centrifuged at 16,000g for 10 min at 4°C to remove the remaining cell debris. The supernatant was stored at -80°C until DNA extraction. The cfDNA was isolated from 4 ml of plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen GmbH) according to the lyse, bind, wash, elute steps, and 20 μL of AVE Buffer was used to elute every 4 ml of plasma sample, and stored at -80°C. 20 μL of cfDNA purification solution was treated with a polypeptide freeze-drying powder vacuum freeze-drying machine LGJ-10FD (Henan Xingling Instrument Equipment Co., Ltd.) into a freeze-dried powder form to improve the cfDNA concentration. The cfDNA lyophilized powder can be stored at -20°C, and diluted with ddH2O when performing Methylight or MSER-RPA-CRISPR / Cas12a reactions. The clinical samples used in this study were taken from the Affiliated Tumor Hospital of Harbin Medical University.

[0180] 2. Detection method

[0181] The method for detecting plasma cfDNA samples of clinical breast cancer patients includes the following steps:

[0182] 1) Extract cfDNA from plasma samples or gDNA from tissue samples of breast cancer patients and benign tumor patients according to 1;

[0183] 2) Selectively cut unmethylated DNA with methylation-sensitive restriction enzymes (MSRE, HpaII / BstUI / SsiI): 20 μL system, 10×CutSmart buffer 2 μL, 10 U MSRE 1 μL, template DNA (cfDNA or gDNA) 17 μL. 37°C, reaction for 15 min; 60°C, 10 min to terminate enzyme digestion, and retain the methylation target as the subsequent amplification template;

[0184] 3) Use the methylated cfDNA from breast cancer patient plasma samples or gDNA from tissue samples as a template to prepare a 30 μL RPA-CRISPR / Cas12a reaction system, as follows:

[0185] TLR5: Add 18 μL rehydration buffer, 1 μL each of 10 μM upstream / downstream primers, 3 μL 10× CRISPR reaction buffer, 1.5 μL 10 μM single-stranded fluorescent reporter molecule (FQ-ssDNA), 1.5 μL 50 nM crRNA, 1.5 μL 50 nM Cas12a, 1 μL methylated cfDNA from plasma samples of breast cancer patients and benign tumor patients after enzyme digestion prepared in step (3), and 1.5 μL 280 nM MgOAc to the PCR tube containing the premixed enzyme lyophilized pellet. Finally, add RNase-free water to 30 μL.

[0186] OTP: Add 18 μL rehydration buffer, 1 μL each of 10 μM upstream / downstream primers, 3 μL 10× CRISPR reaction buffer, 1.5 μL 10 μM single-stranded fluorescent reporter molecule (FQ-ssDNA), 1.5 μL 70 nM crRNA, 1.5 μL 40 nM Cas12a, 1 μL methylated cfDNA from plasma samples of breast cancer patients and benign tumor patients after enzyme digestion prepared in step (3), and 1.5 μL 280 nM MgOAc to the PCR tube containing the premixed enzyme lyophilized pellet. Finally, add RNase-free water to 30 μL.

[0187] TSHZ3: Add 18 μL rehydration buffer, 1 μL each of 10 μM upstream / downstream primers, 3 μL 10× CRISPR reaction buffer, 1.5 μL 10 μM single-stranded fluorescent reporter molecule (FQ-ssDNA), 1.5 μL 50 nM crRNA, 1.5 μL 50 nM Cas12a, 1 μL methylated cfDNA from plasma samples of breast cancer patients and benign tumor patients after enzyme digestion prepared in step (3), and 1.5 μL 280 nM MgOAc to the PCR tube containing the premixed enzyme lyophilized pellet. Finally, add RNase-free water to 30 μL.

[0188] FEZF2: Add 18 μL rehydration buffer, 1 μL each of 10 μM upstream / downstream primers, 3 μL 10× CRISPR reaction buffer, 1.5 μL 10 μM single-stranded fluorescent reporter (FQ-ssDNA), 1.5 μL 60 nM crRNA, 1.5 μL 40 nM Cas12a, 1 μL methylated cfDNA from plasma samples of breast cancer patients and benign tumor patients after enzyme digestion prepared in step (3), and 1.5 μL 280 nM MgOAc to the PCR tube containing the premixed enzyme lyophilized pellet. Finally, add RNase-free water to 30 μL.

[0189] 4) Detection was performed using the RPA-CRISPR / Cas12a reaction system prepared in step 3), and then visual observation was performed under a blue light transilluminator.

[0190] 3. Results

[0191] This part uses the MSRE-RPA-CRISPR / Cas12a detection system to detect clinical patient tissue samples and plasma samples, to verify the detection of methylation of actual clinical samples by the MSRE-RPA-CRISPR / Cas12a fluorescence detection system. The specific results are as follows:

[0192] To evaluate the clinical applicability of the four methylation sites TLR5, OTP, TSHZ3 and FEZF2, 15 patient tissue samples (cancer tissue and normal breast tissue adjacent to benign tumor in patients) and corresponding blood samples were included in this study, and were systematically verified by pyrosequencing. The detailed methylation levels of the sequencing sites are shown in Table 3. Kota Ouchi et al. used Illumina Infinium Human Methylation450 methylation chip (Illumina) to comprehensively evaluate the DNA methylation state. They used β≥0.3 (30% methylation level) as the standard for determining the methylation positivity of the site. The β value of the Infinium methylation chip means that β = M / (M+U+100), M represents the signal intensity of the methylated probe (Methylated probe intensity), U represents the signal intensity of the unmethylated probe (Unmethylated probe intensity), and 100 is the offset. Therefore, the β value directly reflects the proportion of methylated DNA molecules in the total DNA. Pyrosequencing calculates the peak ratio of cytosine (C) and thymine (T) from the sequencing peak chart, and directly outputs the methylation percentage. Both of them quantify the proportion of methylated DNA molecules, and the threshold logic can be directly migrated. Therefore, in the research of this application, the methylation degree of the site ≥30% is determined to be methylation positive. Further analysis by Student's t-test (P<0.001) shows that the methylation degree of the TLR5 methylation site in normal tissue is significantly higher than that in breast cancer tissue; the methylation degree of the other three methylation sites in cancer tissue is significantly higher than that in normal breast tissue (P<0.001), proving its clinical reliability as a biomarker for breast cancer. It is worth noting that the pyrosequencing results of this time found that the methylation degree of the TSHZ3 methylation site in cancer tissue and normal breast tissue was higher than 30%, and we had reason to determine that the site was in a methylation state in cancer tissue and normal breast tissue, and there was a significant difference in the methylation degree. Figure 17

[0193] ​Based on the above results, this study further used the optimized MSRE-RPA-Cas12a detection system to perform methylation analysis on 15 patient tissue samples (flow chart as shown in the figure). Figure 18 ), and observe the fluorescence under blue light at the end of the reaction ( Figure 19 The results showed that the detection sensitivity of the detection system for TLR5 site was 100% and the specificity was 88.89% ( Figure 20 The detection sensitivity of OTP site was 100% and the specificity was 75% ( Figure 20 The detection sensitivity of TSHZ3 site was 100% ( Figure 20 C); the detection sensitivity of FEZF2 site was 100% and the specificity was 100% ( Figure 20 D in the figure); the sensitivity of Methylight in detecting TLR5 sites in the same sample was 100% and the specificity was 88.89% ( Figure 21 A and E in ); the sensitivity of OTP site was 85.7% and the specificity was 75% ( Figure 21 B and F in Figure 3); the sensitivity of TSHZ3 locus was 100% ( Figure 21 C and G in ); the sensitivity of FEZF2 locus was 90% and the specificity was 100% ( Figure 21 To further explore the liquid biopsy capability of the sensor, the paired plasma cfDNA of the 15 patients was further tested for 4 target fragments MSRE-RPA-CRISPR / Cas12a ( Figure 22 ), the methylation-positive results were completely consistent with the corresponding tissues, while Methylight missed one positive sample each when detecting OTP and FEZF2 target sites ( Figure 23 ). It is speculated that the reasons for missed detection may be: bisulfite treatment is a harsh chemical operation that will severely degrade DNA, resulting in the DNA of these two samples being of poorer quality and severely fragmented after treatment, and ultimately failing PCR amplification of Methylight. Plasma cfDNA itself is a highly fragmented apoptotic product, with a length centered around a 166bp nucleosome unit and containing tumor-specific ultra-short fragments (<150bp). Target site loss is more likely to occur after bisulfite treatment. The specific target MSRE-RPA-CRISPR / Cas12a detection method we constructed eliminates the need for sulfite conversion and has significant advantages in preserving the integrity of short fragments of cfDNA. The above results show that the four-target MSRE-RPA-CRISPR / Cas12a detection system constructed in this study is not only suitable for methylation detection in tissue samples, but also shows excellent clinical application potential in the field of liquid biopsy.

[0194] The above merely is the preferred embodiment of the present application, it should be pointed out that, for ordinary skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A breast cancer methylation detection system, characterized in that: Includes at least one of System 1 or System 2 shown below: The system 1 comprises: a methylation-sensitive restriction endonuclease, Cas12a (Cpf1), a probe 1, and a primer set targeting at least one of the target fragments of nucleotide sequences such as SEQ ID NO: 1 to SEQ ID NO: 4, and a crRNA combination; The system 2 includes: a methylation-sensitive restriction endonuclease, Cas12a (Cpf1), and a primer set and a probe 2 targeting at least one of the target fragments of the nucleotide sequence such as SEQ ID NO: 1 to SEQ ID NO: 4; The methylation-sensitive restriction endonuclease includes: HpaII, BstUI or SsiI; The nucleotide sequence of the probe 1 is shown in SEQ ID NO:

49.

2. The breast cancer methylation detection system according to claim 1, characterized in that: In the system 1, The combination of the primer set and crRNA targeting the target fragment with a nucleotide sequence as shown in SEQ ID NO: 1 includes: an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 2, a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 3, and a crRNA with a nucleotide sequence as shown in SEQ ID NO: 4; The combination of the primer set and crRNA targeting the target fragment with a nucleotide sequence as shown in SEQ ID NO: 2 includes: an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 10, a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 11, and a crRNA with a nucleotide sequence as shown in SEQ ID NO: 12; The combination of the primer set and crRNA targeting the target fragment with a nucleotide sequence as shown in SEQ ID NO: 3 includes: an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 18, a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 19, and a crRNA with a nucleotide sequence as shown in SEQ ID NO: 20; The combination of the primer set and crRNA targeting the target fragment of the nucleotide sequence as shown in SEQ ID NO: 4 includes: an upstream primer of the nucleotide sequence as shown in SEQ ID NO: 26, a downstream primer of the nucleotide sequence as shown in SEQ ID NO: 27, and a crRNA of the nucleotide sequence as shown in SEQ ID NO:

28.

3. The breast cancer methylation detection system according to claim 1, characterized in that: In the system 2, The combination of the primer set and crRNA targeting the target fragment of the nucleotide sequence as shown in SEQ ID NO: 1 includes: an upstream primer of the nucleotide sequence as shown in SEQ ID NO: 2, a downstream primer of the nucleotide sequence as shown in SEQ ID NO: 3, and a probe of the nucleotide sequence as shown in SEQ ID NO: 5; The combination of the primer set and crRNA targeting the target fragment with a nucleotide sequence as shown in SEQ ID NO: 2 includes: an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 10, a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 11, and a probe with a nucleotide sequence as shown in SEQ ID NO: 13; The combination of the primer set and crRNA targeting the target fragment with a nucleotide sequence as shown in SEQ ID NO: 3 includes: an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 18, a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 19, and a probe with a nucleotide sequence as shown in SEQ ID NO: 21; The combination of the primer set and crRNA targeting the target fragment of the nucleotide sequence as shown in SEQ ID NO: 4 includes: an upstream primer of the nucleotide sequence as shown in SEQ ID NO: 26, a downstream primer of the nucleotide sequence as shown in SEQ ID NO: 27, and a probe of the nucleotide sequence as shown in SEQ ID NO:

29.

4. The breast cancer methylation detection system according to claim 2, characterized in that: The concentration ratio of Cas12a (Cpf1) to crRNA is: (1-4): (1-7).

5. A kit, characterized in that Includes DNA extraction reagents, RNA extraction reagents, purification reagents, DNA polymerase and dNTP.

6. Use of at least one of the following A) to B) in breast cancer methylation detection: A), the breast cancer methylation detection system according to any one of claims 1 to 4; B) The kit according to claim 5.

7. A method for detecting methylation in breast cancer, characterized in that: The method comprises detecting the sample using at least one of the following a) to b): a) The breast cancer methylation detection system according to any one of claims 1 to 4; b) The kit according to claim 5.

8. The method according to claim 7, characterized in that The steps include: Step 1: The sample DNA is digested with a methylation-sensitive restriction endonuclease to obtain a digestion product; Step 2, the enzyme digestion product is reacted in the presence of an upstream primer, a downstream primer, probe 1, crRNA, Cas12a (Cpf1), dNTP and MgOAc to obtain a reaction product; Step 3: Excite the fluorescence of the reaction product and observe it.

9. The method according to claim 9, characterized in that In step 1, The enzyme digestion system includes: 8-12 U of methylation-sensitive restriction endonuclease, 0.5-1.5 ng of sample DNA and enzyme digestion buffer; The enzyme digestion conditions are 37° C., 30 to 40 minutes.

10. The method according to claim 8, characterized in that In step 2, The concentration of Cas12a (Cpf1) is 2 to 2.5 nM; The concentration of the crRNA is 2.5 μM to 3.5 μM; The reaction conditions are 39° C. and 60 min.

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