A DNA methylation detection method based on recombinase polymerase amplification and CRISPR / Cas12a coupling

Abstract

The present invention discloses a DNA methylation detection method based on recombinase polymerase amplification and CRISPR / Cas12a coupling. By utilizing the specificity of HhaI, methylated and non-methylated DNA can be effectively distinguished. Meanwhile, RPA combined with the high amplification ability of Lambda enzyme can produce a large amount of ssDNA, and then the obtained ssDNA can be recognized by Cas12a. No PAM site is required in the recognition process. In addition, the high specificity and self-amplification ability of CRISPR / Cas12a further improve the sensing performance, so that the detection method of the present invention has high sensitivity and selectivity, good anti-interference ability and repeatability.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a DNA methylation detection method based on recombinase polymerase amplification and CRISPR / Cas12a coupling. Background Art

[0002] Epigenetic research has penetrated into various fields, and has achieved remarkable achievements and significant breakthroughs in disease mechanisms, diagnosis and treatment, drug development, and plant and animal trait improvement. DNA methylation is the most characteristic and common epigenetic modification, which is mainly the covalent binding between the 5th carbon atom of cytosine on genomic DNA and the methyl group. Cytosine is thus modified to 5-methylcytosine. DNA methylation is related to gene expression and gene stability. Unlike gene mutation, DNA methylation modification is a reversible modification, which makes it an important potential target for disease treatment. At present, the progress of DNA methylation research shows that many genes encoding drug metabolizing enzymes, drug transporters, nuclear receptors and drug targets are affected by DNA methylation, and drug development targeting DNA methylation has also received more and more attention. Clinical trial results also show that DNA methylation drug treatment can reduce adverse reactions and stabilize drug efficacy. Therefore, the detection and research of DNA methylation is conducive to the development of new drugs and the discovery of more drug targets.

[0003] At present, a variety of molecular detection methods have been developed for analyzing DNA methylation, including methylation-sensitive restriction endonuclease (MSRE)-polymerase chain reaction (PCR), bisulfite sequencing and mass spectrometry. These methods are mainly based on the principle of bisulfite conversion or methylation-sensitive restriction endonuclease (MSRE) digestion. The bisulfite conversion reaction is considered to be the gold standard method for determining DNA methylation, mainly by converting unmethylated cytosine into uracil through bisulfite. However, this process needs to be carried out under acidic and high temperature conditions, which will lead to DNA degradation and detection failure. The technology based on the MSRE principle provides a method for determining DNA methylation that does not rely on bisulfite. MSRE is a class of restriction endonucleases whose recognition sites are sensitive to methylated bases, so methylated and unmethylated sequences can be distinguished, and the reaction is carried out under mild conditions to avoid DNA degradation. However, the content of methylated DNA from cancer cells is very low, even as low as 0.1%, and a large amount of DNA is unmethylated. Therefore, the sensitivity and accuracy of DNA methylation detection based on MSRE digestion need to be further improved. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a DNA methylation detection method based on recombinase polymerase amplification and CRISPR / Cas12a coupling, so as to solve the problems that the prior art easily leads to DNA degradation and detection failure in DNA methylation detection, and has insufficient sensitivity and accuracy in detecting low concentrations of methylated DNA.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A DNA methylation detection method based on recombinase polymerase amplification and CRISPR / Cas12a coupling, characterized in that it comprises the following steps:

[0007] (1) Transcription and synthesis of crRNA: 2 μL, 10 μM T7 promoter, 2 μL, 10 μM crRNA template and 16 μL enzyme-free water were prepared into a mixture, annealed at 95°C for 5 min, and slowly cooled to room temperature; 10 μL, 10 mM nucleoside triphosphate and 1.5 μL T7 RNA polymerase were added, and the transcription reaction was carried out at 37°C for 16 hours; then 2 μL of deoxyribonuclease I was added to the above reaction solution, incubated at 37°C for 2 hours to degrade the crRNA template; after degradation, the product was purified using a miRNA purification kit, and quantified to obtain crRNA;

[0008] (2) Methylation treatment of DNA target genes and plasmids: DNA target genes and plasmids are both double-stranded. 2 μL of 32 mM S-adenosylmethionine, 5 μL of NEB biological buffer 2, 2 μL of 100 U of CpG methyltransferase, 100 nM DNA target gene or 2 μg of target plasmid and enzyme-free water are mixed into a 50 μL reaction solution, and then incubated at 37°C for 5 hours; 2 μL of 100 U of CpG methyltransferase and 1 μL of 32 mM S-adenosylmethionine are added, and the reaction is continued at 37°C for 12 hours, and then the reaction is terminated after reacting at 65°C for 20 minutes to obtain the treated methylated DNA or methylated plasmid. The methylated DNA is used as the electrophoresis verification target, and the methylated plasmid is used as the target DNA for detection;

[0009] (3) Endonuclease cleavage of target DNA: 2 μL of target DNA treated in step (2), 1 μL of NEB restriction endonuclease buffer, and 10 U of HhaI enzyme were mixed, reacted at 37°C for 10 to 70 minutes, and then heated to 65°C and incubated for 25 minutes to terminate the digestion, thereby obtaining a digestion product;

[0010] (4) RPA and generation of single-stranded DNA products: amplify the digestion product of step (3) using an RPA kit to obtain a double-stranded DNA amplification product, and then obtain ssDNA under Lambda endonuclease treatment;

[0011] (5) CRISPR / Cas12a detection: 9 μL of enzyme-free water, 2 μL of Cas12 enzyme reaction buffer, 2 μL of 1 μM crRNA obtained in step (1), 2 μL of 1 μM Cas12a, 1 μL of 10 μM fluorescent probe and 4 μL of the amplification product obtained in step (4) were mixed, incubated at 37° C. for 10 to 50 minutes, and 80 μL of double distilled water was added to perform fluorescence detection analysis on the obtained substance to obtain the detection result of the target DNA.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] 1. The present invention provides a new method for detecting DNA methylation without PAM site based on nuclease-assisted recombinase polymerase amplification and CRISPR / Cas12a (E-PfRPA / Cas). Compared with traditional methylation-specific PCR, RPA based on nuclease HhaI has mild reaction conditions and fast detection speed, and shows good performance in DNA methylation detection. It can accurately detect low-concentration methylation levels and has good application prospects.

[0014] 2. In the method described in the present invention, the specificity of HhaI is utilized to effectively distinguish methylated and unmethylated DNA. At the same time, RPA combined with the high amplification ability of Lambda enzyme can produce a large amount of ssDNA, and then the obtained ssDNA can be recognized by Cas12a. No PAM site is required during the recognition process, thereby avoiding the dependence of CRISPR / Cas12a on PAM sites; and the high specificity and self-amplification ability of CRISPR / Cas12a further improve the sensing performance, so that the detection method described in the present invention has high sensitivity and selectivity, good anti-interference ability and repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A is a schematic diagram of the E-PfRPA / Cas strategy for detecting DNA methylation in the present invention; Figure 1 B is a diagram showing its application in lateral flow analysis.

[0016] Figure 2 A is the PAGE image of HhaI digestion, lane 1: RAFFS1A-mC / HhaI, lane 2: RAFFS1A-C / HhaI; Figure 2B is the PAGE image of RPA and Lambda exonuclease treatment, lane 3: RAFFS1A-mC / HhaI / RPA; lane 4: RAFFS1A-C / HhaI / RPA; lane 5: RAFFS1A-C / HhaI / RPA / Lambda; lane 6: RAFFS1A-mC / HhaI / RPA / Lambda; Figure 2C is a map of the recognition site of HhaI in the RAFFS1A sequence.

[0017] Figure 3 A is the fluorescence spectra of different samples based on the E-PfRPA / Cas system; Figure 3 B is the LFA detection image of different samples based on the E-PfRPA / Cas system.

[0018] Figure 4 The result diagram after optimizing the method of the present invention is as follows: Figure 4 A is the cleavage time of CRISPR / Cas12a, Figure 4 B is the digestion time of HhaI, Figure 4 C is the amplification time of RPA, Figure 4 D is the concentration of Lambda.

[0019] Figure 5 A is the fluorescence spectra of the target RAFFS1A-mC at different concentrations from 0fM to 20pM; Figure 5 B is the fluorescence response of RAFFS1A-mC at different concentrations ranging from 2 fM to 20 pM, wherein the inset is the linear relationship between the fluorescence intensity (ΔF) and the logarithm of the RAFFS1A-mC concentration in the range of 2 fM to 20 pM; the error bars represent the standard deviation of three independent measurements.

[0020] Figure 6 A is the fluorescence spectrum of the methylation level in the range of 0 to 100%; Figure 6 B is the linear fitting curve between the input methylation level and the measured methylation level; Figure 6 C is the fluorescence spectra of different targets (RASSF1A-mC, RASSF1A-C, random DNA) at a concentration of 2 pM; Figure 6 D is the fluorescence response of different targets (RASSF1A-mC, RASSF1A-C, random DNA) at 2 pM concentration; the error bars represent the standard deviation of three independent measurements.

[0021] Figure 7 A is the fluorescence response of the six experimental groups at a concentration of 2 pM. Figure 7 B is the corresponding fluorescence value, and the error bars represent the standard deviation of three independent measurements.

[0022] Figure 8A is a schematic diagram of the lateral flow method for detecting methylated DNA and unmethylated DNA; Figure 8 B is the LFA band results at different concentrations of RASSF1A-mC from 20 fM to 20 pM; Figure 8 C is the LFA band results of different DNA targets.

[0023] Fig. 9 A is the fluorescence spectrum of genomic DNA of MCF-7 cancer cells and normal LO2 cells at a concentration of 200 ng / μL; Fig. 9 B shows the fluorescence response value of genomic DNA of MCF-7 cancer cells and normal LO2 cells at a concentration of 200 ng / μL, and shows the LFA band results of genomic DNA of MCF-7 and LO2 cells at a concentration of 500 ng / μL; the error bars represent the standard deviation of three independent measurements. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0025] 1. A DNA methylation detection method based on recombinase polymerase amplification coupled with CRISPR / Cas12a

[0026] The present invention has found through research that the existing various molecular detection methods for analyzing DNA methylation have many shortcomings in actual use. In order to solve the shortcomings of the existing detection methods, the present invention considers that the methylated DNA content from cancer cells is very low and needs to be amplified. Nucleic acid amplification is a promising target signal amplification strategy. Among the many amplification techniques, rolling circle amplification (RCA), exponential amplification reaction (EXPAR) and recombinase polymerase amplification (RPA) and other isothermal amplification techniques can effectively amplify the target signal. Compared with the traditional polymerase chain reaction (PCR) detection that requires a thermal cycle process, isothermal amplification can be performed under isothermal conditions and has high amplification efficiency. However, the padlock probe required for RCA is relatively expensive, and the background signal easily reduces the sensitivity of the detection; and the specificity and sensitivity of the exponential amplification reaction are insufficient, which limits its further promotion and application in actual clinical detection. For this reason, the present invention uses RPA for isothermal amplification, but this alone is not enough. The present invention considers combining RPA with other tools to further improve its accuracy, sensitivity and selectivity. To this end, clustered regularly interspaced short palinmic repeats and their corresponding Cas protein (CRISPR) / Cas system are considered to combine nucleic acid amplification with CRISPR / Cas system in order to further improve the sensitivity and specificity of detection. However, at present, a variety of technologies have been developed based on different CRISPR / Cas systems, such as SHERLOCK (Cas13), DETECTR (Cas12 or Cas14) and HOLMES (Cas12). For example, in the VA type CRISPR system, CRISPR / Cas12a (Cpf1) exhibits specific recognition ability for double-stranded DNA (dsDNA) and can stimulate its trans-cleavage activity to detect targets. However, these technologies usually require TTTV protospacer adjacent motif (PAM) sequence to locate the target region for double-stranded recognition, but single-stranded recognition does not require it. Therefore, the present invention adopts a new method based on nuclease-assisted recombinase polymerase amplification combined with CRISPR / Cas12a to develop a PAM-free site (E-PfRPA / Cas) to sensitively measure DNA methylation. In the present invention, for different cancers, methylation often occurs in the promoter region of genes related to them. Taking breast cancer as an example, methylation of the RASSF1A promoter region is considered to be an effective biomarker for breast cancer; similarly, for different cancers, the selected promoter will be different. In order to detect the methylation of RASSF1A, a methylation-sensitive restriction endonuclease is used to digest unmethylated DNA containing a restriction endonuclease site.When the recognition site is methylated, the target DNA will not be cut and the structure remains intact, thus triggering the RPA reaction and obtaining a large amount of product within minutes. At the same time, in order to achieve PAM-free recognition of CRISPR / Cas12a, the front primer of RPA was 5' phosphate modified, because Lambda exonuclease can recognize and degrade sequences containing 5'-phosphate, thereby releasing a large amount of ssDNA, so that the obtained ssDNA can be recognized by CRISPR / Cas12a without PAM sites, activating its trans-cutting activity for fluorescence detection and lateral flow test strips. The E-PfRPA / Cas method has high sensitivity and specificity and can be used for the detection of actual samples.

[0027] Therefore, the method of the present invention is specifically as follows:

[0028] (1) Transcription and synthesis of crRNA: 2 μL T7 promoter (10 μM), 2 μL crRNA template (10 μM) and 16 μL enzyme-free water were prepared into a mixture, annealed at 95°C for 5 min, and slowly cooled to room temperature; 10 μL nucleoside triphosphate (10 mM) and 1.5 μL T7 RNA polymerase were added, and the transcription reaction was carried out at 37°C for 16 hours; then 2 μL deoxyribonuclease I was added to the above reaction solution, incubated at 37°C for 2 hours to degrade the crRNA template; after degradation, the product was purified using a miRNA purification kit, and quantified to obtain crRNA;

[0029] (2) Methylation treatment of DNA target gene and plasmid: Both DNA target gene and plasmid are double-stranded. Mix 2 μL of S-adenosylmethionine (32 mM), 5 μL of NEB biological buffer 2, 2 μL of CpG methyltransferase (100 U), 100 nM DNA target gene (or 2 μg target plasmid) and enzyme-free water into 50 μL reaction solution, and then incubate at 37°C for 5 hours; add 2 μL of CpG methyltransferase (100 U) and 1 μL of S-adenosylmethionine (32 mM), continue incubation at 37°C for 12 hours, and then terminate the reaction after reacting at 65°C for 20 minutes to obtain the treated methylated DNA target gene (or methylated plasmid). Use the methylated DNA target gene as the electrophoresis verification target and the methylated plasmid as the target DNA for detection;

[0030] (3) Endonuclease cleavage of target DNA: 2 μL of target DNA treated in step (2), 1 μL of NEB restriction endonuclease buffer, and 10 U of HhaI enzyme were mixed, reacted at 37°C for 10 to 70 minutes, and then heated to 65°C and incubated for 25 minutes to terminate the digestion, thereby obtaining a digestion product;

[0031] (4) RPA and generation of single-stranded DNA (ssDNA) products: The digestion product of step (3) was amplified using an RPA kit to obtain a double-stranded DNA (dsDNA) amplification product, wherein the amplification step is as follows: 29.5 μL of RPA reaction buffer was added to the RPA lyophilized powder to prepare an RPA reaction solution; then 4 μL of the digestion product obtained in step (3) was taken, 5.9 μL of RPA reaction solution, 0.5 μL of reverse primer (10 μM), 0.5 μL of forward primer (10 μM) and 1 μL of magnesium acetate (280 mM) were added thereto, and the mixture was reacted at 42° C. for 15 to 35 minutes to obtain a reaction product; then 2 μL of Lambda endonuclease (0.5 U / μL to 4 U / μL) and 2 μL of Lambda buffer were added to the reaction product, and the mixture was reacted at 37° C. for 20 minutes to obtain a ssDNA product.

[0032] (5) CRISPR / Cas12a detection: 9 μL of enzyme-free water, 2 μL of Cas12 enzyme reaction buffer, 2 μL of crRNA (1 μM) obtained in step (1), 2 μL of Cas12a (1 μM), 1 μL of fluorescent probe (10 μM) and 4 μL of amplification product obtained in step (4) were mixed, incubated at 37° C. for 10 to 50 minutes, and 80 μL of double distilled water was added to perform fluorescence detection analysis on the obtained substance to obtain the detection result of the target DNA.

[0033] In the specific implementation, in step (5), the fluorescent probe is HEX-DNA-BHQ1 fluorescent probe, and the fluorescence intensity is measured by a fluorescence spectrometer under 490nm excitation to obtain the detection result of the target DNA. When the target DNA is subjected to instant detection by lateral flow analysis, in step (5), the fluorescent probe is the lateral flow probe Bio-DNA-FAM, and the lateral flow test strip is inserted into the substance obtained in step (5) for 2 minutes to obtain the detection result of the target DNA.

[0034] 2. Embodiment

[0035] The present invention takes breast cancer as an example, and the methylation of the RASSF1A promoter region is considered to be an effective biomarker for breast cancer, which is detected by the following method.

[0036] 1. Materials and reagents

[0037] The RPA kit was obtained from TwistDx. HiScribe T7 high-efficiency RNA synthesis kit, HhaI endonuclease, Lambda exonuclease, and CpG methyltransferase (M.SssI) were purchased from New England BioLabs, Beijing, China. The miRNA purification kit was from Beijing Tiangen. HybriDetect test strips (MGHD 1) were provided by Milenia Biotec GmbH. All DNA sequences and plasmids were synthesized by Shanghai Sangon Biotech Co., Ltd. Ezup column animal genomic DNA purification kit, SanPrep column PCR product purification kit, and PAGE-related reagents (Acryl / Bis 30% Solution (29:1), nucleic acid dyes, TBE buffer, TE buffer, DEPC-treated water, loading buffer, and DNA molecular weight standard markers were all from Shanghai Sangon Biotech Co., Ltd.

[0038] Table 1 DNA sequences used in the examples of the present invention

[0039]

[0040] Note: RAFFS1A-C, RAFFS1A, Random 1, Random 2 and Random 3 are all double-stranded DNA. "P" refers to the phosphate group modification.

[0041] 2. Preparation of crRNA and methylation targets

[0042] crRNA was synthesized by T7 transcription in vitro. First, 2 μL of T7 promoter (10 μM), 2 μL of crRNA template (10 μM) and 16 μL of enzyme-free water were prepared into a mixture, annealed at 95 °C for 5 min, and slowly cooled to room temperature; 10 μL of nucleoside triphosphate (10 mM) and 1.5 μL of T7 RNA polymerase were added, and the transcription reaction was carried out at 37 °C for 16 hours; then 2 μL of deoxyribonuclease I was added to the above reaction solution, incubated at 37 °C for 2 hours to degrade the crRNA template; after degradation, the product was purified with a miRNA purification kit and quantified with NanoDrop 2000C (Thermo Fisher). The obtained crRNA was analyzed by gel electrophoresis.

[0043] The DNA target gene and plasmid were treated with CpG methyltransferase, and the treatment steps were as follows: 2 μL of S-adenosylmethionine (32 mM), 5 μL of NEB biological buffer 2, 2 μL of CpG methyltransferase (100 U), 100 nM DNA target gene (or 2 μg target plasmid) and enzyme-free water were mixed into a 50 μL reaction solution, and then incubated at 37°C for 5 hours; 2 μL of CpG methyltransferase (100 U) and 1 μL of S-adenosylmethionine (32 mM) were added, and the incubation continued at 37°C for 12 hours, and then the reaction was terminated after 20 minutes at 65°C to obtain the methylated DNA target gene and methylated plasmid. The methylated DNA target gene was used as the electrophoresis verification target, and the methylated plasmid was used as the target DNA for detection;

[0044] 3. HhaI endonuclease cleaves target DNA

[0045] The target DNA (RAFFS1A-C and RAFFS1A-mC) was digested with HhaI enzyme in a total reaction volume of 10 μL. The specific steps are as follows: add 2 μL of target DNA, 1 μL of NEB restriction endonuclease buffer, 10 U of HhaI and enzyme-free water, react at 37°C for 50 minutes, and then heat at 65°C for 25 minutes to terminate the digestion.

[0046] 4. RPA and ssDNA generation

[0047] The digestion product was amplified using the RPA kit. First, 29.5 μL of reaction buffer was added to the RPA lyophilized powder to prepare the RPA reaction solution; then 4 μL of the digestion product was added to 5.9 μL of RPA reaction solution, 0.5 μL (reverse primer (10 μM), 0.5 μL forward primer (10 μM) and 1 μL magnesium acetate (280 mM), and reacted at 42°C for 25 minutes. In order to obtain ssDNA, 2 μL Lambda endonuclease (2U / μL) and 2 μL Lambda endonuclease buffer were added to the above solution and reacted at 37°C for 20 minutes.

[0048] 5. Electrophoresis analysis

[0049] The reaction principle of the method was verified by non-denaturing 10% polyacrylamide gel. Because the plasmid sample gene was too long, a 125bp DNA target gene double strand was synthesized as the target for electrophoresis analysis. The reacted sample was mixed with 6X loading buffer at room temperature for 2 minutes and then moved to the gel well. The electrophoresis was performed in 1 XTBE buffer with a voltage of 90V and an electrophoresis time of 50 minutes. Finally, the electrophoresis gel was reacted in a nucleic acid dye for 15 minutes and the image was collected using a JENA UVsolo Imager.

[0050] 6. CRISPR / Cas12a detection

[0051] Fluorescence spectroscopy and lateral flow detection are based on the trans-cleavage activity of Cas12a, so the Cas12a reaction process is as follows, with a total reaction volume of 20 μL: 9 μL enzyme-free water, 2 μL Cas12 enzyme reaction buffer, 2 μL crRNA (1 μM), 2 μL Cas12a (1 μM), 1 μL fluorescent probe (10 μM HEX-DNA-BHQ1) and 4 μL reaction product, incubated at 37 ° C for 30 minutes, and then 80 μL of double distilled water was added, and the fluorescence intensity was measured with a fluorescence spectrometer (PerkinElmer) under 490 nm excitation. For lateral flow detection, the fluorescent probe was replaced by a lateral flow probe (10 μM Bio-DNA-FAM). The other reaction steps are consistent with the above steps. The test strip was then inserted into the reaction solution for 2 minutes, and the results were finally photographed with a camera.

[0052] 7. Cell culture and genomic DNA preparation

[0053] Human breast cancer cells (MCF-7) and normal human liver cells (LO2) were selected as real samples. To prepare genomic DNA, these cells were first cultured in 90% RPMI 1640 medium (Hyclone, USA) and 10% fetal bovine serum (Solarbio, China). When the cell density reached 80% of the culture flask, genomic DNA was extracted and purified using the Ezup column animal genomic DNA purification kit. The obtained genomic DNA was then used as the target DNA to verify the practical application capability of the method.

[0054] 3. Discussion on the Detection Principle and Results

[0055] 1. E-PfRPA / Cas detection principle

[0056] The principle of the detection method of the present invention is shown in the attached Figure 1 From the attached Figure 1As can be seen from A, methylated DNA and unmethylated DNA are first treated with HhaI exonuclease, which can recognize and digest the "-GCGC-" site. Therefore, unmethylated DNA will be digested by HhaI. In the present invention, the promoter of the RAFFS1A gene contains multiple -GCGC- restriction sites. Therefore, unmethylated RAFFS1A is completely digested by HhaI. In contrast, methylated RAFFS1A has no effect and the structure remains intact. The structurally intact methylated RAFFS1A is used to trigger the RPA amplification reaction. In the PRA system, the recombinase binds to the primer to locate the homologous sequence in the double strand and amplifies the target region exponentially. Since the 5' end of the positive primer has a phosphate modification, the sense strand in all amplified double strands has a 5'-phosphate. Lambda exonuclease can degrade the 5'-phosphate modified sequence of the double strand. Therefore, a large amount of single-stranded ssDNA can be obtained by Lambda treatment. At the same time, nonspecific amplification can be avoided under Lambda treatment, and the detection background is effectively filtered. Since the generated ssDNA is partially complementary to crRNA, the trans-cleavage activity of Cas12a is activated, and Cas12a can arbitrarily cut the nearby fluorescent reporter probe (HEX-DNA-BHQ1) to release fluorescent signals.

[0057] In addition, the method of the present invention can also be used for real-time detection of lateral flow analysis (LFA) by changing the reporter probe (Bio-DNA-FAM). Figure 1 As shown in B, biotin and FAM are modified at both ends of the reporter probe to enable it to bind to AuNPs-anti-FAM. Due to the presence of streptavidin in the control line area (line C), AuNPs-anti-FAM / Bio-DNA-FAM can be captured by binding of biotin to streptavidin, so a red band appears on line C. When methylated targets are present, they can initiate a series of reactions to produce a large amount of ssDNA. The trans-cleavage activity of Cas12a is then activated, thereby shearing the Bio-DNA-FAM reporter gene. The sheared reporter probe containing AuNPs can be captured by the rabbit anti-human serum albumin antibody on the test line (line T), and a red stripe will appear on line T. In contrast, in the unmethylated target area, there is only a red band on line C. Based on the detection principle of the sensor, DNA methylation can be successfully distinguished and applied to the point-of-care platform.

[0058] 2. Feasibility of the E-PfRPA / Cas strategy adopted in the present invention

[0059] The feasibility of the E-PfRPA / Cas strategy was verified by electrophoresis, fluorescence detection and LFA detection. Figure 2 A and Figure 2B is the electrophoresis image. The length of methylated RAFFS1A and unmethylated RAFFS1A is 125 bp. Unmethylated RAFFS1A contains the "-GCGC-" site of HhaI. After reacting with HhaI, RAFFS1A is digested into different fragments. Figure 2 As can be seen in A, a digested band can be observed in lane 2, which is consistent with Figure 2 The schematic diagram is consistent with that of C. Some of the digested sequences are too short to stain clearly or run out of the electrophoresis plate, such as the 5 bp and 7 bp bands, which cannot be seen in lane 2. The last low-mobility band is the incompletely digested RAFFS1A, which corresponds to the methylated RAFFS1A in lane 1, which is protected from endonuclease digestion by the methyl modification. These results indicate that the methylated RAFFS1A and the unmethylated RAFFS1A have been successfully recognized by HhaI. Figure 2 B is the electrophoresis result of RPA reaction and Lambda exonuclease treatment. After amplification, an obvious band appears in lane 3. Due to the interference of single-stranded binding protein and recombinase, the amplified band is wide. The other two bands are the forward primer and the reverse primer. For unmethylated RAFFS1A, there is also RPA product in lane 4 due to incomplete digestion and nonspecific amplification. However, when Lambda exonuclease is added, only the methylated target can generate ssDNA (lane 5), while the unmethylated target is digested and no ssDNA is produced.

[0060] To further validate the CRISPR / Cas12a-based DNA methylation detection, the fluorescence spectrum and LFA results were as follows Figure 3 A and Figure 3 As shown in B. Figure 3In A, only methylated RAFFS1A (RAFFS1A-mC) produces strong fluorescence (curve c), while the fluorescence of unmethylated RAFFS1A (RAFFS1A-C) (curve b) and the blank group (curve a) is negligible. The above results show that RAFFS1A-mC can avoid HhaI cleavage through 5mC modification, thereby initiating the RPA reaction to generate a large amount of DNA double strands. After the addition of Lambda, DNA sequences with 5'-phosphate ends can be recognized and degraded, leaving a large amount of ssDNA. Therefore, the obtained ssDNA can activate the trans-cutting activity of Cas12a and release fluorescent signals. In contrast, RAFFS1A-C is digested by HhaI, and HhaI cannot trigger the following series of reactions. In the LFA test, only the experimental group with RAFFS1A-mC showed an obvious band on the test line, which was due to AuNPs-anti-FAM coupling. The above results indicate that the E-PfRPA / Cas method can successfully detect DNA methylation with low background interference, high sensitivity and accuracy.

[0061] 3. Optimizing the method of the present invention

[0062] In order to improve the sensitivity of this method, important parameters such as the cleavage time of Cas12a, the digestion time of HhaI, the amplification time of RPA, and the concentration of Lambda were optimized. First, the shearing time of Cas12a was studied. Figure 4 As shown in A, the fluorescence intensity increases with the increase of Cas12a cleavage time and reaches a peak at 30 minutes, so the Cas12a cleavage time in the next experiment is 30 minutes. HhaI is related to the background signal, and incomplete digestion will lead to high background signals. Set a high concentration of 50pM DNA target (RAFFS1A-mC and RAFFS1A-C) and a high concentration of enzyme content (10U) to ensure complete digestion. Figure 4 As shown in Figure B, as the digestion time increases from 10 minutes to 70 minutes, the background signal decreases, and the fluorescence signal is completely quenched after 50 minutes of HhaI treatment. Therefore, the digestion of unmethylated DNA requires 50 minutes of Hhal treatment. Amplification time is also an important parameter and is closely related to sensitivity. The fluorescence intensity of the RPA reaction is measured after an amplification time of 15 to 35 minutes, and a saturated fluorescence signal appears after 25 minutes ( Figure 4 C) In addition, the concentration of Lambda enzyme was optimized for rapid detection. Figure 4 As shown in D, the fluorescence response is concentration-dependent and reaches saturation at 2 U / μL. Therefore, 2 U / μL of Lambda was selected as the optimal condition.

[0063] 4. Study on the performance of E-PfRPA / Cas in detecting DNA methylation

[0064] To investigate the performance of the proposed method, the concentration range of the method for RAFFS1A-mC detection was measured under optimal conditions. Figure 5 As shown in A, from 0fM to 20pM, as the concentration of RAFFS1A-mC increases, the fluorescence response gradually increases. There is a good linear relationship between the fluorescence intensity (ΔF) and the logarithm of the RAFFS1A-mC concentration from 2fM to 20pM ( Figure 5 B illustration). The fluorescence intensity (ΔF) is the value after subtracting the fluorescence intensity of the blank group. The linear fitting equation is ΔF = 241.3LogC + 623.3 (R = 0.994), C is the concentration of RAFFS1A-mC (pM). The detection limit (LOD) was calculated to be 0.98fM. Compared with the DNA methylation detection method in the prior art, the method of the present invention has a lower detection limit and a wider detection range (Table 2). These excellent performances may be due to the following reasons: (1) The specific recognition of HhaI enables methylated and unmethylated DNA to be effectively distinguished; (2) The high amplification ability of RPA combined with Lambda enzyme can produce a large amount of ssDNA; (3) The obtained ssDNA can be recognized by Cas12a without the need for a PAM site; and the high specificity and self-amplification ability of CRISPR / Cas12a further improve the sensing performance.

[0065] Table 2 Comparison with other methylation detection methods

[0066]

[0067] Existing technology shows that 5mC accounts for 2-7% of cytosine in the mammalian genome, and most genes are in an unmethylated state. Therefore, in the presence of a large number of unmethylated samples, it is necessary to distinguish the methylated regions. First, the concentration of the unmethylated target (RAFFS1A-C) was set to 20pM, and then different concentrations of the methylated target (RAFFS1A-mC) were added to the unmethylated (RAFFS1A-C) reaction system. Figure 6 As shown in A, as the methylation level increases, the fluorescence signal gradually increases from 0% to 100%, and there is a good linear relationship between the input methylation level and the measured methylation level. The linear fitting equation is y=1.1x+0.41 (R=0.999), y is the measured methylation level, and x is the input methylation level. It is worth noting that a methylation level of 0.05% (10fM) can be identified, indicating that the E-PfRPA / Cas strategy adopted in the present invention has high accuracy and sensitivity.

[0068] Selectivity and repeatability are the key to the sensing system. Therefore, random DNA such as RASSF1A-C was first used as an interfering substance to study the selectivity of the method of the present invention for site-specific methylation detection. Figure 6 C and Figure 6 D shows that with the presence of RASSF1A-mC, an obvious fluorescence peak appeared. At the same concentration of RASSF1A-mC (2pM), the fluorescence response was weaker than that of RASSF1A-C and random DNA. In addition, the fluorescence response of 5 parallel experimental groups and the control group was tested to examine the repeatability ( Figure 7 ). There was no significant fluctuation in fluorescence values ​​between the groups. The relative standard deviation (RSD) was 2.28%. The above results show that the detection method of the present invention has good anti-interference ability and repeatability.

[0069] 5. Lateral flow assay (LFA) for DNA methylation detection

[0070] Lateral flow test strips are intuitive and portable, and therefore have good practical application potential for establishing a naked eye detection platform. Therefore, the present invention integrates the E-PfRPA / Cas method into a lateral flow assay (LFA) strip. Figure 8 A is a schematic diagram of the principle of lateral flow detection of methylated DNA and unmethylated DNA. According to the detection principle of the strips, when the methylated target (RASSF1A-mC) is present, there are two red strips on the C line and the T line. In the unmethylated target (RASSF1A-C), only one red strip appears. Then the sensitivity of LFA based on the E-PfRPA / Cas method was studied. Figure 8 As shown in B, on the T line, as the concentration of RASSF1A-mC increases from 0fM to 20pM, the color of the band changes from colorless to dark. However, the color on the C line shows the opposite trend. RASSF1A-mC as low as 20fM can be clearly identified. In addition, Figure 8 C It can be seen that only RASSF1A-mC can induce red on the test line. Random DNA such as RASSF1A-C only shows red on the control line. These results show that the LFA based on the E-PfRPA / Cas method used in the present invention has high sensitivity and selectivity and has good application prospects in areas with limited resources.

[0071] 6. Real sample testing

[0072] In order to evaluate the practicality of the E-PfRPA / Cas detection method, 20-fold diluted human serum was added to the target solution to simulate a complex biological environment. The dilution target concentrations were 20fM, 200fM and 2pM, respectively. The diluted target was determined by the E-PfRPA / Cas method. The results are shown in Table 3, and the recovery rate was 97% to 112.4%. The RSD was 2.05% to 4.66%, which is suitable for the detection of complex samples. In addition, data show that methylation of the RASSF1A promoter is closely related to breast cancer, and the methylated region (site 50340771-site 50340676) can affect the expression of cytokeratin 19 (CK19) mRNA, thereby leading to the occurrence of tumors. Therefore, in order to further evaluate the reliability of the method described in the present invention in practical applications, the E-PfRPA / Cas detection was applied to real genomic DNA samples. The detection of genomic DNA of normal cells (LO2) and breast cancer cells (MCF-7) based on this method was studied. As Fig. 9 A and Fig. 9 As shown in B, under the same amount of genomic DNA (200 ng / μL), the fluorescence reaction of the genomic DNA of MCF-7 cells was significantly higher than that of the genomic DNA of normal cells (LO2), and under the same amount of genomic DNA (500 ng / μL), the genomic DNA of MCF-7 cells also showed a red band on the T line, while the genomic DNA of normal cells (LO2) only showed red on the control line. The above results show that the method of the present invention has a high sensitivity for the determination of cell DNA methylation.

[0073] Table 3 DNA methylation detection in human serum samples (RASSF1A-mC)

[0074] Add target concentration Detection and calculation of concentration Recovery rate (%) RSD(%) 20fM 19.4fM 97 4.66 200fM 224.7fM 112.4 4.63 2pM 2.18pM 109 2.05

[0075] 7. Conclusion

[0076] In summary, the present invention provides a new method for detecting DNA methylation without PAM sites based on endonuclease-assisted recombinase polymerase amplification and CRISPR / Cas12a (E-PfRPA / Cas). Compared with traditional methylation-specific PCR, the HhaI-based RPA reaction conditions are mild and the detection speed is fast. The trans-cleavage activity of CRISPR / Cas12a is triggered in the absence of a PAM site. The results show that the method of the present invention exhibits good performance in DNA methylation detection and can be combined with lateral flow strips to achieve POC detection. The fluorescence detection limit is 0.98fM, which can distinguish methylation levels as low as 0.05%. By lateral flow method, methylated DNA of 20fM can be clearly identified by the naked eye. In addition, E-PfRPA / Cas can detect genomic DNA methylation of cancer cells. Based on the programmability of crRNA, the E-PfRPA / Cas method can be further used to establish a universal platform for multiple methylation sites and even nucleic acid detection, which has a good application prospect.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the technical solution. Those skilled in the art should understand that those modifications or equivalent substitutions of the technical solution of the present invention that do not depart from the purpose and scope of the technical solution should be included in the scope of the claims of the present invention. Sequence Listing <110> Chongqing University; Three Gorges Hospital Affiliated to Chongqing University <120> A DNA methylation detection method based on recombinase polymerase amplification and CRISPR / Cas12a coupling <160> 12 <210> 1 <211> 125 <212> DNA <213> RAFFS1A-C (embedded in pUC-SP plasmid) <400> cattgagctg cgggagctgg cacccgctgg gcgcgctggg aagggccgca cccggctgga 60 gcgtgccaac gcgctgcgca tcgcgcgggg caccgcgtgc aaccccacac ggcagctggt 120 ccctg 125 <210> 2 <211> 125 <212> DNA <213> RAFFS1A <400> cattgagctg cgggagctgg cacccgctgg gcgcgctggg aagggccgca cccggctgga 60 gcgtgccaac gcgctgcgca tcgcgcgggg caccgcgtgc aaccccacac ggcagctggt 120 ccctg 125 <210> 3 <211> 30 <212> DNA <213> RPA forward primer <400> cattgagctg cgggagctgg cacccgctgg 30 <210> 4 <211> 32 <212> DNA <213> Post-RPA Primer <400> cagggaccag ctgccgtgtg gggttgcacg cg 32 <210> 5 <211> 7 <212> DNA <213> Fluorescent probes <400> tattatt 7 <210> 6 <211> 8 <212> DNA <213> Lateral flow probe <400> ttattatt 8 <210> 7 <211> 25 <212> DNA <213> T7 promoter <400> gaaattaata cgactcacta taggg 25 <210> 8 <211> 66 <212> RNA <213> crRNA values <400> acgcgctgcg catcgcgcgg atctacactt agtagaaatt accctatagt gagtcgtatt aatttc 66 <210> 9 <211> 41 <212> DNA <213> crRNA <400> uaauuucuac uaaguguaga uccgcgcgau gcgcagcgcg u <210> 10 <211> 80 <212> DNA <213> Random <400> 60. ccacatgtcg gtcacgtgcg cccacaccca gccaatcggc gggctcccga cgggaatggg gagcgccctg gtccgcatcc 80 <210> 11 <211> 40 <212> DNA <213> Random <400> tggctggtgg gcagcgggtc gcgcggaggg cagcggcgag <210> 12 <211> 66 <212> DNA <213> Random <400> cagttcctgc atgggcggca tgaaccgggg gcccatcctc accatcatca cactggaaga ctccag 66

Claims

1. A composition for detecting DNA methylation based on recombinase polymerase amplification and CRISPR / Cas12a coupling, characterized in that: The composition includes RAFFS1A-C, RAFFS1A, RPA front primer, RPA rear primer, fluorescent probe, lateral flow probe, T7 promoter, crRNA template, crRNA; wherein RAFFS1A-C is embedded in the PUC-SP plasmid; the sequence of the composition is as follows:

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