A method for constructing nucleic acid sequences for cfDNA multiplex gene methylation detection and application thereof

Abstract

The application discloses a nucleic acid sequence construction method for cfDNA multiplex gene methylation detection and application thereof. The construction method comprises the following steps: using a methylation-sensitive restriction endonuclease to perform enzyme cutting on to-be-detected cfDNA to obtain enzyme cutting fragments, using a ligase to connect an adapter to a nick end of the enzyme cutting fragments to form an adapter product; and using the adapter product as a template to perform PCR amplification to construct a recombined nucleic acid sequence for target gene methylation site detection. The construction method can be used for cfDNA level methylation qPCR analysis and low-abundance sample quantitative analysis, has small sample demand, has a wide methylation analysis coverage, and has high methylation enrichment efficiency.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a method for constructing nucleic acid sequences for detecting multiple gene methylation of cfDNA and its application. Background Technology

[0002] DNA methylation, as one of the core mechanisms of epigenetic modification, plays a crucial role in gene expression regulation, cell differentiation, and disease development. Particularly in the field of oncology, hypermethylation of specific gene promoter regions is an important early molecular event. Therefore, developing sensitive, accurate, and efficient DNA methylation detection technologies is of great significance for early cancer screening, diagnosis, and prognostic monitoring.

[0003] Circulating cell-free DNA (cfDNA), especially circulating tumor DNA (ctDNA) derived from tumors, has become a highly promising target for liquid biopsy. However, cfDNA is present in very low concentrations in blood, is highly fragmented, and often contains a large amount of wild-type background DNA, which poses a significant challenge to the detection of low-abundance methylation signals.

[0004] Currently, mainstream DNA methylation detection technologies are mainly based on the following principles: 1. Bisulfite treatment combined with sequencing / PCR: This is the current gold standard. It converts unmethylated cytosine (C) to uracil (U) using bisulfite, while methylated C remains unchanged, and then identifies it through subsequent sequencing or specific PCR. However, this method has significant drawbacks, including severe DNA damage, extremely high sample loss rate (usually >90%), difficulty in achieving 100% conversion efficiency, and reduced sequence complexity. 2. Methylation-sensitive restriction endonuclease (MSRE) method: This method utilizes the characteristic that MSREs (such as HhaI, HpaII, AciI, etc.) can only cleave unmethylated recognition sequences. It digests unmethylated DNA through enzymatic digestion, thereby enriching methylated fragments. This method avoids chemical damage, but its inherent limitations include: limited coverage, incomplete background digestion, and the simplification of traditional MSRE-qPCR methods. Other technologies, such as methods based on the enrichment of methylation-binding proteins and emerging enzymatic transformation methods (such as APOBEC enzymatic transformation), may have problems such as high cost, complex operation, and immature technology, and are difficult to promote in clinical practice for the detection of multiple methylation of low-abundance cfDNA.

[0005] Therefore, there is an urgent need to develop a novel strategy for constructing a cfDNA methylation detection method that can maximize the preservation and utilization of limited cfDNA templates and achieve simultaneous, sensitive, and quantitative detection of multiple target gene methylation sites through efficient enrichment and labeling methods, making it suitable for clinical applications such as liquid biopsy. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a method for constructing nucleic acid sequences for cfDNA multiplex gene methylation detection and its application. By using methylation-sensitive restriction endonucleases to cleave unmethylated sites, negative screening of unmethylated sequences is achieved. Simultaneously, specific sticky end ligators generated by enzyme digestion are used to achieve specific enrichment and labeling of methylated sequences.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for constructing a nucleic acid sequence for detecting multiple gene methylation in cfDNA. The method includes: digesting the cfDNA to be tested with a methylation-sensitive restriction endonuclease to obtain a digested fragment; using a ligase to connect an adapter to the cut end of the digested fragment to form an adapter product; and performing PCR amplification using the adapter product as a template to construct a recombinant nucleic acid sequence for detecting methylation sites of a target gene.

[0008] This invention employs unmethylated linkers (first and second linkers) for the analysis of methylation sites and the construction of a methylation detection method. The unmethylated linkers are digested with methylation-sensitive restriction endonucleases to generate sticky ends, which are then introduced using ligases. The methylation-sensitive restriction endonucleases recognize specific sites containing methylated C bases and perform enzymatic digestion flanking the recognition site. The unmethylated linkers are added using unmethylation-related endonucleases capable of generating sticky ends, including using methylation-sensitive restriction endonucleases to generate sticky ends. The methylation-sensitive restriction endonucleases recognize and digest specific sites containing unmethylated C bases. For example, the 5' end CCGC sequence of the linker corresponding to the methylation-sensitive endonuclease in the examples ensures unidirectional ligation between the linker and the nucleic acid fragment to be analyzed. Simultaneously, the CG linker, under the action of the methylation-sensitive endonuclease AciI, prevents the linker from self-ligating. When using methylation-sensitive restriction endonucleases, this invention identifies partially methylated sites, i.e., indirect identification that is not digested by the sensitive enzyme. Methylation-sensitive restriction endonucleases can digest unmethylated sequences to avoid interference, while the unmethylated CpG sites of the restriction endonucleases can be identified to generate sticky ends. Furthermore, one enzyme can be applied to multiple gene sequences, thus improving the detection coverage of methylated CpG sites.

[0009] Preferably, the methylation-sensitive restriction endonuclease includes AciI and / or HhaI.

[0010] Preferably, the base sequence protruding from the 5' end of the adhesive end of the connector is CG.

[0011] Preferably, the ligase comprises T4 DNA ligase.

[0012] Preferably, the source of the cfDNA to be tested includes biological samples and / or artificially synthesized samples; the biological samples include any one of plasma, serum, tissue or cells.

[0013] Secondly, the present invention provides the application of the method for constructing nucleic acid sequences for cfDNA multiplex gene methylation detection described in the first aspect in the preparation of methylation detection products.

[0014] Thirdly, the present invention provides a method for detecting methylation of cervical cancer-related genes, the method comprising the following steps: (1) The cfDNA sample to be tested is processed using the method for constructing nucleic acid sequences for the detection of multiple gene methylation of cfDNA described in the first aspect to obtain recombinant nucleic acid sequences; (2) Using the recombinant nucleic acid sequence as a template, quantitative multiple methylation-specific PCR amplification and detection were performed using primers and fluorescent probes for detecting cervical cancer-related genes; (3) Determine the methylation status of the gene based on the amplification detection results.

[0015] Preferably, the cervical cancer-related genes include any one or a combination of at least two of PAX1, SOX1, RXFP3.1, RXFP3.2 or ZNF671.

[0016] Preferably, the nucleic acid sequence of the primer includes the sequences shown in SEQ ID NO.1-SEQ ID NO.10; the nucleic acid sequence of the fluorescent probe includes the sequences shown in SEQ ID NO.11-SEQ ID NO.15.

[0017] Primer F1 (SEQ ID NO.1): CCAAAGGGCCGCAGTGAC.

[0018] Primer R1 (SEQ ID NO.2): GAGTGTCCTCCACGTCAATCTC.

[0019] Primer 2F (SEQ ID NO.3): GGCTCTGACGTTACCTTGC.

[0020] Primer 2R (SEQ ID NO.4): CTTCCTCCTCCCTCCTCTGG.

[0021] Primer 3F (SEQ ID NO.5): GAATGCGATCTTGCGCGCTTTC.

[0022] Primer 3R (SEQ ID NO.6): GTAGCGGACATTTTGTTTCTGT.

[0023] Primer 4F (SEQ ID NO.7): CCTCATCCAAGCAGTCCC.

[0024] Primer 4R (SEQ ID NO.8): GAATGCGATCTTGCGCTC.

[0025] Primer 5F (SEQ ID NO.9): GCGATCTTTGCGCCGCCTTG.

[0026] Primer 5R (SEQ ID NO.10): CGTCTCTCCGCGGTTGTC.

[0027] Probe 1 (SEQ ID NO.11): CACGCCGGAGACGCGC.

[0028] Probe 2 (SEQ ID NO.12): CAGGTGGAAGGCGCCCCGC.

[0029] Probe 3 (SEQ ID NO.13): GTGGGCCGCAGGT.

[0030] Probe 4 (SEQ ID NO.14): GGGGCCGCTCGCTCCC.

[0031] Probe 5 (SEQ ID NO.15): GCCAGCGGCTCTCACC.

[0032] Preferably, the fluorescent probe has a fluorescent group at its 5' end and a quenching group at its 3' end.

[0033] Preferably, the fluorescent group includes any one or a combination of at least two of FAM, HEX, VIC or ROX.

[0034] Preferably, the quenching group includes any one or a combination of at least two of BHQ1, BHQ2, or MGB.

[0035] Preferably, the quantitative multiple methylation-specific PCR amplification system includes a template, primers, fluorescent probes, and qPCR premix.

[0036] Preferably, the concentration of primers in the system is 5-10 μM (e.g., 5 μM, 8 μM or 10 μM), and the concentration of fluorescent probes is 5-10 μM (e.g., 5 μM, 8 μM or 10 μM).

[0037] Preferably, the quantitative multiple methylation-specific PCR amplification procedure includes: (1) pre-denaturation at 93-95℃ (e.g., 93℃, 94℃, or 95℃) for 30-40 s (e.g., 30 s, 35 s, or 40 s); (2) denaturation at 93-95℃ (e.g., 93℃, 94℃, or 95℃) for 5-10 s (e.g., 5 s, 8 s, or 10 s); (3) annealing at 55-60℃ (e.g., 55℃, 58℃, or 60℃) for 20-30 s (e.g., 20 s, 25 s, or 30 s); extension at 55-60℃ (e.g., 55℃, 58℃, or 60℃) for 20-30 s (e.g., 20 s, 25 s, or 30 s), for a total of 40-45 cycles (e.g., 40 cycles, 42 cycles, or 45 cycles).

[0038] The specific point values ​​that can be selected from the above 93-95℃ range include 93℃, 94℃, or 95℃.

[0039] Compared with the prior art, the present invention has the following beneficial effects: (1) The method for methylation detection that does not rely on sodium bisulfite provided by the present invention has a small sample requirement and can be used for quantitative analysis of methylation level of plasma cfDNA and qualitative analysis of disease progression by combining multiple gene sites. Since the coverage of single gene methylation site analysis is limited, the present invention combines multiple methylation sites of multiple genes and uses two enzymes to ensure relatively high detection site coverage, which has practical value in the field of cfDNA methylation analysis. (2) This invention focuses on methyl-sensitive restriction endonucleases. By introducing non-methylated site adapters, it aims to link as many non-methylated site adapters upstream of methylated sites as possible to the nucleic acid molecules to be tested that have methylated site-specific adapters connected downstream of methylated sites. This maximizes the analysis efficiency of methylated sites. Introducing sequencing adapters at any end of the methylated site simplifies the methylation enrichment process and expands the range of methylation recognition. (3) The construction method provided by the present invention, combined with subsequent detection, can achieve a sensitivity of 0.00001% for methylation detection of cervical cancer-related genes such as ZNF671, and has excellent detection capability for low abundance cfDNA samples. Attached Figure Description

[0040] Figure 1 This is a schematic diagram illustrating how methylation sites inhibit the action of methylation-sensitive restriction endonucleases in Example 1. Figure 2 This is a schematic diagram illustrating the working principle of the methylation-sensitive endonuclease product generating CG cleavage sticky ends in Example 1; Figure 3This is a schematic diagram illustrating the working principle of the disappearance of the original restriction site after the CG linker is ligated to the methylation-sensitive endonuclease product in Example 1. Figure 4 The graph shows the detection sensitivity results for the PAX1 gene. Figure 5 The graph shows the detection sensitivity results for the SOX1 gene. Figure 6 The graph shows the detection sensitivity results for gene RXFP3.1; Figure 7 The graph shows the detection sensitivity results for gene RXFP3.2; Figure 8 The graph shows the detection sensitivity results for the ZNF671 gene. Figure 9 Gel electrophoresis images of templates with different methylation ratios; Figure 10 This is a graph showing the accuracy test results of the construction method of the present invention. Detailed Implementation

[0041] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0042] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0043] The specific sequences of the artificial DNA template sequence, AciI adapter, and HhaI adapter used in the following examples are as follows: (1) Artificial DNA template 1: the positive strand is shown in SEQ ID NO.16, and the reverse strand is shown in SEQ ID NO.17.

[0044] SEQ ID NO.16: 5'-GGGGCGCTGGGGCGCAGTGACGGGAACCAATGAGCTGCCAACTCGCGCGTCTCCGGCTGACTGCCGAGATTGACGTGGAGGACACGTC-3'.

[0045] SEQ ID NO.17: 5'-GACGTGTCCTCCACGTCAATCTCGGCAGTCACGCCGGAGACGCGCGAGTTGGCAGCTCATTGGTTCCCGTCACTGCGCCCCAGCGCCCC-3'.

[0046] (2) Artificial DNA template 2: the positive strand is shown in SEQ ID NO.18, and the reverse strand is shown in SEQ ID NO.19.

[0047] SEQ ID NO.18: 5'-GGGGCTGCTGCGCGTTACCTTGCTGCAAGCGGGGCGCCTTCCACCTGGCTGGGGGTCTGCGCCACAGTTTGGTCCAGAGGAGGGAGGAGGAAG-3'.

[0048] SEQ ID NO.19: 5'-CTTCCTCCTCCCTCCTCTGGACCAAAACTGTGGCGCAGACCCCCAGCCAGGTGGAAGGCGCCCCGCTTGCAGCAAGGTAACGCGCAGCAGCCCC-3'.

[0049] (3) Artificial DNA template 3: the positive strand is shown in SEQ ID NO.20, and the reverse strand is shown in SEQ ID NO.21.

[0050] SEQ ID NO.20: 5'-CCTCATCCAAGCAGTCCCAGAGGAAACGGATAAAGGTATTTGAAAGGGAGCGAGCGGCCAAATCGCACAATTGAGCGGCTGGGGGAG-3'.

[0051] SEQ ID NO.21: 5'-CTCCCCCAGCCGCTCAATTGTGCGATTTGGGGCCGCTCGCTCCCTTTCAAATACCTTTATCCGTTTCCTCTGGGACTGCTTGGATGAGG-3'.

[0052] (4) Artificial DNA template 4: the positive strand is shown in SEQ ID NO.22, and the reverse strand is shown in SEQ ID NO.23.

[0053] SEQ ID NO.22: 5'-TCCTTGGGGCCCGCCGCCTTGGCCGTGCAAGTGCCACCGTAACTGGTGAGAGCCGCTGGCAACCCACCCGGAGTTGACAACCGCGGAGAGACG-3'.

[0054] SEQ ID NO.23: 5'-CGTCTCTCCGCGGTTGTCAACTCCGGGTGGGTTGCCAGCGGCTCACCAGTTACGGTGGCACTTGCACGGCCAAGGCGGCGGGCCCCAAGGA-3'.

[0055] (5) Artificial DNA template 5: the positive strand is shown in SEQ ID NO.24, and the reverse strand is shown in SEQ ID NO.25.

[0056] SEQ ID NO.24: 5'-CTCCTCCGCGCTTTCCCAACACCTCCACCTGCGGCCCACACAAGCGTTACAGAACCCCGGCCAGGGACAGCCTGACAGAAACAAAATGTCCGCTAC-3'.

[0057] SEQ ID NO.25: 5'-GTAGCGGACATTTTGTTTCTGTCAGGCTGTCCCTGGCCGGGGTTCTGTAACGCTTGTGTGGGCCGCAGGTGGAGGTGTTGGGGAAAGCGCGGAGGAG-3'.

[0058] (6) AciI connector: positive chain as shown in SEQ ID NO.26, negative chain as shown in SEQ ID NO.27.

[0059] AciI connector - SEQ ID NO.26: 5'-CGCAAGATCGCATTCATGCGTCTTCACCTGGA-3'.

[0060] AciI connector - SEQ ID NO.27: 5'-TCCAGGTGAAGACGCATGAATGCGATCTTG-3'.

[0061] (7) HhaI connector: positive chain as shown in SEQ ID NO.28, anti chain as shown in SEQ ID NO.29.

[0062] HhaI connector - SEQ ID NO.28: 5'-GCCTTTGGGGCTGTGGTTCATTTGGCTCTGA-3'.

[0063] HhaI connector - SEQ ID NO.29: 5'-TCAGAGCCAAATGAACCACAGCCCCAAAGGGCCG-3'. Example 1

[0064] This embodiment provides a kit for detecting cervical cancer gene cfDNA methylation. The kit includes primers and fluorescent probes. The primers include 5 pairs of primers, with nucleic acid sequences as shown in SEQ ID NO.1-SEQ ID NO.10. The fluorescent probes include 5 corresponding nucleic acid sequences, with nucleic acid sequences as shown in SEQ ID NO.11-SEQ ID NO.15. The kit also includes Q5, T4 DNA ligase buffer, Q811 ChamQ Geno SNP Master Mix, rCutsmart buffer, AciI enzyme, HhaI enzyme, T4 DNA ligase, and DEPC H2O. Example 2

[0065] This embodiment analyzes the detection efficiency of methylation-sensitive restriction endonuclease-quad primer ligation-quantitative PCR (MSRE-4TL-qPCR method) for methylated cfDNA of cervical cancer genes PAX1, SOX1, RXFP3.1, RXFP3.2, and ZNF671.

[0066] Based on the DMIWD database, genes with high methylation levels and CpG sites in cervical cancer were identified. Nucleic acid fragments (excluding) methylated CpG sites, namely PAX1, SOX1, RXFP3.1, RXFP3.2, and ZNF671, were synthesized using Ribo. These fragments were then digested with AciI or HhaI, and adapters were ligated using T4 DNA Ligase to obtain the artificially synthesized DNA for testing. Details are as follows: (1) Enzyme digestion The systems for enzymatic digestion using methylation-sensitive restriction endonucleases AciI or HhaI are shown in Table 1.

[0067] Table 1 10×Cutsmart buffer 2 AciI or HhaI 1 (100 ng / μL) Artificial DNA template (1-5) 10 ddH2O 7 The enzyme digestion procedure is as follows: digestion at 37℃ for 60 min; enzyme denaturation at 65℃ for 25 min; and storage at 4℃.

[0068] The recognition sites and cleavage diagrams of methylation-sensitive restriction endonucleases are shown below. Figure 1 and Figure 2 .

[0069] (2) Connecting joint The connection reaction systems for connecting the AciI connector or the HhaI connector are shown in Table 2.

[0070] Table 2 10×T4 DNA ligase buffer 2 T4 DNA ligase 1 (50 ng / μL) enzyme digestion product 2 AciI connector or HhaI connector 5 ddH2O 10 The reaction procedure for the ligation adapter reaction is as follows: 25℃, 1 h; 65℃ for 20 min to denature the T4 DNA ligase and inactivate it; 4℃ for storage.

[0071] See the schematic diagram of the connection connector reaction principle. Figure 3 .

[0072] (3) PCR reaction The PCR reaction system is shown in Table 3.

[0073] Table 3 2× Q5 Master Mix 10 (5 ng / μL) The ligation product obtained in the previous step 1 Primers F1-F5 1 Primers R1-R5 1 ddH2O 7 Note: The ligation product in this example is the template DNA ligation product, and the amount used is 1 μL. If it is the plasma cfDNA ligation product, the amount used is 5 μL.

[0074] The PCR reaction procedure is as follows: (1) 98℃ for 30 s, 1 cycle; (2) 98℃ for 5 s, 50℃ for 10 s, 72℃ for 20 s, 25 cycles; (3) 72℃ for 2 min, 1 cycle, 4℃ for storage.

[0075] (4) qPCR reaction The qPCR reaction system is shown in Table 4.

[0076] Table 4 2×ChamQ Geno SNP Master Mix 10 The amplification product obtained from the previous PCR step 1 Primers F1-F5 1 Primers R1-R5 1 Probes P1-P5 1 ddH2O 6 The qPCR reaction program is as follows: (1) 95℃ for 30 s, 1 cycle; (2) 95℃ for 10 s, 60℃ for 30 s, 45 cycles; (3) 72℃ for 30 s, 1 cycle.

[0077] PAX1-methylated and unmethylated DNA (DNA was artificial DNA template 1, positive strand as shown in SEQ ID NO.16, reverse strand as shown in SEQ ID NO.17) were mixed with 100% methylated, 100% unmethylated, and 50% methylated plus 50% unmethylated DNA to prepare mixed DNA with different methylation levels. The DNA was digested with HhaI enzyme (the digestion system is shown in Table 1), and incubated at 37℃ for 60 min. The digested products were then ligated with HhaI adapters (the ligation system is shown in Table 2), and incubated at 25℃ for 60 min. The ligated products were then subjected to PCR amplification (PCR reaction system and temperature are shown in Table 3). Subsequently, the DNA was amplified with 1, 10, 100, 1000, 10000, and 10... 5 10 6 After a 1:1 dilution, the next step of the qPCR reaction mainly targets the ligated methylated recombinant DNA fragment for fluorescence signal detection. The system configuration is shown in Table 4. The sensitivity of PAX1 methylated DNA detection is determined by observing the fluorescence values. The results are as follows: Figure 4 As shown, the sensitivity of the method of the present invention is 0.001%.

[0078] SOX1-methylated and unmethylated DNA (DNA was artificial DNA template 2, positive strand as shown in SEQ ID NO.18, reverse strand as shown in SEQ ID NO.19) were mixed with 100% methylated, 100% unmethylated, and 50% methylated plus 50% unmethylated DNA to prepare mixed DNA with different methylation levels. The DNA was digested with HhaI enzyme (the digestion system is shown in Table 1), and incubated at 37℃ for 60 min. The digested products were ligated with HhaI adapters (the ligation system is shown in Table 2), and incubated at 25℃ for 60 min. The ligated products were then subjected to PCR amplification (PCR reaction system and temperature are shown in Table 3). Subsequently, the DNA was amplified with 1, 10, 100, 1000, 10000, and 10... 5 10 6 10 7 After a 1:1 dilution, the next step of the qPCR reaction mainly targets the ligated methylated recombinant DNA fragment for fluorescence signal detection. The system configuration is shown in Table 4. The sensitivity of SOX1 methylation detection is assessed by observing the fluorescence value. Results are as follows: Figure 5 As shown, the sensitivity of the method of the present invention is 0.0001%.

[0079] Methylated and unmethylated DNA (using artificial DNA template 3, positive strand as shown in SEQ ID NO. 20, reverse strand as shown in SEQ ID NO. 21) of RXFP3.1 were mixed with 100% methylated and 100% unmethylated DNA to prepare mixed DNA with different methylation levels. The DNA was digested with AciI (the digestion system is shown in Table 1) and incubated at 37℃ for 60 min. The digested product was then ligated with an AciI adapter (the ligation system is shown in Table 2) and incubated at 25℃ for 60 min. The ligated product was then subjected to PCR amplification (PCR reaction system and temperature are shown in Table 3). Subsequently, the DNA was diluted 1, 10, 100, 1000, and 10000 times. The next step, qPCR, mainly targeted the ligated methylated recombinant DNA fragment for fluorescence signal detection (the system is shown in Table 4). The sensitivity of RXFP3.1 methylation detection was observed by measuring the fluorescence value. The results are as follows: Figure 6 As shown, the sensitivity of the method of the present invention is 0.2%.

[0080] Methylated and unmethylated DNA (using artificial DNA template 4, positive strand as shown in SEQ ID NO. 22, reverse strand as shown in SEQ ID NO. 23) of RXFP3.2 were mixed at 100% methylation, 100% unmethylation, and 50% methylation plus 50% unmethylation to prepare mixed DNA with different methylation levels. The mixtures were digested with AciI (enzyme system shown in Table 1), incubated at 37℃ for 60 min, and then ligated with AciI adapters (enzyme system shown in Table 2), incubated at 25℃ for 60 min. The ligation products were then subjected to PCR amplification (PCR reaction system and temperature shown in Table 3). Subsequently, the amplification was performed at concentrations of 1, 10, 100, 1000, 10000, and 10... 5 10 6 After a 1:1 dilution, the next step of the qPCR reaction mainly targets the ligated methylated recombinant DNA fragment for fluorescence signal detection. The system configuration is shown in Table 4. The sensitivity of RXFP3.2 methylation detection is assessed by observing the fluorescence values. Results are as follows: Figure 7 As shown, the sensitivity of the method of the present invention is 0.001%.

[0081] ZNF671 methylated and unmethylated DNA (DNA was artificial DNA template 5, positive strand as shown in SEQ ID NO. 24, reverse strand as shown in SEQ ID NO. 25) were mixed with 100% methylation and 100% unmethylation respectively to prepare mixed DNA with different methylation levels. The DNA was digested with AciI enzyme (the digestion system is shown in Table 1), and incubated at 37℃ for 60 min. The digested products were ligated with AciI adapters (the ligation system is shown in Table 2), and incubated at 25℃ for 60 min. The ligated products were then subjected to PCR amplification (PCR reaction system and temperature are shown in Table 3). Subsequently, 1, 10, 100, 1000, 10000, and 10... 5 10 6 10 7 10 8 After a 1:1 dilution, the next step of the qPCR reaction mainly targets the ligated methylated recombinant DNA fragment for fluorescence signal detection. The system configuration is shown in Table 4. The sensitivity of ZNF671 methylation detection is assessed by observing the fluorescence values. Results are as follows: Figure 8 As shown, the sensitivity of the method of the present invention is 0.00001%.

[0082] In summary, based on different dilution ratios, it can be seen that the larger the dilution factor, the smaller the detected Ct value. Compared with methylation, methylation has a relatively smaller detected Ct value at the same proportion of non-methylation. Figures 4-8 ). Example 3

[0083] This embodiment performs specificity verification.

[0084] Methylated and unmethylated DNA (SEQ ID NO. 7) of genes RXFP3.1, RXFP3.2, and ZNF671 were mixed at ratios of 0:10, 2:8, 4:6, 6:4, 8:2, and 10:0 to prepare DNA with different methylation ratios (0%, 20%, 40%, 60%, 80%, and 100%). Genes with the same methylation ratio were simultaneously amplified by qPCR. The qPCR reaction system and temperature are shown in Table 4. The DNA reaction products from the five wells were then validated by agarose gel electrophoresis. The specificity of this method for screening and detecting methylation was verified by observing the intensity of the DNA bands. The results are shown below. Figure 9As shown, the electrophoretic bands of five groups of DNA detection were compared: 0% (0:10), 20% (2:8), 40% (4:6), 60% (6:4), 80% (8:2), and 100% (10:0) methylated DNA. Based on the brightness of the electrophoretic bands, it can be determined that the targeting efficiency and specificity of methylated DNA are higher; that is, the fluorescence value of 0% methylated DNA is the lowest, while the fluorescence value of 100% methylated DNA is the highest. Qualitative observation of the electrophoretic band brightness confirms that this method has specific enrichment and detection efficacy for methylated DNA. The methylation content indicates that in mixed DNA with different proportions of methylated and unmethylated DNA, a higher methylation ratio results in more sensitive detection and more specifically amplified bands. Example 4

[0085] This embodiment tests the accuracy of the method for constructing nucleic acid sequences for detecting multiple gene methylation of cfDNA according to the present invention.

[0086] This embodiment uses quantitative methylation data (Ct value and methylation ratio) of the five target genes (PAX1, SOX1, RXFP3.1, RXFP3.2, and ZNF671) measured in Example 2 as feature variables, and the sample status (disease / health) of clinically diagnosed cervical cancer patients and healthy controls as response variables. Using SPSS statistical software, a logistic regression model was fitted using maximum likelihood estimation. The model can be used to calculate the risk probability of disease in the samples. In this embodiment, a joint prediction model incorporating methylation data of three genes, RXFP3.1, RXFP3.2, and ZNF671, was constructed, and its diagnostic efficacy was evaluated using receiver operating characteristic (ROC) curves. The results are as follows: Figure 10 As shown, the data obtained by the method of the present invention has extremely high detection accuracy in disease identification.

[0087] In summary, the construction method of this invention can be used for methylation qPCR analysis at the cfDNA level, with a detection sensitivity of 0.00001% to 0.2% for multiple genes. It can also construct diagnostic models based on multi-gene data, and the area under the curve (AUC) of its combined prediction model can reach over 0.8, indicating that it has high sensitivity, high accuracy and broad application potential in quantitative analysis of low-abundance samples and cervical cancer screening.

[0088] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for constructing a nucleic acid sequence for detecting multiplex gene methylation of cfDNA, characterized in that, The construction method includes: digesting the target cfDNA with a methylation-sensitive restriction endonuclease to obtain a digested fragment; using a ligase to connect an adapter to the cut end of the digested fragment to form an adapter product; and using the adapter product as a template for PCR amplification to construct a recombinant nucleic acid sequence for detecting methylation sites of the target gene.

2. The method for constructing a nucleic acid sequence for cfDNA multiplex gene methylation detection according to claim 1, characterized in that, The methylation-sensitive restriction endonucleases include AciI and / or HhaI.

3. The method for constructing a nucleic acid sequence for cfDNA multiplex gene methylation detection according to claim 1 or 2, characterized in that, The 5' end of the adhesive end of the connector has a protruding base sequence of CG.

4. The method for constructing a nucleic acid sequence for cfDNA multiplex gene methylation detection according to any one of claims 1-3, characterized in that, The ligase includes T4 DNA ligase.

5. The method for constructing a nucleic acid sequence for cfDNA multiplex gene methylation detection according to any one of claims 1-4, characterized in that, The source of the cfDNA to be tested includes biological samples and / or artificially synthesized samples; the biological samples include any one of plasma, serum, tissue or cells.

6. The method for constructing the nucleic acid sequence for cfDNA multiplex gene methylation detection according to any one of claims 1-5 is used in the preparation of methylation detection products.

7. A method for detecting methylation of cervical cancer-related genes, characterized in that, The method includes the following steps: (1) The cfDNA sample to be tested is processed using the method for constructing the nucleic acid sequence for cfDNA multiple gene methylation detection according to any one of claims 1-5 to obtain the recombinant nucleic acid sequence; (2) Using the recombinant nucleic acid sequence as a template, quantitative multiple methylation-specific PCR amplification and detection were performed using primers and fluorescent probes for detecting cervical cancer-related genes; (3) Determine the methylation status of the gene based on the amplification detection results.

8. The method for detecting cervical cancer-related gene methylation according to claim 7, characterized in that, The cervical cancer-related genes include any one or a combination of at least two of PAX1, SOX1, RXFP3.1, RXFP3.2 or ZNF671.

9. The method for detecting cervical cancer-related gene methylation according to claim 7 or 8, characterized in that, The nucleic acid sequence of the primer includes the sequences shown in SEQ ID NO.1-SEQ ID NO.10; the nucleic acid sequence of the fluorescent probe includes the sequences shown in SEQ ID NO.11-SEQ ID NO.15; the 5' end of the fluorescent probe contains a fluorescent group, and the 3' end contains a quenching group; the fluorescent group includes any one or a combination of at least two of FAM, HEX, VIC, or ROX; the quenching group includes any one or a combination of at least two of BHQ1, BHQ2, or MGB.

10. The method for detecting cervical cancer-related gene methylation according to any one of claims 7-9, characterized in that, The quantitative multiple methylation-specific PCR amplification system includes a template, primers, a fluorescent probe, and a qPCR premix; the concentration of the primers in the system is 5-10 μM, and the concentration of the fluorescent probe is 5-10 μM. The quantitative multiple methylation-specific PCR amplification procedure includes: (1) pre-denaturation at 93-95℃ for 30-40 s; (2) denaturation at 93-95℃ for 5-10 s; (3) annealing at 55-60℃ for 20-30 s; extension at 55-60℃ for 20-30 s, for a total of 40-45 cycles.

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