Method for evaluating DNA methylation degree of target gene and application

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for evaluating the DNA methylation degree of a target gene and application thereof.The method comprises the steps that firstly, a PCR technology is utilized, methylated DNA fragments of different degrees are prepared through basic group doping, quantitative PCR detection is conducted, the corresponding relation between Ct and the DNA methylation degree is established, a curve is drawn, and the methylation degree of the target gene is evaluated by detecting the Ct value of the target gene in a sample; the evaluation on the methylation degree of the gene is realized. The invention establishes a novel and more convenient method for detecting DNA methylation difference, and can identify methylation degrees of target gene segments in different samples, or judge differences among groups based on the methylation degree differences of target genes among different groups. A lung cancer and colorectal cancer blood sample experiment proves that the method is simple and convenient to operate, short in detection time, accurate and reliable in result, suitable for detection of all types of methylated DNA fragments and extremely high in application value.

Description

[0001] This application is a divisional application with an application date of November 5, 2021, application number 202111305138.9, and invention name "A DNA methylation detection method based on quantitative PCR technology and its kit". Technical Field

[0002] The invention belongs to the field of biotechnology, and specifically relates to a method for evaluating the degree of DNA methylation of a target gene and its application. Background Art

[0003] DNA methylation has become an important part of molecular biology research and a new favorite for disease detection. Epigenetic studies have found that DNA epigenetic modification plays an important role in gene regulation, and gene regulation is largely caused by gene epigenetic modification, among which DNA methylation is a hot spot in epigenetic modification research. For a long time, gene abnormalities have mainly focused on the study of their structural changes, but the structural changes of genes are usually located after the abnormalities of gene epigenetic regulation, and the sites and frequencies of their occurrence are very uncertain, and they are more sporadic. In addition, in the early stages of tumorigenesis, the proportion of DNA with abnormal gene structure is very low, and its detection becomes very difficult. Studies have shown that compared with changes in DNA structure, in the detection of gene abnormalities, the scope and proportion of gene epigenetic modification changes are larger, and they occur earlier. Detection of them can greatly improve the sensitivity of judgment. Among them, gene methylation has become a highlight of epigenetic detection due to its strong operability, and is used in all aspects of molecular biology research. Therefore, the development of a new method for efficient and sensitive DNA methylation detection will undoubtedly greatly promote the progress of epigenetic and molecular biology research.

[0004] Currently, commonly used DNA methylation detection methods include: high performance liquid chromatography-ultraviolet (HPLC-UV), DNA methylation-specific detection method (DNA methylation-specific detection method, DNA ... Due to the large difference in conversion rate caused by sodium sulfite, if excessive treatment leads to DNA breakage, subsequent analysis cannot be performed; if insufficient treatment leads to incomplete conversion, subsequent test results will vary greatly, thus resulting in different heterogeneity of gene structure caused by sodium sulfite treatment, which has a great impact on the judgment of results and causes inaccurate results; in the analysis of the converted DNA structure, if MSP (methylation-specific PCR) technology is used, the target of methylation detection is limited to the area covered by the primers, and the methylation in the sequence between the upstream and downstream primers cannot be detected. At the same time, in this process, two sets of primers need to be designed for whether cytosine is methylated or not; the high-resolution melting curve method is to judge whether DNA is methylated by observing the changes in the melting curve after PCR amplification. Due to the complex heterogeneity of gene structure caused by sodium sulfite treatment, the HRM curve is messy and even cannot be judged; the high-throughput sequencing detection method first needs to build a library for the DNA to be tested, which not only has requirements on the amount of DNA, but also causes the loss of some information during the library construction process. Other factors include high costs and long cycles. Another method that does not require sulfite treatment is the DNA methylation detection method, which uses a restriction enzyme that is sensitive to methylation within the DNA. If the PCR region is not analyzed in combination, the site cannot be detected, that is, this method does not have the broad spectrum of methylated DNA detection. Therefore, it is urgent to establish a highly specific and sensitive DNA methylation detection method to break through the bottleneck of methylation detection. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a method and application for evaluating the degree of DNA methylation of a target gene, which is simple, rapid and has accurate and reliable results.

[0006] The present invention provides a method for evaluating the degree of DNA methylation of a target gene, comprising the following steps:

[0007] 1) The quantitative PCR reaction system was prepared using the Taq enzyme premix system, primers and probes for quantitative PCR detection of the target gene, and the target gene was subjected to high temperature denaturation amplification and low temperature denaturation amplification respectively to obtain the Ct of high temperature denaturation amplification. X-HT and Ct of lower temperature denaturation amplification X-LH ;

[0008] 2) Ct X-LH and the Ct X-HT Substitute into formula I to obtain the △Ctx of the target gene;

[0009] △Ctx=Ct X-LH –Ct X-HT Formula I

[0010] Among them, △Ctx represents the detection value of gene X; Ct X-LH Indicates the Ct value of gene X amplified under lower temperature denaturation conditions; Ct X-HT It represents the Ct value of X gene amplified under higher temperature denaturation conditions, and X gene represents a target gene;

[0011] 3) using PCR technology and reagents for drawing reference curves, preparing methylated DNA fragments of different degrees by base incorporation, diluting and using them as methylated DNA templates for PCR amplification, and operating according to the methods of steps 1) and 2) to obtain ΔCtx' of different methylated DNA fragments, obtaining the linear relationship between different proportions of methylated cytosine and ΔCtx', and drawing a reference curve;

[0012] 4) comparing the ΔCtx of the target gene in step 2) with the ΔCtx' in the reference curve in step 3) to obtain the ratio of methylated cytosine in the target gene, thereby determining the degree of DNA methylation of the target gene;

[0013] The reaction conditions of the higher temperature denaturation amplification are as follows: 95°C for 5 min; 94°C for 5 s, 60-62°C for 15 s, 72°C for 30 s, 45 cycles;

[0014] The reaction conditions of the lower temperature denaturation amplification are as follows: 85-88°C for 15s, 60-62°C for 15s, 72°C for 30s, 18 cycles; 94°C for 5s, 60-62°C for 15s, 72°C for 30s, 27 cycles;

[0015] The reagents for drawing the reference curve include dNTP reagents containing methylated cytosine in different proportions; the dNTP reagents containing methylated cytosine in different proportions include dATP, dTTP, dGTP and reagent A in equimolar concentrations; the reagent A is 5'-m-dCTP and / or dCTP.

[0016] Preferably, in the reagent A, the molar ratio of 5'-m-dCTP to dCTP includes 1:0, 1:(1-30) and 0:1.

[0017] Preferably, the target gene includes but is not limited to one or more of the following genes:

[0018] APC, SHOX2, Apobec3B, P53, AID, HOXD12, Alu, SDC2, WDR17, and ADHFE1.

[0019] Preferably, the reagents used to draw the reference curve also include common PCR amplification reagents.

[0020] The present invention also provides the application of the method described in the above technical solution in identifying the methylation degree of the target gene in a sample and distinguishing the DNA methylation changes of two research groups.

[0021] The present invention also provides a method for distinguishing two research groups based on DNA methylation changes in a target gene combination, comprising the following steps:

[0022] After obtaining the ΔCt of each target gene of the two research groups by the method described in the above technical solution, the ΔCt of each target gene corresponding to the samples of research group 1 and the samples of research group 2 is statistically analyzed to obtain the threshold and weight value of the combined detection of the target genes, and complete the modeling of the combined detection of the target genes; the target genes include two or more target genes;

[0023] The weight value is obtained by substituting the ΔCt of each target gene into Formula II to calculate the weight value of the multi-target gene joint detection of each sample;

[0024] Weight value of multi-target gene joint detection = a1×△Ct x1 +a2×△Ct x2 +......+an×△Ct xn Formula II Wherein, a1, a2, and an represent the corresponding coefficients obtained in the statistical analysis of each target gene;

[0025] Verification of the difference between the two groups: The multi-target gene weight values ​​of the samples of study group 1 and study group 2 that did not participate in the modeling were obtained according to the above method, and then compared with the above thresholds respectively. The samples with weight values ​​lower than the threshold were low-methylation samples, and the samples with weight values ​​higher than the threshold were high-methylation samples. The differences between the two groups were further compared by statistical analysis;

[0026] Determination of the difference between the two groups: The mean of the weight values ​​of the samples of study group 1 and study group 2 will be calculated, and a T-test analysis will be performed. When the mean of the weight values ​​of the samples of study group 1 and the mean of the weight values ​​of the samples of study group 2 are significantly different, it indicates that the DNA methylation difference between the two groups is significant, which means that the two groups can be identified through methylation detection of the target gene combination.

[0027] The present invention also provides an application of the method described in the above technical solution to prepare a product for distinguishing cancer patients from non-cancer patients;

[0028] The cancer includes lung cancer and / or colorectal cancer;

[0029] The target genes for lung cancer include HOXD12, AID and Alu;

[0030] The target genes for colorectal cancer include SDC2, WDR17, ADHFE1 and Alu.

[0031] Beneficial effects:

[0032] The present invention provides a method for evaluating the degree of DNA methylation of a target gene, using quantitative PCR technology, preparing methylated DNA fragments of different degrees by base incorporation, and then performing quantitative PCR detection on the fragments, establishing a relationship between ΔCt and the degree of DNA methylation (the higher the degree of DNA methylation, the greater the ΔCt), and establishing a method for evaluating the degree of DNA methylation of the target gene by detecting the ΔCt of the target gene. The method provided by the present invention is simple to operate, has a short detection time, and does not detect a single methylation site, but detects the DNA methylation target area to be detected as a whole, so that the detection result is accurate and reliable, and is applicable to the detection of all types of methylated DNA fragments, and has a very high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The difference in melting of DNA with different methylation ratios under low-temperature denaturation conditions;

[0034] Figure 2 The △Ct change curve of SHOX2 gene fragments with different methylation degrees, where the abscissa is the molar ratio of 5'-m-dCTP and dCTP, and the ordinate is △Ct;

[0035] Figure 3 It is the fitted logarithmic curve for the relationship analysis between SHOX2 gene methylation and detection value;

[0036] Figure 4 The △Ct change curve of P53 gene fragments with different methylation degrees, where the horizontal axis is the molar ratio of 5'-m-dCTP and dCTP, and the vertical axis is △Ct;

[0037] Figure 5 It is the fitted logarithmic curve for the relationship analysis between P53 gene methylation and detection value;

[0038] Figure 6 The △Ct change curve of APC gene fragments with different methylation degrees, where the abscissa is the molar ratio of 5'-m-dCTP and dCTP, and the ordinate is △Ct;

[0039] Figure 7 It is the fitted logarithmic curve for the relationship analysis between APC gene methylation and detection value;

[0040] Figure 8 The △Ct change curve of the Apobec3B gene fragment with different methylation degrees, where the abscissa is the molar ratio of 5'-m-dCTP and dCTP, and the ordinate is △Ct;

[0041] Fig. 9 It is the fitted logarithmic curve for the relationship analysis between Apobec3B gene methylation and detection value;

[0042] Fig.10 The △Ct change curve of AID gene fragments with different methylation degrees, where the abscissa is the molar ratio of 5'-m-dCTP and dCTP, and the ordinate is △Ct;

[0043] Fig.11 It is the fitted logarithmic curve for the relationship analysis between AID gene methylation and detection value;

[0044] Fig.12 The △Ct change curve of HOXD12 gene fragments with different methylation degrees, where the abscissa is the molar ratio of 5'-m-dCTP and dCTP, and the ordinate is △Ct;

[0045] Fig.13 It is the fitted logarithmic curve for the relationship analysis between HOXD12 gene methylation and detection value;

[0046] Fig.14 Analysis of the test results of lung cancer samples and normal subjects, where a: box plot; b: ROC curve analysis;

[0047] Fig.15 The △Ct change curve of SDC2 gene fragments with different methylation degrees, where the abscissa is the molar ratio of 5'-m-dCTP and dCTP, and the ordinate is △Ct;

[0048] Fig.16 It is the fitted logarithmic curve for the relationship analysis between SDC2 gene methylation and detection value;

[0049] Fig.17The △Ct change curve of WDR17 gene fragments with different methylation degrees, where the abscissa is the molar ratio of 5'-m-dCTP and dCTP, and the ordinate is △Ct;

[0050] Fig.18 It is the fitted logarithmic curve for the relationship analysis between WDR17 gene methylation and detection value;

[0051] Fig.19 The △Ct change curve of ADHFE1 gene fragments with different methylation degrees, where the abscissa is the molar ratio of 5'-m-dCTP and dCTP, and the ordinate is △Ct;

[0052] Fig. 20 It is the fitted logarithmic curve for the relationship analysis between ADHFE1 gene methylation and detection value;

[0053] Fig.21 Analysis of the test results of colorectal cancer samples and normal subjects, where a: box plot; b: ROC curve analysis. DETAILED DESCRIPTION

[0054] The present invention provides a kit for detecting DNA methylation based on quantitative PCR technology, comprising the following components:

[0055] Taq enzyme premix system, primers and probes for target gene quantitative PCR detection, and reagents for Taq enzyme activity quality control.

[0056] The present invention realizes the detection of DNA methylation based on quantitative PCR technology. The detection mechanism is shown in Figure 1 , utilizing the effect of methylation on the physical and chemical properties of DNA, the differences in DNA methylation are converted into differences in the amount of effective PCR templates, and then detected through quantitative PCR.

[0057] The kit of the present invention has no special restrictions on the object to be detected, and is applicable to gene fragments with different methylation degrees. In the embodiment of the present invention, the feasibility of the verification method of APC, SHOX2, Apobec3B, and P53 is respectively adopted, and APC is used as an example to illustrate. The verification method of the APC gene is preferably as follows: according to the detection principle, PCR primers containing a certain number of CG island sequences in the amplified fragment are designed, and the target gene is amplified by the method of ordinary PCR, and the primer specificity is verified. After obtaining a unique specific band, the target region fragment is PCR amplified, and in this process, different methylated DNA fragments are prepared by the incorporation of methylated cytosine in different proportions, and then the prepared DNA is used as a template (different methylated DNA fragments and different dilutions are combined), and qPCR detection is performed under higher temperature and lower temperature conditions, respectively, and the two Ct values ​​obtained are obtained by difference to obtain ΔCt value. Confirmed: ΔCt value is only related to the degree of methylation of DNA, and has nothing to do with the concentration of DNA, that is, ΔCt value is positively correlated with the degree of DNA methylation. Further, the DNA fragments with lower methylation ratios are detected and compared with their corresponding non-methylated DNA fragments to find the limit values ​​of methylation detection of different genes, so that the method can quantitatively evaluate the DNA methylation degree of each target gene. The preparation of target genes with different methylation degrees is achieved by adding dNTP reagents containing methylated cytosines of different proportions during PCR amplification. The dNTP reagents containing methylated cytosines of different proportions preferably include equimolar concentrations of dATP, dTTP, dGTP and reagent A. The concentrations of dATP, dTTP, dGTP and reagent A are independently preferably 2.5mM. The reagent A is 5'-m-dCTP and / or dCTP. In the reagent A, the molar ratio of 5'-m-dCTP and dCTP preferably includes 1:0, 1:(1-30) and 0:1, wherein 1:(1-30) represents the ratio of multiple groups among 1:1, 1:2, 1:3...1:29 and 1:30. Different types of target genes have different cytosine ratios when drawing reference curves.The quantitative PCR detection primer sequence of APC is an upstream primer sequence as shown in SEQ ID NO.1, and a downstream primer sequence as shown in SEQ ID NO.2. The quantitative PCR detection probe of APC is a nucleotide sequence as shown in SEQ ID NO.3; the quantitative PCR detection primer sequence of SHOX2 is an upstream primer sequence as shown in SEQ ID NO.4, the downstream primer sequence as shown in SEQ ID NO.5, and the quantitative PCR detection probe sequence of SHOX2 is a sequence as shown in SEQ ID NO.6; the quantitative PCR detection primer sequence of Apobec3B is an upstream primer sequence as shown in SEQ ID NO.7, and a downstream primer sequence as shown in SEQ ID NO.8. The quantitative PCR detection probe sequence of Apobec3B is a sequence as shown in SEQ ID NO.9; the quantitative PCR detection primer sequence of P53 is an upstream primer sequence as shown in SEQ ID NO.10, the downstream primer sequence as shown in SEQ ID NO.11, and the quantitative PCR detection probe sequence of P53 is a sequence as shown in SEQ ID NO.12.

[0058] In the present invention, the kit also includes DNA methylation detection of tumor samples. In the embodiment of the present invention, when DNA methylation detection is performed on lung cancer samples, plasma free DNA from normal people and lung cancer patients is first extracted, and AID, HOXD12 and Alu are detected as target genes. In this process, primers are first designed for the target gene, requiring a certain number of CG island sequences to be included in the amplification target region, and then different candidate primers are used to amplify the target gene of the template DNA respectively, and the specificity of the candidate primers is evaluated, and the primers that amplify the specific bands are selected as primers for quantitative PCR. The quantitative PCR detection primers of AID include an upstream primer sequence with a nucleotide sequence such as SEQ ID NO.13, a downstream primer sequence such as SEQ ID NO.14, and a probe sequence for quantitative PCR detection of AID such as a sequence shown in SEQ ID NO.15. When the AID is used for methylated DNA detection, the reagent A to be prepared for drawing the reference curve includes 1:0, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6 and 0:1. The primer sequence for quantitative PCR detection of HOXD12 is as shown in SEQ ID NO.16, the upstream primer sequence is as shown in SEQ ID NO.17, and the probe sequence for quantitative PCR detection of HOXD12 is as shown in SEQ ID NO.18. When HOXD12 is used for methylated DNA detection, the reagent A to be prepared for drawing the reference curve includes 1:0, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, 1:10, 1:20 and 0:1. The primer for quantitative PCR detection of Alu includes a nucleotide sequence, such as the upstream primer sequence shown in SEQ ID NO.19, the downstream primer sequence shown in SEQ ID NO.20, and the probe sequence for quantitative PCR detection of Alu is as shown in SEQ ID NO.21.

[0059] In the present invention, when DNA methylation detection is performed on colorectal cancer samples, free plasma DNA from normal people and colorectal cancer patients is first extracted, and SDC2, WDR17 and ADHFE1 are detected as target genes. The primer sequence for quantitative PCR detection of SDC2 is such as the upstream primer sequence shown in SEQ ID NO.22, the downstream primer sequence shown in SEQ ID NO.23, and the probe sequence for quantitative PCR detection of SDC2 is such as the sequence shown in SEQ ID NO.24. When the SDC2 is used for methylated DNA detection, the reagent A to be prepared for drawing the reference curve includes 1:0, 1:1, 1:2, 1:4, 1:6, 1:8, 1:10 and 0:1. The primer sequence for quantitative PCR detection of WDR17 is such as the upstream primer sequence shown in SEQ ID NO.25, the downstream primer sequence shown in SEQ ID NO.26, and the probe sequence for quantitative PCR detection of WDR17 is such as the sequence shown in SEQ ID NO.27. When the WDR17 is used for methylated DNA detection, the reagent A to be prepared for drawing the reference curve includes 1:0, 1:1, 1:2, 1:4, 1:6, 1:8, 1:10, 1:20, 1:30 and 0:1. The primer sequence for quantitative PCR detection of ADHFE1 is such as the upstream primer sequence shown in SEQ ID NO.28, the downstream primer sequence shown in SEQ ID NO.29, and the probe sequence for quantitative PCR detection of ADHFE1 is such as the sequence shown in SEQ ID NO.30. When the ADHFE1 is used for methylated DNA detection, the reagent A to be prepared for drawing the reference curve includes 1:0, 1:1, 1:2, 1:4, 1:6, 1:8, 1:10 and 0:1. The primer sequence for quantitative PCR detection of Alu is such as the upstream primer sequence shown in SEQ ID NO.31, the downstream primer sequence shown in SEQ ID NO.32, and the probe for quantitative PCR detection of Alu is a nucleotide sequence such as the sequence shown in SEQ ID NO.33.

[0060] The present invention provides the use of the kit in evaluating the degree of DNA methylation of a target gene. The direct purpose of the use of the present invention is to verify the feasibility of the method in tumor identification.

[0061] The present invention provides a method for evaluating the degree of DNA methylation of a target gene, comprising the following steps:

[0062] 1) The primers and probes in the kit are used to prepare a quantitative PCR reaction system, and the reaction system is subjected to high temperature denaturation amplification and low temperature denaturation amplification to obtain the Ct amplified under the high temperature denaturation condition. X-HT and Ct amplified under lower temperature denaturation conditions X-LH ;

[0063] 2) Change Ct in step 1) X-LH and Ct X-HT Substitute into formula I to obtain the △Ctx of the target gene;

[0064] △Ctx=Ct X-LH –Ct X-HT Formula I

[0065] Among them, △Ctx represents the detection value of gene X; Ct X-LH Indicates the Ct value of gene X amplified under low denaturation conditions; Ct X-HT It represents the Ct value of gene X amplified under higher temperature denaturation conditions, where X represents a target gene;

[0066] 3) using PCR technology and reagents for drawing reference curves, preparing methylated DNA fragments of different degrees by base incorporation, diluting and using them as methylated DNA templates for PCR amplification, and operating according to the methods of steps 1) and 2) to obtain ΔCtx' of different methylated DNA fragments, obtaining the linear relationship between different proportions of methylated cytosine and ΔCtx', and drawing a reference curve;

[0067] 4) Compare the ΔCtx of the target gene in step 2) with the ΔCtx' in the reference curve in step 3) to obtain the actual proportion of methylated cytosine in the target gene, thereby determining the degree of DNA methylation of the target gene.

[0068] The present invention uses the primers and probes in the kit to prepare a reaction system for quantitative PCR detection, and respectively performs high temperature denaturation amplification and low temperature denaturation amplification to obtain Ct amplified under high temperature denaturation conditions. X-HT and Ct amplified under lower temperature denaturation conditions X-LH .

[0069] In the present invention, the reaction system of quantitative PCR detection is as follows:

[0070]

[0071]

[0072] In the present invention, the reaction conditions of higher temperature denaturation amplification are preferably as follows: pre-denaturation at 95°C for 5 min; 94°C for 5 s, 60°C for 15 s, 72°C for 30 s, 45 cycles. The reaction conditions of lower temperature denaturation amplification are preferably as follows: 85°C for 15 s, 60°C for 15 s, 72°C for 30 s, 18 cycles; 94°C for 5 s, 60°C for 15 s, 72°C for 30 s, 27 cycles. Appropriate adjustments can be made according to the length of the amplified fragment and the CG content.

[0073] Get Ct X-LH and Ct X-HT, the present invention will Ct X-LH and Ct X-HT Substitute into formula I to obtain the △Ctx of the target gene;

[0074] △Ctx=Ct X-LH –Ct X-HT Formula I

[0075] Among them, △Ctx represents the detection value of gene X; Ct X-LH Indicates the Ct value of gene X amplified under low temperature denaturation conditions; Ct X-HT It represents the Ct value of gene X amplified under high temperature denaturation conditions, where X represents a target gene.

[0076] The present invention utilizes PCR technology and reagents for drawing a reference curve, prepares methylated DNA fragments of different degrees by base incorporation, dilutes the fragments and uses them as methylated DNA templates for PCR amplification, operates according to the methods of step 1) and step 2), obtains ΔCtx' of different methylated DNA fragments, obtains the linear relationship between different proportions of methylated cytosine and ΔCtx', and draws a reference curve.

[0077] In the present invention, the method of drawing the reference curve is the same as above and will not be described in detail here.

[0078] After obtaining the reference curve, the present invention compares the ΔCtx of the target gene with the ΔCtx' in the reference curve to obtain the ratio of methylated cytosine in the target gene, thereby determining the degree of DNA methylation of the target gene.

[0079] The present invention provides the use of the kit or the method for detecting DNA methylation in identifying the methylation degree in the target region of the target gene in the sample to be tested, and in different groups, identifying the research group by detecting the difference in DNA methylation in the target gene combination. The direct purpose of the application of the present invention is to achieve the purpose of distinguishing groups by identifying methylated DNA in different groups.

[0080] In the present invention, when the target genes preferably include two or more, the method for distinguishing DNA methylation changes between two research groups comprises the following steps:

[0081] After obtaining the ΔCt of each target gene, the ΔCt of each target gene corresponding to the samples of group 1 and group 2 was statistically analyzed to obtain the threshold for distinguishing the two groups and the weight value calculation formula II for the joint detection of target genes, and the establishment of the model was completed. The ΔCt of each target gene was substituted into formula II to calculate the weight value of each sample;

[0082] The weight value of the target gene = a1 × △Ct x1 +a2×△Ct x2+......+an×△Ct xn Formula II

[0083] Among them, a1, a2, and an are the corresponding coefficients obtained from the regression analysis of each target gene; x1, x2, and xn are different target genes.

[0084] Verification of samples from two groups: The samples from group 1 and group 2 that were not involved in the modeling were tested according to the above method and the weight values ​​were calculated. Then, they were compared with the threshold values. Samples with weight values ​​lower than the threshold values ​​were considered to be hypomethylated samples, and samples with weight values ​​higher than the threshold values ​​were considered to be hypermethylated samples. The differences between the two groups were then compared through statistical analysis.

[0085] Statistical analysis of the differences between the two groups: Calculate the mean of the weighted values ​​of the samples in group 1 and the mean of the weighted values ​​of the samples in group 2. After T-test analysis, when the mean of the weighted values ​​of the samples in group 1 and the mean of the weighted values ​​of the samples in group 2 show a significant difference, it indicates that the methylation difference of the target gene combination between the two groups is significant.

[0086] In the embodiment of the present invention, the two groups include normal plasma samples and cancer (determined by pathological test results) plasma samples.

[0087] First, plasma free DNA was extracted and tested according to the above method. After the ΔCt of each target gene was obtained, the ΔCt of each target gene corresponding to the normal plasma sample and the cancer plasma sample was statistically analyzed to obtain the threshold and weight value calculation formula II for the joint detection of the target gene. After modeling was completed, the ΔCt of each target gene was substituted into formula II to calculate the weight value of each cancer sample;

[0088] The weight value of the target gene = a1 × △Ct x1 +a2×△Ct x2 +......+an×△Ct xn Formula II

[0089] Among them, a1, a2, and an are the corresponding coefficients obtained in the regression analysis of each target gene;

[0090] Using normal plasma samples and cancer (pathological results are clear) plasma samples as the standard, the weight values ​​of the samples not involved in the modeling were compared with the threshold, and the number of samples above or below the threshold in the two groups was counted. The results showed that the weight values ​​of the vast majority of normal samples were lower than the threshold, while the weight values ​​of the vast majority of cancer samples were higher than the threshold. Normal samples with weight values ​​higher than the threshold are false positives, while cancer samples with weight values ​​lower than the threshold are false negatives;

[0091] The mean of the weighted values ​​of each cancer plasma sample and the mean of the weighted values ​​of the normal plasma samples are calculated. After T-test analysis, when the mean of the weighted values ​​of the cancer plasma samples and the mean of the weighted values ​​of the normal plasma samples are significantly different, it indicates that the method is suitable for identifying cancer patients. The calculation method of the threshold is preferably calculated by statistical analysis.

[0092] The following is a detailed description of a method for evaluating the degree of DNA methylation of a target gene provided by the present invention and its application in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0093] Example 1

[0094] Method for establishing methylation detection method

[0095] This example study found that the physical and chemical properties of methylated DNA have significant changes compared to non-methylated DNA. Based on this phenomenon, a new methylation detection method was explored. The basic principle is that under certain conditions, the difference in DNA methylation is converted into a difference in the amount of PCR effective template, and then the detection is completed by quantitative PCR. In the modeling process, PCR is used to prepare methylated DNA fragments of different degrees through base incorporation, and then the methylation difference is detected to establish the corresponding relationship between the detection value and the degree of methylation. The specific method is as follows:

[0096] 1) Experimental Materials

[0097] Reagents: Taq enzyme premix system (2×Premix Ex Taq TM ) and Taq TM Reagents were purchased from TaKaRa BioInc; 5'-m-dCTP was purchased from New England Biolabs, USA; T / A vector was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. and NEB; primers and probes were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0098] Target genes: In the exploration of new detection methods, we selected the promoter regions of different genes (APC, Apobec3B, SHOX2) and P53 exon region (exon 4 region) fragments already available in the laboratory.

[0099] 2) Preparation of methylated DNA fragments (PCR incorporation method): In the process of preparing methylated DNA fragments, methylated cytosine and cytosine are prepared in different proportions (no methylation, full methylation, and different degrees of methylation). In this process, 5'-m-dCTP (5'-methylated cytosine) and dCTP are first mixed in different proportions, and then dATP, TTP and dGTP are added respectively to perform PCR amplification to verify the amplification specificity of each primer. In this way, the preparation of different methylated DNA fragments is completed.

[0100] 3) PCR reaction: including the composition of the reaction mixture and reaction conditions.

[0101] (1) Preparation of DNA methylation templates with different ratios

[0102] Preparation of dNTPs containing different proportions of methylated cytosine (see Table 1): 5'-m-dCTP: dCTP was prepared at molar concentrations of 1:0, 1:(1-30) and 0:1, respectively, and then further mixed with equal proportions of dATP, dTTP and dGTP to complete the preparation of dNTPs containing different proportions of methylated cytosine (each base concentration is 2.5 mM). The primer sequences are shown in Table 3.

[0103] Table 1 PCR reaction mixture containing 5′-m-dCTP (20 μL reaction system)

[0104]

[0105]

[0106] PCR reaction conditions (20 μL reaction system): pre-denaturation at 95°C for 5 min, followed by a 3-step amplification program: 94°C for 5 s, 60°C for 15 s, 72°C for 30 s, and 45 cycles.

[0107] (2) Detection of methylated DNA fragments

[0108] The preparation of the reaction mixture is shown in Table 2.

[0109] Table 2 qPCR reaction system

[0110]

[0111] Table 3 Primer and probe sequences

[0112]

[0113] Note: The length of the amplified fragment of the APC (promoter) target region is 148bp; the length of the amplified fragment of the SHOX2 (promoter) target region is 174bp; the length of the amplified fragment of the Apobec3B (promoter) target region is 135bp; the length of the amplified fragment of the P53 (exon 4) target region is 180bp.

[0114] PCR instrument: Macrostone SLAN 96P.

[0115] Reaction conditions:

[0116] (1) High temperature denaturation (HT): 4 genes (APC / Apobec3B / / P53 / SHOX2): pre-denaturation at 95°C for 5 min, followed by a 3-step amplification program: 94°C for 5 s, 60°C for 15 s, 72°C for 30 s, for 45 cycles.

[0117] (2) Low temperature denaturation (LT): 4 genes (APC / Apobec3B / P53 / SHOX2): 85°C for 15 s, 60°C for 15 s, 72°C for 30 s, for 18 cycles, followed by the following cycling program: 94°C for 5 s, 60°C for 15 s, 72°C for 30 s, for 27 cycles.

[0118] Appropriate adjustments can be made based on the length of the amplified fragment and the CG content.

[0119] (3) Calculation of ΔCt

[0120] After the data in the PCR results are exported, they are calculated according to the following formula I to obtain the corresponding values.

[0121] △Ct x =Ct X-LH –Ct X-HT Formula I

[0122] Among them, △Ct x is the detection value of gene X; Ct X-LH Indicates the Ct value of gene X amplified under low temperature denaturation conditions; Ct X-HT It represents the Ct value of gene X amplified under high temperature denaturation conditions.

[0123] 4) Experimental results

[0124] (1) ΔCt is independent of template concentration: The prepared different methylated DNA fragments were diluted in a gradient manner and used as templates for quantitative PCR analysis under two temperature conditions, where Ct X-HT Defined as Ct1, Ct X-LH It is defined as Ct2, and the difference between Ct2 and Ct1 is defined as ΔCt.

[0125] The following are the detection results of different target gene promoter fragments:

[0126] The detection results of SHOX2 gene fragments are shown in Table 4.

[0127] Table 4 Detection and analysis of SHOX2 gene fragments at different concentrations and different methylation ratios (molar concentrations)

[0128]

[0129]

[0130] The results showed that although the Ct values ​​(Ct1 and Ct2) under different template concentrations showed corresponding changes, the ΔCt values ​​were almost exactly the same, while the ΔCt values ​​of different methylation were significantly different, indicating that the ΔCt value was only related to the degree of DNA methylation and had nothing to do with the concentration of DNA, and that the ΔCt value was positively correlated with the degree of DNA methylation.

[0131] (2) ΔCt is positively correlated with the degree of methylation of the template

[0132] According to the aforementioned methylation detection method, different DNA fragments with different methylation degrees were detected, and the differences in ΔCt values ​​were compared. The results are shown in Tables 5 to 8 and Figure 2 to Figure 9 shown.

[0133] Table 5 Detection of different degrees of methylation of SHOX2 gene fragments

[0134] 5'-m-dCTP: dCTP 1：1 1：2 1：4 1：6 1：8 1：10 0：1 Ct2 20.31 17.36 15.79 15.47 15.59 14.12 14.16 Ct1 15.09 14.9 14.73 14.99 15.08 14.06 14.12 ΔCt 5.22 2.46 1.06 0.48 0.51 0.06 0.04

[0135] Table 6 Detection of different degrees of methylation of P53 gene fragments

[0136] 5'-m-dCTP: dCTP 1：0 1：1 1：2 1：4 1：6 1：8 1：10 1：20 1：30 0：1 Ct2 24.43 22.28 21.17 19.89 19.47 18.51 18.09 17.00 16.94 16.71 Ct1 18.75 16.74 16.33 16.33 16.10 16.14 15.91 15.29 15.70 15.35 ΔCt 5.68 5.54 4.84 3.56 3.37 2.37 2.18 1.71 1.24 1.36

[0137] Table 7 Detection of different degrees of methylation of APC gene fragments

[0138] 5'-m-dCTP: dCTP 1：2 1：4 1：6 1：8 0：1 Ct2 21.87 19.73 17.33 16.7 16.2 Ct1 15.83 16.09 15.88 15.78 16.18 ΔCt 6.04 3.64 1.45 0.92 0.02

[0139] Table 8 Detection of different degrees of methylation of Apobec3B gene fragments

[0140] 5'-m-dCTP: dCTP 1：1 1：2 1：4 1：6 1：8 1：10 1：20 1：30 0：1 Ct2 26.03 24.34 22.72 21.76 22.62 20.71 19.73 19.68 18.85 Ct1 20.35 18.97 18.41 18.06 19.18 17.28 17.10 17.21 17.04 ΔCt 5.68 5.37 4.31 3.7 3.44 3.43 2.63 2.47 1.81

[0141] The above results show that this method shows significant differences in the above four different DNA methylation fragments, and it is inferred that this detection method is suitable for the detection of all methylated DNA fragments. Comparison of DNA fragments with different methylation ratios and their corresponding non-methylated DNA fragments shows that the detection limit values ​​of methylation fragments of different genes are different.

[0142] Example 2

[0143] Lung cancer cf / ctDNA methylation detection method

[0144] 1) Identification of lung cancer target genes:

[0145] HOXD12 gene: Through the analysis of TCGA database, it was found that the methylation of HOXD12 gene was the most different between normal tissue and lung cancer tissue. The random forest statistical analysis model showed that it made the greatest contribution to the identification of lung cancer. HOXD12 is a member of the homeobox gene family, which plays an important regulatory role in the morphogenesis process. Current studies have shown that different members of this family play an important role in the occurrence and development of tumors.

[0146] Cytosine deaminase gene (AID): Studies have shown that cytosine deaminase converts cytosine into uracil by deaminating it. Abnormal expression of this gene can lead to various mutations in DNA and is an important factor in genomic variation. Studies have shown that AID is abnormally highly expressed in many tumors. Cell experiments have shown that overexpression of this gene can significantly increase the frequency of genomic mutations. Transgenic animals have shown that overexpression of AID can produce time-dependent dysplasia and then form tumors. There have been a lot of reports on the relationship between this gene and lung cancer, but they mainly focus on the study of its expression differences, while there are few reports on its methylation differences.

[0147] Studies have shown that although the two genes are generally demethylated in lung cancer, they both show significant hypermethylation in the CG island region of the promoter. Therefore, when designing primers, in addition to considering the specificity of the primers, it is also necessary to consider the number of CG islands and the C / G content contained in the target area to be amplified.

[0148] Alu gene fragment (highly repetitive sequence): It is the sequence with the highest copy number in the human genome. Due to the different metabolism between tumors and normal people, the total amount of the genome in the peripheral blood of normal people and tumor patients is very different. Therefore, in the comparison of the total ctDNA concentration between normal people and lung cancer patients, the Alu gene copy number can be used as a proxy for the total amount of the genome for quantitative detection.

[0149] 2) Experimental Materials and Methods

[0150] Sample sources: Normal group samples: from the physical examination personnel of Beijing Jiaotong University Hospital; lung cancer group samples: from the laboratory of the Tumor Center of the First Medical Center of the General Hospital of the People's Liberation Army. We randomly selected 42 normal samples and 53 lung cancer samples.

[0151] Design of primers and probes: Primer software Primer 5 was used to design the promoter regions of AID and HOXD12 genes, and parameters such as the number of CG islands, C / G content, and length of the amplified fragment were referred to; the conserved region sequence in the Alu gene was designed.

[0152] Table 9 Target gene primers and probe sequences

[0153]

[0154] Note: PCR fragment lengths of target gene amplification target regions: AID (promoter region): 145 bp; HOXD12 (promoter region): 155 bp; Alu (gene body): 111 bp.

[0155] Primer and probe synthesis: provided by Sangon Biotech (Shanghai) Co., Ltd.

[0156] Plasma free DNA extraction kit: free DNA detection kit from Meiji Biotechnology.

[0157] Identification of target gene copy number: Alu primers were used to amplify and quantify the extracted DNA samples to find highly specific target areas. In this process, it was found that the Ct value of both normal people and lung cancer patients did not exceed 20. In order to keep the unit consistent, we subtracted the Ct value of Alu from 20 to obtain its corresponding ΔCt value: ΔCt = 20-Ct.

[0158] PCR instrument: Macrostone SLAN 96P.

[0159] PCR reaction conditions: (1) High temperature denaturation (HT): 3 gene fragments (AID / HOXD12 / Alu): pre-denaturation at 95°C for 5 min, followed by a 3-step amplification program: 94°C for 5 s, 60°C for 15 s, 72°C for 30 s, for 45 cycles. (2) Low temperature denaturation (LT): 2 gene fragments (AID / HOXD12): 85°C for 15 s, 60°C for 15 s, 72°C for 30 s, for 18 cycles, followed by the following cycling program: 94°C for 5 s, 60°C for 15 s, 72°C for 30 s, for 27 cycles.

[0160] 3) According to the different ratio methylation detection results of different genes, the reference curve of target gene methylation target region is established. The different ratio methylation detection results of different genes are shown in Tables 10 to 11 and Figure 10 to Figure 13 shown.

[0161] Table 10 AID gene methylation detection data at different ratios

[0162] 5'-m-dCTP: dCTP 1：0 1：1 1：2 1：4 1：6 0：1 Ct2 22.54 22.71 20.16 20.94 23.26 23.11 Ct1 19.26 20.88 19.51 20.71 23.11 23.05 ΔCt 3.28 1.83 0.65 0.23 0.15 0.06

[0163] Table 11 HOXD12 gene methylation detection data at different ratios

[0164]

[0165] 4) Qualitative determination of differences between the two groups (target gene combination and weight value analysis)

[0166] Modeling analysis: Extract plasma free DNA from the two groups and test according to the above method, then subtract the Ct values ​​of the AID and HOXD12 target genes under the two conditions to obtain the ΔCt value (Ct2-Ct1). The ΔCt value of Alu is the fixed value 20 minus the Ct value of its detection. The ΔCt values ​​of the three genes of normal people and lung cancer patients were statistically analyzed to obtain the weight value formula and threshold for the joint detection of the three genes (AID, HOXD12, Alu), and the sensitivity and specificity of the detection were analyzed by ROC curve. Modeling is completed. The weight value calculation formula of the sample is as follows:

[0167] Weight value = 1.325 × ΔCt AID +2.084×ΔCt HOXD12 +2.152×ΔCt Alu

[0168] Threshold = 12.468.

[0169] Verification analysis: The samples of normal people and lung cancer patients that were not involved in the modeling were tested according to the above method, and then the test results of each sample were compared with the threshold to obtain the threshold-based judgment results of lung cancer and normal samples (see Table 12). The test data of normal people and lung cancer samples are shown in Appendix 20 (the gray font in the table indicates a false positive) and Table 21 (the gray font in the table indicates a false negative). The evaluation results were subjected to T test analysis, and the results are shown in Table 13.

[0170] Table 12 Evaluation results of lung cancer and normal samples based on thresholds

[0171]

[0172] Table 13 T-test analysis of lung cancer and normal samples

[0173]

[0174] The statistical results showed that the mean values ​​of the weights between normal people and lung cancer patients were significantly different (P value = 0.000), which indicated that the target gene DNA methylation was significantly different between lung cancer samples and normal samples.

[0175] Sensitivity analysis: There were 51 patients with lung cancer, of whom 6 had false negatives;

[0176] Sensitivity = (51-6) / 51 = 88.23%;

[0177] Specificity analysis: There were 44 normal subjects, of whom 6 were false positives;

[0178] Specificity = (44-6) / 44 = 86.36%;

[0179] The matching rate = [(51-6)+(44-6)] / (51+44) = 87.3%.

[0180] Further analysis of sensitivity and specificity (ROC curve) showed that the area under the curve (AUC) accounted for 0.96, indicating that the sensitivity and specificity of the detection technology are both high. The distribution of weight values ​​in the normal and lung cancer groups, as well as the ROC curve for sensitivity and specificity analysis are shown in Figure 2. Fig.14 .

[0181] Example 3

[0182] cf / ctDNA methylation detection in rectal cancer

[0183] 1) Identification of colorectal cancer target genes:

[0184] Through the analysis of the TCGA database, it was found that the methylation of the three genes SDC2 / WDR17 / ADHFE1 was the most different between normal tissues and colorectal cancer tissues. Further analysis by the random forest statistical model showed that the methylation of the three genes contributed greatly to the identification of colorectal cancer. Subsequently, the methylation of the promoter region of the three genes was detected according to the aforementioned method. In this process, as mentioned above, when designing PCR primers, in addition to considering the specificity of the primers, it is also necessary to consider the number of CG islands contained in the target area to be amplified and the C / G content. In addition, the Alu gene fragment was selected as a proxy for the total amount of free DNA in peripheral blood for quantitative detection.

[0185] Table 14 Target gene primers and probe sequences

[0186]

[0187] Note: PCR fragment length of target gene amplification target region: WDR17 (promoter region): 124bp; SDC2 (promoter region): 107bp; ADHFE1 (promoter region): 157bp; Alu (gene body): 111bp.

[0188] 2) Materials and methods:

[0189] Sample sources: The normal group samples came from the physical examination personnel of Beijing Jiaotong University Hospital; the colorectal cancer group samples came from the laboratory of the Tumor Center of the First Medical Center of the General Hospital of the People's Liberation Army. 54 normal samples and 55 colorectal cancer samples were randomly selected.

[0190] Primer design: Primer software Primer 5 was used to design the promoter region of SDC2 / WDR17 / ADHFE1 gene, and parameters such as the number of CG islands and the length of the amplified fragment were referred to; the conserved region sequence in the Alu gene was designed.

[0191] Primer and probe synthesis: provided by Sangon Biotech (Shanghai) Co., Ltd.

[0192] Plasma free DNA extraction kit: free DNA detection kit from Meiji Biotechnology.

[0193] Identification of target gene copy number: Alu primers were used to amplify and quantify the extracted DNA samples to find highly specific target areas. In this process, we found that the Ct values ​​of both normal people and colorectal cancer patients did not exceed 20. In order to keep the units consistent, we subtracted the Ct value of Alu from 20 to obtain its corresponding ΔCt value: ΔCt = 20-Ct.

[0194] PCR instrument: Macrostone SLAN 96P.

[0195] PCR reaction conditions: (1) High temperature denaturation (HT): 4 gene fragments (SDC2 / WDR17 / ADHFE1 / Alu): pre-denaturation at 95°C for 5 min, followed by a 3-step amplification program: 94°C for 5 s, 62°C for 15 s, 72°C for 30 s, for 45 cycles. (2) Low temperature denaturation (LT): WDR17 / ADHFE1 gene fragment: 86°C for 15 s, 62°C for 15 s, 72°C for 30 s, for 16 cycles, followed by a cycling program: 94°C for 5 s, 60°C for 15 s, 72°C for 30 s, for 29 cycles. SDC2 gene fragment: 88°C for 15 s, 62°C for 15 s, 72°C for 30 s, for 16 cycles, followed by a cycling program: 94°C for 5 s, 60°C for 15 s, 72°C for 30 s, for 29 cycles.

[0196] 3) Draw the target gene methylation target region reference curve according to the methylation detection results of different target genes, where the methylation detection results of different target genes are shown in Tables 15 to 17 and Figures 15 to 20 shown.

[0197] Table 15 SDC2 gene methylation detection data at different ratios

[0198]

[0199] Table 16 WDR17 gene methylation detection data at different ratios

[0200]

[0201]

[0202] Table 17 ADHFE1 gene methylation detection data at different ratios

[0203]

[0204] 4) Determination of differences between two groups (target gene combination and weight value analysis)

[0205] Modeling analysis: Extract free DNA from the plasma of the two groups and detect it according to the above method, then subtract the Ct values ​​of the target gene under the two conditions to obtain the ΔCt value (Ct2-Ct1). The ΔCt value of Alu is the fixed value 20 minus the Ct value of its detection. The ΔCt values ​​of the four genes of normal people and colorectal cancer patients were statistically analyzed to obtain the weight value formula and threshold for the joint detection of the four genes, and the sensitivity and specificity were obtained through ROC curve analysis to complete the modeling. The weight value calculation formula III of the sample is as follows: Weight value = 3.35×ΔCt SDC2 +0.99×ΔCt WDR17 +0.933×ΔCt ADHFE1 +1.677×ΔCt Alu Formula III Threshold = 12.173.

[0206] Verification analysis: The samples of normal people and colorectal cancer patients that were not involved in the modeling were tested according to the above method, and then the test results of each sample were compared with the threshold. The test results of colorectal cancer patients are shown in Table 22 (grayscale shading indicates false negatives), and the test results of normal people are shown in Table 23 (grayscale shading indicates false positives).

[0207] Table 18 Threshold-based evaluation results of two samples (colorectal cancer and normal people)

[0208]

[0209] Sensitivity = 85.9%, specificity = 94.4%; consistency = 90.1%.

[0210] T test analysis was performed based on the evaluation results. The results are shown in Table 19.

[0211] Table 19 T test analysis of test results

[0212] Group Number of cases Mean Standard Deviation Standard error of the mean Colorectal cancer 57 16.6525 5.20192 0.68901 Normal people 54 9.3080 2.35822 0.31513

[0213] The statistical results showed that the mean weight values ​​of normal people and lung cancer patients were significantly different (P value

[0214] =0.000), which indicates that the target gene DNA methylation has significant differences between lung cancer samples and normal samples.

[0215] Further analysis of sensitivity and specificity (ROC curve see Fig.21 ), and its area under the curve (AUC) accounted for 0.97, indicating that the sensitivity and specificity of this detection technology were high.

[0216] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

[0217] Table 20 Test data of normal human samples

[0218]

[0219] Table 21 Detection data of lung cancer samples

[0220]

[0221] Table 22 Colorectal cancer patient test data

[0222]

[0223] Table 23 Normal people test data

[0224]

Claims

1. A method for evaluating the degree of DNA methylation of a target gene, characterized in that: The following steps are involved: 1) The quantitative PCR reaction system was prepared using the Taq enzyme premix system, primers and probes for quantitative PCR detection of the target gene, and the target gene was subjected to high temperature denaturation amplification and low temperature denaturation amplification respectively to obtain the Ct of high temperature denaturation amplification. X-HT and Ct of lower temperature denaturation amplification X-LH ; 2) Ct X-LH and the Ct X-HT Substitute into formula I to obtain the △Ctx of the target gene; △Ctx=Ct X-LH –Ct X-HT Formula I Among them, △Ctx represents the detection value of gene X; Ct X-LH Indicates the Ct value of gene X amplified under lower temperature denaturation conditions; Ct X-HT It represents the Ct value of X gene amplified under higher temperature denaturation conditions, and X gene represents a target gene; 3) using PCR technology and reagents for drawing reference curves, preparing methylated DNA fragments of different degrees by base incorporation, diluting and using them as methylated DNA templates for PCR amplification, and operating according to the methods of steps 1) and 2) to obtain ΔCtx' of different methylated DNA fragments, obtaining the linear relationship between different proportions of methylated cytosine and ΔCtx', and drawing a reference curve; 4) comparing the ΔCtx of the target gene in step 2) with the ΔCtx' in the reference curve in step 3) to obtain the actual ratio of methylated cytosine in the target gene, thereby determining the degree of DNA methylation of the target gene; The reaction conditions of the higher temperature denaturation amplification are as follows: 95°C for 5 min; 94°C for 5 s, 60-62°C for 15 s, 72°C for 30 s, 45 cycles; The reaction conditions of the lower temperature denaturation amplification are as follows: 85-88°C for 15s, 60-62°C for 15s, 72°C for 30s, 18 cycles; 94°C for 5s, 60-62°C for 15s, 72°C for 30s, 27 cycles; The reagents for drawing the reference curve include dNTP reagents containing methylated cytosine in different proportions; the dNTP reagents containing methylated cytosine in different proportions include dATP, dTTP, dGTP and reagent A in equimolar concentrations; the reagent A is 5'-m-dCTP and / or dCTP.

2. The method according to claim 1, characterized in that: In the reagent A, the molar ratio of 5'-m-dCTP to dCTP includes 1:0, 1:(1-30) and 0:

1.

3. The method according to claim 1, characterized in that The target gene includes but is not limited to one or more of the following genes: APC, SHOX2, Apobec3B, P53, AID, HOXD12, Alu, SDC2, WDR17, and ADHFE1.

4. The method according to claim 1, characterized in that The reagents used to draw the reference curve also include common PCR amplification reagents.

5. Use of the method according to any one of claims 1 to 4 in identifying the methylation degree of a target gene in a sample and distinguishing between two research groups.

6. A method for distinguishing two research populations based on DNA methylation changes in a target gene combination, characterized in that: The following steps are involved: After obtaining the ΔCt of each target gene of the two research groups by the method described in any one of claims 1 to 4, statistically analyzing the ΔCt of each target gene corresponding to the samples of research group 1 and research group 2 to obtain the threshold and weight value of the joint detection of the target genes, completing the modeling of the combined detection of the target genes, and obtaining the threshold and weight value of the joint detection of the target genes; the target genes include two or more target genes; The weight value is obtained by substituting the ΔCt of each target gene into Formula II to calculate the weight value of the multi-target gene joint detection of each sample; Weight value of multi-target gene joint detection = a1×△Ct x1 +a2×△Ct x2 +......+an×△Ct xn Formula II Among them, a1, a2, and an represent the corresponding coefficients obtained in the statistical analysis of each target gene; Verification of the difference between the two groups: The multi-target gene weight values ​​of the samples of study group 1 and study group 2 that did not participate in the modeling were obtained according to the above method, and then compared with the above thresholds respectively. The samples with weight values ​​lower than the threshold were low-methylation samples, and the samples with weight values ​​higher than the threshold were high-methylation samples. The differences between the two groups were further compared by statistical analysis; Determination of the difference between the two groups: The mean of the weight values ​​of the samples of study group 1 and study group 2 will be calculated, and a T-test analysis will be performed. When the mean of the weight values ​​of the samples of study group 1 and the mean of the weight values ​​of the samples of study group 2 are significantly different, it indicates that the DNA methylation difference between the two groups is significant, which means that the two groups can be identified through methylation detection of the target gene combination.

7. Use of the method according to any one of claims 1 to 4 or the method according to claim 6 to prepare a product for distinguishing cancer patients from non-cancer patients; The cancer includes lung cancer and / or colorectal cancer; The target genes for lung cancer include HOXD12, AID and Alu; The target genes for colorectal cancer include SDC2, WDR17, ADHFE1 and Alu.

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