A quantitative digital PCR detection reagent for detecting pan-solid tumor multi-gene promoter methylation and application thereof

Abstract

The application discloses a quantitative digital PCR detection reagent for detecting promoter methylation of pan-solid tumor multi-genes and application, which comprises methylation-specific primer probe pairs and unmethylation-specific primer probe pairs for detecting CDKN2A, RASSF1A, RARbeta2 and SHOX2 genes. The application adopts the digital PCR technology to detect pan-solid tumor multi-genes (CDKN2A, RASSF1A, RARbeta2 and SHOX2), has greater tumor discrimination, and has important significance for tumor screening and risk assessment, early diagnosis, staging and typing, prognosis judgment and treatment detection. The method has higher sensitivity, higher accuracy and absolute quantification, is not affected by amplification efficiency, can more efficiently detect extremely low abundance (0.1%) promoter methylation, and is suitable for screening of a large number of clinical samples.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene detection, in particular to a digital PCR detection reagent for detecting promoter methylation of multiple genes of pan-solid tumors and application thereof. BACKGROUND

[0002] Biomarkers are very promising in improving the clinical routine cancer treatment. So far, several markers from genetics, proteomics and epigenetics have been applied in clinical studies. DNA methylation as a powerful, well-studied epigenetic marker provides broad insights into transcriptional regulation, embryonic development, genomic stability and chromatin structure. DNA methylation patterns alterations play a key role in the process of tumorigenesis. Therefore, in many clinical applications, DNA methylation is used as a biomarker for early detection, diagnosis and individual treatment, usually with precise characterization of methylation at specific regions or single base level.

[0003] Tumorigenesis is a complex process involving multiple genes and multiple links, and genetic changes caused by the disorder of related gene functions are closely related thereto. In clinical applications, most of the established biomarkers only rely on the methylation difference of a certain number of CpG sites to determine whether a disease is present or not. DNA methylation detection at specific sites is a commonly used method in the diagnosis and treatment of conventional cancer. However, detecting a single gene site to determine the benign or malignant of a tumor is defective in that: firstly, the maximum sensitivity of detection may be consistent with the methylation frequency of a specific gene CpG site; secondly, in some cases, there may be high methylation of the same gene locus in non-tumor tissues. Therefore, attempting to detect the methylation of multiple gene promoters at the same time may make up for the deficiency.

[0004] The method widely used at present is the combination of bisulfite conversion and fluorescent PCR, that is, after the detection sample is subjected to bisulfite conversion, the unmethylated cytosine (C) is converted into uracil (U), and the methylated cytosine (C) remains unchanged, so that methylation / unmethylation is changed into C / T polymorphism, combined with fluorescent PCR method, specific probe is designed to detect the state of C / T of the target site to determine whether the sample is methylated.

[0005] CN109072310A discloses an in vitro method for determining whether an individual has a precancerous lesion or cancer, which comprises determining the presence or absence of one or more methylation markers of a set of methylation markers in a urine sample of the individual; and determining whether the individual has a precancerous lesion or cancer based on detection of the presence or absence of the one or more methylation markers in the urine sample, wherein the presence of the one or more methylation markers indicates that the individual has a precancerous lesion or cancer. The method uses fluorescent quantitative PCR for qualitative detection of urine, and cannot perform absolute quantitative detection of methylation level.

[0006] Therefore, developing a quantitative detection reagent capable of simultaneously detecting methylation of multiple gene promoters has become one of the technical problems urgently to be solved in the field. SUMMARY

[0007] The purpose of the present application is to provide a quantitative digital PCR detection reagent for detecting methylation of multiple gene promoters of pan-solid tumors and application thereof.

[0008] The research team of the inventors found that, by using dPCR amplification technology and designing multiple gene (CDKN2A, RASSF1A, RARbeta2 and SHOX2) promoter region-specific methylation and unmethylation detection primer probes, accurate detection of samples with different degrees of disease can be performed, and the method has high specificity and sensitivity, greatly eliminates the interference of false negatives, and has clinical significance for early diagnosis, long-term monitoring and prognosis of tumors.

[0009] The technical solutions adopted by the present application are as follows:

[0010] A quantitative digital PCR detection reagent for detecting methylation of multiple gene promoters of pan-solid tumors comprises upstream primer 1, downstream primer 1 and probe 1 for specific amplification of CDKN2A gene promoter methylation, and unmethylated upstream primer 2, downstream primer 2 and probe 2; and upstream primer 3, downstream primer 3 and probe 3 for specific amplification of RASSF1A gene promoter methylation, and unmethylated upstream primer 4, downstream primer 4 and probe 4; and upstream primer 5, downstream primer 5 and probe 5 for specific amplification of RARbeta2 gene promoter methylation, and unmethylated upstream primer 6, downstream primer 6 and probe 6; and upstream primer 7, downstream primer 7 and probe 7 for specific amplification of SHOX2 gene promoter methylation, and unmethylated upstream primer 8, downstream primer 8 and probe 8.

[0011] The nucleotide sequences of the upstream primer 1 and the downstream primer 1 and the specific probe 1 are shown in SEQ ID No. 1-3, the nucleotide sequences of the upstream primer 2 and the downstream primer 2 and the specific probe 2 are shown in SEQ ID No. 4-6, the nucleotide sequences of the upstream primer 3 and the downstream primer 3 and the specific probe 3 are shown in SEQ ID No. 7-9, the nucleotide sequences of the upstream primer 4 and the downstream primer 4 and the specific probe 4 are shown in SEQ ID No. 10-12, the nucleotide sequences of the upstream primer 5 and the downstream primer 5 and the specific probe 5 are shown in SEQ ID No. 13-15, the nucleotide sequences of the upstream primer 6 and the downstream primer 6 and the specific probe 6 are shown in SEQ ID No. 16-18, the nucleotide sequences of the upstream primer 7 and the downstream primer 7 and the specific probe 7 are shown in SEQ ID No. 19-21, and the nucleotide sequences of the upstream primer 8 and the downstream primer 8 and the specific probe 8 are shown in SEQ ID No. 22-24.

[0012] The methylation-specific probe carries a detection marker, and the detection marker includes 5' end label FAM fluorescence and 3' end label BHQ1 fluorescence.

[0013] The unmethylated-specific probe carries a detection marker, and the detection marker includes 5' end label HEX fluorescence and 3' end label BHQ1 fluorescence.

[0014] The addition amount of any primer is 1.4 μL, and the addition amount of any probe is 0.6 μL.

[0015] The reaction concentration of any primer is 700 nM, and the reaction concentration of any probe is 300 nM.

[0016] A quantitative digital PCR detection kit for detecting promoter methylation of multiple genes of pan-solid tumors, comprising the quantitative digital PCR detection reagent described above, specifically comprising: (1) a first container, and upstream primer 1, downstream primer 1, probe 1 and upstream primer 2, downstream primer 2, probe 2 located in the container; (2) a second container, and upstream primer 3, downstream primer 3, probe 3 and upstream primer 4, downstream primer 4, probe 4 located in the container; (3) a third container, and upstream primer 5, downstream primer 5, probe 5 and upstream primer 6, downstream primer 6, probe 6 located in the container; (4) a fourth container, and upstream primer 7, downstream primer 7, probe 7 and upstream primer 8, downstream primer 8, probe 8 located in the container; (5) a matched reagent.

[0017] The matched reagent includes a digital PCR reaction enzyme reagent, a positive control, a negative control, and ultrapure water, etc.

[0018] A digital PCR detection method for detecting promoter methylation of multiple genes of a general tumor: using the detection reagent or detection kit of the present application, a digital PCR detection is performed on a detection sample. Specifically, the following steps are included:

[0019] (1) providing a detection sample genomic DNA, which is a product converted by EZ DNA Methylation TM Kit;

[0020] (2) using specific primers and probes, a digital PCR reaction is performed on the converted product as a template, so as to obtain amplification products of CDKN2A, RASSF1, RARβ2, and SHOX2 genes;

[0021] (3) fluorescence signal collection and analysis are performed on the amplification products, so as to obtain methylation and unmethylation copy number contents of CDKN2A, RASSF1A, RARβ2, and SHOX2 genes;

[0022] (4) according to the methylation and unmethylation copy number contents, a methylation ratio is calculated according to a formula:

[0023]

[0024] The reaction system of the digital PCR reaction includes: a sample to be detected: 0-50 ng / μL; 2×ddPCR SuperMix for Probes (Bio-Rad Laboratories) 10 μL;

[0025] The upstream primer 1 and the downstream primer 1: 1.4 μL, the probe 1: 0.6 μL, the upstream primer 2 and the downstream primer 2: 1.4 μL, the probe 2: 0.6 μL;

[0026] The upstream primer 3 and the downstream primer 3: 1.4 μL, the probe 3: 0.6 μL, the upstream primer 4 and the downstream primer 4: 1.4 μL, the probe 4: 0.6 μL;

[0027] The upstream primer 5 and the downstream primer 5: 1.4 μL, the probe 5: 0.6 μL, the upstream primer 6 and the downstream primer 6: 1.4 μL, the probe 6: 0.6 μL;

[0028] The upstream primer 7 and the downstream primer 7: 1.4 μL, the probe 7: 0.6 μL, the upstream primer 8 and the downstream primer 8: 1.4 μL, the probe 8: 0.6 μL.

[0029] PCR reaction conditions: 95℃ pre-denaturation 10 min, then 94℃ denaturation 30 sec, 55℃ annealing 60 sec, 40 cycles, finally 98℃ extension 10 min, 4℃ storage.

[0030] The application is based on a methylation detection method of bisulfite modification. After the DNA detection sample is converted by bisulfite, unmethylated C is converted into U, while methylated C remains unchanged. After PCR amplification, U is converted into T. The methylation level of the sample is detected by the binding of a specific probe to the bisulfite conversion product.

[0031] Compared with the prior art, the application has the following prominent effects:

[0032] (1) The application adopts digital PCR (dPCR) technology to detect the promoter regions of CDKN2A, RASSF1A, RARβ2 and SHOX2 four genes, which can accurately and effectively calculate the methylation level of the sample, and has important significance for tumor screening and risk assessment, early diagnosis, staging and typing, prognosis judgment and treatment detection.

[0033] CDKN2A gene is a multiple tumor suppressor, which is involved in cell cycle regulation, mainly responsible for inhibiting cell cycle-dependent kinase complex in G1 / S cell cycle, effectively blocking cell proliferation and differentiation. A large amount of evidence shows that the high methylation of CDKN2A gene promoter CpG island makes the gene transcription silencing an important cause of tumor occurrence, and is observed in various cancers. RASSF1A gene is a tumor suppressor that interacts with Cdc20, an activator of the anaphase promoting complex, inhibits the activity of the complex, and regulates apoptosis and microtubule dynamics during mitosis. Its epigenetic inactivation is observed in 53%-71% of solid tumors and epithelial cancers. RARβ2 gene is a major developmental process that regulates cell growth and differentiation, and is involved in retinoid anti-proliferation and differentiation. It mainly plays a tumor suppressor role in lung, breast and gynecological tumors. SHOX2 gene is a common transcription factor that regulates gene expression and participates in organ skeletal development. Its promoter hypermethylation silences the gene at the transcription level, thereby inhibiting the expression of downstream genes, thereby promoting cell carcinogenesis. At present, the methylation status of SHOX2 promoter is mainly applied to lung cancer detection, and certain achievements have been made accordingly. SHOX2 methylation as a biomarker is also expected to be confirmed in other cancers, such as ovarian cancer, prostate cancer, and glioma.

[0034] (2) Compared with the DNA methylation technology using fluorescent PCR detection in the prior art, the quantitative digital PCR detection reagent of the application can perform absolute quantification on the sample, the quantitative result is in the form of the copy number of the target gene, the deviation of quantifying the methylation level is minimized, the detection result is more intuitive without relying on the judgment of the amplification curve, the internal reference gene and the Ct value, the subjective factors of artificial judgment are avoided, the false negative is reduced, the detection flux is high, it is suitable for clinical large sample, and the detection result with higher sensitivity, higher stability and repeatability is consistent with the clinical test data, which assists the clinician to make appropriate decision on disease diagnosis and treatment, has broad application prospect and industrialization prospect.

[0035] The quantitative digital PCR detection reagent for detecting pan-solid tumor multi-gene promoter methylation and the application thereof described in the application will be further described below in combination with the description of the drawings and specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 The figure is the annealing temperature optimization result graph of the CDKN2A gene digital PCR method.

[0037] Figure 2 The figure is the annealing temperature optimization result graph of the RASSF1A gene digital PCR method.

[0038] Figure 3 The figure is the annealing temperature optimization result graph of the RARβ2 gene digital PCR method.

[0039] Figure 4 The figure is the annealing temperature optimization result graph of the SHOX2 gene digital PCR method.

[0040] Figure 5 The figure is the reaction system optimization result graph of the CDKN2A gene digital PCR method.

[0041] Figure 6 The figure is the reaction system optimization result graph of the RASSF1A gene digital PCR method.

[0042] Figure 7 The figure is the reaction system optimization result graph of the RARβ2 gene digital PCR method.

[0043] Figure 8 The figure is the reaction system optimization result graph of the SHOX2 gene digital PCR method.

[0044] Figure 9 The figure is the linear equation obtained by detecting different methylation level samples of the CDKN2A gene.

[0045] Figure 10 The figure is the linear equation obtained by detecting different methylation level samples of the RASSF1A gene.

[0046] Figure 11 Linear equation for RARβ2 detection of different methylation level samples.

[0047] Figure 12 Linear equation for SHOX2 detection of different methylation level samples.

[0048] Figure 13 CDKN2A detection of different sample-specific amplification results map.

[0049] Figure 14 RASSF1A detection of different sample-specific amplification results map.

[0050] Figure 15 RARβ2 detection of different sample-specific amplification results map.

[0051] Figure 16 SHOX2 detection of different sample-specific amplification results map. DETAILED DESCRIPTION

[0052] I. Design four kinds of gene promoter region-specific methylation and unmethylation primer probe pairs

[0053] According to NCBI and UCSC biological software analysis, the CDKN2A gene promoter region (21974733-21974802), the RASSF1A gene promoter region (50340818-50340892), the RARβ2 gene promoter region (25428325-25428417) and the SHOX2 gene promoter region (158103517-158103624) are selected as the target sequences; the bisulfite modified fully methylated DNA sequence is used as the template combined with Methprimer and Primer express software to design specific methylation primer probe pairs and unmethylation primer probe pairs.

[0054] The specific methylation primer probe pairs and the unmethylation primer probe pairs include the upstream primer 1, the downstream primer 1 and the probe 1 for specific amplification of the CDKN2A gene methylation and the unmethylation upstream primer 2, the downstream primer 2 and the probe 2; and the upstream primer 3, the downstream primer 3 and the probe 3 for specific amplification of the RASSF1A gene methylation and the unmethylation upstream primer 4, the downstream primer 4 and the probe 4; and the upstream primer 5, the downstream primer 5 and the probe 5 for specific amplification of the RARβ2 gene methylation and the unmethylation upstream primer 6, the downstream primer 6 and the probe 6; and the upstream primer 7, the downstream primer 7 and the probe 7 for specific amplification of the SHOX2 gene methylation and the unmethylation upstream primer 8, the downstream primer 8 and the probe 8.

[0055] The nucleotide sequences of upstream primer 1, downstream primer 1, and specific probe 1 are shown in SEQ ID Nos. 1-3, the nucleotide sequences of upstream primer 2, downstream primer 2, and specific probe 2 are shown in SEQ ID Nos. 4-6, the nucleotide sequences of upstream primer 3, downstream primer 3, and specific probe 3 are shown in SEQ ID Nos. 7-9, the nucleotide sequences of upstream primer 4, downstream primer 4, and specific probe 4 are shown in SEQ ID Nos. 10-12, the nucleotide sequences of upstream primer 5, downstream primer 5, and specific probe 5 are shown in SEQ ID Nos. 13-15, the nucleotide sequences of upstream primer 6, downstream primer 6, and specific probe 6 are shown in SEQ ID Nos. 16-18, the nucleotide sequences of upstream primer 7, downstream primer 7, and specific probe 7 are shown in SEQ ID Nos. 19-21, and the nucleotide sequences of upstream primer 8, downstream primer 8, and specific probe 8 are shown in SEQ ID Nos. 22-24. Table 1 below shows this.

[0056] Table 1

[0057]

[0058]

[0059] 2. Optimizing the amplification conditions of primer-probe pairs specific for methylated and unmethylated promoter regions of four genes

[0060] The working concentration of the primers for CDKN2A gene was set to 500 nM, and the working concentration of the probe was set to 250 nM ( Figure 1 ), and set the annealing temperatures to 50℃, 53℃, 55℃, 58℃, and 60℃ respectively.

[0061] The working concentration of the primers for RASSF1A gene was set to 500 nM, and the working concentration of the probe was set to 250 nM ( Figure 2 ), and set the annealing temperatures to 50℃, 53℃, 55℃, 58℃, and 60℃ respectively.

[0062] The working concentration of the primers for RARβ2 gene was set to 500 nM, and the working concentration of the probe was set to 250 nM ( Figure 3 ), and set the annealing temperatures to 50℃, 53℃, 55℃, 58℃, and 60℃ respectively.

[0063] The working concentration of the primers for SHOX2 gene was set to 500 nM, and the working concentration of the probe was set to 250 nM ( Figure 4 ), and set the annealing temperatures to 50℃, 53℃, 55℃, 58℃, and 60℃ respectively.

[0064] According to the one-dimensional scatter plot of the amplification results ( Figures 1-4 ), good amplification was achieved at the five annealing temperatures. At 55°C, the negative droplet clusters were well separated from the positive droplet clusters, the droplet number was high, and the quantitative repeatability was good. Therefore, 55°C was determined to be the optimal annealing temperature.

[0065] The primer and probe concentrations were optimized by orthogonal experiments. The primer concentrations were set at 500nM, 700nM, and 900nM, and the probe concentrations were set at 200nM, 300nM, and 400nM, respectively. Figures 5-8 When the primer concentration remained constant and the probe concentration increased from 200 nM to 400 nM, the fluorescence intensity of both negative and positive droplets increased, and the separation between the negative and positive droplets increased. When the probe concentration remained constant and the primer concentration increased from 500 nM to 900 nM, the fluorescence intensity of the negative droplets remained unchanged, while the fluorescence intensity of the positive droplets initially increased significantly and then leveled off. Consequently, the separation between the negative and positive droplets also increased and then remained constant. However, the dispersion of the positive droplets increased accordingly with increasing probe concentration. Taking into account the reproducibility of the quantitative results, the working concentrations of the methylation primers and probes for the CDKN2A, RASSF1A, RARβ2, and SHOX2 genes were determined to be 700 nM and 300 nM, respectively. The working concentrations of the unmethylation primers and probes for the CDKN2A, RASSF1A, RARβ2, and SHOX2 genes were also determined to be 700 nM and 300 nM, respectively.

[0066] The digital PCR detection method for detecting methylation of promoters of multiple genes in pan-solid tumors specifically includes the following steps:

[0067] (1) Providing a test sample genomic DNA, wherein the test sample genomic DNA is subjected to EZ DNA Methylation TM Kit transformation products;

[0068] (2) using the transformed product as a template, using specific primers and probes, to perform a digital PCR reaction, thereby obtaining amplified products of CDKN2A, RASSF1, RARβ2, and SHOX2 genes;

[0069] (3) collecting and analyzing the fluorescence signals of the amplified products to obtain the methylated and unmethylated copy number contents of the CDKN2A, RASSF1A, RARβ2, and SHOX2 genes;

[0070] (4) Calculate the methylation ratio according to the methylated and unmethylated copy number contents according to the formula:

[0071]

[0072] The final system of the droplet digital PCR reaction is: 0-50 ng / μL of the sample to be tested, 10 μL of 2x ddPCR SuperMix for Probes (Bio-Rad Laboratories), 1.4 μL of methylation upstream and downstream primers, 0.6 μL of methylation probes, 1.4 μL of unmethylation upstream and downstream primers, 0.6 μL of unmethylation probes, 2 μL of DNA template, and ddH2O to make up 20 μL.

[0073] The ddPCR reaction program is: 95°C pre-denaturation for 10 min, 94°C denaturation for 30 s, 55°C annealing for 60 s, 40 cycles, 98°C extension for 10 min, and 4°C storage.

[0074] III. Study on the linear range of the digital PCR detection method for the promoter regions of the four genes

[0075] When the digital PCR method is used to detect the concentration of nucleic acids, it is necessary to ensure that the concentration of the nucleic acid sample is within the detection range of the digital PCR method. Samples with different methylation levels of 100%, 20%, 4%, 1%, 0.5%, 0.2%, 0.1%, and 0.05% are prepared, and digital PCR reactions are performed, with 3 parallel tests for each gradient. The linear relationship between the copy number and the quality is established, and the correlation coefficient R2≥99.0% is used as the evaluation standard for good linear relationship.

[0076] As shown in FIG. 1, Figures 9-12 The results show that X in the figure is the logarithmic value of the different methylation proportions prepared by the weight method, and Y is the logarithmic value of the different methylation proportions determined by the digital PCR method. It can be seen that the linear relationship of the digital PCR method established by the experiment is good, and the R2 of the CDKN2A, RASSF1A, RARβ2, and SHOX2 genes is 99.99%, 99.93%, 99.99%, and 99.99%, respectively.

[0077] Then, the detection limit of the digital PCR detection method for the four genes of CDKN2A, RASSF1A, RARβ2, and SHOX2 is studied.

[0078] According to the requirements of the relevant specifications, the target DNA with known concentration is gradiently diluted, and each concentration is measured repeatedly for 3-12 times. The coefficient of variation (CV) corresponding to each concentration is calculated, and the quantitative limit of the method is determined according to the CV<25%. The quantitative limits of the CDKN2A, RASSF1A, and SHOX2 genes are all 0.99%, and the quantitative limit of the RARβ2 gene is 0.50% (Tables 2-5).

[0079] The lowest methylation level concentration was repeatedly detected, and the positive droplet detection result was greater than 95%, so the detection limit of the four genes CDKN2A, RASSF1A, RARβ2 and SHOX2 was determined to be 0.10%.

[0080] Table 1 Quantitative limit results of CDKN2A gene

[0081] Methylation level (%) by weight method Methylation level (%) by ddPCR CV (%) 100.00 100.00 4.85 20.02 19.01 6.24 3.94 3.59 6.59 0.99 0.81 12.76 0.50 0.43 43.04 0.20 0.18 53.52 0.10 0.10 56.08

[0082] Table 2 Quantitative limit results of RASSF1A gene

[0083] Methylation level (%) by weight method Methylation level (%) by ddPCR CV (%) 100.00 100.00 1.36 20.02 17.40 3.05 3.94 3.60 15.23 0.99 1.12 16.47 0.50 0.41 43.34 0.20 0.18 47.74 0.10 0.10 66.16

[0084] Table 3 Quantitative limit results of RARβ2 gene

[0085] Methylation level (%) by weight method Methylation level (%) by ddPCR CV (%) 100.00 100.00 1.66 20.02 18.98 4.71 3.94 3.53 6.78 0.99 0.85 7.29 0.50 0.56 14.34 0.20 0.20 59.43 0.10 0.10 62.85

[0086] Table 4 Quantitative limit results of SHOX2 gene

[0087]

[0088]

[0089] IV. Specificity verification by using ddPCR technology to detect different types of samples

[0090] To verify the specificity of the established method, CDKN2A gene methylation primer probe (upstream primer 1, downstream primer 1 and probe 1) and unmethylated primer probe (upstream primer 2, downstream primer 2 and probe 2) were used, RASSF1A gene methylation primer probe (upstream primer 3, downstream primer 3 and probe 3) and unmethylated primer probe (upstream primer 4, downstream primer 4 and probe 4) were used, RARβ2 gene methylation primer probe (upstream primer 5, downstream primer 5 and probe 5) and unmethylated primer probe (upstream primer 6, downstream primer 6 and probe 6) were used, SHOX2 gene methylation primer probe (upstream primer 7, downstream primer 7 and probe 7) and unmethylated primer probe (upstream primer 8, downstream primer 8 and probe 8) were used, and cross experiments were performed using methylated genomic DNA and unmethylated genomic DNA as templates, and the detection results are shown in Table 5. Figures 13-16

[0091] The methylation digital PCR method of CDKN2A gene only has positive amplification in the DNA sample which has methylation, and the unmethylated DNA sample is negative; similarly, its unmethylated digital PCR method only has positive amplification in the DNA sample which has no methylation. Figure 13

[0092] ​​The methylation digital PCR method of the RASSF1A gene has positive amplification only in the DNA sample which is methylated, and the unmethylated DNA sample is negative; similarly, the unmethylated digital PCR method has positive amplification only in the DNA sample which is not methylated. Figure 14

[0093] The methylation digital PCR method of the RARβ2 gene has positive amplification only in the DNA sample which is methylated, and the unmethylated DNA sample is negative; similarly, the unmethylated digital PCR method has positive amplification only in the DNA sample which is not methylated. Figure 15

[0094] The methylation digital PCR method of the SHOX2 gene has positive amplification only in the DNA sample which is methylated, and the unmethylated DNA sample is negative; similarly, the unmethylated digital PCR method has positive amplification only in the DNA sample which is not methylated. Figure 16 ) It is shown that the established methylation and unmethylation digital PCR methods have good specificity.

[0095] From the above experimental results, it can be seen that:

[0096] (1) The detection method of the present application has high sensitivity, and can more efficiently detect extremely low abundance (0.1%) DNA promoter methylation, and is suitable for screening a large number of samples in clinic.

[0097] (2) The method of the present application adopts methylation and unmethylation specific primer probes in the promoter region of multiple genes (CDKN2A, RASSF1A, RARβ2 and SHOX2), and needs to perform digital PCR detection after the genome DNA of the pan-solid tumor is converted by bisulfite, and the early detection of pan-solid tumor patients, monitoring of minimal residual disease, prognosis and drug response prediction are calculated by the methylation proportion formula.

[0098] (3) The method of the present application adopts the methylation and unmethylation specific primer probe method of multiple genes (CDKN2A, RASSF1A, RARβ2 and SHOX2), and four pairs of methylation primer probes and four pairs of unmethylation primer probes are respectively combined on the DNA sequences of different genes, which has greater tumor discrimination and effectively eliminates false positive and false negative results caused by single gene primer probe amplification, and has higher reliability compared with the existing single gene detection technology.

[0099] (4) In the field, the method has high sensitivity and objective stability, and can be used in other technical uncertain areas.

[0100] ​​(5) The present application adopts digital PCR, which can directly give the copy number of methylated and unmethylated DNA, and can be used for monitoring drug reaction and evaluating treatment effect of pan-solid tumor patients after drug administration.

[0101] The above-described embodiments are merely intended to describe the preferred embodiments of the present application, and are not intended to limit the scope of the present application, and various modifications and improvements to the technical solutions of the present application made by those skilled in the art without departing from the design spirit of the present application shall fall within the protection scope of the present application.

Claims

1. A quantitative digital PCR detection reagent for detecting multiple gene promoter methylation of pan-solid tumor, characterized in that, The upstream primer 1, the downstream primer 1 and the probe 1 for specifically amplifying the promoter methylation of the CDKN2A gene and the unmethylated upstream primer 2, the downstream primer 2 and the probe 2; and the upstream primer 3, the downstream primer 3 and the probe 3 for specifically amplifying the promoter methylation of the RASSF1A gene and the unmethylated upstream primer 4, the downstream primer 4 and the probe 4; The upstream primer 5, the downstream primer 5 and the probe 5 for specifically amplifying the promoter methylation of the RARβ2 gene and the unmethylated upstream primer 6, the downstream primer 6 and the probe 6; and the upstream primer 7, the downstream primer 7 and the probe 7 for specifically amplifying the promoter methylation of the SHOX2 gene and the unmethylated upstream primer 8, the downstream primer 8 and the probe 8; The nucleotide sequences of the upstream primer 1, the downstream primer 1 and the specific probe 1 are shown in SEQ ID No. 1-3, the nucleotide sequences of the upstream primer 2, the downstream primer 2 and the specific probe 2 are shown in SEQ ID No. 4-6, the nucleotide sequences of the upstream primer 3, the downstream primer 3 and the specific probe 3 are shown in SEQ ID No. 7-9, the nucleotide sequences of the upstream primer 4, the downstream primer 4 and the specific probe 4 are shown in SEQ ID No. 10-12, the nucleotide sequences of the upstream primer 5, the downstream primer 5 and the specific probe 5 are shown in SEQ ID No. 13-15, the nucleotide sequences of the upstream primer 6, the downstream primer 6 and the specific probe 6 are shown in SEQ ID No. 16-18, the nucleotide sequences of the upstream primer 7, the downstream primer 7 and the specific probe 7 are shown in SEQ ID No. 19-21, and the nucleotide sequences of the upstream primer 8, the downstream primer 8 and the specific probe 8 are shown in SEQ ID No. 22-24.

2. The quantitative digital PCR detection reagent according to claim 1, characterized in that: The methylation-specific probe carries a detection marker, and the detection marker includes 5' end label FAM fluorescence and 3' end label BHQ1 fluorescence.

3. The quantitative digital PCR assay reagent of claim 1, wherein: The unmethylated-specific probe carries a detection marker, and the detection marker includes 5' end label HEX fluorescence and 3' end label BHQ1 fluorescence.

4. The quantitative digital PCR detection reagent according to any one of claims 1-3, characterized in that: The addition amount of any primer is 1.4 μL, and the addition amount of any probe is 0.6 μL; the reaction concentration of any primer is 700 nM, and the reaction concentration of any probe is 300 nM.

5. A quantitative digital PCR detection kit for detecting promoter methylation of multiple genes in pan-cancer, characterized in that, The quantitative digital PCR detection reagent according to any one of claims 1-4.

6. The quantitative digital PCR detection kit according to claim 5, characterized in that, The quantitative digital PCR detection reagent according to any one of claims 1-4. (1) the first container and upstream primer 1, downstream primer 1, probe 1 and upstream primer 2, downstream primer 2, probe 2 located in the container; (2) the second container and upstream primer 3, downstream primer 3, probe 3 and upstream primer 4, downstream primer 4, probe 4 located in the container; (3) the third container and upstream primer 5, downstream primer 5, probe 5 and upstream primer 6, downstream primer 6, probe 6 located in the container; (4) the fourth container and upstream primer 7, downstream primer 7, probe 7 and upstream primer 8, downstream primer 8, probe 8 located in the container; (5) the matched reagent.

7. The test kit according to claim 6, characterized in that The matched reagent comprises digital PCR reaction enzyme reagent, positive control, negative control and ultrapure water.

8. A digital PCR method for detecting multiple gene promoter methylation of pan-solid tumors for non-diagnostic purposes, characterized by: The detection sample is detected by digital PCR using the detection reagent of any one of claims 1-4 or the detection kit of any one of claims 5-7.

9. The digital PCR detection method according to claim 8, characterized in that, The method comprises the following steps: (1) providing a detection sample genomic DNA, wherein the detection sample genomic DNA is the EZ DNA Methylation TM Kit transformed product; (2) using specific primers and probes, performing digital PCR reaction with the transformed product as a template, thereby obtaining the amplification product of CDKN2A, RASSF1, RARβ2 and SHOX2 genes; (3) collecting and analyzing the fluorescence signal of the amplification product, thereby obtaining the methylation and unmethylation copy number content of CDKN2A, RASSF1A, RARβ2 and SHOX2 genes; (4) calculating the methylation ratio according to the methylation and unmethylation copy number content according to the formula:

10. The digital PCR detection method of claim 9, wherein: The reaction system of the digital PCR reaction comprises: 0-50 ng / μL of the sample to be detected; 10 μL of 2×ddPCR SuperMix for Probes; wherein 1.4 μL of upstream primer 1 and downstream primer 1, 0.6 μL of probe 1, 1.4 μL of upstream primer 2 and downstream primer 2, 0.6 μL of probe 2; or 1.4 μL of upstream primer 3 and downstream primer 3, 0.6 μL of probe 3, 1.4 μL of upstream primer 4 and downstream primer 4, 0.6 μL of probe 4; or 1.4 μL of upstream primer 5 and downstream primer 5, 0.6 μL of probe 5, 1.4 μL of upstream primer 6 and downstream primer 6, 0.6 μL of probe 6; or 1.4 μL of upstream primer 7 and downstream primer 7, 0.6 μL of probe 7, 1.4 μL of upstream primer 8 and downstream primer 8, 0.6 μL of probe 8; The PCR reaction conditions are as follows: 95℃ pre-denaturation for 10 min; then 94℃ denaturation for 30 sec, 55℃ annealing for 60 sec, 40 cycles; finally 98℃ extension for 10 min, 4℃ storage.

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