A primer probe combination and kit for detecting methylation of cervical cancer-related genes

Abstract

The present invention discloses a primer-probe combination and a kit for detecting methylation of cervical cancer-related genes. Based on the cervical cancer-related genes RAB3C, ZNF93, and PAX1, the present invention designs a primer-probe combination for detecting methylation of the cervical cancer-related genes, and on this basis provides a kit for detecting methylation of cervical cancer-related genes. Utilizing the primer-probe combination and kit for detecting methylation of cervical cancer-related genes described herein, cervical cancer and high-grade precancerous lesions can be detected to assess the risk of cervical cancer. The kit has the advantages of good detection specificity, high detection sensitivity, and accurate test results. Furthermore, the kit is simple to operate and non-invasive, making it suitable for initial screening of cervical cancer and providing a reference for early diagnosis of cervical cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene methylation detection, and more specifically, relates to a primer-probe combination and a kit for detecting methylation of cervical cancer-related genes. Background Art

[0002] Cervical cancer is a common gynecological malignancy, ranking second in incidence among female malignant tumors in my country and posing a serious threat to women's health. Cervical intraepithelial neoplasia (CIN) is a collective term for precancerous lesions closely related to invasive cervical cancer, and its development is linked to infection with the human papillomavirus (HPV). Because cervical precancerous lesions and early-stage cervical cancer lack obvious symptoms, cervical cancer screening is considered the most effective measure to reduce cervical cancer incidence and mortality.

[0003] Cervical cancer screening is generally performed through cervical cytology screening for initial screening, and confirmed through cervical biopsy histopathology. Cervical cytology screening primarily involves staining cervical exfoliated cells, observing changes in cell morphology, and then determining whether the cervical cells are cancerous. This method has high specificity but low sensitivity. Cytology results require interpretation by a professional pathologist under a microscope, which is affected by subjective factors, and cells tend to accumulate together, leading to missed diagnoses and misdiagnoses. Furthermore, due to the shortage of pathologists, this method is difficult to popularize in low-resource areas. Therefore, providing a simple and effective kit and method for detecting cervical cancer to conduct cervical cancer risk assessment will facilitate early diagnosis of cervical cancer, allowing for timely and effective treatment and reducing the incidence and mortality of cervical cancer.

[0004] DNA methylation is a key epigenetic modification that plays a crucial role in regulating gene expression patterns and genomic stability. By detecting methylation in genes associated with cervical cancer, cervical cancer and cervical precancerous lesions can be sensitively and specifically detected, allowing for cervical cancer risk assessment. For example, a patent has been developed to detect the development and progression of cervical cancer by detecting methylation in the cervical cancer-related genes PAX1, FAM19A4, and miR1242. However, this sensitivity still needs to be further improved. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the above-mentioned existing technologies and provide a primer probe combination and a kit for detecting methylation of cervical cancer-related genes.

[0006] The first object of the present invention is to provide a use of a reagent for detecting methylation of the RAB3C and / or ZNF93 genes in the preparation of a product for detecting the risk of cervical cancer.

[0007] The second object of the present invention is to provide a primer probe combination for detecting methylation of cervical cancer-related genes.

[0008] The third object of the present invention is to provide a kit for detecting methylation of cervical cancer-related genes.

[0009] The fourth object of the present invention is to provide the use of the primer-probe combination or the kit in the preparation of a product for detecting the risk of cervical cancer.

[0010] The fifth object of the present invention is to provide a kit for detecting the risk of cervical cancer.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention uses primers and probes designed to detect RAB3C and ZNF93 gene methylation to test samples of cervical cancer and high-grade cervical precancerous lesions (cervical intraepithelial neoplasia (CIN) stages 2 and 3). The results show that detecting RAB3C and ZNF93 gene methylation can detect cervical cancer and its high-grade precancerous lesions and assess the risk of cervical cancer. Therefore, the present invention claims the use of reagents for detecting RAB3C and / or ZNF93 gene methylation in the preparation of products for detecting the risk of cervical cancer.

[0013] As an optional embodiment, the reagent for detecting RAB3C gene methylation is a primer pair and a probe for detecting RAB3C gene methylation, the sequence of the primer pair is shown in SEQ ID NO.1-2, and the sequence of the probe is shown in SEQ ID NO.3.

[0014] As an optional embodiment, the reagent for detecting ZNF93 gene methylation is a primer pair and a probe for detecting ZNF93 gene methylation, the sequence of the primer pair is shown in SEQ ID NO.4-5, and the sequence of the probe is shown in SEQ ID NO.6.

[0015] The present invention also designs a methylation detection primer-probe combination with good detection specificity and high sensitivity based on the promoter sequences of cervical cancer-related genes RAB3C, ZNF93, and PAX1. The primer-probe combination includes the following components:

[0016] (1) A primer pair and a probe for detecting methylation of the RAB3C gene, wherein the sequences of the primer pair are shown in SEQ ID NOs. 1 to 2, and the sequence of the probe is shown in SEQ ID NO. 3;

[0017] (2) A primer pair and a probe for detecting ZNF93 gene methylation, wherein the sequences of the primer pair are shown in SEQ ID NOs. 4 to 5, and the sequence of the probe is shown in SEQ ID NO. 6;

[0018] (3) A primer pair and a probe for detecting PAX1 gene methylation, wherein the sequences of the primer pair are shown in SEQ ID NOs. 7 to 8, and the sequence of the probe is shown in SEQ ID NO. 9.

[0019] The present invention also designs a primer pair and a probe for detecting the internal reference gene ACTB. The sequences of the primer pair are shown in SEQ ID NOs. 10 to 11, and the sequence of the probe is shown in SEQ ID NO. 12.

[0020] The 5' end of the probe of the present invention is labeled with a reporter fluorescent group, and the 3' end of the probe is labeled with a quencher fluorescent group; the reporter fluorescent group is selected from one of FAM, JOE, VIC, HEX, ROX, CY3, and CY5, and the quencher fluorescent group is selected from one of BHQ, TAMRA, and MGB.

[0021] Specifically, the 5' end of the probe for detecting RAB3C gene methylation is labeled with a reporter fluorescent group FAM, and the 3' end is labeled with a quencher fluorescent group BHQ; the 5' end of the probe for detecting ZNF93 gene methylation is labeled with a reporter fluorescent group ROX, and the 3' end is labeled with a quencher fluorescent group BHQ; the 5' end of the probe for detecting PAX1 gene methylation is labeled with a reporter fluorescent group CY5, and the 3' end is labeled with a quencher fluorescent group BHQ; the 5' end of the probe for detecting ACTB gene methylation is labeled with a reporter fluorescent group JOE, and the 3' end is labeled with a quencher fluorescent group BHQ.

[0022] The present invention also claims to protect the use of the primer-probe combination and the primer pair and probe for detecting the internal reference gene ACTB in detecting methylation of cervical cancer-related genes or in preparing a kit for detecting methylation of cervical cancer-related genes.

[0023] Specifically, the cervical cancer-related genes are RAB3C, ZNF93 and PAX1 genes.

[0024] The present invention also provides a kit for detecting methylation of cervical cancer-related genes, which comprises the primer and probe combination of the present invention.

[0025] Specifically, the kit comprises primer pairs and probes shown in SEQ ID NOs. 1 to 12.

[0026] Specifically, the cervical cancer-related genes are RAB3C, ZNF93 and PAX1 genes.

[0027] The kit for detecting methylation of cervical cancer-related genes described in the present invention also includes reagents required for fluorescent PCR reaction, positive quality control products and negative quality control products; the positive quality control product is human methylated genomic DNA, and the negative quality control product is human unmethylated genomic DNA.

[0028] The primer-probe combination and kit for detecting methylation of cervical cancer-related genes described herein can be used to detect cervical cancer risk and conduct cervical cancer risk assessment, and have good detection specificity, high sensitivity, and accurate detection results. Therefore, the present invention also claims protection for the use of the primer-probe combination or kit for detecting methylation of cervical cancer-related genes in the preparation of a product for detecting cervical cancer risk.

[0029] The present invention also provides a kit for detecting the risk of cervical cancer, wherein the kit contains the primer probe combination for detecting methylation of cervical cancer-related genes.

[0030] Specifically, the primer probe combination or kit for detecting methylation of cervical cancer-related genes described in the present invention can be used to detect cervical intraepithelial neoplasia (CIN) stage 2, cervical intraepithelial neoplasia stage 3 and cervical cancer, and to detect the risk of cervical cancer; although it cannot specifically distinguish which stage of cervical intraepithelial neoplasia the tested sample is in or whether it has developed into cervical cancer, the use of the primer probe combination or kit described in the present invention for detection has the advantages of being simple, non-invasive, having good specificity, high sensitivity, and accurate results. It can be used for initial screening of cervical cancer. If the test result is positive, further testing and confirmation can be carried out through methods such as cervical biopsy tissue pathology to avoid excessive screening.

[0031] Specifically, the steps for detecting the risk of cervical cancer are as follows:

[0032] S1. Extract DNA from the sample to be tested;

[0033] S2. performing sulfite conversion on the DNA obtained in step S1;

[0034] S3. Performing real-time fluorescence quantitative PCR amplification on the sulfite-converted DNA sample using the primer pairs and probes shown in SEQ ID NOs. 1 to 12;

[0035] S4. Fluorescence signal detection and result determination.

[0036] Specifically, the sample in step S1 is human cervical exfoliated cells, vaginal secretions, cervical tissue or a combination thereof; preferably human cervical exfoliated cells.

[0037] Specifically, the extraction of the DNA of the sample to be tested in step S1 includes sample lysis, DNA binding, DNA washing and elution.

[0038] Specifically, the sulfite conversion in step S2 includes sulfite conversion, binding, first washing, desulfonation, second washing, third washing, drying and elution.

[0039] Specifically, the reaction system of the real-time fluorescence quantitative PCR amplification reaction in step S3 includes: PCR buffer, dNTP, MgCl2, TaqDNA polymerase and ddH2O. The final concentration of each primer in the reaction system is 200nM, the final concentration of each probe is 100nM, and other reagents are added according to conventional methods and dosages.

[0040] Specifically, the reaction conditions of the real-time fluorescence quantitative PCR amplification reaction in step S3 are: denaturation at 95° C. for 5 min; denaturation at 95° C. for 5 s, annealing and extension at 55° C. for 35 s, and 45 cycles.

[0041] After the real-time fluorescence quantitative PCR amplification reaction is completed, the PCR amplification results are analyzed using the analysis software supporting the fluorescence PCR instrument. The difference between the Ct value of the amplified fluorescence curve and the Ct value of the internal reference gene ACTB detection primer and probe, that is, the △Ct value, is calculated and the results are judged using the △Ct value.

[0042] Specifically, the method for determining the risk of cervical cancer is as follows: calculating the difference between the Ct value of the gene where the amplification curve appears and the Ct value of the internal reference gene ACTB, that is, the ΔCt value; when ΔCt≦9, the test result is positive, indicating a high risk of cervical cancer; when ΔCt>9 or not detected, the test result is negative, indicating a low risk of cervical cancer; if there is only one ΔCt value, the result is determined based on this ΔCt value; if multiple ΔCt values ​​appear, the result is determined based on the smallest ΔCt value.

[0043] The present invention has the following beneficial effects:

[0044] The present invention designs a primer-probe combination based on the cervical cancer-associated genes RAB3C, ZNF93, and PAX1 for detecting methylation of these genes. Furthermore, a kit for detecting methylation of cervical cancer-associated genes is provided. The primer-probe combination and kit for detecting methylation of cervical cancer-associated genes described herein can be used to detect cervical cancer and its high-grade precancerous lesions, thereby assessing the risk of cervical cancer. This method has the advantages of good detection specificity, high detection sensitivity, and accurate test results. Furthermore, it is simple to operate and non-invasive, making it suitable for initial screening of cervical cancer and providing a reference for early diagnosis of cervical cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the amplification curve of RAB3C gene methylation detection.

[0046] Figure 2This is the detection amplification curve of ZNF93 gene methylation.

[0047] Figure 3 This is the detection amplification curve of PAX1 gene methylation.

[0048] Figure 4 This is the detection amplification curve of mixed methylation of RAB3C, ZNF93 and PAX1 genes. DETAILED DESCRIPTION

[0049] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0050] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0051] Example 1 Design of primers and probes

[0052] Based on the promoter sequences of cervical cancer-related genes RAB3C, ZNF93, and PAX1, the present invention designed multiple pairs of primers and probes according to primer design principles, and then obtained primer-probe combinations for detecting methylation of cervical cancer-related genes with good detection specificity and high sensitivity. The present invention also designed primer pairs and probes for detecting the internal reference gene ACTB. The sequences of some of the designed primers and probes are shown in Table 1:

[0053] Table 1 Primer and probe sequence list

[0054]

[0055]

[0056] Among them, the 5' end of the probe for detecting RAB3C gene methylation is labeled with a reporter fluorescent group FAM, and the 3' end is labeled with a quencher fluorescent group BHQ; the 5' end of the probe for detecting ZNF93 gene methylation is labeled with a reporter fluorescent group ROX, and the 3' end is labeled with a quencher fluorescent group BHQ; the 5' end of the probe for detecting PAX1 gene methylation is labeled with a reporter fluorescent group CY5, and the 3' end is labeled with a quencher fluorescent group BHQ; the 5' end of the probe for detecting ACTB gene methylation is labeled with a reporter fluorescent group JOE, and the 3' end is labeled with a quencher fluorescent group BHQ.

[0057] The present invention combined the above primers and probes and performed sensitivity and specificity tests respectively. The results are shown in Table 2:

[0058] Table 2 Sensitivity and specificity test results of primer and probe combinations

[0059]

[0060]

[0061] Based on the results of various tests, the present invention finally selected the primer pairs and probes shown in SEQ ID NOs. 1 to 11 for combination to prepare a kit for detecting methylation of cervical cancer-related genes.

[0062] Example 2 Kit for detecting methylation of cervical cancer-related genes

[0063] Based on the primer pair and probe combination obtained in Example 1, the present invention provides a kit for detecting methylation of cervical cancer-related genes (RAB3C, ZNF93 and PAX1), wherein the kit contains the primer pairs and probes shown in SEQ ID NOs. 1 to 12, and also contains reagents required for fluorescent PCR reaction, positive quality control products and negative quality control products; the positive quality control product is human methylated genomic DNA, and the negative quality control product is human unmethylated genomic DNA.

[0064] Example 3

[0065] The detection method for detecting methylation of cervical cancer-related genes using the primer-probe combination for detecting methylation of cervical cancer-related genes described in Example 1 of the present invention or the kit described in Example 2 is as follows:

[0066] 1. Materials, Reagents, and Instruments

[0067] The DNA extraction kit used in the present invention was purchased from QIAGEN; the sulfite conversion kit was purchased from QIAGEN; the PCR buffer, dNTP, and TaqDNA polymerase were purchased from Takara; MgCl2 was purchased from Sigma; and the fluorescence quantitative PCR instrument was ABI7500.

[0068] 2. Sample Preparation

[0069] Cervical exfoliated cells from patients with cervical cancer were used as test samples, cervical exfoliated cells from normal healthy people were used as negative controls, human methylated genomic DNA was used as a positive quality control, and human unmethylated genomic DNA was used as a negative quality control.

[0070] 3. DNA Extraction

[0071] 1. Add 0.2 mL of sample, 0.5 mL of nucleic acid lysis adsorption buffer, and 10 μL of magnetic beads to a 1.5 mL centrifuge tube, vortex to mix, and place the centrifuge tube at 56°C for 10 minutes;

[0072] 2. Place the centrifuge tube in a magnetic rack for 2 minutes, remove all the waste liquid, add 0.5 mL of wash solution A, and mix well to ensure that the magnetic beads are completely resuspended;

[0073] 3. Place the centrifuge tube in a magnetic rack for 2 minutes, remove all the waste liquid, and use a 10-100 μL pipette tip to remove as much residual liquid as possible. Move the centrifuge tube to a non-magnetic test tube rack, open the tube cap, and dry at room temperature for 5 minutes.

[0074] 4. Add 40 μL of elution buffer, cover the tube tightly, vortex to mix and resuspend the magnetic beads, and incubate the centrifuge tube at 56°C for 5 minutes;

[0075] 5. Place the centrifuge tube in a magnetic rack for 2 minutes and transfer all the eluate to a new 0.2 mL PCR tube.

[0076] 4. Sulfite Conversion

[0077] 1. Add 40 μL of extracted DNA to a 0.2 mL PCR tube, then add 110 μL of sulfite solution. Cap the tube tightly, vortex to mix, and briefly centrifuge. Place the tube in a standard PCR instrument for reaction. The reaction conditions are: 95°C for 5 minutes, 60°C for 10 minutes, 95°C for 5 minutes, and 60°C for 10 minutes.

[0078] 2. Transfer the DNA solution after the reaction to a new 1.5 mL centrifuge tube, add 600 μL of binding buffer and 10 μL of magnetic beads, vortex to mix, and let it stand at room temperature for 5 minutes;

[0079] 3. Place the centrifuge tube in a magnetic rack for 2 minutes, remove all the discarded liquid, add 500 μL of wash solution, and vortex to mix to ensure that the magnetic beads are completely resuspended;

[0080] 4. Place the centrifuge tube in a magnetic rack for 2 minutes, remove all the discarded liquid, add 500 μL of desulfurization solution, vortex to mix to ensure that the magnetic beads are completely resuspended, and let it stand at room temperature for 15 minutes;

[0081] 5. Place the centrifuge tube in a magnetic rack for 2 minutes, remove all the discarded liquid, add 500 μL of washing solution, and vortex to mix to ensure that the magnetic beads are completely resuspended;

[0082] 6. Place the centrifuge tube in a magnetic rack for 2 minutes, remove all the discarded liquid, add 500 μL of washing solution, and vortex to mix to ensure that the magnetic beads are completely resuspended;

[0083] 7. Place the centrifuge tube in a magnetic rack for 2 minutes, remove all the waste liquid, and use a 10-100 μL pipette tip to remove as much residual liquid as possible. Move the centrifuge tube to a non-magnetic test tube rack, open the tube cap, and dry at room temperature for 2 minutes.

[0084] 8. Add 50 μL of elution buffer, cap the tube tightly, vortex to mix and resuspend the magnetic beads, and incubate the centrifuge tube at 56°C for 5 minutes;

[0085] 9. Place the centrifuge tube in a magnetic rack for 2 minutes, and transfer all the eluate to a new centrifuge tube for later use.

[0086] 5. PCR reaction

[0087] 1. Preparation of PCR Reaction Solution

[0088] The present invention uses the detection primers and probes of a single gene and the primer-probe combination to perform fluorescent quantitative PCR reaction. The prepared PCR reaction solution systems are shown in Tables 3 to 6 respectively:

[0089] Table 3 RAB3C gene methylation detection reaction solution

[0090]

[0091]

[0092] Table 4 ZNF93 gene methylation detection reaction solution

[0093] Components of a single PCR reaction Amount of a single PCR reaction Primer shown in SEQ ID NO.4 (10 μM) 0.5μL Primer shown in SEQ ID NO.5 (10 μM) 0.5μL Probe shown in SEQ ID NO.6 (10 μM) 0.25 μL Primer shown in SEQ ID NO.10 (10 μM) 0.5μL Primer shown in SEQ ID NO.11 (10 μM) 0.5μL Probe shown in SEQ ID NO.12 (10 μM) 0.25 μL Taq DNA polymerase (5M / μL) 0.5μL dNTP (10mM) 0.5μL <![CDATA[MgCl2(25mM)]]> 4 μL PCR buffer (10×) 5μL <![CDATA[ddH2O]]> 7.5 μL Total 20 μL

[0094] Table 5 PAX1 gene methylation detection reaction solution

[0095]

[0096]

[0097] Table 6 RAB3C+ZNF93+PAX1 methylation detection reaction solution

[0098]

[0099]

[0100] 2. Add sample

[0101] Add 20 μL of prepared PCR reaction solution and 5 μL of sample DNA to the prepared PCR reaction tubes, cap the tubes tightly, and centrifuge briefly at low speed; the addition of negative and positive quality controls is the same as that of the samples.

[0102] 3. Fluorescence quantitative PCR detection

[0103] 1) Fluorescence channel selection: Select the fluorescence channel based on the fluorescent group carried by the probe. In this example, FAM, JOE, ROX, and CY5 are selected for each sample, for a total of 4 channels. Set the reference fluorescence (Passive Reference) to none;

[0104] 2) The reaction conditions were set as shown in the following table (reaction volume was set to 25 μL):

[0105]

[0106] 6. Results Analysis

[0107] 1. PCR Results Analysis

[0108] After the reaction is completed, the results are automatically saved and analyzed using the instrument software. If any of the three genes RAB3C, ZNF93, and PAX1 has an amplification curve during PCR amplification, its ΔCt value is calculated. The ΔCt value is the difference between the Ct value of any of the three genes RAB3C, ZNF93, and PAX1 and the Ct value of the ACTB gene. The size of the difference reflects the relative quantification between the tested gene and the internal reference gene.

[0109] 2. Determination of test results

[0110] The smallest △Ct value among the three △Ct values ​​of RAB3C, ZNF93 and PAX1 is used as the judgment standard. When △Ct≦9, the result is positive, indicating a high risk of cervical cancer; when △Ct>9 or ND, the result is negative, indicating a low risk of cervical cancer, where ND is the abbreviation for "Not Detected", which means "not detected".

[0111] 3. Test results

[0112] The methylation of RAB3C, ZNF93 and PAX1 genes was not detected in normal samples, and the results were all negative. They were all detected in cervical cancer samples, and the results were all positive. The amplification curves of RAB3C, ZNF93, PAX1 and the mixed detection of the three genes are shown in the following order: Figures 1 to 4 As shown by Figures 1 to 4 The results show that the primer-probe combination of the present invention can successfully detect methylated RAB3C, ZNF93, and PAX1 genes. The Ct values ​​and judgment results of the mixed detection of RAB3C, ZNF93, PAX1 and the three genes of the present invention are shown in Tables 7 to 10 respectively:

[0113] Table 7 RAB3C methylation detection results

[0114]

[0115] Table 8 ZNF93 methylation detection results

[0116]

[0117] Table 9 PAX1 methylation detection results

[0118]

[0119]

[0120] Table 10 RAB3C+ZNF93+PAX1 methylation detection results

[0121]

[0122] From the results shown in Tables 7 to 10, it can be seen that the methylation detection primers and probes for RAB3C, ZNF93 and PAX1 genes of the present invention can be used to detect cervical cancer.

[0123] Example 4 Actual sample detection

[0124] In this example, the kit described in Example 2 of the present invention was used to detect clinical samples of cervical cancer, CIN III, CIN II, and CIN I using the detection method described in Example 3 to test the sensitivity and specificity of the primers and probes for detecting methylation of cervical cancer-related genes described in the present invention for detecting cervical cancer and its high-grade precancerous lesions. The experimental results are shown below:

[0125] Table 11 RAB3C detection results

[0126]

[0127] Table 12 ZNF93 detection results

[0128]

[0129] Table 13 PAX1 detection results

[0130]

[0131]

[0132] Table 14 RAB3C+ZNF93+PAX1 detection results

[0133]

[0134] The results shown in Tables 11 to 14 show that, compared with detecting one gene alone, the combined detection of the three genes has a higher sensitivity for detecting cervical cancer, CINⅢ and CINⅡ.

[0135] As demonstrated in the above examples, the primer-probe combination and kit for detecting methylation of cervical cancer-related genes described herein can be used to assess cervical cancer risk, providing a reference for early diagnosis of cervical cancer. Compared to conventional cervical cancer diagnostic methods, the primer-probe combination or kit described herein offers the advantages of high sensitivity and specificity for cervical cancer detection, simple operation, and the ability to perform noninvasive early diagnosis of human cervical cancer.

[0136] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of a primer-probe combination for detecting methylation of cervical cancer-related genes in the preparation of a product for detecting the risk of cervical cancer, characterized in that: The primer-probe combination includes the following components: (1) A primer pair and a probe for detecting methylation of the RAB3C gene, wherein the sequences of the primer pair are shown in SEQ ID NOs. 1 and 2, and the sequence of the probe is shown in SEQ ID NO. 3; (2) A primer pair and a probe for detecting ZNF93 gene methylation, wherein the sequences of the primer pair are shown in SEQ ID NOs. 4 to 5, and the sequence of the probe is shown in SEQ ID NO. 6; (3) A primer pair and a probe for detecting PAX1 gene methylation, wherein the sequences of the primer pair are shown in SEQ ID NOs. 7 to 8, and the sequence of the probe is shown in SEQ ID NO. 9; The primer-probe combination further includes a primer pair and a probe for detecting the internal reference gene ACTB, the sequences of the primer pair are shown in SEQ ID NOs. 10-11, and the sequence of the probe is shown in SEQ ID NO. 12; The 5' end of the probe in the primer-probe combination is labeled with a reporter fluorescent group, and the 3' end of the probe in the primer-probe combination is labeled with a quencher fluorescent group; the reporter fluorescent group is selected from one of FAM, JOE, VIC, HEX, ROX, CY3, and CY5, and the quencher fluorescent group is selected from one of BHQ, TAMRA, and MGB.

2. Use of a kit for detecting methylation of cervical cancer-related genes in the preparation of a product for detecting the risk of cervical cancer, characterized in that: The cervical cancer-related genes are RAB3C, ZNF93 and PAX1 genes; the kit comprises the primer-probe combination described in claim 1.

3. The use according to claim 2, characterized in that The kit also includes reagents required for fluorescent PCR reaction, positive quality control products and negative quality control products; the positive quality control product is human methylated genomic DNA, and the negative quality control product is human unmethylated genomic DNA.

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