Cervical cancer risk assessment detection system and kit
By combining ARMS-PCR and capillary electrophoresis, the problems of efficiency, accuracy, and cost in existing cervical cancer susceptibility gene detection technologies have been solved, and a high-throughput cervical cancer risk assessment detection system has been constructed.
Patent Information
- Application Number
- CN202511485760.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-17
- Publication Date
- 2026-01-13
AI Technical Summary
Existing technologies are insufficient to achieve efficient, accurate, low-cost, and high-throughput cervical cancer susceptibility gene testing. Traditional methods are cumbersome, costly, and prone to errors, making it difficult to meet the needs of simultaneous testing of multiple genes and multiple loci.
ARMS-PCR technology combined with capillary electrophoresis was used to design specific primers to detect multiple SNP sites associated with cervical cancer susceptibility. High-resolution multi-fragment synchronous separation was achieved by combining capillary electrophoresis, and a SNP site multiplex amplification detection system was constructed.
It achieves efficient, accurate, and low-cost simultaneous detection of multiple genes and multiple sites, with high throughput and speed, suitable for large-scale sample testing, and the detection results are highly accurate.
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Figure CN121320535A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of nucleic acid detection technology, and in particular to a cervical cancer risk assessment detection system and kit. Background Technology
[0002] Studies have confirmed that the development of cervical cancer is the result of the combined effects of genetic and environmental factors, with genetic factors playing a crucial role in individual susceptibility. Single nucleotide polymorphisms (SNPs) in specific genes are an important molecular basis for genetic susceptibility. Numerous studies have shown that SNP site variations in multiple genes are closely related to the risk of cervical cancer: MTHFR, a key enzyme gene for folate metabolism, can have its SNP site variations affect folate metabolism efficiency, thereby interfering with DNA synthesis and repair processes and increasing the risk of cervical cancer; GSTM1, an important detoxification and metabolism gene, can have its site variations weaken the body's ability to clear harmful substances, leading to the accumulation of harmful substances and inducing malignant transformation of cervical cells; and p53, a classic tumor suppressor gene, can have its tumor suppressor function impaired by SNP site mutations, resulting in the loss of regulation of abnormal cell proliferation.
[0003] Accurate detection of the aforementioned susceptibility gene SNP loci is crucial for early risk assessment, screening, and prevention of cervical cancer. Current clinical detection technologies have significant limitations: traditional Sanger sequencing, while highly accurate, is cumbersome and has low throughput, making it difficult to meet the needs of simultaneous multi-gene, multi-locus detection; quantitative real-time PCR can rapidly detect a small number of loci, but when detecting multiple genes simultaneously, cross-interference between primers can lead to inaccurate results; agarose gel electrophoresis has low resolution, cannot accurately distinguish SNP genotypes, and has a high false-positive rate; while next-generation sequencing provides rich genetic information, it is expensive, complex, has a long testing cycle, and is difficult to analyze data, hindering its large-scale deployment in primary healthcare institutions. Based on this situation, there is an urgent need in this field for an efficient, accurate, low-cost, and high-throughput cervical cancer susceptibility gene detection system to provide better technical support for early risk assessment of cervical cancer. Summary of the Invention
[0004] The purpose of this invention is to disclose a cervical cancer risk assessment and detection system and kit to solve one or more technical problems existing in the existing methods and to provide at least one beneficial option or create conditions.
[0005] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of this invention is to provide a SNP site multiplex amplification detection system.
[0006] A second aspect of the present invention is to provide a kit containing the SNP site multiplex amplification detection system described in the first aspect of the present invention.
[0007] A third aspect of the present invention is to provide the application directions of the SNP site multiplex amplification detection system described in the first aspect of the present invention or the kit described in the second aspect of the present invention.
[0008] The SNP site multiplex amplification detection system of the first aspect of the present invention includes a primer combination for genotyping detection of 10 SNP sites, wherein the SNP sites include rs1801133, rs25487, rs1799782, rs861539, rs4646903, rs1042522, rs4025935, rs11556218, rs361525, and rs4150407. The rs1801133 site is a SNP site in the MTHFR gene, associated with the folic acid metabolism pathway; the rs25487 and rs1799782 sites are SNP sites in the XRCC1 gene, the rs861539 site is a SNP site in the XRCC3 gene, and the rs4150407 site is a SNP site in the ERCC1 gene, all three of which are associated with DNA damage repair function; the rs4025935 site is a SNP site in the GSTM1 gene, and the rs4646903 site is a SNP site in the CYP1A1 gene, both of which are associated with detoxification metabolism; the rs11556218 site is a SNP site in the IL-16 gene, and the rs361525 site is a SNP site in the TNF-α gene, both of which are associated with immune regulation; and the rs1042522 site is a SNP site in the P53 gene, associated with the tumor suppressor pathway. The SNP site multiplex amplification detection system can simultaneously detect multiple gene SNP sites closely related to cervical cancer susceptibility, and features high detection efficiency, fast speed, low cost, and suitability for large-scale sample testing.
[0009] In some embodiments of the present invention, the primer combination includes primers with nucleotide sequences as shown in SEQ ID No:1~29.
[0010] In some embodiments of the present invention, in the primer set for detecting the same SNP site, at least one primer has a fluorescent group modified at its 5' end. Preferably, the fluorescent group is selected from FAM, HEX, TAMRA, SUM, or VIG.
[0011] In some embodiments of the present invention, specific primer combination information is shown in Table 1.
[0012] Table 1. Primer combination information for SNP site multiplex amplification detection system
[0013] In some embodiments of the present invention, the total volume of the SNP site multiplex amplification detection system is 10.0 μL, including 4.0 μL of PCR premix, 2.0 μL of primer combination, 1.0 μL of genomic DNA, and the remainder is made up by ddH2O.
[0014] In some embodiments of the present invention, the PCR premix includes Taq DNA polymerase, dNTPs, MgCl2, and PCR buffer.
[0015] In some embodiments of the present invention, the amplification program of the SNP site multiplex amplification detection system is as follows: pre-denaturation at 95°C for 2 minutes; denaturation at 94°C for 30 seconds; annealing at 60°C for 1 minute; extension at 72°C for 1 minute, for a total of 30 cycles; final extension at 72°C for 10 minutes; and maintenance at 4°C.
[0016] The kit described in the second aspect of the present invention includes the SNP site multiplex amplification detection system.
[0017] In some embodiments of the present invention, the kit further includes a positive standard, which is a mixture of wild-type and mutant sequence fragments of rs1801133, rs25487, rs1799782, rs861539, rs4646903, rs1042522, rs4025935, rs11556218, rs361525 and rs4150407 sites.
[0018] In some embodiments of the present invention, the method of using the reagent kit includes the following steps: (1) Collect the samples and extract genomic DNA using the Chelex-100 method; (2) Using the combined primers, mix with PCR premixed mix, sample genomic DNA and ddH2O to form a reaction system, and perform PCR amplification according to the described multiplex amplification reaction procedure; (3) Take 1.0 μL of the amplification product and 0.3 μL of internal standard SIZ and add them to 10.0 μL of formamide. After denaturation at 95°C for 3 minutes, incubate on ice for 3 minutes. Then, use a genetic analyzer to perform capillary electrophoresis detection and use analysis software to analyze the detection results.
[0019] The third aspect of the invention describes the use of the SNP site multiplex amplification detection system described in the first aspect of the invention or the kit described in the second aspect of the invention to detect tissues, body fluids or excrement that have been removed from the human body, thereby obtaining information on intermediate results and indicating whether the tested subject is at risk of cervical cancer infection.
[0020] This invention is based on ARMS-PCR technology. Using specific primers targeting specific SNP sites, it can accurately identify wild-type and mutant genotypes, offering advantages of high specificity and sensitivity. Capillary electrophoresis, with its high resolution (able to distinguish fragments differing by 1-2 bp in length), enables simultaneous and efficient separation of multiple fragments, combining high throughput and speed. Combining ARMS-PCR and capillary electrophoresis is expected to integrate the advantages of both, overcoming the shortcomings of existing technologies in accuracy, throughput, cost, and ease of operation. This will lead to the construction of a highly efficient, accurate, low-cost, and high-throughput SNP site multiplex amplification detection system, providing superior technical support for early risk assessment of cervical cancer. Attached Figure Description
[0021] Figure 1 This is the genotyping map of the primer combinations screened in Example 1; Figure 2 This is the genotyping pattern of the rs1799782 site before modification in Example 1; Figure 3 This is the genotyping map of the rs4646903 site before modification in Example 1; Figure 4 This is the genotyping pattern before modification of the rs25487 site in Example 1; Figure 5 This is the chromatogram of the positive standard in Example 3; Figure 6 This is a typing map of an oral sample from Example 4. Detailed Implementation
[0022] Unless otherwise specified, the molecular biology experimental methods described in the following examples were performed in accordance with Molecular Cloning: A Laboratory Manual (3rd Edition) or the product instructions. Unless otherwise specified, the biological materials used in these methods are commercially available.
[0023] Example 1: ARMS genotyping primer design for SNP site multiplex amplification detection system By comprehensively analyzing the distribution frequency of SNP sites in various genes across different populations and the strength of their association with the risk of cervical cancer, 10 key SNP sites from 9 genes were ultimately identified as detection targets. These include: MTHFR (rs1801133) in the folic acid metabolism pathway; XRCC1 (rs25487, rs1799782), XRCC3 (rs861539), and ERCC1 (rs4150407) related to DNA damage repair; GSTM1 (rs4025935) and CYP1A1 (rs4646903) related to detoxification metabolism; IL-16 (rs11556218) and TNF-α (rs361525) related to immune regulation; and P53 (rs1042522) related to the tumor suppressor pathway. All of these sites have been validated by multiple independent studies and show a significant statistical association with the occurrence and development of cervical cancer (P<0.05), making them suitable for simultaneous multi-target detection of single nucleotide polymorphisms for cervical cancer risk assessment.
[0024] The designed ARMS primers were synthesized and systematically tested and screened to ensure the accuracy and stability of genotyping. Using artificially synthesized DNA fragments containing the corresponding SNP loci and flanking sequences as templates, and double-distilled water as a blank control, ARMS-PCR amplification experiments were performed using the synthesized primers. The amplification products were detected using an ABI 3130XL genetic analyzer, and the primers' ability to distinguish between different genotypes was determined based on the position, height, and area of the peaks in the capillary electrophoresis pattern.
[0025] After multiple rounds of testing and screening, primer combinations with high amplification efficiency, strong specificity, and the ability to accurately distinguish between wild-type and mutant alleles were identified and can be used to construct subsequent multiplex detection systems. Information on the final primer combinations with good genotyping performance is shown in Table 1, and the primer screening map is available in [link to primer screening map]. Figure 1 .
[0026] Characteristics of primer amplification for genotyping of gene loci related to metabolic pathways, such as Figure 1 As shown in section (A): the primer amplification spectrum of the MTHFR gene at the rs1801133 site has a length range of 166~170 bp, and the C-type peak position is about 4 bp larger than the T-type peak position; the primer amplification spectrum of the GSTM1 gene at the rs4025935 site has a length range of 187~190 bp, and the functional peak position is about 2 bp larger than the deletion peak position; the primer amplification spectrum of the CYP1A1 gene at the rs4646903 site has a length range of 233~237 bp, and the T-type peak position is about 4 bp larger than the C-type peak position.
[0027] Amplification characteristics of genotyping primers for DNA damage repair-related loci, such as Figure 1As shown in section (B): The amplification spectrum of the XRCC1 gene at the rs25487 site has a length range of 105~111 bp, with the G-type peak position being 6 bp larger than the A-type peak position; the amplification spectrum of the XRCC1 gene at the rs1799782 site has a length range of 127~130 bp, with the T-type peak position being 3 bp larger than the C-type peak position; the amplification spectrum of the XRCC3 gene at the rs861539 site has a length range of 140~146 bp, with the T-type peak position being approximately 4 bp larger than the C-type peak position; the amplification spectrum of the ERCC1 gene at the rs4150407 site has a length range of 220~224 bp, with the C-type peak position being approximately 4 bp larger than the T-type peak position.
[0028] Amplification characteristics of primers for genotyping gene loci related to immune regulation, such as Figure 1 As shown in section (C): the primer amplification spectrum of the IL-16 gene at the rs11556218 site has a length range of 75~77 bp, and the T-type peak position is about 2 bp larger than the G-type peak position; the primer amplification spectrum of the TNF-α gene at the rs361525 site has a length range of 264~266 bp, and the C-type peak position is about 2 bp larger than the T-type peak position.
[0029] Characteristics of primer amplification for genotyping of tumor suppressor pathways, such as Figure 1 As shown in section (D): the primer amplification spectrum of the P53 gene at the rs1042522 site ranges from 179 to 184 bp, and the C-type peak position is about 5 bp larger than the G-type peak position.
[0030] The screening process needs to meet core factors such as high specificity, high amplification efficiency, and no interference.
[0031] High specificity means that the primer must bind to the target genotype template only for amplification, ensuring clear genotyping. Taking the rs1799782 locus as an example, the initial primers for its wild-type (C genotype) were set to 5'-GGATGTCTTGTTGATCCG-3' (SEQ ID No:30, target fragment 156 bp). However, the amplification results of this primer set were as follows... Figure 2 As shown, both wild-type (C-genotype) and mutant (T-genotype) samples amplified bands, making genotype differentiation impossible. Analysis revealed the problem lay in the wild-type primer's lack of an effective mismatch at the 3' end; a single base mismatch with the T-genotype template allowed for amplification. The redesigned primers introduced a mismatched base near the 3' end, significantly improving specificity for the target fragment and achieving accurate genotyping.
[0032] High amplification efficiency requires highly sensitive primers to ensure the amplification of low concentrations of the target DNA sequence. Taking the rs4646903 site as an example, the initial primer for its wild-type (T-genotype) was set to 5'-GAGACTCGTGTGAGCACA-3' (SEQ ID No: 31, target fragment 233 bp). The amplification results of the primer set are as follows... Figure 3 As shown, although there was no amplification in mutant (C-genotype) samples, the amplification peak for wild-type (T-genotype) samples was very low, posing a risk of low sensitivity. In the case of low-quality templates, insufficient amplification efficiency may occur. Analysis revealed that the problem lay in a six-base mismatch at the 3' end of the primer with other regions of the target site, ΔG = -11.0 kcal / mol, which significantly reduced amplification efficiency. The redesigned primers, with ΔG ≥ -5.0 kcal / mol, significantly improved amplification efficiency.
[0033] Non-interference means that the primers must avoid non-target amplification products to prevent interference with genotyping. Taking the rs25487 locus as an example, the primers for the wild-type (A genotype) were initially designed as 5'-GGCGTGTGAGGCCTTACCTCT-3' (SEQ ID No:32, target fragment 105 bp), and the primers for the mutant (G genotype) were initially designed as 5'-GGTTGGCGTGTGAGGCCTTACCTCT-3' (SEQ ID No:33, target fragment 111 bp). The amplification results of the primer sets are as follows... Figure 4 As shown, when both are added to the reaction system, a non-target product appears at approximately 388 bp, interfering with genotype determination. Analysis revealed that the hybrid peak was caused by cross-primer pairing. The mutant primer at rs25487 (G genotype) paired unexpectedly with the downstream primer at rs1042522, amplifying a non-target product of approximately 388 bp.
[0034] Example 2: Establishment of a multiplex fluorescently labeled allele typing system for simultaneous detection of 10 SNP loci The SNP genotyping primers successfully validated in Example 1 were added one by one to the same system for testing to construct a fluorescently labeled multiplex amplification system. Since the amplification product fragment size and primer amplification efficiency differ for each SNP site, the concentration of each primer needed to be finely optimized. Through multiple experiments, the optimal concentration ratio of each primer in the same reaction system was determined to avoid competitive inhibition between primers and ensure effective amplification of each SNP site. Simultaneously, other components in the PCR reaction system, such as DNA template concentration, dNTP concentration, and DNA polymerase dosage, were optimized to ensure the high efficiency and stability of the entire amplification reaction. To determine the ideal annealing and extension temperatures, gradient PCR technology was used to conduct temperature gradient experiments within a preset temperature range. After the above optimization, the various parameters in the multiplex amplification were determined, ensuring that the amplification products met the requirements of equilibrium and specificity. The primer concentrations (as shown in Table 1), reaction system (Table 2), and reaction procedure (Table 3) of the final multiplex amplification system are shown below. The PCR premix was purchased from Sangon Biotech (Shanghai) Co., Ltd., product number B110006-0001.
[0035] Table 2. Reaction system for multiplex amplification
[0036] Table 3. Reaction Procedure for Multiplex Amplification
[0037] Example 3: Preparation of positive standards To ensure the accuracy and reliability of the detection system, positive standards were prepared for each SNP locus. The positive standards for this detection system were obtained by mixing the DNA sequences of 20 detection targets (wild-type and mutant) at each SNP locus in equal proportions.
[0038] The positive standard was subjected to PCR amplification and capillary electrophoresis analysis according to the reaction system and procedure determined in Example 2. The resulting complexation site detection pattern is shown in [Figure 2]. Figure 5 By comparing the capillary electrophoresis patterns of the test sample and the positive standard, potential problems during the experiment can be identified in a timely manner, such as amplification failure, non-specific amplification, and abnormal detection signals, thereby ensuring the reliability of the test results.
[0039] Example 4: Application Verification of the Detection System DNA was extracted from 10 oral epithelial cell samples from female volunteers using the Chelex-100 method. The specific steps were as follows: A saliva card was placed in a sterile 1.5 mL centrifuge tube using a 1.0 mm aperture punch, and 50 μL of 5% Chelex-100 was added, followed by shaking for a few seconds; the tube was incubated at 56°C for 30 minutes, followed by shaking for a few seconds; then incubated at 98°C for 10 minutes, followed by shaking for a few seconds; finally, the tube was centrifuged at 12000 rpm for 3 minutes, and the supernatant was collected as the extracted genomic DNA. Amplification was performed according to the reaction system and procedure described in Example 3. The amplified products were analyzed using a 3130XL genetic analyzer; simultaneously, the samples were sent to a sequencing company for Sanger sequencing.
[0040] One oral epithelial cell sample was selected for specific analysis. The detection spectrum of this sample using the SNP site multiplex amplification detection system established in this invention is as follows. Figure 6 .
[0041] Table 4. Sanger sequencing results of an oral cavity sample.
[0042] The results showed that the rs1801133 locus was genotyped as CT; rs25487 as AG; rs1799782 as TT; rs861539 as CC; rs1042522 as GG; rs4025935 as deletion; rs4646903 as CC; rs11556218 as TT; rs361525 as TC; and rs4150407 as CT. The Sanger sequencing results for this sample are shown in Table 4. Comparison of capillary electrophoresis results and Sanger sequencing results showed complete consistency, indicating that the kit developed in this study has good accuracy.
[0043] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.
Claims
1. A SNP site multiplex amplification detection system, characterized in that, It includes primer combinations for genotyping detection of 10 SNP sites, including rs1801133, rs25487, rs1799782, rs861539, rs4646903, rs1042522, rs4025935, rs11556218, rs361525, and rs4150407.
2. The SNP site multiplex amplification detection system according to claim 1, characterized in that, The primer sets include: primer sets with nucleotide sequences as shown in SEQ ID No: 1-3 for detecting the rs1801133 site; primer sets with nucleotide sequences as shown in SEQ ID No: 4-6 for detecting the rs25487 site; primer sets with nucleotide sequences as shown in SEQ ID No: 7-9 for detecting the rs1799782 site; primer sets with nucleotide sequences as shown in SEQ ID No: 10-12 for detecting the rs861539 site; primer sets with nucleotide sequences as shown in SEQ ID No: 13-15 for detecting the rs4646903 site; primer sets with nucleotide sequences as shown in SEQ ID No: 16-18 for detecting the rs1042522 site; primer sets with nucleotide sequences as shown in SEQ ID No: 19-20 for detecting the rs4025935 site; primer sets with nucleotide sequences as shown in SEQ ID No: 21-23 for detecting the rs11556218 site; and primer sets with nucleotide sequences as shown in SEQ ID No: 1-3 for detecting the rs1801133 site. Primer sets shown in ID No: 24~26 are used to detect the rs361525 site; primer sets with nucleotide sequences shown in SEQ ID No: 27~29 are used to detect the rs4150407 site.
3. The SNP site multiplex amplification detection system according to claim 2, characterized in that, The concentrations of the primers with the nucleotide sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, and SEQ ID No. 11 are 0.10 μmol / L and 0.05 μmol / L, respectively. μmol / L; the concentration of primers with nucleotide sequences as shown in SEQ ID No. 12 is 0.07 μmol / L; the concentration of primers with nucleotide sequences as shown in SEQ ID No. 13 is 0.04 μmol / L; the concentration of primers with nucleotide sequences as shown in SEQ ID No. 14 is 0.03 μmol / L; the concentration of primers with nucleotide sequences as shown in SEQ ID No. 15 is 0.03 μmol / L; the concentration of primers with nucleotide sequences as shown in SEQ ID No. 16 is 0.07 μmol / L; the concentration of primers with nucleotide sequences as shown in SEQ ID No. 17 is 0.07 μmol / L; the concentration of primers with nucleotide sequences as shown in SEQ ID No. 18 is 0.07 μmol / L; the concentration of primers with nucleotide sequences as shown in SEQ ID No. 19 is 0.12 μmol / L; the concentration of primers with nucleotide sequences as shown in SEQ ID No. 20 is 0.12 μmol / L; the concentration of primers with nucleotide sequences as shown in SEQ ID No. 21 is 0.06 μmol / L; the concentration of primers with nucleotide sequences as shown in SEQ ID No. 12 is 0.07 μmol / L; the concentration of primers with nucleotide sequences as shown in SEQ ID No. 13 is 0.04 μmol / L; the concentration of primers with nucleotide sequences as shown in SEQ ID No. 14 is 0.03 μmol / L; the concentration of primers with nucleotide sequences as shown in SEQ ID No. 15 is 0.03 μmol / L; the concentration of primers with nucleotide sequences as shown in SEQ ID No. 16 is 0.07 μmol / L; the concentration of primers with nucleotide sequences as shown in SEQ ID No. 17 is 0.07 μmol / L; the concentration of primers with nucleotide sequences as The concentration of primer No. 22 is 0.03 μmol / L; the concentration of primer with nucleotide sequence as shown in SEQ ID No. 23 is 0.04 μmol / L; the concentration of primer with nucleotide sequence as shown in SEQ ID No. 24 is 0.07 μmol / L; the concentration of primer with nucleotide sequence as shown in SEQ ID No. 25 is 0.07 μmol / L; and the concentration of primer with nucleotide sequence as shown in SEQ ID No. 26 is 0.The concentration of primers with nucleotide sequences as shown in SEQ ID No. 27 is 0.13 μmol / L; the concentration of primers with nucleotide sequences as shown in SEQ ID No. 28 is 0.08 μmol / L; and the concentration of primers with nucleotide sequences as shown in SEQ ID No. 29 is 0.10 μmol / L.
4. The SNP site multiplex amplification detection system according to claim 2, characterized in that, At least one primer in each primer set has a fluorescent group modified at its 5' end.
5. The SNP site multiplex amplification detection system according to claim 4, characterized in that, The fluorescent group is selected from FAM, HEX, TAMRA, SUM, or VIG.
6. The SNP site multiplex amplification detection system according to any one of claims 1 to 5, characterized in that, The total volume of the detection system is 10.0 μL, including 4.0 μL of PCR premix, 2.0 μL of primer combination, 1.0 μL of genomic DNA, and the remainder is made up by ddH2O.
7. The SNP site multiplex amplification detection system according to claim 6, characterized in that, The PCR premix includes Taq DNA polymerase, dNTPs, MgCl2, and PCR buffer.
8. A reagent kit, characterized in that, Includes the SNP site multiplex amplification detection system as described in any one of claims 1 to 7.
9. The reagent kit according to claim 8, characterized in that, It also includes positive standards, which are composed of wild-type and mutant sequence fragments of rs1801133, rs25487, rs1799782, rs861539, rs4646903, rs1042522, rs4025935, rs11556218, rs361525 and rs4150407.
10. The use of the SNP site multiplex amplification detection system according to any one of claims 1 to 7 or the kit according to any one of claims 8 to 9 in obtaining information on intermediate results of reducing the risk of cervical cancer.