Cervical cancer high-risk shunt kit based on MassARRAY methylation detection

Abstract

The invention discloses a high-risk cervical cancer shunt kit based on MassARRAY methylation detection. The kit comprises an amplification primer combination, an extension primer combination, a multiple PCR (Polymerase Chain Reaction) reaction system and an extension reaction system, wherein the amplification primer combination is used for detecting methylation levels of five CpG sites of PAX1, SOX1, MIR124-2 and FAM19A4 genes. According to the kit, hydrosulfite conversion is carried out on DNA of a cervical or near-cervical cell sample, then a MassARRAY technology is combined, specific methylation detection primers are designed, amplification, extension and mass spectrometry are carried out on five CpG sites, and the methylation level is calculated according to methylation C peak HEIGHT data and unmethylated T peak HEIGHT data obtained through mass spectrometry; and finally, predicting the disease risk of the subject according to a methylation detection result. The gene methylation result provided by the invention perfects epigenetics information of gene detection, has the characteristics of large flux, high accuracy, low cost and the like, and can assist clinicians in shunting HPV positive subjects, reduce TCT leak detection and avoid excessive medical treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of methylation detection, and in particular relates to a high-risk cervical cancer triage kit based on MassARRAY methylation detection. Background Art

[0002] Cervical cancer is the second most common gynecological malignancy in the world, and ranks fourth in both morbidity and mortality among all common cancers. There are about 140,000 new cases of cervical cancer in my country each year, of which about 30,000 to 40,000 die. Over the past 30 years, the incidence and mortality of cervical cancer in developed countries have been declining, while in China, they have been on the rise. The main reason for this is the neglect of screening.

[0003] At present, the main cervical cancer screening technologies include direct visual colposcopy, cytology-based thin-layer cell testing (TCT), and molecular biology-based human papillomavirus (HPV) testing. Both colposcopy and TCT have a large degree of missed detection. Epidemiological and molecular biological studies have shown that persistent infection with high-risk HPV is the main cause of cervical cancer. High-risk HPV testing can make up for the shortcomings of colposcopy and TCT screening to a certain extent and improve the sensitivity of screening. However, about 90% of high-risk HPV infections are automatically cleared within 12 months, and only a few progress to cervical cancer. HPV testing also leads to overdiagnosis of the CIN2 population. This requires the use of other methods and markers to screen and divert high-risk populations.

[0004] DNA methylation is an epigenetic modification that can cause changes in chromatin structure, DNA conformation, DNA stability, and DNA-protein interactions without changing the DNA sequence, and has an important regulatory effect on gene expression. Hypomethylation of the promoter region of oncogenes leads to activation, and hypermethylation of the promoter region of tumor suppressor genes leads to inactivation, which also affects the occurrence and development of cervical cancer. At present, the genes whose methylation level changes can cause cervical cancer and have been studied more include paired box gene 1 (PAX1), sex determining region Y box protein 1 (SOX1), zinc finger protein 582 (ZNF582), etc.

[0005] At present, there are three main types of DNA methylation detection technologies according to their detection principles: based on methylation-sensitive endonucleases, based on sulfite treatment to convert unmethylated base C into base U, and based on other treatments to convert unmethylated base C into base U. The detection methods mainly include whole genome methylation sequencing (WGBS), reduced methylation sequencing (RRBS), methylated DNA immunoprecipitation sequencing (MeDIP-Seq), gene chip detection technology (MicroArray), methylation-specific PCR method (MSP), etc. Due to the high cost of whole genome sequencing, MassARRAY has more advantages in terms of cost, experimental operability, analysis complexity, and accuracy for the detection of representative CpG sites in the target area. At present, there are few studies on cervical cancer methylation in Asians. Therefore, it is very necessary to establish a methylation detection method and analysis model suitable for Chinese people. Summary of the invention

[0006] To solve the above problems, the purpose of the present invention is to detect the methylation levels of CpG sites of PAX1, SOX1, MIR124-2, and FAM19A4 genes based on the MassARRAY method. Through the analysis model, a more comprehensive understanding of the changes in the human body is provided, and the genetic testing information is improved from the perspective of epigenetics, which can assist doctors in diagnosis, divert high-risk groups for cervical cancer, and avoid over-medicalization.

[0007] To achieve the above object, the technical solution of the present invention is as follows:

[0008] (1) Collect exfoliated cell samples by cervical scraping or vaginal swab near the cervical opening (1 / 3 of the vaginal wall);

[0009] (2) Extracting DNA from the sample, then performing bisulfite conversion, and then testing the concentration of single-stranded DNA (ssDNA);

[0010] (3) Using the bisulfite-converted DNA as a template, multiple PCR amplification reactions were performed to obtain methylated and unmethylated amplified products. The PCR primer pool is a mixture of specific target detection fragments and quality control fragment primers, which can simultaneously amplify methylated and unmethylated fragments;

[0011] (4) subjecting the product of the amplification reaction in step (3) to a shrimp alkaline phosphatase (SAP) reaction to remove excess deoxyribonucleoside triphosphates;

[0012] (5) subjecting the product of step (4) to an extension reaction to obtain an extension product of the detected CpG site, wherein the methylated product is a C peak and the unmethylated product is a T peak;

[0013] (6) purifying the product of the extension reaction by resin and desalting;

[0014] (7) spotting the desalted product on a mass spectrometer chip matrix for co-crystallization, then transferring the crystal into a vacuum tube of a mass spectrometer, and using laser excitation for MassARRAY time-of-flight mass spectrometry detection;

[0015] (8) Calculate the methylation rate based on the HEIGHT data of the methylated C peak and the HEIGHT data of the unmethylated T peak obtained by MassARRAY time-of-flight mass spectrometry. Based on this, subjects who are HPV-positive and have cytology results of ASCUS / ASC-H / LSIL are shunted to reduce missed TCT and unnecessary colposcopy. Subjects with colposcopy results of CIN2 and CIN3 are shunted to predict the risk of cervical cancer and prevent overtreatment.

[0016] The kit provided by the present invention comprises a multiplex PCR reaction system, a SAP reaction system and an extension reaction system. The multiplex PCR reaction system comprises PCR Buffer, MgCl2, dNTPs, PCR primer Pool and PCR EpiTaq enzyme.

[0017] In the embodiment of the present invention, the PCR primer Pool is a mixture of specific target detection fragments and quality control fragment primers. The length of the amplified fragment is 100 to 400 bp, and the quality control is the imprinted gene H19.

[0018] Furthermore, the sequence of the PCR primer Pool in the multiplex PCR reaction system is as follows:

[0019]

[0020]

[0021] In an embodiment of the present invention, the extension reaction system includes Buffer Plus, Termination Mix, extension primer mixture and iPLEX Enzyme.

[0022] Further, the sequence of the extension primer mixture is as follows:

[0023] UEP1 TaGGGAGGTTGTAGIGTG SEQ ID NO:13 UEP2 cTTTTTTAGGATTTAGGATTGGGT SEQ ID NO:14 UEP3 gGTTAAAGAGGTaGAaGGTaGGGGT SEQ ID NO:15 UEP4 gGTGTAGAaGGTGaGTGTGTTTG SEQ ID NO:16 UEP5 GaGAGGTTGTAGAGGTGATGGGT SEQ ID NO:17 UEP6 TTGGGAGAGTTTGTGAGGT SEQ ID NO:18

[0024] As an improvement of the present invention, the same extension primer in step (5) can extend the C peak of the methylated product and the T peak of the unmethylated product. The extension primer mixture is an extension primer mixture that matches the sequence of the amplified product of the target detection gene and the amplified product of the quality control in step (3).

[0025] Furthermore, the HEIGHT data of the methylated C peak and the HEIGHT data of the unmethylated T peak in step (8) can be used to calculate the methylation rate of the CpG site according to the following formula:

[0026] CpG site methylation rate = C peak HEIGHT value / (C peak HEIGHT value + T peak HEIGHT value)

[0027] The methylation rates of the five CpG sites were calculated, and the positive and negative results were determined based on the calculated results. The disease risk determination method was as follows: when the number of positives among the five CpG sites was ≥1, it indicated that the subject had a high risk of cervical cancer and required further diagnosis and treatment; when all five CpG sites were negative, it indicated that the subject had a low risk of cervical cancer and regular follow-up examinations were required.

[0028]

[0029] The beneficial effects of the present invention are as follows: a MassARRAY-based methylation level detection kit provided by the present invention innovatively combines multiplex PCR and MassARRAY time-of-flight mass spectrometry for simultaneous amplification of methylated and unmethylated fragments, and each well can detect 5 CpG sites of cervical cancer-related PAX1, SOX1, MIR124-2, and FAM19A4 genes, and 384 samples can be detected simultaneously, with the characteristics of high throughput, high accuracy, and low cost. The results of gene methylation levels provide a more comprehensive view of human body changes, improve gene detection information from the perspective of epigenetics, further divert HPV-positive subjects, provide a basis for doctors' diagnosis and treatment plans, and avoid excessive medical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a scatter plot of the MassARRAY quality control site results of the present invention.

[0031] Figure 2 It is a scatter plot of the MassARRAY detection site results of the present invention.

[0032] Figure 3 It is a peak diagram of the MassARRAY detection site results of the present invention. DETAILED DESCRIPTION

[0033] The present invention will be further explained below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0034] The main abbreviations and key terms involved in the present invention are explained as follows:

[0035] PCR: Polymerase Chain Reaction;

[0036] TCT: Thinprep Cytologic Test, liquid-based thin-layer cytology examination;

[0037] HPV: Human Papillomavirus;

[0038] CIN: Cervical Intraepithelial Neoplasia;

[0039] LSIL: Low Grade Squamous Intraepithelial Lesion, low grade squamous intraepithelial lesion;

[0040] HSIL: High Grade Squamous Intraepithelial Lesion, high grade squamous intraepithelial lesion;

[0041] WGBS: Whole Genome Bisulfite Sequencing, also known as whole genome methylation sequencing, is a method that combines Bisulfite with a high-throughput sequencing platform to sequence methylation levels across the entire genome of species with a reference genome.

[0042] RRBS: Reduced Representation Bisulfite Sequencing, simplified methylation sequencing, this method is mainly used for methylation research in CpG-rich regions. It enriches the fragments of CCGG sites on genomic DNA by restriction enzyme digestion, and performs single-base resolution methylation sequencing in CpG-rich regions of the genome through Bisulfite treatment and high-throughput sequencing technology;

[0043] MeDIP-Seq: Methylated DNA immunoprecipitation sequencing, which is a large-scale chromosome or genome purification technology used to enrich DNA methylation sequences in molecular biology;

[0044] MSP: Methylation-specific PCR, a technique for detecting methylation at specific sites;

[0045] UEP: Unextended primer, extended primer;

[0046] SAP: Shrimp Alkaline Phosphatase, a high specific activity, thermolabile recombinant alkaline phosphatase;

[0047] NILM: Negative for intraepithelial lesion or malignancy, no intraepithelial lesions and malignant changes were found;

[0048] ASCUS: Atypical squamous cells of undetermined significance;

[0049] ASC-H: Atypical squamous cells-cannot exclude high-grade squamous intraepithelial lesion, atypical squamous epithelial cells that cannot exclude high-grade squamous intraepithelial lesion;

[0050] QC: Quality control, quality control.

[0051] Example 1

[0052] 1. Detection of 5 CpG sites of PAX1, SOX1, MIR124-2, and FAM19A4 genes:

[0053] (1) Preparation of DNA samples

[0054] The exfoliated cell samples were collected by cervical scraping or vaginal swab near the cervical opening (1 / 3 of the vaginal wall), and human genomic DNA was extracted using a commercial DNA extraction kit. The quality of the extracted DNA was tested by Nano300, and the OD260 / OD280 must be between 1.8-2.0 and the concentration must be >5ng / μl. The qualified DNA was placed at 4℃ for later use.

[0055] (2) Bisulfite conversion

[0056] Human genomic DNA was converted with bisulfite using a Zymo kit (Cat. No. D5006). The DNA quality was tested with Qubit ssDNA reagent and the concentration was > 2.5 ng / μl. The conversion product with qualified quality was stored at -20°C for later use.

[0057] (3) PCR amplification reaction

[0058] The multiplex PCR reaction system used in the present application contains PCR Buffer, MgCl2, dNTPs and PCREpiTaq enzyme (purchased from Takara, product number R110A), and PCR primer Pool.

[0059] In an embodiment of the present invention, the preferred CpG sites of PAX1 are CpG1 (chr20: 21705670) and CpG2 (chr20: 21706091), the CpG site of SOX1 is CpG3 (chr13: 112067399), the CpG site of MIR124-2 is CpG4 (chr8: 64378957), the CpG site of FAM19A4 is CpG5 (chr3: 68932353), and the reference genome for the position information is hg38.

[0060] In an embodiment of the present invention, the PCR amplification primer combination designed according to the CpG site is shown in Table 1, and the amplification primer combination includes specific amplification primers for 2 CpG sites of the PAX1 gene (SEQ ID NO: 1-4), specific amplification primers for 1 CpG site of the SOX1 gene (SEQ ID NO: 5-6), specific amplification primers for 1 CpG site of the MIR124-2 gene (SEQ ID NO: 7-8), specific amplification primers for 1 CpG site of the FAM19A4 gene (SEQ ID NO: 9-10), and specific amplification primers for the CpG site of the quality control H19 (SEQ ID NO: 11-12). The above amplification primers are mixed at a final concentration of 0.5 μM and prepared into a PCR primer Pool. The PCR amplification reaction system is shown in Table 2, and the reaction procedure is shown in Table 3.

[0061] Table 1. PCR amplification primer sequences

[0062]

[0063]

[0064] Table 2. PCR amplification reaction system

[0065] Reagents Single pore volume / μl <![CDATA[ddH2O]]> 0.8 10×PCRBuffer 0.5 <![CDATA[MgCl2]]> 0.8 dNTPMix 0.25 PCR Primer Pool 1 PCREpiTaq 0.2 DNA template 2 Total volume 5

[0066] Table 3. PCR amplification reaction program

[0067]

[0068] (4) SAP reaction

[0069] The SAP reaction system contains SAP enzyme and SAP Buffer (purchased from Agena BioScience, catalog number 10141). After PCR amplification, 2ul of SAP reaction solution is added to the amplified product. The SAP reaction system is shown in Table 4, and the reaction procedure is shown in Table 5.

[0070] Table 4. SAP reaction system

[0071] Reagents Single pore volume / μl <![CDATA[ddH2O]]> 1.53 SAPBuffer 0.17 SAP enzyme 0.30 Total volume 2

[0072] Table 5. SAP reaction procedure

[0073] temperature time Number of cycles 37℃ 40min 1 85℃ 5min 1

[0074] (5) Extension reaction

[0075] The extension reaction system used in this application contains Buffer Plus, Termination Mix and iPLEXEnzyme (purchased from Agena BioScience, Cat. No. 10141), and an extension primer mixture. The preferred extension primer combination sequence in this application is shown in Table 6, and the extension primer is a specific extension primer (SEQ ID NO: 13-14) for 2 CpG sites of the PAX1 gene, a specific extension primer (SEQ ID NO: 15) for 1 CpG site of the SOX1 gene, a specific extension primer (SEQ ID NO: 16) for 1 CpG site of the MIR124-2 gene, a specific extension primer (SEQ ID NO: 17) for 1 CpG site of the FAM19A4 gene, and a specific extension primer (SEQ ID NO: 18) for the CpG site of the quality control H19. The extension primers (UEP) are mixed at a final concentration of 5 μM to prepare a UEP mixture. After the SAP reaction is completed, 2ul of extension reaction solution is added to the SAP product. The extension reaction system is shown in Table 7, and the reaction procedure is shown in Table 8.

[0076] Table 6. Extension primer sequences

[0077] UEP1 TaGGGAGGTTGTAGIGTG SEQ ID NO:13 UEP2 cTTTTTTAGGATTTAGGATTGGGT SEQ ID NO:14 UEP3 gGTTAAAGAGGTaGAaGGTaGGGGT SEQ ID NO:15 UEP4 gGTGTAGAaGGTGaGTGTGTTTG SEQ ID NO:16 UEP5 GaGAGGTTGTAGAGGTGATGGGT SEQ ID NO:17 UEP6 TTGGGAGAGTTTGTGAGGT SEQ ID NO:18

[0078] Table 7. Extension reaction system

[0079] Reagents Single pore volume / μl <![CDATA[ddH2O]]> 0.619 10×BufferPlus 0.2 TerminationMix 0.2 UEP Mixture 0.94 iPLEX ProEnzyme 0.041 Total volume 2

[0080] Table 8. Extension reaction program

[0081]

[0082] (6) Desalination

[0083] Add 19 ul of ultrapure water to the product of the extension reaction, put it into the MassARRAY-CPM machine, set the machine to add 10 μl of resin, perform desalting purification, and let it stand to settle.

[0084] (7) Mass spectrometry

[0085] MassARRAY-CPM automatically spots the product on a chip and moves it into a vacuum tube, where it is excited by a laser and subjected to time-of-flight mass spectrometry.

[0086] 2. Data analysis (calculation of methylation values, gradient standard detection)

[0087] Use Typer software to open the experimental results, click View--Plate Data Pane to export the data table, obtain the HEIGHT values ​​of the methylated C peak and the unmethylated T peak, and calculate the methylation rate according to the following formula. The results are shown in Table 9.

[0088] CpG site methylation rate = C peak HEIGHT value / (C peak HEIGHT value + T peak HEIGHT value)

[0089] Table 9. Methylation rates calculated by MassARRAY method

[0090]

[0091] 3. Quality control standards

[0092] The scatter plot of the CpG site detection results of QC is shown in Figure 1 The methylation rate range of QC is 40-66%. If it exceeds the range, the experiment is considered unqualified. Therefore, S1 and S2 samples are qualified and data analysis can continue. S3 sample is unqualified and needs to be redone. The qualified samples are corrected according to the QC results. The corrected methylation rate results are shown in Table 10.

[0093] Correction factor = QC methylation rate / 50%

[0094] Table 10. Methylation rates after QC correction

[0095]

[0096]

[0097] 4. Product performance indicators

[0098] The positive reference was prepared by mixing methylated and non-methylated human gDNA standards (purchased from Takara, catalog number 3522 / 3521) in different proportions (1%, 5%, 10%). All five CpG sites of the three positive references were positive. Ten negative references were tested, and all five CpG sites were negative.

[0099] Example 2

[0100] Seven HPV-negative and 14 high-risk HPV-positive subjects were tested. The sample information is shown in Table 11 and the scatter plot of the test results is shown in Figure 2 , see the peak diagram example Figure 3 According to the formula, the methylation rate and positive and negative results are shown in Table 12. According to the test results of each CpG site, the risk assessment level of the sample is obtained, as shown in Table 13.

[0101] Table 11. Sample information

[0102]

[0103] Table 12. Test results

[0104]

[0105]

[0106]

[0107]

[0108] Table 13. Risk Assessment

[0109]

[0110] Seven HPV-negative and 14 high-risk HPV-positive subjects were tested to assess the risk of cervical cancer and compare it with clinical information. The American Society for Colposcopy and Cervical Pathology (ASCCP) recommends the risk of CIN3 and above lesions as the primary clinical endpoint. Most CIN2 lesions can gradually regress naturally, and CIN3 subjects are more likely to progress to cervical cancer. From the five biopsy CIN3 samples, the missed detection rate of TCT results was very high, three of which were ASCUS and one was NILM. Among the results of methylation prediction, there were four high risks and one low risk, with a sensitivity of 80%, which was highly consistent with the clinical situation. Compared with TCT, methylation results can better predict whether the subject needs treatment. Among the two CIN1, six CIN2 and one CIN2-3 samples, three samples were predicted to be high risk, indicating that the subjects may need more active observation or treatment, while low-risk subjects can be observed in stages. In clinical applications, most CIN2 subjects also underwent local cervical excision surgery. Methylation testing can assess the risk of cervical cancer through non-invasive sampling, playing a role in high-risk diversion. Doctors can re-formulate diagnosis and treatment plans based on risk levels to avoid over-medicalization. The results of gene methylation levels provide a more comprehensive understanding of human changes, improve genetic testing information from an epigenetic perspective, and are more conducive to assisting doctors in clinically determining the course of cervical cancer.

[0111] The present invention develops a high-risk cervical cancer triage kit based on MassARRAY methylation detection, which uses a MassARRAY-based method to detect the methylation levels of CpG sites of PAX1, SOX1, MIR124-2, and FAM19A4 genes. Through the analysis model, a more comprehensive understanding of human body changes is provided, and genetic detection information is improved from the perspective of epigenetics, which helps to assist doctors in diagnosis, triage high-risk groups for cervical cancer, and avoid over-medicalization.

[0112] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0113] It should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made on the basis of the above embodiments without departing from the principles of the present invention. These improvements and modifications all fall within the scope of protection of the claims of the present invention.

Claims

1. A high-risk cervical cancer triage kit based on MassARRAY methylation detection, characterized in that: The kit comprises an amplification primer combination, an extension primer combination, a multiplex PCR reaction system, and an extension reaction system for detecting the methylation levels of five CpG sites of PAX1, SOX1, MIR124-2 and FAM19A4 genes.

2. A high-risk cervical cancer triage kit based on MasaARRAY methylation detection according to claim 1, characterized in that: The amplification primer combination includes specific amplification primers for detecting the methylation levels of 5 CpG sites of PAX1, SOX1, MIR124-2 and FAM19A4 genes: The specific amplification primers for the CpG1 site of the PAX1 gene are shown in SEQ ID NO: 1-2. The specific amplification primers for the CpG2 site of the PAX1 gene are shown in SEQ ID NO: 3-4. The specific amplification primers for the CpG3 site of the SOX1 gene are shown in SEQ ID NO: 5-6. The specific amplification primers for the CpG4 site of the MIR124-2 gene are shown in SEQ ID NOs: 7-8. The specific amplification primers for the CpG5 site of the FAM19A4 gene are shown in SEQ ID NOs: 9-10.

3. A high-risk cervical cancer triage kit based on MassARRAY methylation detection according to claim 1, characterized in that: The extended primer combination includes specific extended primers for detecting the methylation levels of 5 CpG sites of PAX1, SOX1, MIR124-2 and FAM19A4 genes: The specific extension primer for the CpG1 site of the PAX1 gene is shown in SEQ ID NO: 13, The specific extension primer for the CpG2 site of the PAX1 gene is shown in SEQ ID NO: 14, The specific extension primer for the CpG3 site of the SOX1 gene is shown in SEQ ID NO: 15, The specific extension primer for the CpG4 site of the MIR124-2 gene is shown in SEQ ID NO: 16, The specific extension primer for the CpG5 site of the FAM19A4 gene is shown in SEQ ID NO:

17.

4. A high-risk cervical cancer triage kit based on MassARRAY methylation detection according to claim 1, characterized in that: The kit comprises an amplification primer combination for detecting the quality control CpG site, as shown in SEQ ID NO: 11-12; an extension primer combination for detecting the quality control CpG site, as shown in SEQ ID NO: 18, and the quality control is the CpG site of the imprinted gene H19.

5. The high-risk cervical cancer triage kit based on MassARRAY methylation detection according to claim 1, characterized in that: The multiplex PCR reaction system comprises PCR Buffer, MgCl2, dNTPs, PCR EpiTaq enzyme, and a PCR primer Pool in which the primers of SEQ ID NO: 1-12 as described in claims 2 and 4 are mixed at a final concentration of 0.5 μM.

6. A high-risk cervical cancer triage kit based on MassARRAY methylation detection according to claim 1, characterized in that: The extension reaction system comprises Buffer Plus, Termination Mix, iPLEX Enzyme and an extension primer mixture in which the primers of SEQ ID NOs: 13-18 as described in claims 3 and 4 are mixed at a final concentration of 5 μM.

7. A high-risk cervical cancer triage kit based on MassARRAY methylation detection according to claim 5, characterized in that: The PCR reaction conditions of the PCR reaction system of the kit are as follows:

8. The high-risk cervical cancer triage kit based on MassARRAY methylation detection according to claim 6, characterized in that: The extension reaction conditions of the kit extension reaction system are as follows:

9. Use of the reagent system and reaction conditions described in any one of claims 1 to 8 in the preparation of a reagent for high-risk triage of cervical cancer.

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