Primer probe combination for detection of high-grade squamous intraepithelial lesion and cervical cancer and use thereof

Abstract

The application provides a primer probe combination for detection of high-grade squamous intraepithelial lesion (HSIL) and cervical cancer and use thereof. The primer probe combination comprises a PCR primer probe combination for SOX1 methylation detection, and comprises a first primer probe combination, a second primer probe combination or a third primer probe combination. The primer probe combination provided by the application can cover HSIL and cervical cancer, and can distinguish HSIL and cervical cancer from LSIL (low-grade squamous intraepithelial lesion) and healthy people, so that early intervention and timely treatment of HSIL patients can be realized, and the final formation of cervical cancer caused by missed detection of HSIL can be effectively avoided, and reasonable diversion management of high-risk human papilloma virus (HPV) positive infection persons can be realized, and a basis for determining whether the positive infection persons need to be subjected to colposcopy and / or histopathological examination is provided.

Description

Technical Field

[0001] This application relates to the field of gene detection technology, specifically to a primer-probe combination for detecting high-grade squamous intraepithelial lesions and cervical cancer, and its uses. Background Technology

[0002] Cervical cancer is a malignant tumor that occurs in the cervix. Its development is a long, slow, and progressive disease process. Cervical cancer is mostly caused by persistent infection with high-risk human papillomavirus (HPV). Cervical epithelial cells gradually undergo abnormal proliferation from a normal state, forming cervical squamous intraepithelial lesions (HSIL). This stage is classified into CIN1, CIN2, and CIN3 according to severity. CIN1 is mostly a low-grade lesion with the possibility of spontaneous remission, while CIN2 and CIN3 belong to high-grade HSIL. If not intervened in time, the abnormal cells will gradually break through the epithelial basement membrane and infiltrate deeper layers, eventually developing into invasive cervical cancer, which can spread to surrounding tissues and metastasize to distant sites. Due to this long precancerous stage, regular cervical screening can effectively prevent its transformation into cancer.

[0003] Current technologies typically employ cytological testing and gene methylation detection for screening. However, cytological testing suffers from low sensitivity, a high false negative rate, and is heavily influenced by the experience of the testing personnel. Gene methylation detection includes both single-marker and multi-marker combinations. For single-marker detection, most protocols focus solely on cervical cancer detection, failing to identify high-risk individuals in the precancerous stage. This leads to missed diagnoses of numerous HSIL patients, delaying intervention and increasing the risk of progression to cervical cancer. For multi-marker combinations, multiple target genes are usually incorporated to compensate for the diagnostic efficacy of a single marker. This can result in non-specific amplification, causing a decrease in sensitivity and specificity.

[0004] Therefore, how to effectively diagnose cervical cancer, high-grade squamous intraepithelial lesions, and low-grade squamous intraepithelial lesions at an early stage, and how to triage and manage high-risk human papillomavirus-positive individuals to achieve early detection and intervention of cervical cancer and precancerous lesions are urgent technical problems that need to be solved. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the purpose of this application is to provide a primer-probe combination for the detection of high-grade squamous intraepithelial lesions and cervical cancer, and its uses.

[0006] To achieve the above objectives, the embodiments of this application propose the following technical solutions: In a first aspect, embodiments of this application provide a primer-probe combination for detecting high-grade squamous intraepithelial lesions and cervical cancer, the primer-probe combination comprising: PCR primer-probe combinations for SOX1 methylation detection include a first primer-probe combination, a second primer-probe combination, or a third primer-probe combination. The first primer-probe combination includes an upstream primer as shown in SEQ ID NO.1, a downstream primer as shown in SEQ ID NO.2, and a fluorescent probe as shown in SEQ ID NO.3; The second primer-probe combination includes an upstream primer as shown in SEQ ID NO.4, a downstream primer as shown in SEQ ID NO.5, and a fluorescent probe as shown in SEQ ID NO.6; The third primer-probe combination includes an upstream primer as shown in SEQ ID NO.7, a downstream primer as shown in SEQ ID NO.8, and a fluorescent probe as shown in SEQ ID NO.9.

[0007] In one embodiment, the primer-probe combination further includes an internal standard first primer-probe combination, an internal standard second primer-probe combination, or an internal standard third primer-probe combination for detecting the internal reference gene GAPDH. The internal standard first primer-probe combination includes an upstream primer as shown in SEQ ID NO.10, a downstream primer as shown in SEQ ID NO.11, and a fluorescent probe as shown in SEQ ID NO.12; The internal standard second primer-probe combination includes an upstream primer as shown in SEQ ID NO.13, a downstream primer as shown in SEQ ID NO.14, and a fluorescent probe as shown in SEQ ID NO.15; The internal standard third primer-probe combination includes an upstream primer as shown in SEQ ID NO.16, a downstream primer as shown in SEQ ID NO.17, and a fluorescent probe as shown in SEQ ID NO.18.

[0008] In one embodiment, the 5' end of the fluorescent probe contains a fluorescent group; The fluorescent group is selected from FAM, VIC, HEX, NED, ROX, TET, JOE, TAMRA, CY3 or CY5.

[0009] In one embodiment, the 3' end of the fluorescent probe contains a quenching group; The quenching group is selected from MGB, BHQ-1, BHQ-2, BHQ-3, TAMRA, or DABCYL.

[0010] Secondly, embodiments of this application propose the use of the primer-probe combination as described in the first aspect in the preparation of a cervical high-grade squamous intraepithelial lesion and cervical cancer gene methylation detection kit.

[0011] Thirdly, embodiments of this application provide a cervical high-grade squamous intraepithelial lesion and cervical cancer gene methylation detection kit, the detection kit comprising the primer-probe combination described in the first aspect.

[0012] Fourthly, embodiments of this application propose a non-disease diagnostic method for detecting high-grade squamous intraepithelial lesions of the cervix and cervical cancer gene methylation, using the detection kit described in the third aspect, wherein the detection method includes: Obtain the DNA of the sample to be tested; The DNA sample to be tested was subjected to bisulfite conversion treatment to obtain the converted DNA; Using the transformed DNA as a template, fluorescent PCR amplification was performed using a combination of PCR primers and probes. The fluorescence signal was detected and the results were determined.

[0013] As one implementation method, the procedure for the fluorescent PCR amplification reaction is as follows: React at 94-96℃ for 4-8 minutes, and repeat 1-2 times. The reaction is carried out at a temperature of 94-96℃ for 12-20s, and at a temperature of 58-68℃ for 25-35s, for 18-22 cycles. The reaction was carried out at 94-96℃ for 8-15s, and at 55-65℃ for 25-35s, for 38-42 cycles, and fluorescence was collected.

[0014] Fifthly, embodiments of this application disclose the use of a reagent for detecting SOX1 gene methylation in the preparation of products for detecting high-grade squamous intraepithelial lesions and cervical cancer.

[0015] In one implementation, the SOX1 gene methylation is used as a biomarker.

[0016] Compared with the prior art, the embodiments of this application have at least the following beneficial effects: The primer-probe combination provided in this embodiment is designed for SOX1-specific methylation detection sites and high-grade squamous intraepithelial lesion (HSIL) and cervical cancer-specific target sites. Through targeted optimization using nucleic acid recognition structures, standardized and differentiated detection thresholds can be constructed at the nucleic acid molecular level. This primer-probe combination exhibits significant detection specificity and sensitivity, accurately distinguishing the target population from individuals with low-grade squamous intraepithelial lesion (LSIL) and healthy individuals within the same detection system. Current cervical screening methods often suffer from problems such as ambiguous lesion grading, difficulty in molecularly defining LSIL and HSIL, and the susceptibility to false positives in healthy individuals. The primer-probe combination in this embodiment can establish a clear detection threshold difference at the nucleic acid molecular level, effectively distinguishing HSIL and cervical cancer samples from LSIL and healthy samples. This overcomes the limitations of conventional testing in distinguishing cervical lesion grades, enabling accurate grading of cervical health status, low-grade lesions, high-grade lesions, and cancer. This effectively avoids unnecessary surgeries (such as cervical conization) caused by misdiagnosing LSIL as HSIL, while also preventing the misdiagnosis of HSIL as LSIL and missing the optimal intervention time. This facilitates early intervention and timely treatment for HSIL patients, effectively avoiding missed detections and the eventual formation of cervical cancer. Furthermore, it provides a basis for the rational triage and management of high-risk human papillomavirus (HPV) positive individuals and for determining whether positive individuals require colposcopy and / or histological examination.

[0017] Additional aspects and advantages of this disclosure will be set forth in part in the description which follows, and will become apparent from the description or may be learned by practice of this disclosure. Detailed Implementation

[0018] The technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the embodiments described below are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0019] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0020] It should also be understood that the terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of this application. As used in the embodiments of this application and the appended claims, unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" are intended to include the plural forms.

[0021] The following details the PCR primer-probe combination for detecting gene methylation in endometrial cancer in this embodiment and its application.

[0022] First, the primer-probe combination for detecting high-grade squamous intraepithelial lesions and cervical cancer in the first aspect of this embodiment will be described.

[0023] Primer-probe combination for the detection of high-grade squamous intraepithelial lesions and cervical cancer.

[0024] In current technologies, cervical cancer often develops gradually from cervical squamous intraepithelial lesions. Among these, high-grade squamous intraepithelial lesions (SILs) are precancerous lesions with a high risk of malignant transformation. Early and accurate identification and differentiation of these lesions are crucial for preventing disease progression and protecting health. Current clinical cervical screening methods mainly involve liquid-based cytology and broad-spectrum high-risk human papillomavirus (HPV) testing. However, these traditional screening methods have many inherent limitations. For example, cytology screening relies on microscopic observation of cell morphology by medical personnel to determine lesions, which is easily affected by external factors such as vaginal inflammation, local tissue irritation, cell degeneration, and sampling impurities. Human interpretation is highly subjective and can easily lead to unclear lesion grading. Furthermore, traditional screening can only detect viral infection status and does not rely on lesion-specific molecular targets for detection. It cannot clearly distinguish between low-grade and high-grade squamous intraepithelial lesions at the molecular level, making it difficult to accurately determine whether cells have shown a trend towards malignant transformation.

[0025] Furthermore, routine viral testing cannot effectively distinguish between transient, self-limiting infections and persistent pathogenic infections. Coupled with interference factors such as abnormal physiological states and residual viral fragments from previous infections, false positive results are highly likely in health screening populations. Current screening technologies are prone to missed cases and misdiagnosis, hindering early intervention and timely treatment of patients with high-grade lesions, failing to curb cervical cancer at its source, and making it difficult to scientifically triage and manage high-risk HPV-positive individuals. They also cannot provide accurate and reliable references for clinically determining whether further colposcopy and histological biopsy are necessary. Overall, the screening and diagnostic efficacy is insufficient, and the limitations in clinical application are quite prominent.

[0026] Therefore, this embodiment proposes a primer-probe combination for detecting high-grade squamous intraepithelial lesions and cervical cancer. Specifically, this embodiment detects the methylation status of the SOX1 gene.

[0027] The primer-probe combination proposed in this embodiment includes: PCR primer-probe combinations for SOX1 methylation detection include a first primer-probe combination, a second primer-probe combination, or a third primer-probe combination.

[0028] Specifically, the first primer-probe combination includes an upstream primer as shown in SEQ ID NO.1, a downstream primer as shown in SEQ ID NO.2, and a fluorescent probe as shown in SEQ ID NO.3; the second primer-probe combination includes an upstream primer as shown in SEQ ID NO.4, a downstream primer as shown in SEQ ID NO.5, and a fluorescent probe as shown in SEQ ID NO.6; and the third primer-probe combination includes an upstream primer as shown in SEQ ID NO.7, a downstream primer as shown in SEQ ID NO.8, and a fluorescent probe as shown in SEQ ID NO.9.

[0029] As those skilled in the art will know, the SOX1 gene belongs to the SRY-box transcription factor family and is involved in the regulation of cell differentiation. Its promoter methylation leads to gene silencing, which results in the loss of its inhibitory effect on abnormal cell proliferation. It can be detected in early cancer and is a marker for early warning. Furthermore, SOX1 methylation is an early and critical event in the development of cervical cancer, spanning the entire process from HSIL (CIN2 and CIN3) to cervical cancer.

[0030] Based on this, this application employs a primer-probe combination for detecting high-grade squamous intraepithelial lesions (SILs) and cervical cancer at the SOX1-specific methylation detection site. Through targeted optimization design using a special nucleic acid recognition structure, standardized and differentiated detection thresholds can be constructed at the nucleic acid molecular level, forming a quantitatively unified judgment standard. Relying on this differentiated threshold system, the primer-probe combination of this application can efficiently and accurately define and classify high-grade squamous intraepithelial lesions and cervical cancer samples from low-grade squamous intraepithelial lesion samples and normal healthy population samples. This effectively overcomes the technical shortcomings of existing conventional detection methods, such as insufficient lesion grade differentiation and blurred grading boundaries, achieving accurate grading and identification of cervical health status, low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and cervical cancer.

[0031] Specifically, this embodiment, through precise molecular-level identification capabilities, can effectively distinguish low-grade lesions from high-grade precancerous lesions with molecular abnormalities, accurately identify healthy individuals without lesions from those at risk of developing lesions, significantly reduce false-positive results caused by factors such as inflammatory interference, abnormal cell morphology, and residual viral fragments, and significantly reduce the probability of missed or misdiagnosed high-grade lesions. Based on the above-mentioned precise molecular detection results, clinicians can identify patients with high-grade squamous intraepithelial lesions of the cervix as early as possible, and promptly carry out early intervention and symptomatic treatment, effectively blocking the progression of lesions to cervical cancer and reducing the risk of cervical cancer from the source.

[0032] Meanwhile, the standardized molecular detection data output by this primer-probe combination can provide objective and reliable risk assessment for high-risk human papillomavirus (HPV) positive individuals, which helps to achieve refined triage management of infected individuals in clinical practice, accurately determine whether the tested population needs further colposcopy and histological biopsy, avoid unnecessary invasive examinations for low-risk individuals and reduce over-diagnosis, and ensure that high-risk lesion individuals receive timely diagnosis and intervention, thus comprehensively improving the scientificity, accuracy and practicality of the entire process of early screening, risk stratification and clinical diagnosis and treatment of cervical diseases.

[0033] In summary, this application targets the SOX1 specific methylation detection site and optimizes the design through a special nucleic acid recognition structure to achieve accurate identification of high-grade squamous intraepithelial lesions, cervical cancer and cervical health status, as well as low-grade squamous intraepithelial lesions. This improves the scientific rigor, accuracy, and practicality of early screening, risk stratification, and the entire clinical diagnosis and treatment process for cervical diseases.

[0034] In one embodiment, the PCR primer-probe combination further includes an internal standard first primer-probe combination, an internal standard second primer-probe combination, or an internal standard third primer-probe combination for detecting internal reference genes. The internal standard first primer-probe combination includes an upstream primer as shown in SEQ ID NO.10, a downstream primer as shown in SEQ ID NO.11, and a fluorescent probe as shown in SEQ ID NO.12; The internal standard second primer-probe combination includes an upstream primer as shown in SEQ ID NO.13, a downstream primer as shown in SEQ ID NO.14, and a fluorescent probe as shown in SEQ ID NO.15; The internal standard third primer-probe combination includes an upstream primer as shown in SEQ ID NO.16, a downstream primer as shown in SEQ ID NO.17, and a fluorescent probe as shown in SEQ ID NO.18.

[0035] This embodiment designs upper and lower primers and fluorescent probes with the nucleotide sequence described above for the internal reference gene GAPDH. The upper and lower primers and fluorescent probes provided in this embodiment can achieve quality control of samples and the PCR amplification system, identifying unqualified clinical samples and abnormal amplification reactions, and avoiding invalid test results due to basic experimental problems. Secondly, it can effectively correct nucleic acid differences between clinical samples, offsetting individual deviations in nucleic acid extraction efficiency, template loading amount, and nucleic acid integrity of cervical exfoliated cell samples, establishing a data reference benchmark for SOX1 methylation target detection. Furthermore, this internal standard primer-probe combination can assist in accurately interpreting amplification signals, effectively distinguishing between SOX1 gene unmethylation (true low-risk / healthy individuals) and no signal (false negative) caused by nucleic acid degradation or amplification failure, reducing misjudgments from the source. Finally, this internal standard primer-probe combination can improve the system architecture of the aforementioned primer-probe combination, integrating target detection and quality control systems to ensure the validity and reliability of SOX1 methylation detection results, providing a qualified data foundation for subsequent clinical risk assessment, patient triage, and treatment decisions.

[0036] Specifically, the detection genes and corresponding detection channels in this embodiment are shown in Table 1 below.

[0037] Table 1:

[0038] In one embodiment, the 5' end of the fluorescent probe contains a fluorescent group; the fluorescent group is selected from FAM, VIC, HEX, NED, ROX, TET, JOE, TAMRA, CY3 or CY5.

[0039] The 3' end of the fluorescent probe contains a quenching group; the quenching group is selected from MGB, BHQ-1, BHQ-2, BHQ-3, TAMRA, or DABCYL.

[0040] It is understood that this embodiment is based on the combination design of the 5' end fluorescent group and the 3' end quencher group. It can utilize the changing rules of fluorescence quenching and signal release to establish a detection signal system that can be recognized by the real-time fluorescence quantitative PCR instrument, realize the real-time monitoring of the PCR amplification process, and finally complete the specific detection of the methylation status of the SOX1 gene.

[0041] This embodiment provides a variety of fluorescent groups with different emission spectra, supports simultaneous detection of multiple channels in a single tube, and can realize the separate acquisition of signals of SOX1 methylation target probe and the above-mentioned internal reference gene probe, eliminating the need for separate tube experiments, simplifying the experimental process and saving clinical samples.

[0042] In this embodiment, the fluorescent probe described above is used. When the probe is intact, the 5' fluorescent group and the 3' quencher group are very close in space, resulting in fluorescence resonance energy transfer (FRET). The energy generated by the excited fluorescent group is directly transferred to the quencher group, and the energy is dissipated as heat, making it impossible to detect a valid fluorescence signal. During PCR amplification, the probe specifically binds to the SOX1 methylated target DNA template region through complementary base pairing. As Taq DNA polymerase extends along the template strand, its exonuclease activity gradually cleaves the probe bound to the template, separating the 5' fluorescent group from the 3' quencher group. When the spatial distance between them increases, the FRET effect disappears, and the fluorescent group releases characteristic fluorescence upon excitation by the light source, allowing the instrument to stably capture the fluorescence signal. Therefore, this embodiment can detect the methylation status of the SOX1 gene by detecting the fluorescence intensity in the reaction system.

[0043] Therefore, this embodiment, targeting the SOX1 specific methylation detection site, employs a primer-probe combination for detecting high-grade squamous intraepithelial lesions (SILs) and cervical cancer. Through targeted optimization design using a special nucleic acid recognition structure, standardized and differentiated detection thresholds can be constructed at the nucleic acid molecular level, forming a quantitatively unified judgment standard. Based on this differentiated threshold system, the primer-probe combination of this application can efficiently and accurately clearly define and classify high-grade squamous intraepithelial lesions and cervical cancer samples from low-grade squamous intraepithelial lesion samples and normal healthy population samples. This effectively overcomes the technical defects of insufficient lesion grade differentiation and blurred grading boundaries in existing conventional detection methods, achieving accurate grading and identification of cervical health status, low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and cervical cancer.

[0044] In summary, this embodiment targets the SOX1 specific methylation detection site and uses a special nucleic acid recognition structure for targeted optimization design to achieve accurate identification of high-grade squamous intraepithelial lesions, cervical cancer, cervical health status, and low-grade squamous intraepithelial lesions. This improves the scientific rigor, accuracy, and practicality of the entire process of early screening, risk stratification, and clinical diagnosis and treatment of cervical diseases.

[0045] Next, the use of the primer-probe combination in the second aspect of this embodiment will be explained.

[0046] This embodiment illustrates the use of the primer-probe combination as described in the first aspect in the preparation of a kit for detecting high-grade squamous intraepithelial lesions and cervical cancer gene methylation.

[0047] The SOX1 methylation detection PCR primer and probe combination provided in this embodiment can be effectively used to prepare a kit for detecting high-grade squamous intraepithelial lesions (HSIL, including CIN2 and CIN3 stages) and cervical cancer gene methylation. This kit is mainly intended for diverse clinical applications such as early screening for cervical diseases, risk stratification of high-risk HPV-positive individuals, auxiliary diagnosis of suspected cervical lesions, and monitoring of postoperative recurrence of cervical lesions.

[0048] This embodiment leverages the characteristic of SOX1 gene methylation as a key epigenetic marker in the early stages of cervical lesion development. Utilizing the high specificity, excellent anti-interference ability, and high detection accuracy of the aforementioned primer-probe combination, the prepared kit effectively avoids false positives caused by factors such as inflammatory interference, abnormal cell morphology, and residual HPV fragments in cervical samples. It accurately distinguishes between healthy individuals, benign inflammatory lesions, low-grade cervical lesions, and high-grade precancerous lesions and cervical cancer with molecular abnormalities, significantly reducing the probability of missed or misdiagnosed high-grade lesions.

[0049] Meanwhile, the kit can output standardized and quantifiable molecular detection data, providing an objective basis for the refined triage management of high-risk clinical populations. It can accurately determine whether the tested population needs to undergo further colposcopy and histological biopsy, effectively avoiding unnecessary invasive examinations for low-risk populations and reducing over-diagnosis and treatment. It can also ensure timely diagnosis and early intervention for high-risk lesions, blocking the progression of cervical lesions to cervical cancer from the source. This significantly improves the overall scientificity and accuracy of cervical disease screening, risk stratification, and clinical diagnosis and treatment, and is suitable for large-scale cervical cancer screening and routine clinical diagnosis.

[0050] Therefore, this embodiment can be based on the primer and probe combination described in the first aspect to prepare a methylation detection kit for high-grade squamous intraepithelial lesions and cervical cancer genes. This methylation detection kit has the advantages of accurately distinguishing between HSIL and cervical cancer, LSIL and healthy individuals, while ensuring sensitivity and specificity, thus comprehensively improving the scientificity, accuracy and practicality of the entire process of early screening, risk stratification and clinical diagnosis and treatment of cervical diseases.

[0051] Next, the detection kit of the third aspect of this embodiment will be described.

[0052] The cervical high-grade squamous intraepithelial lesion and cervical cancer gene methylation detection kit of this embodiment includes the primer-probe combination described in the first aspect.

[0053] The cervical high-grade squamous intraepithelial lesion and cervical cancer gene methylation detection kit provided in this embodiment includes the SOX1 methylation detection PCR primer and probe combination disclosed in the first aspect of this embodiment. Relying on the technical advantages of the primer and probe combination of high specificity, high anti-interference and high detection accuracy, a gene methylation detection kit with extremely high stability and practicality is constructed.

[0054] The above-mentioned kit is used for targeted screening and auxiliary identification of high-grade squamous intraepithelial lesions and cervical cancer. Specifically, it accurately detects the methylation status and methylation expression level of the SOX1 gene in the test biological sample to achieve molecular-level identification and risk indication of high-risk cervical lesions. Based on the biological characteristic that SOX1 gene methylation is highly positively correlated with the degree of cervical lesion progression, it provides core molecular evidence for clinical screening of cervical diseases.

[0055] It is understood that the kit in this embodiment is compatible with a variety of common clinical biological test samples. The types of samples that can be selected include, but are not limited to, cervical histological sections, cervical biopsy samples, paraffin-embedded cervical tissue, clinical cervical exfoliated cells, and any combination of the above samples. The sample compatibility is wide and can meet the usage requirements of different clinical sampling scenarios and different testing needs.

[0056] As a preferred embodiment of this example, the preferred sample type for this kit is cervical exfoliated cells. This sample is a non-invasive or minimally invasive sampling sample, which has the advantages of simple sampling operation, high patient acceptance, strong repeatability, and suitability for large-scale population screening. It can better adapt to the core application scenarios of clinical cervical cancer screening and follow-up monitoring of high-risk groups.

[0057] It is important to note that this kit is a gene molecular auxiliary detection reagent. The methylation detection results it outputs do not constitute independent diagnostic evidence for diseases and should not be used as the sole criterion for judging a patient's condition, evaluating treatment efficacy, or confirming a diagnosis. In actual clinical application, it is necessary to combine the patient's clinical symptoms, past medical history, imaging examination results, routine pathological examination results, and other supporting tumor detection indicators to conduct a comprehensive and integrated analysis and evaluation of the patient's condition.

[0058] For example, even if the test kit shows a positive result for SOX1 methylation, it is still necessary to integrate the patient's multi-dimensional clinical data, exclude interfering factors, identify the nature and degree of the lesion, in order to accurately complete the disease assessment and formulate a scientific and symptomatic follow-up and treatment plan, effectively avoid the interpretation bias caused by a single test result, and ensure the accuracy and standardization of clinical diagnosis and treatment.

[0059] As a preferred embodiment, the above detection kit is usually also provided with a complete set of auxiliary reagent system for experiments, specifically including PCR reaction reagents (also referred to as PCR MIX), which can provide a standardized and stable reaction environment for the fluorescent quantitative PCR amplification reaction of the target gene, and guarantee the amplification efficiency and detection stability.

[0060] Meanwhile, in order to simplify the pre-processing operation of clinical samples, improve the quality of sample conversion and purification, and facilitate medical staff to complete nucleic acid extraction and purification of clinical samples and DNA bisulfite conversion, the kit usually also comes with nucleic acid extraction or purification reagents and bisulfite conversion agent.

[0061] For example, the nucleic acid extraction or purification reagent can be the *Nucleic Acid Extraction or Purification Reagent (Universal Type)* produced by Anhiu Dajian Medical Technology Co., Ltd. (Medical Device Filing Certificate No.: Wanwu Xiebei 20190002). The bisulfite conversion agent can be the *Nucleic Acid Extraction or Purification Reagent (Spin Column Type)* produced by Anhui Dajian Medical Technology Co., Ltd. (Medical Device Filing Certificate No.: Wanwu Xiebei 20200003); for the specific operation procedures of the above reagents, please refer to the corresponding instructions.

[0062] Generally, the primer-probe combination is mixed with Tris-EDTA buffer (hereinafter referred to as TE, pH=8.0) to obtain a primer-probe mixture (also referred to as PCR primer-probe mixture).

[0063] Illustratively, in the kit of this example, the final concentrations and dosages of each component are shown in the following Table 2 and Table 3.

[0064] Table 2: PCR primer-probe mixture

[0065] Table 3: PCR MIX

[0066] In summary, based on the primer-probe combination of the first aspect, the kit of the present example can quickly and sensitively detect the methylation status of the SOX1 gene promoter region by designing specific primers and probes, realize accurate graded identification of cervical health status, low-grade squamous intraepithelial lesion, high-grade squamous intraepithelial lesion and cervical cancer, provide objective and reliable risk assessment basis for high-risk human papillomavirus positive infected persons, help clinically complete the fine triage management of infected persons, and accurately determine whether the detected population needs further colposcopy and histological pathological biopsy. It not only avoids low-risk populations receiving unnecessary invasive examinations and reduces overtreatment and overdiagnosis, but also ensures that people with high-risk lesions get timely diagnosis and intervention, and comprehensively improves the scientificity, accuracy and practicability of the whole process of early screening, risk stratification and clinical diagnosis and treatment of cervical diseases.

[0067] The detection method of the fourth aspect in this embodiment will be described below.

[0068] This embodiment further illustrates the detection method based on the detection kit from the third aspect. The detection method includes: (1) Extract DNA from cervical exfoliated cells to obtain DNA samples to be tested.

[0069] In step (1), cervical exfoliated cells collected clinically are selected as the biological samples to be tested. The nucleic acid extraction or purification reagents provided with the kit are used to lyse, adsorb nucleic acid, wash and elute the cervical exfoliated cell samples to effectively remove residual proteins, polysaccharides, inflammatory impurities, cell debris and PCR inhibitors from the samples. The purified DNA samples are obtained with high purity and good integrity, providing qualified nucleic acid templates for subsequent bisulfite conversion and PCR amplification reactions, and avoiding detection errors caused by impurities.

[0070] (2) The DNA of the sample to be tested is converted by bisulfite to obtain the converted DNA (or BisDNA).

[0071] In step (2), the extracted DNA sample is modified and transformed using the bisulfite conversion agent provided with the kit to obtain transformed DNA (BisDNA). The bisulfite conversion is a core pretreatment step for gene methylation detection. It specifically deaminates unmethylated cytosine in the genome into uracil, while the methylated cytosine stably retains its original base structure. Through the specific differences in base sequences, it effectively distinguishes between methylated and unmethylated sites, laying the foundation for subsequent primer and probe-specific amplification and recognition of methylated templates.

[0072] (3) Using the transformed DNA as a template, fluorescent PCR amplification reaction was performed using a combination of PCR primers and probes, and the fluorescence signal was detected and the result was determined.

[0073] In step (3), using BisDNA that has undergone bisulfite conversion as an amplification template, the specific PCR primer and probe combination, internal standard primer and probe combination, and PCR reaction reagent (PCR MIX) of this embodiment are added to prepare a standardized PCR amplification reaction system, which is then placed in a real-time quantitative PCR instrument for real-time quantitative amplification reaction.

[0074] During PCR amplification, the instrument collects real-time changes in the fluorescence signal of the reaction system, monitoring the amplification curves of the target gene and the internal reference gene throughout the process. After the reaction, the effectiveness of the sample and reaction system is determined by combining the amplification results of the internal reference gene. Then, based on the presence or absence of fluorescence signal of the SOX1 target gene, the morphology of the amplification curve, and the Ct value, the methylation status and methylation level of the SOX1 gene in the sample to be tested are accurately determined, ultimately achieving auxiliary screening and risk assessment for high-grade squamous intraepithelial lesions and cervical cancer.

[0075] In step (3), the procedure for the fluorescent PCR amplification reaction is as follows: First stage: React at 94-96℃ for 4-8 min, cycle 1-2 times; In the first stage, react at 94-96℃ for 4-8 min, cycle 1-2 times to complete template pre-denaturation, completely untangle the double-stranded DNA structure, and eliminate the interference of template secondary structure on amplification.

[0076] The second stage involves reacting at 94-96℃ for 12-20 seconds, then at 58-68℃ for 25-35 seconds, for a total of 18-22 cycles. This completes the initial specific amplification of the target fragment and enriches the target gene template.

[0077] The third stage involves reacting at 94-96℃ for 8-15 seconds, then at 55-65℃ for 25-35 seconds, for 38-42 cycles, during which fluorescence is collected. Fluorescence signals are collected synchronously after each cycle in this stage to record the real-time fluorescence amplification dynamics of the target gene and the internal reference gene.

[0078] Preferably, the procedure for the above-mentioned fluorescent PCR amplification reaction is as follows: The reaction was carried out at 95℃ for 5 minutes, and the cycle was repeated once. The reaction was carried out at 95℃ for 15 seconds and at 64℃ for 30 seconds, and the cycle was repeated 20 times. The reaction was carried out at 95℃ for 10 seconds, then at 58℃ for 31 seconds, for 40 cycles, and fluorescence was collected.

[0079] It should be noted that during the above sampling process: (1) Centrifuge the preservation solution containing cervical exfoliated cells, carefully remove the supernatant, resuspend the sediment in a small amount of preservation solution, and then transfer it to an EP tube. Cell sample storage time: no more than 7 days at room temperature, 12 months at -20±5℃, and long-term storage at -70°C and below.

[0080] (2) The extracted DNA can be stored at -20±5℃ for no more than 6 months, or at -70℃ for long-term storage.

[0081] (3) Samples treated with bisulfite should be tested as soon as possible, at -20±5℃ for no more than 3 days, and repeated freeze-thaw cycles should be avoided.

[0082] The sample storage conditions are as follows: at room temperature, the sample can be stored for no more than 7 days; at -20℃±5℃, the sample can be stored for 12 months; and at -70℃ or below, the sample can be stored for a long time.

[0083] In summary, the detection method of this embodiment can detect the methylation status of the SOX1 gene based on the detected fluorescence intensity. Based on the methylation status of the SOX1 gene promoter region, it can efficiently and accurately distinguish and classify cervical high-grade squamous intraepithelial lesion and cervical cancer samples from low-grade squamous intraepithelial lesion samples and normal healthy population samples. This effectively overcomes the technical defects of existing conventional detection methods, such as insufficient differentiation of lesion grades and blurred grading boundaries, and achieves accurate grading and identification of cervical health status, low-grade squamous intraepithelial lesion, high-grade squamous intraepithelial lesion, and cervical cancer.

[0084] Next, the use of the reagent for detecting SOX1 gene methylation in the fifth aspect of this embodiment in the preparation of endometrial cancer detection products will be explained.

[0085] The use of reagents for detecting SOX1 gene methylation in the preparation of products for detecting high-grade squamous intraepithelial lesions and cervical cancer.

[0086] This embodiment further proposes the use of reagents for detecting SOX gene methylation in the preparation of cervical high-grade squamous intraepithelial lesion and cervical cancer detection products. The reagents include PCR primer-probe combinations, which are described in the first aspect.

[0087] This embodiment uses SOX1 gene methylation as a biomarker. Based on the highly specific and sensitive SOX1 gene methylation detection reagent of this embodiment, detection products for screening and auxiliary diagnosis of high-grade squamous intraepithelial lesions and cervical cancer can be prepared on a large scale.

[0088] As mentioned earlier, abnormal methylation of the SOX1 gene is an early and key epigenetic driver event in the development of cervical lesions, stably persisting throughout the entire progression from HSIL (CIN2, CIN3) to cervical cancer, and the methylation level is significantly positively correlated with the degree of malignant progression. Based on the advantages of the primer-probe combination in this embodiment—high specificity, resistance to inflammatory interference, and resistance to interference from residual HPV fragments—the resulting detection product can accurately identify abnormal SOX1 gene methylation associated with cervical lesions, effectively distinguishing between healthy individuals, benign inflammatory lesions, low-grade lesions, and high-risk high-grade precancerous lesions and cervical cancer. This significantly reduces the risk of false positives and missed detections, providing objective and standardized molecular evidence for early cervical cancer screening, risk stratification of high-risk HPV-positive individuals, lesion progression assessment, and clinical triage. It effectively guides the selection of colposcopic biopsies and clinical intervention programs, reducing overdiagnosis, missed diagnosis, and misdiagnosis, and improving the accuracy and standardization of overall cervical disease diagnosis and treatment.

[0089] Meanwhile, abnormal SOX1 gene methylation can also serve as an important molecular marker for the development and progression of endometrial cancer. Therefore, the reagent for detecting SOX1 gene methylation described in this embodiment can also be used to prepare products for auxiliary detection, screening, or risk assessment of endometrial cancer. This application expands the clinical application scenarios of this reagent, enabling molecular screening of lesions related to two major malignant tumors of the female reproductive tract using a single reagent system. This significantly enhances the application value and clinical universality of the reagent, providing a new and efficient molecular detection method for early screening and diagnosis of gynecological malignancies in women.

[0090] The following section provides further explanation of the results analysis methods and quality control standards for the testing methods.

[0091] This study analyzed the methylation level of the SOX1 gene based on the amplification curves and cycle number threshold (Ct value) of fluorescent PCR. Higher methylation levels indicated potentially lower SOX1 gene expression and a higher malignant potential for high-grade squamous intraepithelial lesions and cervical cancer.

[0092] Threshold setting: The threshold can be set automatically by the instrument or manually adjusted according to the instrument's instruction manual. Set the threshold to the linear portion of the fluorescence logarithmic plot and read the Ct value from the software. If there is no gene amplification, the Ct value is set to 40.

[0093] Determination of reagent kit validity: If the kit control meets the criteria listed in Table 4, and the test sample is measured in the same PCR reaction along with the negative and positive controls, the PCR reaction is considered valid.

[0094] Table 4: Determination of Kit Validity

[0095] Determination of test validity: If the Ct value of the internal control gene is ≤25 and the amplification curve is S-shaped, the analysis can continue; if the Ct value of the internal control gene is >25 or there is no amplification curve, the analysis cannot continue and the test needs to be repeated.

[0096] Test result determination: Please refer to Table 5 for the standard for determining the test results.

[0097] Table 5: Judgment of Test Results

[0098] It should be noted that the above test results are for clinical reference only. The selection of individualized treatment for patients should be based on a comprehensive consideration of their symptoms / signs, medical history, other laboratory tests, and treatment response.

[0099] The present disclosure is further illustrated below with reference to embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure.

[0100] Unless otherwise specified, all materials, reagents and instruments used in the following embodiments are commercially available.

[0101] The present disclosure will be further described below with reference to specific embodiments.

[0102] Example 1: This embodiment identifies HSIL and cervical cancer-related hypermethylated genes through literature review. Specific PCR primer-probe combinations for detecting SOX1 gene methylation are designed targeting the SOX1-specific methylation detection site. Specific primers and probes for the internal reference gene GAPDH are also designed. The specific PCR primer-probe combinations and the internal reference gene's internal standard primer-probe combinations are shown in Table 6 below.

[0103] Table 6:

[0104] The methylation status of the SOX1 gene in HSIL and cervical cancer samples was detected based on the primer and probe design described above. In this embodiment, cervical exfoliated cells were used as the test samples (117 samples, including 77 control samples (cervicitis and other benign cervical diseases and LSIL) and 40 positive samples (including 31 HSIL samples and 9 cervical cancer samples).

[0105] The specific testing steps include: (1) Extraction of sample DNA: Take a DNA sample to be tested (cervical exfoliated cells), and use the nucleic acid extraction or purification reagent from Anhui Medical Technology Co., Ltd. (Medical Device Filing Number: Wan Wu Xie Bei 20190002) to extract high-purity DNA.

[0106] The specific steps are as follows: (a) Take 500 µL of the pretreated cervical exfoliated cell suspension, add 500 µL of lysis buffer and 30 µL of proteinase K, mix thoroughly, and incubate at 60°C for 1 hour until the sample is completely lysed; (b) Centrifuge briefly, leave it at room temperature for 10 min, then add 200 µL of isopropanol, vortex and shake, and mix thoroughly; (c) Centrifuge briefly, add all the liquid from step 3 into the adsorption column (place the adsorption column in a collection tube), centrifuge at 12000 rpm for 1 min, and discard the waste liquid. (Note: If there is too much digestive solution, it can be added to the adsorption column in multiple times, 600 μL each time); (d) Add 600 µL of washing buffer I into the adsorption column once, centrifuge at 12000 rpm for 30 s, discard the waste liquid; (e) Add 600 µL of washing buffer II into the adsorption column once, centrifuge at 12000 rpm for 30 s, discard the waste liquid; (f) Add another 600 µL of washing buffer II into the adsorption column once, centrifuge at 12000 rpm for 30 s, discard the waste liquid, then centrifuge at 12000 rpm for 3 min; (g) Uncap the lid, and air-dry for 2 min in a fume hood; (h) Elution: Add 50 µL of elution buffer into the adsorption column, stand at room temperature for 3 min, then centrifuge at 12000 rpm for 2 min, collect the DNA, and store at -20°C.

[0107] (2) Bisulfite conversion and purification treatment: Subsequently, the DNA extracted in the above steps is subjected to bisulfite conversion and purification treatment to obtain Bis-DNA.

[0108] In this example, the bisulfite conversion treatment is carried out using the nucleic acid extraction or purification reagent (spin column type) produced by Anhui Dajian Medical Technology Co., Ltd. (Product Filing Number: Wan Wu Xie Bei 20200003); the specific steps are as follows: (a) Take 45 µL of the DNA sample to be tested into a new 1.5 mL centrifuge tube, add 5 µL of conversion buffer, and incubate at a constant temperature of 37°C in a metal bath for 15 min; (b) After incubation is completed, add 100 µL of the prepared conversion solution to each sample, mix and centrifuge briefly, and incubate in a metal bath at 50°C protected from light for 12-16 hours; (c) Incubate the sample on ice (0-4°C) for 10 min; (d) Place the adsorption column in the collection tube and add 400 μL of binding solution to the adsorption column; (e) Add the sample from step c into the adsorption column (containing the binding solution), tighten the cap and invert the tube several times to mix, centrifuge at full speed (14000 rpm) for 30 seconds, and discard the waste liquid. (f) Add 100 μL of washing solution to the adsorption column, centrifuge at full speed for 30 s, and discard the waste liquid; (g) Add 200 μL of desulfurization solution to the adsorption column, incubate at room temperature (25℃) for 20 min, then centrifuge at full speed for 30 s and discard the waste liquid; (h) Add 200 μL of washing solution to the adsorption column, centrifuge at full speed for 30 s, repeat adding 200 μL of washing solution, centrifuge at full speed for 30 s, and discard the waste liquid and collection tube. (i) Place the adsorption column into a 1.5 mL sterile centrifuge tube, add 30 μL of elution buffer to the middle of the adsorption membrane, elute the transformed DNA, centrifuge at full speed for 1 min, collect Bis-DNA, and store at -20 °C.

[0109] Next, after obtaining Bis-DNA, using the Bis-DNA converted by bisulfite as a template, PCR reaction reagents, PCR primer and probe combinations for SOX1, PCDH8 and PAX1 gene methylation and internal control primer and probe combinations for internal reference genes (as shown in Table 6) were added to perform fluorescent PCR amplification reaction.

[0110] The PCR reaction system is shown in Table 7.

[0111] Table 7: PCR reaction system

[0112] The specific composition of PCR MIX is shown in Table 3.

[0113] Mix the reaction system in Table 7 thoroughly, centrifuge briefly, and aliquot 22 μL into 0.2 mL PCR reaction tubes. Place the PCR reaction tubes into the corresponding fluorescence PCR instrument (ABI 7500 fluorescence PCR amplification instrument), record the sample placement order, and then perform methylation fluorescence quantitative PCR amplification detection.

[0114] Please refer to Table 8 for the procedure of the fluorescent PCR amplification reaction in this embodiment.

[0115] Table 8: PCR Reaction Procedure

[0116] In this embodiment, 117 samples were tested under the condition that the negative and positive controls met the validity criteria of the kit. Among them, there were 77 benign lesion samples (cervicitis and other benign cervical diseases and LSIL) and 40 cervical cancer and HSIL samples (SOX1 detection site), including 31 HSIL samples and 9 cervical cancer samples. The sensitivity and specificity of the above gene methylation detection results were analyzed. The comparison between the detection results and the clinical results is shown in Table 9.

[0117] Table 9: Comparison of test reagent results with "clinical diagnosis" results

[0118] The sensitivity test results for HSIL and cervical cancer are shown in Table 10.

[0119] Table 10: Sensitivity test results for HSIL and cervical cancer

[0120] According to the test results in Table 10, this embodiment included 31 pathologically confirmed HSIL samples. The detection method in this embodiment detected 28 positive cases and 3 negative cases, with a sensitivity of 90.32%. This embodiment also included 9 pathologically confirmed cervical cancer samples. The detection method in this embodiment detected 8 positive cases and 1 negative case, with a sensitivity of 88.89%. This embodiment achieved a high sensitivity of nearly 90% for both HSIL and cervical cancer, indicating that it has an extremely low risk of missing high-grade precancerous lesions and malignant lesions of the cervix. It can reliably identify high-risk samples at critical stages of disease progression, providing a reliable molecular basis for early clinical intervention.

[0121] The primer-probe combination in this embodiment is specifically optimized for the SOX1 gene methylation-specific sequence. Through highly specific primers and modified probes, a unique nucleic acid recognition structure is employed to achieve high affinity binding to methylated templates and single-base-level sequence differentiation. This allows for precise identification of SOX1 gene methylation sites (this design can stably identify SOX1 methylated fragments in HSIL / cervical cancer samples, even when methylation abundance is low), while effectively excluding the influence of unmethylated sequences and low-abundance interfering fragments. This precise molecular-level targeting directly ensures efficient detection of methylated templates in HSIL and cervical cancer samples, forming the crucial basis for achieving the high sensitivity of 90.32% and 88.89% in this embodiment, and guaranteeing stable identification of malignant lesions.

[0122] According to the test results in Table 9, the specific primers provided in this embodiment exhibit a sensitivity of 90.00%, a specificity of 97.40%, an overall concordance rate of 94.87%, and a Kappa value of 0.8847. The overall concordance rate represents the proportion of consistency between the reagent results and the clinical diagnostic results, reflecting the overall reliability of the test results. A Kappa value of 0.8847 indicates a high degree of consistency between the reagent results and the clinical diagnostic results. Specifically, the overall concordance rate of 94.87% and the Kappa value of 0.8847 demonstrate that the test results of this embodiment are highly consistent with the clinical diagnostic standards, proving the stability and repeatability of the standardized judgment system. This high consistency effectively avoids errors caused by subjective interpretation in traditional testing, ensuring the uniformity of test results from different operators and different batches, and providing a reliable foundation for large-scale clinical application.

[0123] Secondly, the high sensitivity of 90.00% indicates that this embodiment can identify the vast majority of HSIL and cervical cancer samples, avoiding the missed detection problems caused by atypical cell morphology and inflammatory interference in traditional morphological testing. This high detection rate can help clinicians identify HSIL patients as early as possible, enabling timely early intervention and symptomatic treatment, effectively blocking the progression of lesions to cervical cancer, and reducing the risk of cervical cancer from the source. The high specificity of 97.40% indicates that this embodiment has an extremely low false positive rate for low-grade lesions and healthy individuals, with a false positive rate of only about 2.6%, which can effectively reduce false positive results caused by factors such as inflammatory interference and residual HPV fragments. This means that low-risk individuals will not be misjudged as high-risk and will not need to undergo unnecessary colposcopy and histopathological biopsy, significantly reducing overdiagnosis.

[0124] Therefore, the combination of high sensitivity and high specificity allows the test reagent in this embodiment to efficiently and accurately define and classify HSIL / cervical cancer samples from low-grade squamous intraepithelial lesion samples and normal healthy population samples. This clear molecular-level distinction provides an objective and reliable risk assessment basis for high-risk HPV-positive individuals, helping clinicians to achieve refined triage management of infected individuals: positive results indicate high-risk lesions requiring further examination; negative results indicate low risk, allowing for regular follow-up, thus improving the scientific rigor and accuracy of clinical diagnosis and treatment. High overall concordance rate and Kappa value verify the reliability and reproducibility of the reagent results, and standardized judgment criteria avoid subjective interpretation errors, ensuring comparability of test results from different laboratories and operators. This stable and reliable testing system is suitable for the entire process of cervical disease, from early screening and risk stratification to treatment intervention. It is suitable for large-scale population-wide cervical cancer screening and can also be used for auxiliary diagnosis and postoperative follow-up of suspected outpatient cases, comprehensively improving the scientific rigor, accuracy, and practicality of the entire clinical diagnosis and treatment process for cervical diseases.

[0125] In summary, this embodiment uses SOX1 gene methylation as a biomarker. Targeting the SOX1 gene methylation detection site, the primer-probe combination for detecting high-grade squamous intraepithelial lesions (GILs) and cervical cancer employs a targeted optimization design with a special nucleic acid recognition structure. This allows for the construction of standardized and differentiated detection thresholds at the nucleic acid molecular level, forming a quantitatively unified judgment standard. This efficiently and accurately distinguishes and categorizes GIL and cervical cancer samples from low-grade squamous intraepithelial lesion samples and normal healthy population samples, thereby improving the scientific rigor, accuracy, and practicality of early cervical disease screening, risk stratification, and the entire clinical diagnosis and treatment process.

[0126] The technical solutions provided by the embodiments of this application have been described in detail above. Specific examples have been used in this document to illustrate the principles and implementation methods of the embodiments of this application. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A primer-probe combination for detecting high-grade squamous intraepithelial lesions and cervical cancer, characterized in that, The primer-probe combination includes: PCR primer-probe combinations for SOX1 methylation detection include a first primer-probe combination, a second primer-probe combination, or a third primer-probe combination. The first primer-probe combination includes an upstream primer as shown in SEQ ID NO.1, a downstream primer as shown in SEQ ID NO.2, and a fluorescent probe as shown in SEQ ID NO.3; The second primer-probe combination includes an upstream primer as shown in SEQ ID NO.4, a downstream primer as shown in SEQ ID NO.5, and a fluorescent probe as shown in SEQ ID NO.6; The third primer-probe combination includes an upstream primer as shown in SEQ ID NO.7, a downstream primer as shown in SEQ ID NO.8, and a fluorescent probe as shown in SEQ ID NO.

9.

2. The primer-probe combination for detecting high-grade squamous intraepithelial lesions and cervical cancer according to claim 1, characterized in that, The primer-probe combination also includes an internal standard first primer-probe combination, an internal standard second primer-probe combination, or an internal standard third primer-probe combination for detecting the internal reference gene GAPDH. The internal standard first primer-probe combination includes an upstream primer as shown in SEQ ID NO.10, a downstream primer as shown in SEQ ID NO.11, and a fluorescent probe as shown in SEQ ID NO.12; The internal standard second primer-probe combination includes an upstream primer as shown in SEQ ID NO.13, a downstream primer as shown in SEQ ID NO.14, and a fluorescent probe as shown in SEQ ID NO.15; The internal standard third primer-probe combination includes an upstream primer as shown in SEQ ID NO.16, a downstream primer as shown in SEQ ID NO.17, and a fluorescent probe as shown in SEQ ID NO.

18.

3. The primer-probe combination for detecting high-grade squamous intraepithelial lesions and cervical cancer according to claim 1, characterized in that, The 5' end of the fluorescent probe contains a fluorescent group; The fluorescent group is selected from FAM, VIC, HEX, NED, ROX, TET, JOE, TAMRA, CY3 or CY5.

4. The primer-probe combination for detecting high-grade squamous intraepithelial lesions and cervical cancer according to claim 1, characterized in that, The 3' end of the fluorescent probe contains a quenching group; The quenching group is selected from MGB, BHQ-1, BHQ-2, BHQ-3, TAMRA, or DABCYL.

5. The use of the primer-probe combination according to any one of claims 1 to 4 in the preparation of a kit for detecting high-grade squamous intraepithelial lesions and cervical cancer gene methylation.

6. A kit for detecting gene methylation in high-grade squamous intraepithelial lesions and cervical cancer, characterized in that, The detection kit includes the primer-probe combination as described in any one of claims 1 to 4.

7. A non-disease diagnostic method for detecting high-grade squamous intraepithelial lesions and cervical cancer gene methylation, using the detection kit as described in claim 6, characterized in that, The detection method includes: Obtain the DNA of the sample to be tested; The DNA sample to be tested was subjected to bisulfite conversion treatment to obtain the converted DNA; Using the transformed DNA as a template, fluorescent PCR amplification was performed using a combination of PCR primers and probes. The fluorescence signal was detected and the results were determined.

8. The non-disease diagnostic detection method according to claim 7, further characterized in that the procedure for the fluorescent PCR amplification reaction is as follows: React at 94-96℃ for 4-8 minutes, and repeat 1-2 times. The reaction is carried out at a temperature of 94-96℃ for 12-20s, and at a temperature of 58-68℃ for 25-35s, for 18-22 cycles. The reaction was carried out at 94-96℃ for 8-15s, and at 55-65℃ for 25-35s, for 38-42 cycles, and fluorescence was collected.

9. The use of a reagent for detecting SOX1 gene methylation in the preparation of products for detecting high-grade squamous intraepithelial lesions and cervical cancer, characterized in that, The reagent includes a primer-probe combination, which comprises: PCR primer-probe combinations for SOX1 methylation detection include a first primer-probe combination, a second primer-probe combination, or a third primer-probe combination. The first primer-probe combination includes an upstream primer as shown in SEQ ID NO.1, a downstream primer as shown in SEQ ID NO.2, and a fluorescent probe as shown in SEQ ID NO.3; The second primer-probe combination includes an upstream primer as shown in SEQ ID NO.4, a downstream primer as shown in SEQ ID NO.5, and a fluorescent probe as shown in SEQ ID NO.6; The third primer-probe combination includes an upstream primer as shown in SEQ ID NO.7, a downstream primer as shown in SEQ ID NO.8, and a fluorescent probe as shown in SEQ ID NO.

9.

10. The use according to claim 9, characterized in that, SOX1 gene methylation is used as a biomarker.

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