Application of substance for detecting methylation marker in preparation of product for detecting cervical cancer and / or precancerous lesions of cervical cancer

Abstract

The invention discloses a methylation marker for detecting cervical cancer and / or cervical cancer precancerous lesions and application, and particularly relates to the technical field of molecular biomedicine. According to the application, methylation differences between patients with cervical cancer and / or precancerous lesions of cervical cancer and healthy people are researched through DNA of cervical exfoliated cells of Chinese population, candidate methylation markers with obvious differences are screened out, and by combining LASSO-Logistic modeling, AUC ranking and manual screening methods, the candidate methylation markers with obvious differences are screened out, so that the candidate methylation markers of cervical cancer and / or precancerous lesions of cervical cancer are obtained. 33 optimal methylation markers are screened for the first time to establish a Chinese population cervical cancer and / or cervical precancerous lesion methylation risk prediction method, and the method is suitable for predicting risk assessment of early-stage cervical cancer and / or cervical precancerous lesion occurrence of Chinese population and screening and diagnosis of cervical cancer and / or cervical precancerous lesion.

Description

Technical Field

[0001] This specification relates to the field of molecular biomedicine technology, and particularly to the application of substances for detecting methylation markers in the preparation of products for detecting cervical cancer and / or precancerous lesions of the cervix. Background Art

[0002] Cervical cancer threatens women's health. Among global malignant tumors, the incidence and mortality of cervical cancer both rank fourth. In China, the newly diagnosed and dead cases of cervical cancer account for nearly 20% of the global total respectively.

[0003] Only the persistent infection of high-risk HPV (high-risk human papillomavirus, hrHPV) may lead to cervical lesions, which progress through mild cervical intraepithelial neoplasia (CIN), moderate CIN, and severe CIN, and ultimately develop into cervical cancer. Current screening guidelines recommend HPV DNA testing as the preferred method, or combined with Thinprep cytologic test (TCT). The hrHPV test result is objective and has high repeatability, but it cannot distinguish transient infection from transformational infection, which may lead to an increase in colposcopy referrals and cause anxiety in positive women. Cytological screening methods such as TCT have high specificity, but have limitations such as diagnostic subjectivity and low sensitivity, thus resulting in missed diagnoses.

[0004] In recent years, studies have shown that early epigenetic changes are important features of tumor occurrence and development. DNA methylation detection has gradually become an emerging means for detecting cervical cancer and precancerous lesions of the cervix. Multiple methylated genes such as FAM19A4, Mir124-2, PAX1, ZNF582, SOX1, EPB41L3, etc. have been considered as biomarkers for cervical cancer screening. However, their detection efficacies for cervical cancer and precancerous lesions of the cervix in the Chinese population are generally average. Therefore, it is of important clinical significance to explore and develop more effective and objective biomarkers applicable to the Chinese population. Summary of the Invention

[0005] Aiming at the problem of lacking methylation markers in the Chinese population, reduced representation bisulfite sequencing (RRBS) was used for primary screening of cervical exfoliated cell DNA in the Chinese population, and targeted bisulfite sequencing (TBS) was used for verification. By analyzing the differentially methylated regions between cervical cancer, precancerous lesions of the cervix, and healthy people in the high-throughput sequencing results, a model was constructed to provide new ideas and detection means for the early diagnosis and treatment of cervical cancer and precancerous lesions of the cervix. The main contribution of this application lies in the discovery of these methylation markers, and any methylation level analysis method can be used to detect the methylation level of the methylation markers discovered in this application.

[0006] The present application provides the use of a substance for detecting methylation markers in the preparation of a product for detecting cervical cancer and / or precancerous lesions of the cervix, and the methylation markers include any one of the following genes or their fragments: AMER2, TTC34, NMNAT2, SLC7A14, VSTM2B, SHOX, NKX2-6, LOC728424, ALX3, EN1, PROX1-AS1, LHX4, DCHS2, FOXD3, TBX5-AS1, LOC100420879, CTNND2, ARHGEF4, ZNF536-1, LOC127274910, ST8SIA5, intergenic region 1, ZNF536-2, HTR1F, PDE4B, PROKR2, SFRP4, WEE1P1, VSX1, LINC01210, NOL4, ZNF610 or SLC30A10.

[0007] The present application further provides a product for detecting cervical cancer and / or precancerous lesions of the cervix, and the product includes a substance for detecting methylation markers, and the methylation markers include any one of the following genes or their fragments: AMER2, TTC34, NMNAT2, SLC7A14, VSTM2B, SHOX, NKX2-6, LOC728424, ALX3, EN1, PROX1-AS1, LHX4, DCHS2, FOXD3, TBX5-AS1, LOC100420879, CTNND2, ARHGEF4, ZNF536-1, LOC127274910, ST8SIA5, intergenic region 1, ZNF536-2, HTR1F, PDE4B, PROKR2, SFRP4, WEE1P1, VSX1, LINC01210, NOL4, ZNF610 or SLC30A10.

[0008] The present application further provides a detection device for cervical cancer and / or precancerous lesions of the cervix, including the following modules: a data acquisition module for providing methylation level data of a target marker in a sample to be tested, where the target marker is the methylation marker in the above application; a judgment module for evaluating the situation of cervical cancer and / or precancerous lesions of the cervix based on the methylation level data of the target marker in the sample to be tested.

[0009] The present application further provides a computer-readable storage medium, and the storage medium stores computer instructions, and when the computer instructions are executed by a processor, a method for detecting cervical cancer and / or precancerous lesions of the cervix is implemented, and the method includes: acquiring methylation level data of a target marker in a sample to be tested, where the target marker is the methylation marker in the above application; evaluating the situation of cervical cancer and / or precancerous lesions of the cervix based on the methylation level data of the target marker in the sample to be tested.

[0010] The present application also provides an electronic terminal, which includes: a processor, a memory, an input / output interface, and a communication port; the memory is used to store a computer program, and the processor is used to execute the computer program stored in the memory, so that the terminal executes the method for detecting cervical cancer and / or precancerous lesions of the cervix in the above-mentioned computer-readable storage medium.

[0011] The present application also provides a computer program product, including a computer program, and when the computer program is executed by a processor, it realizes the following steps: obtaining methylation level data of target markers of a sample to be tested, and evaluating the situation of cervical cancer and / or precancerous lesions of the cervix based on the methylation level data of target markers of the sample to be tested.

[0012] The beneficial effects brought by the present application include but are not limited to: the present application conducts precise screening based on cervical lesion samples of different grades in the Chinese population, specifically searches for methylation markers for effectively detecting cervical cancer and precancerous lesions of the cervix in the Chinese population. The screened methylation markers are brand-new methylation markers with clinical potential, and have good sensitivity and specificity, providing a fast, effective, and accurate new way for early screening, auxiliary diagnosis, and evaluation of cervical cancer and precancerous lesions of the cervix. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The present application will be further described by way of exemplary embodiments, and these exemplary embodiments will be described in detail through the drawings. These embodiments are not restrictive, where:

[0014] Figure 1 Shows the R & D process of screening methylation biomarkers for detecting the status of cervical cancer and precancerous lesions of the cervix using methylation sequencing data.

[0015] Figure 2 Shows the differential heat map of the methylation levels of methylation markers between CIN1- and CIN3+ in the reduced-representation bisulfite sequencing in Embodiment 1 of the present application.

[0016] Figure 3 Shows the receiver operating characteristic curve (ROC) graph of screening cervical cancer and precancerous lesions by 20 methylation marker combinations in Embodiment 2 of the present application.

[0017] Figure 4 Shows the receiver operating characteristic curve (ROC) graph of screening cervical cancer and precancerous lesions by methylation markers with AUC Top15 in Embodiment 2 of the present application.

[0018] Figure 5 Shows the receiver operating characteristic curve (ROC) graph of screening cervical cancer and precancerous lesions by 3 manually screened methylation markers in Embodiment 2 of the present application.

[0019] Figure 6 It is a module diagram of a cervical cancer and / or cervical intraepithelial neoplasia detection device according to some embodiments of the present application.

[0020] Figure 7 It is a flowchart of a method for detecting cervical cancer and / or cervical intraepithelial neoplasia according to some embodiments of the present application.

[0021] Figure 8 It is a schematic diagram of the architecture of an electronic terminal 800 according to some embodiments of the present application. Detailed implementation manners

[0022] To more clearly illustrate the technical solutions of the embodiments of this specification, the accompanying drawings required for the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some examples or embodiments of this specification. For those of ordinary skill in the art, without creative efforts, this specification can also be applied to other similar scenarios based on these drawings. Unless obvious from the language context or otherwise stated, the same reference numerals in the figures represent the same structure or operation.

[0023] As shown in this specification and the claims, unless the context clearly indicates an exception, words such as "a", "an", "one", and / or "the" are not specifically singular and may also include plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of the clearly identified steps and elements, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements.

[0024] Flowcharts are used in this specification to illustrate the operations performed by the systems according to the embodiments of this specification. It should be understood that the operations before or after may not be executed precisely in sequence. On the contrary, they can be executed in reverse order or simultaneously. At the same time, other operations can also be added to these processes, or one or several operations can be removed from these processes.

[0025] The present application provides the use of a substance for detecting a methylation marker in the preparation of a product for detecting cervical cancer and / or precancerous lesions of the cervix, and the methylation marker includes any one of the following genes or their fragments: AMER2, TTC34, NMNAT2, SLC7A14, VSTM2B, SHOX, NKX2-6, LOC728424, ALX3, EN1, PROX1-AS1, LHX4, DCHS2, FOXD3, TBX5-AS1, LOC100420879, CTNND2, ARHGEF4, ZNF536-1, LOC127274910, ST8SIA5, intergenic region 1, ZNF536-2, HTR1F, PDE4B, PROKR2, SFRP4, WEE1P1, VSX1, LINC01210, NOL4, ZNF610 or SLC30A10.

[0026] In some embodiments, the region where the intergenic region 1 is located may be Chr10: 23461814-23463239.

[0027] In some embodiments, based on the sequence of the human reference genome Hg19, the methylation markers may include any one of the following genes or their fragments: Chr13:25744938-25746426, Chr1:2705810-2707334, Chr1:183385855-183387356, Chr3:170302295-170303819, Chr19:30018451-30019977, ChrX:590483-592172, Chr8:23566680-23568179, Chr15:30516816-30518367, Chr1:110611042-110612605, Chr2:119607088-119608622, Chr1:214155611-214157113, Chr1:180201754-180203218, Chr4:155410591-155412092, Chr1:63784781-63786348, Chr12:114846786-114848325, Chr1:243052908-243054404, Chr5:11384181-11385604, Chr2:131721013-131722519, Chr19:30865153-30866677, Chr2:137522897-137524244, Chr18:44336420-44337856, Chr10:23461814-23463239, Chr19:30716060-30717060, Chr3:87841226-87842641, Chr1:66257794-66259310, Chr20:5297005-5298538, Chr7:37955552-37957019, Chr4:107146-108577, Chr20:25061403-25062879, Chr3:137489322-137490790, Chr18:31804106-31805604, Chr19:52839052-52840463, Chr1:220100662-220102096.

[0028] "Chr" and the following numbers (1-20) represent specific chromosomes, and the numbers after ":" represent the base positions on the chromosomes. Taking "Chr1:220100662-220102096" as an example, it represents the base sequence between the 220100662nd base and the 220102096th base on human chromosome 1.

[0029] In some embodiments, the cervical pre-cancerous lesion may be a severe cervical pre-cancerous lesion.

[0030] In some embodiments, the sample for product detection may include any one or more of blood, serum, plasma, lymph, urine, cervical scraping cells or tissues, biopsy tissues, surgical tissues or cervical exfoliated cells. In some embodiments, preferably, the sample for product detection may be cervical exfoliated cells.

[0031] In some embodiments, the substance for detecting the methylation marker may include a substance for detecting the methylation level of the methylation marker. In some embodiments, preferably, the substance for detecting the methylation level of the methylation marker may include primer pairs and probes.

[0032] In some embodiments, more preferably, the primer pair and the probe are specific primer pairs and probes corresponding to any one of the following genes or their fragments: Chr13:25744938-25746426, Chr1:2705810-2707334, Chr1:183385855-183387356, Chr3:170302295-170303819, Chr19:30018451-30019977, ChrX:590483-592172, Chr8:23566680-23568179, Chr15:30516816-30518367, Chr1:110611042-110612605, Chr2:119607088-119608622, Chr1:214155611-214157113, Chr1:180201754-180203218, Chr4:155410591-155412092, Chr1:63784781-63786348, Chr12:114846786-114848325, Chr1:243052908-243054404, Chr5:11384181-11385604, Chr2:131721013-131722519, Chr19:30865153-30866677, Chr2:137522897-137524244, Chr18:44336420-44337856, Chr10:23461814-23463239, Chr19:30716060-30717060, Chr3:87841226-87842641, Chr1:66257794-66259310, Chr20:5297005-5298538, Chr7:37955552-37957019, Chr4:107146-108577, Chr20:25061403-25062879, Chr3:137489322-137490790, Chr18:31804106-31805604, Chr19:52839052-52840463, Chr1:220100662-220102096.

[0033] As used herein, "corresponding" refers to the corresponding gene fragment after Bisulfite treatment. After Bisulfite treatment, the corresponding gene fragment will change differently depending on whether it is methylated. Specifically, unmethylated cytosine (C) will be converted to uracil (U) during Bisulfite treatment, while methylated cytosine remains unchanged.

[0034] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker AMER2 gene or its fragment can be as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0035] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker TTC34 gene or its fragment can be as shown in SEQ ID NO.4 and SEQ ID NO.5.

[0036] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker NMNAT2 gene or its fragment can be as shown in SEQ ID NO.7 and SEQ ID NO.8.

[0037] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker SLC7A14 gene or its fragment can be as shown in SEQ ID NO.10 and SEQ ID NO.11.

[0038] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker VSTM2B gene or its fragment can be as shown in SEQ ID NO.13 and SEQ ID NO.14.

[0039] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker SHOX gene or its fragment can be as shown in SEQ ID NO.16 and SEQ ID NO.17.

[0040] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker NKX2-6 gene or its fragment can be as shown in SEQ ID NO.19 and SEQ ID NO.20.

[0041] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker LOC728424 gene or its fragment can be as shown in SEQ ID NO.22 and SEQ ID NO.23.

[0042] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker ALX3 gene or its fragment can be as shown in SEQ ID NO.25 and SEQ ID NO.26.

[0043] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker EN1 gene or its fragment can be as shown in SEQ ID NO.28 and SEQ ID NO.29.

[0044] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker PROX1-AS1 gene or its fragment can be as shown in SEQ ID NO31 and SEQ ID NO.32.

[0045] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker LHX4 gene or its fragment can be as shown in SEQ ID NO.34 and SEQ ID NO.35.

[0046] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker DCHS2 gene or its fragment can be as shown in SEQ ID NO.37 and SEQ ID NO.38.

[0047] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker FOXD3 gene or its fragment can be as shown in SEQ ID NO.40 and SEQ ID NO.41.

[0048] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker TBX5-AS1 gene or its fragment can be as shown in SEQ ID NO.43 and SEQ ID NO.44.

[0049] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker LOC100420879 gene or its fragment can be as shown in SEQ ID NO.46 and SEQ ID NO.47.

[0050] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker CTNND2 gene or its fragment can be as shown in SEQ ID NO.49 and SEQ ID NO.50.

[0051] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker ARHGEF4 gene or its fragment can be as shown in SEQ ID NO.52 and SEQ ID NO.53.

[0052] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker ZNF536-1 gene or its fragment can be as shown in SEQ ID NO.55 and SEQ ID NO.56.

[0053] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker LOC127274910 gene or its fragment can be as shown in SEQ ID NO.58 and SEQ ID NO.59.

[0054] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker ST8SIA5 gene or its fragment can be as shown in SEQ ID NO.61 and SEQ ID NO.62.

[0055] In some embodiments, the nucleotide sequences of the primer pairs for detecting the intergenic region 1 gene of the methylated marker or its fragment can be as shown in SEQ ID NO.64 and SEQ ID NO.65.

[0056] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker ZNF536-2 gene or its fragment can be as shown in SEQ ID NO.67 and SEQ ID NO.68.

[0057] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker HTR1F gene or its fragment can be as shown in SEQ ID NO.70 and SEQ ID NO.71.

[0058] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker PDE4B gene or its fragment can be as shown in SEQ ID NO.73 and SEQ ID NO.74.

[0059] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker PROKR2 gene or its fragment can be as shown in SEQ ID NO.76 and SEQ ID NO.77.

[0060] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker SFRP4 gene or its fragment can be as shown in SEQ ID NO.79 and SEQ ID NO.80.

[0061] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker WEE1P1 gene or its fragment can be as shown in SEQ ID NO.82 and SEQ ID NO.83.

[0062] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker VSX1 gene or its fragment can be as shown in SEQ ID NO.85 and SEQ ID NO.86.

[0063] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker LINC01210 gene or its fragment can be as shown in SEQ ID NO.88 and SEQ ID NO.89.

[0064] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker NOL4 gene or its fragment can be as shown in SEQ ID NO.91 and SEQ ID NO.92.

[0065] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker ZNF610 gene or its fragment can be as shown in SEQ ID NO.94 and SEQ ID NO.95.

[0066] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker SLC30A10 gene or its fragment can be as shown in SEQ ID NO.97 and SEQ ID NO.98.

[0067] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker AMER2 gene or its fragment can be as shown in SEQ ID NO.3.

[0068] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker TTC34 gene or its fragment can be as shown in SEQ ID NO.6.

[0069] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker NMNAT2 gene or its fragment can be as shown in SEQ ID NO.9.

[0070] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker SLC7A14 gene or its fragment can be as shown in SEQ ID NO.12.

[0071] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker VSTM2B gene or its fragment can be as shown in SEQ ID NO.15.

[0072] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker SHOX gene or its fragment can be as shown in SEQ ID NO.18.

[0073] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker NKX2-6 gene or its fragment can be as shown in SEQ ID NO.21.

[0074] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker LOC728424 gene or its fragment can be as shown in SEQ ID NO.24.

[0075] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ALX3 gene or its fragment may be as shown in SEQ ID NO.27.

[0076] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker EN1 gene or its fragment may be as shown in SEQ ID NO.30.

[0077] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker PROX1-AS1 gene or its fragment may be as shown in SEQ ID NO.33.

[0078] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker LHX4 gene or its fragment may be as shown in SEQ ID NO.36.

[0079] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker DCHS2 gene or its fragment may be as shown in SEQ ID NO.39.

[0080] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker FOXD3 gene or its fragment may be as shown in SEQ ID NO.42.

[0081] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker TBX5-AS1 gene or its fragment may be as shown in SEQ ID NO.45.

[0082] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker LOC100420879 gene or its fragment may be as shown in SEQ ID NO.48.

[0083] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker CTNND2 gene or its fragment may be as shown in SEQ ID NO.51.

[0084] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ARHGEF4 gene or its fragment may be as shown in SEQ ID NO.54.

[0085] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ZNF536-1 gene or its fragment may be as shown in SEQ ID NO.57.

[0086] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker LOC127274910 gene or its fragment may be as shown in SEQ ID NO.60.

[0087] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ST8SIA5 gene or its fragment may be as shown in SEQ ID NO.63.

[0088] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker intergenic region 1 gene or its fragment may be as shown in SEQ ID NO.66.

[0089] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ZNF536-2 gene or its fragment may be as shown in SEQ ID NO.69.

[0090] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker HTR1F gene or its fragment may be as shown in SEQ ID NO.72.

[0091] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker PDE4B gene or its fragment may be as shown in SEQ ID NO.75.

[0092] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker PROKR2 gene or its fragment may be as shown in SEQ ID NO.78.

[0093] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker SFRP4 gene or its fragment may be as shown in SEQ ID NO.81.

[0094] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker WEE1P1 gene or its fragment may be as shown in SEQ ID NO.84.

[0095] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker VSX1 gene or its fragment may be as shown in SEQ ID NO.87.

[0096] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker LINC01210 gene or its fragment may be as shown in SEQ ID NO.90.

[0097] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker NOL4 gene or its fragment may be as shown in SEQ ID NO.93.

[0098] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ZNF610 gene or its fragment may be as shown in SEQ ID NO.96.

[0099] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker SLC30A10 gene or its fragment may be as shown in SEQ ID NO.99.

[0100] The present application also provides a product for detecting cervical cancer and / or cervical precancerous lesions, the product comprising a substance for detecting a methylation marker, the methylation marker comprising any one of the following genes or their fragments: AMER2, TTC34, NMNAT2, SLC7A14, VSTM2B, SHOX, NKX2-6, LOC728424, ALX3, EN1, PROX1-AS1, LHX4, DCHS2, FOXD3, TBX5-AS1, LOC100420879, CTNND2, ARHGEF4, ZNF536-1, LOC127274910, ST8SIA5, intergenic region 1, ZNF536-2, HTR1F, PDE4B, PROKR2, SFRP4, WEE1P1, VSX1, LINC01210, NOL4, ZNF610 or SLC30A10.

[0101] In some embodiments, based on the sequence of the human reference genome Hg19, the methylation markers may include any one of the following genes or their fragments: Chr13:25744938-25746426, Chr1:2705810-2707334, Chr1:183385855-183387356, Chr3:170302295-170303819, Chr19:30018451-30019977, ChrX:590483-592172, Chr8:23566680-23568179, Chr15:30516816-30518367, Chr1:110611042-110612605, Chr2:119607088-119608622, Chr1:214155611-214157113, Chr1:180201754-180203218, Chr4:155410591-155412092, Chr1:63784781-63786348, Chr12:114846786-114848325, Chr1:243052908-243054404, Chr5:11384181-11385604, Chr2:131721013-131722519, Chr19:30865153-30866677, Chr2:137522897-137524244, Chr18:44336420-44337856, Chr10:23461814-23463239, Chr19:30716060-30717060, Chr3:87841226-87842641, Chr1:66257794-66259310, Chr20:5297005-5298538, Chr7:37955552-37957019, Chr4:107146-108577, Chr20:25061403-25062879, Chr3:137489322-137490790, Chr18:31804106-31805604, Chr19:52839052-52840463, Chr1:220100662-220102096.

[0102] In some embodiments, the cervical pre-cancerous lesion may be a severe cervical pre-cancerous lesion.

[0103] In some embodiments, the sample for product detection may include any one or more of blood, serum, plasma, lymph, urine, cervical scraping cells or tissues, biopsy tissues, surgical tissues, or cervical exfoliated cells. In some embodiments, preferably, the sample for product detection may be cervical exfoliated cells. In some embodiments, the product may be used to detect DNA in the sample.

[0104] In some embodiments, the product may be any one of a kit, a chip, a membrane strip, a protein array, a composition, or a detection system. In some embodiments, preferably, the product may include one or more of DNA polymerase, deoxynucleotide (dNTP) mixture, buffer solution, primers, probes, sodium bisulfite, positive control, or negative control.

[0105] The term "kit" refers to a packaged collection of related components, such as one or more polynucleotides or compositions, and one or more related materials, such as a delivery device (e.g., a syringe), a solvent, a solution, a buffer, an instruction manual, or a desiccant.

[0106] The term "membrane strip" is a diagnostic tool that utilizes the principle of specific biomolecular recognition. By immobilizing biomolecules such as antigens or antibodies on the membrane, it specifically binds to the substance to be detected in the sample, and qualitatively or quantitatively analyzes the target substance in the sample through visualization or other signal detection means.

[0107] The term "chip" generally refers to a microdevice integrating biosensors and microfluidic technologies. It can perform various operations such as sample preparation, reaction, and detection of biological, chemical, and medical analysis processes at the micro level to achieve rapid and accurate detection of disease-related biomarkers.

[0108] A protein array, also known as a protein microarray, is a high-throughput biotechnology tool that allows simultaneous analysis and study of a large number of proteins. This technology enables rapid protein expression analysis, protein-protein interaction, and protein-small molecule binding studies by orderly arranging thousands of different proteins or protein interaction probes on a solid surface.

[0109] In some embodiments, the substance for detecting methylation markers may include a substance for detecting the methylation level of methylation markers. In some embodiments, preferably, the substance for detecting the methylation level of methylation markers may include primer pairs and probes.

[0110] In some embodiments, more preferably, the primer pair and the probe can be a specific primer pair and probe corresponding to any one of the following genes or their fragments: Chr13:25744938-25746426, Chr1:2705810-2707334, Chr1:183385855-183387356, Chr3:170302295-170303819, Chr19:30018451-30019977, ChrX:590483-592172, Chr8:23566680-23568179, Chr15:30516816-30518367, Chr1:110611042-110612605, Chr2:119607088-119608622, Chr1:214155611-214157113, Chr1:180201754-180203218, Chr4:155410591-155412092, Chr1:63784781-63786348, Chr12:114846786-114848325, Chr1:243052908-243054404, Chr5:11384181-11385604, Chr2:131721013-131722519, Chr19:30865153-30866677, Chr2:137522897-137524244, Chr18:44336420-44337856, Chr10:23461814-23463239, Chr19:30716060-30717060, Chr3:87841226-87842641, Chr1:66257794-66259310, Chr20:5297005-5298538, Chr7:37955552-37957019, Chr4:107146-108577, Chr20:25061403-25062879, Chr3:137489322-137490790, Chr18:31804106-31805604, Chr19:52839052-52840463, Chr1:220100662-220102096.

[0111] As used herein, the term "primer" refers to a naturally occurring oligonucleotide (e.g., a restriction fragment) or a synthetically produced oligonucleotide that can serve as a starting point for the synthesis of a primer extension product, which is complementary to a nucleic acid strand (template or target sequence) under appropriate conditions (e.g., buffer, salt, temperature, and pH) and in the presence of nucleotides and reagents for nucleic acid polymerization (e.g., DNA-dependent or RNA-dependent polymerase). Typically, a set of primers will consist of at least two primers, an "upstream primer" and a "downstream primer", which together define the amplicon (the sequence to be amplified using the primers).

[0112] The term "probe" refers to any molecule that can selectively bind to a target biomolecule (e.g., a nucleic acid sequence that hybridizes to the probe). In some embodiments, the probe can be labeled, e.g., with a fluorophore and a quencher group. In some embodiments, the probe can be a Taqman probe with a fluorescent reporter group added to the 5' end and a fluorescent quencher group added to the 3' end.

[0113] An antibody is a protective protein produced by the body in response to the stimulation of an antigen. In some embodiments, the antibody can be used as a detection reagent to detect the expression of a gene.

[0114] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylation marker AMER2 gene or its fragment can be as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0115] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylation marker TTC34 gene or its fragment can be as shown in SEQ ID NO.4 and SEQ ID NO.5.

[0116] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylation marker NMNAT2 gene or its fragment can be as shown in SEQ ID NO.7 and SEQ ID NO.8.

[0117] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylation marker SLC7A14 gene or its fragment can be as shown in SEQ ID NO.10 and SEQ ID NO.11.

[0118] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylation marker VSTM2B gene or its fragment can be as shown in SEQ ID NO.13 and SEQ ID NO.14.

[0119] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker SHOX gene or its fragment can be as shown in SEQ ID NO.16 and SEQ ID NO.17.

[0120] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker NKX2-6 gene or its fragment can be as shown in SEQ ID NO.19 and SEQ ID NO.20.

[0121] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker LOC728424 gene or its fragment can be as shown in SEQ ID NO.22 and SEQ ID NO.23.

[0122] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker ALX3 gene or its fragment can be as shown in SEQ ID NO.25 and SEQ ID NO.26.

[0123] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker EN1 gene or its fragment can be as shown in SEQ ID NO.28 and SEQ ID NO.29.

[0124] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker PROX1-AS1 gene or its fragment can be as shown in SEQ ID NO31 and SEQ ID NO.32.

[0125] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker LHX4 gene or its fragment can be as shown in SEQ ID NO.34 and SEQ ID NO.35.

[0126] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker DCHS2 gene or its fragment can be as shown in SEQ ID NO.37 and SEQ ID NO.38.

[0127] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker FOXD3 gene or its fragment can be as shown in SEQ ID NO.40 and SEQ ID NO.41.

[0128] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker TBX5-AS1 gene or its fragment can be as shown in SEQ ID NO.43 and SEQ ID NO.44.

[0129] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker LOC100420879 gene or its fragment can be as shown in SEQ ID NO.46 and SEQ ID NO.47.

[0130] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker CTNND2 gene or its fragment can be as shown in SEQ ID NO.49 and SEQ ID NO.50.

[0131] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker ARHGEF4 gene or its fragment can be as shown in SEQ ID NO.52 and SEQ ID NO.53.

[0132] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker ZNF536-1 gene or its fragment can be as shown in SEQ ID NO.55 and SEQ ID NO.56.

[0133] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker LOC127274910 gene or its fragment can be as shown in SEQ ID NO.58 and SEQ ID NO.59.

[0134] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker ST8SIA5 gene or its fragment can be as shown in SEQ ID NO.61 and SEQ ID NO.62.

[0135] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker intergenic region 1 gene or its fragment can be as shown in SEQ ID NO.64 and SEQ ID NO.65.

[0136] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker ZNF536-2 gene or its fragment can be as shown in SEQ ID NO.67 and SEQ ID NO.68.

[0137] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker HTR1F gene or its fragment can be as shown in SEQ ID NO.70 and SEQ ID NO.71.

[0138] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker PDE4B gene or its fragment can be as shown in SEQ ID NO.73 and SEQ ID NO.74.

[0139] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker PROKR2 gene or its fragment may be as shown in SEQ ID NO.76 and SEQ ID NO.77.

[0140] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker SFRP4 gene or its fragment may be as shown in SEQ ID NO.79 and SEQ ID NO.80.

[0141] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker WEE1P1 gene or its fragment may be as shown in SEQ ID NO.82 and SEQ ID NO.83.

[0142] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker VSX1 gene or its fragment may be as shown in SEQ ID NO.85 and SEQ ID NO.86.

[0143] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker LINC01210 gene or its fragment may be as shown in SEQ ID NO.88 and SEQ ID NO.89.

[0144] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker NOL4 gene or its fragment may be as shown in SEQ ID NO.91 and SEQ ID NO.92.

[0145] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker ZNF610 gene or its fragment may be as shown in SEQ ID NO.94 and SEQ ID NO.95.

[0146] In some embodiments, the nucleotide sequences of the primer pairs for detecting the methylated marker SLC30A10 gene or its fragment may be as shown in SEQ ID NO.97 and SEQ ID NO.98.

[0147] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker AMER2 gene or its fragment may be as shown in SEQ ID NO.3.

[0148] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker TTC34 gene or its fragment may be as shown in SEQ ID NO.6.

[0149] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker NMNAT2 gene or its fragment may be as shown in SEQ ID NO.9.

[0150] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the SLC7A14 gene or its fragment as a methylation marker may be as shown in SEQ ID NO.12.

[0151] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the VSTM2B gene or its fragment as a methylation marker may be as shown in SEQ ID NO.15.

[0152] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the SHOX gene or its fragment as a methylation marker may be as shown in SEQ ID NO.18.

[0153] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the NKX2-6 gene or its fragment as a methylation marker may be as shown in SEQ ID NO.21.

[0154] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the LOC728424 gene or its fragment as a methylation marker may be as shown in SEQ ID NO.24.

[0155] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the ALX3 gene or its fragment as a methylation marker may be as shown in SEQ ID NO.27.

[0156] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the EN1 gene or its fragment as a methylation marker may be as shown in SEQ ID NO.30.

[0157] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the PROX1-AS1 gene or its fragment as a methylation marker may be as shown in SEQ ID NO.33.

[0158] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the LHX4 gene or its fragment as a methylation marker may be as shown in SEQ ID NO.36.

[0159] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the DCHS2 gene or its fragment as a methylation marker may be as shown in SEQ ID NO.39.

[0160] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the FOXD3 gene or its fragment as a methylation marker may be as shown in SEQ ID NO.42.

[0161] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker TBX5-AS1 gene or its fragment may be as shown in SEQ ID NO.45.

[0162] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker LOC100420879 gene or its fragment may be as shown in SEQ ID NO.48.

[0163] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker CTNND2 gene or its fragment may be as shown in SEQ ID NO.51.

[0164] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker ARHGEF4 gene or its fragment may be as shown in SEQ ID NO.54.

[0165] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker ZNF536-1 gene or its fragment may be as shown in SEQ ID NO.57.

[0166] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker LOC127274910 gene or its fragment may be as shown in SEQ ID NO.60.

[0167] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker ST8SIA5 gene or its fragment may be as shown in SEQ ID NO.63.

[0168] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker intergenic region 1 gene or its fragment may be as shown in SEQ ID NO.66.

[0169] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker ZNF536-2 gene or its fragment may be as shown in SEQ ID NO.69.

[0170] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker HTR1F gene or its fragment may be as shown in SEQ ID NO.72.

[0171] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylated marker PDE4B gene or its fragment may be as shown in SEQ ID NO.75.

[0172] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker PROKR2 gene or its fragment can be as shown in SEQ ID NO.78.

[0173] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker SFRP4 gene or its fragment can be as shown in SEQ ID NO.81.

[0174] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker WEE1P1 gene or its fragment can be as shown in SEQ ID NO.84.

[0175] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker VSX1 gene or its fragment can be as shown in SEQ ID NO.87.

[0176] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker LINC01210 gene or its fragment can be as shown in SEQ ID NO.90.

[0177] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker NOL4 gene or its fragment can be as shown in SEQ ID NO.93.

[0178] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ZNF610 gene or its fragment can be as shown in SEQ ID NO.96.

[0179] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker SLC30A10 gene or its fragment can be as shown in SEQ ID NO.99.

[0180] The present application also provides a method for diagnosing human cervical cancer and / or cervical precancerous lesions, which uses the above product to detect whether the methylation levels of genes AMER2, TTC34, NMNAT2, SLC7A14, VSTM2B, SHOX, NKX2-6, LOC728424, ALX3, EN1, PROX1-AS1, LHX4, DCHS2, FOXD3, TBX5-AS1, LOC100420879, CTNND2, ARHGEF4, ZNF536-1, LOC127274910, ST8SIA5, intergenic region 1, ZNF536-2, HTR1F, PDE4B, PROKR2, SFRP4, WEE1P1, VSX1, LINC01210, NOL4, ZNF610 or SLC30A10 or their fragments in a test sample are increased relative to healthy individuals.

[0181] In some embodiments, the method may include the following steps: (1) Collect a test sample, extract genomic DNA from the sample, perform Bisulfite treatment to obtain the converted DNA; (2) Prepare a reaction system, which includes the converted genomic DNA of the sample, DNA polymerase, deoxynucleotide (dNTP) mixture, buffer solution, primers, and probes; (3) Perform an amplification reaction; (4) Analyze the results to obtain the methylation levels of genes AMER2, TTC34, NMNAT2, SLC7A14, VSTM2B, SHOX, NKX2-6, LOC728424, ALX3, EN1, PROX1-AS1, LHX4, DCHS2, FOXD3, TBX5-AS1, LOC100420879, CTNND2, ARHGEF4, ZNF536-1, LOC127274910, ST8SIA5, intergenic region 1, ZNF536-2, HTR1F, PDE4B, PROKR2, SFRP4, WEE1P1, VSX1, LINC01210, NOL4, ZNF610 or SLC30A10 or their fragments in the test sample; (5) Determine whether the person corresponding to the test sample has cervical cancer and / or cervical precancerous lesions.

[0182] In some embodiments, the amplification reaction may be quantitative methylation-specific PCR (qMSP). Calculate the ΔCt value of the methylation marker according to the qMSP results. ΔCt 目标基因 = Ct 目标基因 - Ct β-actin .

[0183] The ROC of each gene was obtained using SPSS statistic 21 software, with sensitivity (true positive rate) on the vertical axis and 1 - specificity (false positive rate) on the horizontal axis. The Youden index = sensitivity - (1 - specificity), and the optimal cut-off point (the cut-off value of ΔCt) was determined based on the maximum value of the Youden index.

[0184] In some embodiments, when the ΔCt value of the AMER2 gene or its fragment in the test sample is lower than or significantly lower than the cut-off value of ΔCt, which is 10.80, it can be determined that the person corresponding to the test sample has cervical cancer and / or precancerous lesions of the cervix.

[0185] In some embodiments, when the ΔCt value of the TTC34 gene or its fragment in the test sample is lower than or significantly lower than the cut-off value of ΔCt, which is 7.96, it can be determined that the person corresponding to the test sample has cervical cancer and / or precancerous lesions of the cervix.

[0186] In some embodiments, when the ΔCt value of the NMNAT2 gene or its fragment in the test sample is lower than or significantly lower than the cut-off value of ΔCt, which is 12.17, it can be determined that the person corresponding to the test sample has cervical cancer and / or precancerous lesions of the cervix.

[0187] In some embodiments, when the ΔCt value of the SLC7A14 gene or its fragment in the test sample is lower than or significantly lower than the cut-off value of ΔCt, which is 6.37, it can be determined that the person corresponding to the test sample has cervical cancer and / or precancerous lesions of the cervix.

[0188] In some embodiments, when the ΔCt value of the VSTM2B gene or its fragment in the test sample is lower than or significantly lower than the cut-off value of ΔCt, which is 8.96, it can be determined that the person corresponding to the test sample has cervical cancer and / or precancerous lesions of the cervix.

[0189] In some embodiments, when the ΔCt value of the SHOX gene or its fragment in the test sample is lower than or significantly lower than the cut-off value of ΔCt, which is 8.92, it can be determined that the person corresponding to the test sample has cervical cancer and / or precancerous lesions of the cervix.

[0190] In some embodiments, when the ΔCt value of the NKX2-6 gene or its fragment in the test sample is lower than or significantly lower than the cut-off value of ΔCt, which is 7.62, it can be determined that the person corresponding to the test sample has cervical cancer and / or precancerous lesions of the cervix.

[0191] In some embodiments, when the ΔCt value of the LOC728424 gene or its fragment in the test sample is lower than or significantly lower than the cut-off value of ΔCt, which is 6.48, it can be determined that the person corresponding to the test sample has cervical cancer and / or precancerous lesions of the cervix.

[0192] In some embodiments, when the ΔCt value of the ALX3 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 11.45, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or cervical pre-cancerous lesions.

[0193] In some embodiments, when the ΔCt value of the EN1 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 6.83, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or cervical pre-cancerous lesions.

[0194] In some embodiments, when the ΔCt value of the PROX1-AS1 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 7.25, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or cervical pre-cancerous lesions.

[0195] In some embodiments, when the ΔCt value of the LHX4 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 9.56, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or cervical pre-cancerous lesions.

[0196] In some embodiments, when the ΔCt value of the DCHS2 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 16.68, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or cervical pre-cancerous lesions.

[0197] In some embodiments, when the ΔCt value of the FOXD3 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 15.33, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or cervical pre-cancerous lesions.

[0198] In some embodiments, when the ΔCt value of the TBX5-AS1 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 6.33, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or cervical pre-cancerous lesions.

[0199] In some embodiments, when the ΔCt value of the LOC100420879 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 5.27, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or cervical pre-cancerous lesions.

[0200] In some embodiments, when the ΔCt value of the CTNND2 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 15.37, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or cervical pre-cancerous lesions.

[0201] In some embodiments, when the ΔCt value of the ARHGEF4 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 7.44, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or precancerous lesions of the cervix.

[0202] In some embodiments, when the ΔCt value of the ZNF536-1 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 10.93, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or precancerous lesions of the cervix.

[0203] In some embodiments, when the ΔCt value of the LOC127274910 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 15.35, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or precancerous lesions of the cervix.

[0204] In some embodiments, when the ΔCt value of the ST8SIA5 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 8.72, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or precancerous lesions of the cervix.

[0205] In some embodiments, when the ΔCt value of the intergenic region 1 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 9.50, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or precancerous lesions of the cervix.

[0206] In some embodiments, when the ΔCt value of the ZNF536-2 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 8.83, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or precancerous lesions of the cervix.

[0207] In some embodiments, when the ΔCt value of the HTR1F gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 7.32, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or precancerous lesions of the cervix.

[0208] In some embodiments, when the ΔCt value of the PDE4B gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 9.67, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or precancerous lesions of the cervix.

[0209] In some embodiments, when the ΔCt value of the PROKR2 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 12.04, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or precancerous lesions of the cervix.

[0210] In some embodiments, when the ΔCt value of the SFRP4 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 7.91, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or cervical pre-cancerous lesions.

[0211] In some embodiments, when the ΔCt value of the WEE1P1 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 6.68, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or cervical pre-cancerous lesions.

[0212] In some embodiments, when the ΔCt value of the VSX1 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 4.85, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or cervical pre-cancerous lesions.

[0213] In some embodiments, when the ΔCt value of the LINC01210 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 3.97, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or cervical pre-cancerous lesions.

[0214] In some embodiments, when the ΔCt value of the NOL4 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 7.15, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or cervical pre-cancerous lesions.

[0215] In some embodiments, when the ΔCt value of the ZNF610 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 9.48, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or cervical pre-cancerous lesions.

[0216] In some embodiments, when the ΔCt value of the SLC30A10 gene or its fragment in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt, which is 7.67, it can be determined that the person corresponding to the sample to be tested has cervical cancer and / or cervical pre-cancerous lesions.

[0217] This application also provides a detection device for cervical cancer and / or cervical pre-cancerous lesions, as Figure 6 shown, including the following modules: a data acquisition module 610 and a judgment module 620.

[0218] The data acquisition module 610 is used to provide methylation level data of the target marker in the sample to be tested, and the target marker is the methylation marker in the above application.

[0219] In some embodiments, the ΔCt value of the methylation marker can be calculated based on the methylation level data of the target marker of the sample to be tested. In some embodiments, preferably, the methylation level data can be the Ct value detected by quantitative methylation-specific PCR for the methylation marker. In some embodiments, preferably, the ΔCt value can be the difference between the Ct value of the target marker and the Ct value of the internal reference gene. In some embodiments, more preferably, the internal reference gene can be β-actin.

[0220] A judgment module 620 is configured to evaluate the situation of cervical cancer and / or cervical precancerous lesions based on the methylation level data of the target marker of the sample to be tested. In some embodiments, the situation of cervical cancer and / or cervical precancerous lesions can be evaluated based on the ΔCt value and a preset cut-off value of ΔCt. In some embodiments, preferably, it can be determined whether the sample to be tested is negative or positive based on the ΔCt value and the preset cut-off value of ΔCt. In some embodiments, more preferably, the cut-off value of ΔCt can be the ΔCt value that maximizes the Youden index.

[0221] In some embodiments, when the ΔCt value of the target marker of the sample to be tested is less than or equal to the cut-off value of ΔCt, it can be determined that the sample to be tested is positive. In some embodiments, when the ΔCt value of the target marker of the sample to be tested is greater than the cut-off value of ΔCt, it can be determined that the sample to be tested is negative.

[0222] In some embodiments, "positive" means that the individual corresponding to the sample to be tested is a patient with severe cervical precancerous lesions or cervical cancer, and "negative" means that the individual corresponding to the sample to be tested is a healthy person, a patient with mild cervical precancerous lesions or a patient with moderate cervical precancerous lesions.

[0223] In some embodiments, the sample to be tested can include any one or more of blood, serum, plasma, lymph, urine, cervical scraping cells or tissues, biopsy tissues, surgical tissues or cervical exfoliated cells. In some embodiments, preferably, the sample to be tested can be cervical exfoliated cells.

[0224] The present application also provides a computer-readable storage medium, which stores computer instructions. When the computer instructions are executed by a processor, a method for detecting cervical cancer and / or cervical precancerous lesions is implemented. The flowchart of the method is as Figure 7 shown.

[0225] In step S710, methylation level data of the target marker of the sample to be tested is obtained, and the target marker is the methylation marker in the above application.

[0226] In some embodiments, the ΔCt value of the methylation marker can be calculated based on the methylation level data of the target marker of the sample to be tested. In some embodiments, preferably, the methylation level data can be the Ct value detected by quantitative methylation-specific PCR for the methylation marker. In some embodiments, preferably, the ΔCt value can be the difference between the Ct value of the target marker and the Ct value of the internal reference gene. In some embodiments, more preferably, the internal reference gene can be β-actin.

[0227] In step S720, based on the methylation level data of the target marker of the sample to be tested, the situation of cervical cancer and / or cervical precancerous lesions is evaluated.

[0228] In some embodiments, the situation of cervical cancer and / or cervical precancerous lesions can be evaluated based on the ΔCt value and a preset cut-off value of ΔCt. In some embodiments, preferably, it can be determined whether the sample to be tested is negative or positive based on the ΔCt value and a preset cut-off value of ΔCt. In some embodiments, more preferably, the cut-off value of ΔCt can be the ΔCt value that maximizes the Youden index.

[0229] In some embodiments, when the ΔCt value of the target marker of the sample to be tested is less than or equal to the cut-off value of ΔCt, the sample to be tested can be determined to be positive. In some embodiments, when the ΔCt value of the target marker of the sample to be tested is greater than the cut-off value of ΔCt, the sample to be tested can be determined to be negative.

[0230] In some embodiments, "positive" means that the individual corresponding to the sample to be tested is a patient with severe cervical precancerous lesions or cervical cancer, and "negative" means that the individual corresponding to the sample to be tested is a healthy person, a patient with mild cervical precancerous lesions or a patient with moderate cervical precancerous lesions.

[0231] In some embodiments, the sample to be tested can include any one or more of blood, serum, plasma, lymph, urine, cervical scraping cells or tissues, biopsy tissues, surgical tissues or cervical exfoliated cells. In some embodiments, preferably, the sample to be tested can be cervical exfoliated cells.

[0232] The present application also provides an electronic terminal 800, characterized in that the electronic terminal 800 includes: a processor 810, a memory 820, an input / output interface 830, and a communication port 840; the memory 820 is used to store a computer program, and the processor 810 is used to execute the computer program stored in the memory 820 so that the terminal executes the method for detecting cervical cancer and / or cervical precancerous lesions in the above-mentioned computer-readable storage medium.

[0233] The processor 810 may execute computing instructions (program code) and perform the functions of the detection device described in this application. The computing instructions may include programs, objects, components, data structures, procedures, modules, and functions (where "functions" refer to the specific functions described in this application). For example, the processor 810 may process instructions for evaluating cervical cancer and / or cervical precancerous lesions in a cervical cancer and / or cervical precancerous lesion detection device. In some embodiments, the processor 810 may include a microcontroller, a microprocessor, a reduced instruction set computer (RISC), an application specific integrated circuit (ASIC), an application specific instruction set processor (ASIP), a central processing unit (CPU), a graphics processing unit (GPU), a physics processing unit (PPU), a microcontroller unit, a digital signal processor (DSP), a field programmable gate array (FPGA), an advanced RISC machine (ARM), a programmable logic device, and any circuit and processor capable of performing one or more functions, etc., or any combination thereof. For illustration purposes only, Figure 8 only one processor 810 is described, but it should be noted that this application may include multiple processors.

[0234] The memory 820 may store data / information obtained from any component in the cervical cancer and / or cervical precancerous lesion detection device. In some embodiments, the memory 820 may include a mass storage device, a removable memory, a volatile read and write memory, and a read only memory (ROM), etc., or any combination thereof. Exemplary mass storage devices may include magnetic disks, optical disks, and solid state drives, etc. Removable memories may include flash drives, floppy disks, optical disks, memory cards, USB drives, compact disks, and external hard drives, etc. Volatile read and write memories may include random access memory (RAM). RAM may include dynamic RAM (DRAM), double data rate synchronous dynamic RAM (DDR SDRAM), static RAM (SRAM), thyristor RAM (T-RAM), and zero capacitor (Z-RAM), etc. ROM may include mask ROM (MROM), programmable ROM (PROM), erasable programmable ROM (PEROM), electrically erasable programmable ROM (EEPROM), CD-ROM, and digital versatile disc ROM, etc.

[0235] The input / output interface 830 can be used to input or output signals, data, or information. In some embodiments, the input / output interface 830 can be used to implement the interaction behavior between a user (e.g., the person corresponding to the sample to be tested, the user of the cervical cancer and / or cervical precancerous lesion detection device, etc.) and the processor 710. In some embodiments, the user can input the characteristic information of the person corresponding to the sample to be tested through the input / output interface 830. In some embodiments, the input / output interface 830 can include an input device and an output device. Exemplary input devices can include a keyboard, a mouse, a touch screen, a microphone, etc., or any combination thereof. Exemplary output devices can include a display device, a speaker, a printer, a projector, etc., or any combination thereof. Exemplary display devices can include a liquid crystal display (LCD), a light-emitting diode (LED)-based display, a flat panel display, a curved display, a television device, a cathode ray tube (CRT), etc., or any combination thereof.

[0236] The communication port 840 can be connected to a network for data communication. The connection can be a wired connection, a wireless connection, or a combination of both. Wired connections can include cables, optical fibers, telephone lines, etc., or any combination thereof. Wireless connections can include Bluetooth, WiFi, WiMax, WLAN, ZigBee, a mobile network (e.g., 3G, 4G, or 5G, etc.), etc., or any combination thereof. In some embodiments, the communication port 840 can be a standardized port, such as RS232, RS485, etc. In some embodiments, the communication port 840 can be a specially designed port.

[0237] This application also provides a computer program product, including a computer program, and the method for implementing the following steps when the computer program is executed by a processor: obtaining the methylation level data of the target biomarker of the sample to be tested, and evaluating the situation of cervical cancer and / or cervical precancerous lesions based on the methylation level data of the target biomarker of the sample to be tested.

[0238] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all purchased from conventional biochemical reagent companies unless otherwise specified. For the quantitative tests in the following embodiments, three repeated experiments are set, and the results are averaged.

[0239] The negative population (Normal), mild cervical precancerous lesions (CIN1), moderate cervical precancerous lesions (CIN2), severe cervical precancerous lesions (CIN3), and cervical cancer patients (Cancer) involved in the embodiments all meet the diagnostic criteria. The diagnostic criteria refer to the fifth edition of the World Health Organization (WHO) Classification of Tumours of the Female Reproductive Organs.

[0240] Cervical exfoliated cell specimens, reagent materials used, and their sources in the embodiments:

[0241] 1. Specimen

[0242] The biological samples used in the examples were cervical exfoliated cells collected by the Institute of Clinical Pharmacology of Xiangya Hospital, Central South University from January 2020 to December 2022, and all were samples with known pathological information.

[0243] 2. Main reagents and materials

[0244] The genomic DNA extraction kit for cervical exfoliated cells was HiPure Universal DNA Kit (Magen); the transformation kit was EZ DNA Methylation Gold Kit (ZYMO); the primers and probes used were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; the nuclease-free water, 10×Ex Buffer, Ex Taq HS enzyme, and dNTPs used were purchased from Takara Bio (Dalian) Co., Ltd.

[0245] The analysis flow chart for screening methylation biomarkers for cervical cancer and precancerous lesions of the cervix using methylation sequencing data is as Figure 1 shown.

[0246] Example 1: Screening for candidate methylation markers for cervical cancer and precancerous lesions of the cervix based on reduced representation bisulfite sequencing (RRBS).

[0247] Genomic DNA samples were obtained from cervical exfoliated cells from patients with CIN1- (22 cases), CIN3 (7 cases), and cervical cancer (7 cases) for RRBS detection. The specific method was to extract genomic DNA from cervical exfoliated cell samples using the HiPure Universal DNA Kit (Magen) extraction kit, and evaluate it by agarose gel electrophoresis and the OD260 / 280 ratio.

[0248] Take 100 - 300 ng of genomically DNA that has passed quality inspection. First, perform enzymatic digestion using MspI (methylation-insensitive restriction enzyme). Repair the ends of the digested DNA fragments and ligate adapters. After fragment selection by gel cutting, use the EZ DNA Methylation Gold Kit to perform bisulfite (BS) treatment on the DNA, and perform PCR amplification to obtain a DNA library. After the library passes quality inspection, different libraries are pooled according to the effective concentration and the required number of target reads for sequencing. Evaluate the quality of the sequencing data, and use the Trimming method to truncate the sequencing adapters and low-quality data of the sequencing data to obtain clean data for subsequent analysis. Use the Bsmap software to perform alignment analysis of methylation data to the reference genome. Use the metilene software (Ver0.2-7) to perform differentially methylated regions (DMR) analysis. Use the metilene software (Ver 0.2-7) to perform DMR analysis.

[0249] Group the samples into CIN1- and CIN3+. Define the negative population, CIN1, and CIN2 patients as CIN2-, and CIN3 and cervical cancer patients as CIN3+ patients. Screen the hypermethylated DMRs according to the following conditions: ① annotated regions; ② the average methylation difference between the CIN1- and CIN3+ groups is Top500; ③ the average methylation fold change between the CIN1- and CIN3+ groups is Top500; ④ the methylation level in the CIN1- group < 0.2. A total of 572 DMRs were screened for subsequent verification. The results are shown in Figure 2 .

[0250] Example 2: Screening for methylation markers in cervical cancer and cervical precancerous lesions based on targeted bisulfite sequencing (TBS).

[0251] Genomic DNA samples were obtained from exfoliated cells of 36 negative cases, 35 cases of CIN1, 46 cases of CIN2, 33 cases of CIN3 and 42 cases of cervical cancer for TBS detection. The specific method was as follows: after the samples passed the detection, 1 μg of genomic DNA was taken, and Bisulfite treatment was carried out using the EZ DNAMethylation Gold Kit (Zymo Research). Then, 1 / 20 of the elution product was used as a template, and PCR amplification was carried out for 35 cycles using the KAPA HiFi HotStart Uracil+ReadyMix PCR Kit (Kapa Biosystems, Wilmington, MA, USA). For each sample, the BSP products of multiple genes were pooled together in equal amounts. End repair, addition of "A" and ligation of adapters were carried out, and sequencing was performed on the Illumina platform. The data was processed by trimming to obtain clean data. The clean data was aligned with the amplified target sequence, and the Bsmap software was used for alignment. After the alignment was completed, the python program for calculating methylation in Bsmap was used to calculate the methylation level of CG sites.

[0252] According to p < 0.05 and when the difference in the mean methylation level between the CIN3+ and CIN2- groups was greater than 0.15, a total of 1103 hypermethylated sites were screened. Then, through ① the negative group, the mean methylation level < 0.1; ② CIN3 vs the negative group, p < 0.05; ③ CIN3 vs the CIN2 group, p < 0.10, a total of 125 DMCs were screened for subsequent analysis. LASSO regression analysis was performed using R language. The patient's pathological grade was defined as the outcome variable (CIN2- was defined as 0, CIN3+ was defined as 1), and 125 DMCs were used as the independent variables of the model. After LASSO regression analysis, a total of 20 DMCs were screened. The SPSS statistic 21 software was used to model the 20 differentially methylated markers obtained (Table 1). The formula was:

[0253] Risk score = -7.561 + 5.262 * AMER2 + 5.884 * TTC34 + 8.872 * NMNAT2 + 2.411 * SLC7A14 + 9.925 * VSTM2B - 2.698 * SHOX - 0.077 * NKX2 - 6 - 1 + 6.395 * NKX2 - 6 - 2 + 1.205 * LOC728424 + 0.991 * ALX3 - 1 + 2.192 * ALX3 - 2 + 2.842 * EN1 + 2.1 * PROX1 - AS1 - 1 + 4.308 * PROX1 - AS1 - 2 + 0.924 * LHX4 + 3.01 * DCH S2 + 2.253 * FOXD3 - 1 + 1.212 * FOXD3 - 2 + 2.177 * TBX5 - AS1 + 1.766 * LOC100420879。

[0254] Table 1 Twenty methylation markers for LASSO - Logistic modeling

[0255] Methylation marker Chromosomal location AMER2 Chr13:25744938-25746426 TTC34 Chr1:2705810-2707334 NMNAT2 Chr1:183385855-183387356 SLC7A14 Chr3:170302295-170303819 VSTM2B Chr19:30018451-30019977 SHOX ChrX:590483-592172 NKX2-6-1 Chr8:23566680-23568179 NKX2-6-2 Chr8:23566680-23568179 LOC728424 Chr15:30516816-30518367 ALX3-1 Chr1:110611042-110612605 ALX3-2 Chr1:110611042-110612605 EN1 Chr2:119607088-119608622 PROX1-AS1-1 Chr1:214155611-214157113 PROX1-AS1-2 Chr1:214155611-214157113 LHX4 Chr1:180201754-180203218 DCHS2 Chr4:155410591-155412092 FOXD3-1 Chr1:63784781-63786348 FOXD3-2 Chr1:63784781-63786348 TBX5-AS1 Chr12:114846786-114848325 LOC100420879 Chr1:243052908-243054404

[0256] Using SPSS statistic 21 software, the receiver operating characteristic curve (ROC) of the combination of twenty methylation markers was obtained, with sensitivity (true positive rate) as the vertical coordinate and 1 - specificity (false positive rate) as the horizontal coordinate. Youden index = sensitivity - (1 - specificity), and the optimal critical point (cut - off value of methylation level) was determined according to the maximum value of the Youden index. Figure 3 The area under the curve (AUC) of the composition for predicting the risk of cervical cancer and precancerous lesions of the cervix is shown. Its predicted sensitivity is 86.67%, specificity is 94.87%, and the AUC can reach 0.979.

[0257] Using SPSS statistic 21 software, the ROC and AUC for differentiating CIN2 - and CIN3 + of the sites of 1103 candidate methylation markers were obtained. The AUCs of 1103 hypermethylated sites were ranked, and the top 15 high - AUC sites were screened (Table 2). Figure 4 The ROC of the Top15 methylation markers for predicting the risk of cervical cancer and precancerous lesions of the cervix is shown.

[0258] Table 2 Methylation markers with AUC Top15 and AUC for screening CIN3 +

[0259] Methylation marker Chromosomal location AUC TTC34 Chr1:2705810-2707334 0.870 CTNND2 Chr5:11384181-11385604 0.867 ARHGEF4 Chr2:131721013-131722519 0.866 ZNF536-1 Chr19:30865153-30866677 0.866 LOC127274910 Chr2:137522897-137524244 0.864 ST8SIA5 Chr18:44336420-44337856 0.863 Intergenic region 1 Chr10:23461814-23463239 0.857 ZNF536-2 Chr19:30716060-30717060 0.853 HTR1F Chr3:87841226-87842641 0.851 PDE4B Chr1:66257794-66259310 0.847 PROKR2 Chr20:5297005-5298538 0.846 SFRP4 Chr7:37955552-37957019 0.844 WEE1P1 Chr4:107146-108577 0.844 VSX1 Chr20:25061403-25062879 0.844 LINC01210 Chr3:137489322-137490790 0.840

[0260] For 36 negative cases, 35 cases of CIN1, 46 cases of CIN2, 33 cases of CIN3 and 42 cases of cervical cancer samples, namely 117 cases of CIN2- and 75 cases of CIN3+ samples. Manual analysis was performed on the sites of 1103 candidate methylation markers corresponding to 117 cases of CIN2- and 75 cases of CIN3+ samples to screen methylation markers that could detect CIN3+ samples missed by the previous 35 methylation markers. A total of 3 supplementary methylation markers were obtained, as shown in Table 3. Figure 5 The ROC for the supplementary methylation markers to predict the risk of cervical cancer and precancerous lesions of the cervix is shown.

[0261] Table 3 Supplementary methylation markers and AUC for screening CIN3+

[0262] Methylation marker Chromosome location AUC NOL4 Chr18:31804106-31805604 0.800 ZNF610 Chr19:52839052-52840463 0.663 SLC30A10 Chr1:220100662-220102096 0.749

[0263] Example 3: Quantitative methylation-specific PCR (qMSP) was used to verify the screened methylation markers for cervical cancer and precancerous lesions of the cervix.

[0264] Genomic DNA samples were obtained from exfoliated cells of 19 negative cases, 13 cases of CIN1, 37 cases of CIN2, 33 cases of CIN3 and 44 cases of cervical cancer for qMSP detection to further verify the performance of the screened methylation markers. A total of 33 methylation marker detection systems were successfully established. The specific method was as follows: After the sample was detected to be qualified, 1.5 μL of genomic DNA was taken and Bisulfite treatment was carried out using the EZ DNAMethylation Gold Kit (Zymo Research). The converted product was taken for PCR reaction. The PCR reaction solution was a total of 20 μL, and the composition was as follows: nuclease-free water, 1×PCR buffer, dNTP (0.25 mM), Ex Taq HS enzyme (1 U / reaction), β-actin-QF (0.4 μM), β-actin-QR (0.4 μM), β-actin-P (0.4 μM), target gene-QF (0.45 μM), target gene-QR (0.45 μM), target gene-P (0.45 μM) and the converted product (1.5 μL). The primer sequences and probe sequences are shown in Table 4 and Table 5 respectively. The PCR reaction was carried out according to the following reaction procedure: First, 95°C for 5 min, then 95°C for 15 sec followed by 60°C for 30 sec for a total of 50 cycles. The ΔCt value of the methylation marker was calculated according to the qMSP result. ΔCt 目标基因 = Ct 目标基因 - Ct β-actin .

[0265] The ROC of each gene was obtained using SPSS statistic 21 software, with the sensitivity (true positive rate) as the ordinate and 1 - specificity (false positive rate) as the abscissa. The Youden index = sensitivity - (1 - specificity), and the optimal cut-off point (the cut-off value of ΔCt) was determined based on the maximum value of the Youden index. Table 6 shows the performance of qMSP in verifying the detection of cervical cancer and precancerous lesions of each methylation marker.

[0266] Table 4 Primer sequences

[0267]

[0268]

[0269]

[0270] Table 5 Probe sequences

[0271]

[0272]

[0273] Table 6 Sensitivity and specificity of qMSP method in verifying the detection of CIN3+ by methylation markers

[0274]

[0275]

[0276] The above results indicate that by screening candidate methylation markers for cervical cancer and / or precancerous lesion patients and healthy people from the cervical exfoliated cell DNA of the Chinese population, and combining methods such as LASSO-Logistic modeling, AUC ranking, and manual screening, 33 methylation markers for screening cervical cancer and / or precancerous lesions in the Chinese population were screened out for the first time, and a methylation risk prediction model for cervical cancer and / or precancerous lesions in the Chinese population was established, which is applicable to predicting the risk assessment of the occurrence of early cervical cancer and / or precancerous lesions in the Chinese population, as well as the screening and diagnosis of cervical cancer and / or precancerous lesions, providing a new rapid, effective, and accurate approach for the early screening, auxiliary diagnosis, and evaluation of cervical cancer and / or precancerous lesions.

[0277] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only an example and does not constitute a limitation to this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification, so such modifications, improvements, and corrections still fall within the spirit and scope of the exemplary embodiments of this specification.

[0278] In the meantime, this specification uses specific terms to describe the embodiments of this specification. For example, "an embodiment", "one embodiment", and / or "some embodiments" mean a certain feature, structure, or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that the "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more at different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0279] In some embodiments, numbers are used to describe the components and the quantities of attributes. It should be understood that such numbers used for the description of embodiments are modified by the modifiers "about", "approximate", or "substantially" in some examples. Unless otherwise stated, "about", "approximate", or "substantially" indicate that the said numbers allow a variation of ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and the claims are approximate values, and such approximate values may change according to the characteristics required by individual embodiments. In some embodiments, the numerical parameters should consider the specified significant digits and adopt the method of retaining the general number of digits. Although the numerical ranges and parameters used in some embodiments of this specification to confirm the breadth of their scope are approximate values, in specific embodiments, such numerical settings are made as precise as possible within the feasible range.

[0280] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other deformations may also fall within the scope of this specification. Therefore, by way of example and not limitation, alternative configurations of the embodiments of this specification can be regarded as consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments clearly introduced and described in this specification.

Claims

1. Use of a substance for detecting methylation markers in the preparation of a product for detecting cervical cancer and / or cervical precancerous lesions, wherein the methylation markers include any one of the following genes or fragments thereof: AMER2, TTC34, NMNAT2, SLC7A14, VSTM2B, SHOX, NKX2-6, LOC728424, ALX3, EN1, PROX1-AS1, LHX4, DCHS2, FOXD3, TBX5-AS1, LOC100420879, CTNND2, ARHGEF4, ZNF536-1, LOC127274910, ST8SIA5, intergenic region 1, ZNF536-2, HTR1F, PDE4B, PROKR2, SFRP4, WEE1P1, VSX1, LINC01210, NOL4, ZNF610 or SLC30A10.

2. The use according to claim 1, characterized in that Based on the sequence of human reference genome Hg19, the methylation marker includes any one of the following genes or fragments thereof: Chr13: 25744938-25746426, Chr1: 2705810-2707334, Chr1: 183385855-183387356, Chr3: 170302295-170303819, Chr19: 30018451-30019977, ChrX: 590483-592172, Chr8: 23566680-23568179, Chr r15:30516816-30518367,Chr1:110611042-110612605,Chr2:119607088-119608622,Chr1:214155611-214157113,Chr1:1 80201754-180203218,Chr4:155410591-155412092,Chr1:63784781-63786348,Chr12:114846786-114848325,Chr1:243052 908-243054404, Chr5:11384181-11385604, Chr2:131721013-131722519, Chr19:30865153-30866677, Chr2:137522897-13 C hr1:66257794-66259310, Chr20:5297005-5298538, Chr7:37955552-37957019, Chr4:107146-108577, Chr20:25061403-250 62879, Chr3:137489322-137490790, Chr18:31804106-31805604, Chr19:52839052-52840463, Chr1:220100662-220102096; And / or, the cervical precancerous lesions are severe cervical precancerous lesions; And / or, the samples tested by the product include any one or more of blood, serum, plasma, lymph, urine, cervical scraping cells or tissue, biopsy tissue, surgical tissue or cervical exfoliated cells. Preferably, the samples tested by the product are cervical exfoliated cells.

3. The use according to claim 1, characterized in that The substance for detecting the methylation marker includes a substance for detecting the methylation level of the methylation marker. Preferably, the substance for detecting the methylation level of the methylation marker includes a primer pair and a probe. More preferably, the primer pair and the probe are specific primer pairs and probes corresponding to any one of the following genes or fragments thereof: Chr13:25744938-25746426, Chr1:2705810-2707334, Chr1:183385855-183387356, Chr3:170302295-170303819, Chr19:30018451-30019977, ChrX:590483-592172,Chr8:23566680-23568179,Chr15:30516816-30518367,Chr1:110611042-110612605,Chr2:119607088-11960 8622,Chr1:214155611-214157113,Chr1:180201754-180203218,Chr4:155410591-155412092,Chr1:63784781-63786348,Chr12:11 4846786-114848325, Chr1:243052908-243054404, Chr5:11384181-11385604, Chr2:131721013-131722519, Chr19:30865153-30866 677, Chr2:137522897-137524244, Chr18:44336420-44337856, Chr10:23461814-23463239, Chr19:30716060-30717060, Chr3:87841 226-87842641, Chr1:66257794-66259310, Chr20:5297005-5298538, Chr7:37955552-37957019, Chr4:107146-108577, Chr20:25061 403-25062879, Chr3:137489322-137490790, Chr18:31804106-31805604, Chr19:52839052-52840463, Chr1:220100662-220102096.

4. The use according to claim 1, characterized in that The nucleotide sequences of the primer pair for detecting the methylation marker AMER2 gene or its fragment are shown in SEQ ID NO.1 and SEQ ID NO.2; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker TTC34 gene or its fragment are shown in SEQ ID NO.4 and SEQ ID NO.5; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker NMNAT2 gene or its fragment are shown in SEQ ID NO.7 and SEQ ID NO.8; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker SLC7A14 gene or its fragment are shown in SEQ ID NO.10 and SEQ ID NO.11; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker VSTM2B gene or its fragment are shown in SEQ ID NO.13 and SEQ ID NO.14; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker SHOX gene or its fragment are shown in SEQ ID NO.16 and SEQ ID NO.17; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker NKX2-6 gene or its fragment are shown in SEQ ID NO.19 and SEQ ID NO.20; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker LOC728424 gene or its fragment are shown in SEQ ID NO.22 and SEQ ID NO.23; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker ALX3 gene or its fragment are shown in SEQ ID NO.25 and SEQ ID NO.26; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker EN1 gene or its fragment are shown in SEQ ID NO.28 and SEQ ID NO.29; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker PROX1-AS1 gene or its fragment are shown in SEQ ID NO.31 and SEQ ID NO.32; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker LHX4 gene or its fragment are shown in SEQ ID NO.34 and SEQ ID NO.35; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker DCHS2 gene or its fragment are shown in SEQ ID NO.37 and SEQ ID NO.38; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker FOXD3 gene or its fragment are shown in SEQ ID NO.40 and SEQ ID NO.41; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker TBX5-AS1 gene or its fragment are shown in SEQ ID NO.43 and SEQ ID NO.44; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker LOC100420879 gene or its fragment are shown in SEQ ID NO.46 and SEQ ID NO.47; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker CTNND2 gene or its fragment are shown in SEQ ID NO.49 and SEQ ID NO.50; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker ARHGEF4 gene or its fragment are shown in SEQ ID NO.52 and SEQ ID NO.53; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker ZNF536-1 gene or its fragment are shown in SEQ ID NO.55 and SEQ ID NO.56; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker LOC127274910 gene or its fragment are shown in SEQ ID NO.58 and SEQ ID NO.59; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker ST8SIA5 gene or its fragment are shown in SEQ ID NO.61 and SEQ ID NO.62; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker intergenic region 1 gene or its fragment are shown in SEQ ID NO.64 and SEQ ID NO.65; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker ZNF536-2 gene or its fragment are shown in SEQ ID NO.67 and SEQ ID NO.68; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker HTR1F gene or its fragment are shown in SEQ ID NO.70 and SEQ ID NO.71; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker PDE4B gene or its fragment are shown in SEQ ID NO.73 and SEQ ID NO.74; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker PROKR2 gene or its fragment are shown in SEQ ID NO.76 and SEQ ID NO.77; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker SFRP4 gene or its fragment are shown in SEQ ID NO.79 and SEQ ID NO.80; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker WEE1P1 gene or its fragment are shown in SEQ ID NO.82 and SEQ ID NO.83; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker VSX1 gene or its fragment are shown in SEQ ID NO.85 and SEQ ID NO.86; And / or, the nucleotide sequences of the primer pairs for detecting the methylation marker LINC01210 gene or its fragment are shown in SEQ ID NO.88 and SEQ ID NO.89; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker NOL4 gene or its fragment are shown in SEQ ID NO.91 and SEQ ID NO.92; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker ZNF610 gene or its fragment are shown in SEQ ID NO.94 and SEQ ID NO.95; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker SLC30A10 gene or its fragment are shown in SEQ ID NO.97 and SEQ ID NO.

98.

5. The use according to claim 1, characterized in that The nucleotide sequence of the probe for detecting the methylation level of the methylation marker AMER2 gene or its fragment is shown in SEQ ID NO.3; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker TTC34 gene or its fragment is as shown in SEQ ID NO.6; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker NMNAT2 gene or its fragment is as shown in SEQ ID NO.9; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker SLC7A14 gene or its fragment is as shown in SEQ ID NO.12; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker VSTM2B gene or its fragment is as shown in SEQ ID NO.15; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker SHOX gene or its fragment is as shown in SEQ ID NO.18; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker NKX2-6 gene or its fragment is shown in SEQ ID NO.21; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker LOC728424 gene or its fragment is shown in SEQ ID NO.24; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ALX3 gene or its fragment is shown in SEQ ID NO.27; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker EN1 gene or its fragment is shown in SEQ ID NO.30; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker PROX1-AS1 gene or its fragment is as shown in SEQ ID NO.33; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker LHX4 gene or its fragment is as shown in SEQ ID NO.36; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker DCHS2 gene or its fragment is shown in SEQ ID NO.39; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker FOXD3 gene or its fragment is as shown in SEQ ID NO.42; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker TBX5-AS1 gene or its fragment is as shown in SEQ ID NO.45; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker LOC100420879 gene or its fragment is shown in SEQ ID NO.48; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker CTNND2 gene or its fragment is as shown in SEQ ID NO.51; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ARHGEF4 gene or its fragment is shown in SEQ ID NO.54; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ZNF536-1 gene or its fragment is as shown in SEQ ID NO.57; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker LOC127274910 gene or its fragment is shown in SEQ ID NO.60; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ST8SIA5 gene or its fragment is shown in SEQ ID NO.63; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker intergenic region 1 gene or its fragment is shown in SEQ ID NO.66; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ZNF536-2 gene or its fragment is as shown in SEQ ID NO.69; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker HTR1F gene or its fragment is shown in SEQ ID NO.72; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker PDE4B gene or its fragment is as shown in SEQ ID NO.75; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker PROKR2 gene or its fragment is as shown in SEQ ID NO.78; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker SFRP4 gene or its fragment is shown in SEQ ID NO.81; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker WEE1P1 gene or its fragment is shown in SEQ ID NO.84; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker VSX1 gene or its fragment is as shown in SEQ ID NO.87; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker LINC01210 gene or its fragment is shown in SEQ ID NO.90; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker NOL4 gene or its fragment is as shown in SEQ ID NO.93; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ZNF610 gene or its fragment is shown in SEQ ID NO.96; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker SLC30A10 gene or its fragment is as shown in SEQ ID NO.

99.

6. A product for detecting cervical cancer and / or cervical precancerous lesions, the product comprising a substance for detecting a methylation marker, wherein the methylation marker comprises any one of the following genes or fragments thereof: AMER2, TTC34, NMNAT2, SLC7A14, VSTM2B, SHOX, NKX2-6, LOC728424, ALX3, EN1, PROX1-AS1, LHX4, DCHS2, FOXD3, TBX5-AS1, LOC100420879, CTNND2, ARHGEF4, ZNF536-1, LOC127274910, ST8SIA5, intergenic region 1, ZNF536-2, HTR1F, PDE4B, PROKR2, SFRP4, WEE1P1, VSX1, LINC01210, NOL4, ZNF610 or SLC30A10.

7. The product according to claim 6, characterized in that Based on the sequence of human reference genome Hg19, the methylation marker includes any one of the following genes or fragments thereof: Chr13: 25744938-25746426, Chr1: 2705810-2707334, Chr1: 183385855-183387356, Chr3: 170302295-170303819, Chr19: 30018451-30019977, ChrX: 590483-592172, Chr8: 23566680-23568179, Chr r15:30516816-30518367,Chr1:110611042-110612605,Chr2:119607088-119608622,Chr1:214155611-214157113,Chr1:1 80201754-180203218,Chr4:155410591-155412092,Chr1:63784781-63786348,Chr12:114846786-114848325,Chr1:243052 908-243054404, Chr5:11384181-11385604, Chr2:131721013-131722519, Chr19:30865153-30866677, Chr2:137522897-13 C hr1:66257794-66259310, Chr20:5297005-5298538, Chr7:37955552-37957019, Chr4:107146-108577, Chr20:25061403-250 62879, Chr3:137489322-137490790, Chr18:31804106-31805604, Chr19:52839052-52840463, Chr1:220100662-220102096; And / or, the cervical precancerous lesions are severe cervical precancerous lesions.

8. The product according to claim 6, characterized in that The samples tested by the product include any one or more of blood, serum, plasma, lymph, urine, cervical scraping cells or tissue, biopsy tissue, surgical tissue or cervical exfoliated cells. Preferably, the samples tested by the product are cervical exfoliated cells.

9. The product according to claim 6, characterized in that The product is any one of a kit, a chip, a membrane strip, a protein array, a composition or a detection system. Preferably, the product includes one or more of a DNA polymerase, a deoxynucleotide (dNTP) mixture, a buffer solution, a primer, a probe, sodium bisulfite, a positive control or a negative control.

10. The product according to claim 6, characterized in that The substance for detecting the methylation marker includes a substance for detecting the methylation level of the methylation marker. Preferably, the substance for detecting the methylation level of the methylation marker includes a primer pair and a probe. More preferably, the primer pair and the probe are specific primer pairs and probes corresponding to any one of the following genes or fragments thereof: Chr13:25744938-25746426, Chr1:2705810-2707334, Chr1:183385855-183387356, Chr3:170302295-170303819, Chr19:30018451-30019977, ChrX:590483-592172,Chr8:23566680-23568179,Chr15:30516816-30518367,Chr1:110611042-110612605,Chr2:119607088-11960 8622,Chr1:214155611-214157113,Chr1:180201754-180203218,Chr4:155410591-155412092,Chr1:63784781-63786348,Chr12:11 4846786-114848325, Chr1:243052908-243054404, Chr5:11384181-11385604, Chr2:131721013-131722519, Chr19:30865153-30866 677, Chr2:137522897-137524244, Chr18:44336420-44337856, Chr10:23461814-23463239, Chr19:30716060-30717060, Chr3:87841 226-87842641, Chr1:66257794-66259310, Chr20:5297005-5298538, Chr7:37955552-37957019, Chr4:107146-108577, Chr20:25061 403-25062879, Chr3:137489322-137490790, Chr18:31804106-31805604, Chr19:52839052-52840463, Chr1:220100662-220102096.

11. The product according to claim 6, characterized in that The nucleotide sequences of the primer pair for detecting the methylation marker AMER2 gene or its fragment are shown in SEQ ID NO.1 and SEQ ID NO.2; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker TTC34 gene or its fragment are shown in SEQ ID NO.4 and SEQ ID NO.5; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker NMNAT2 gene or its fragment are shown in SEQ ID NO.7 and SEQ ID NO.8; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker SLC7A14 gene or its fragment are shown in SEQ ID NO.10 and SEQ ID NO.11; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker VSTM2B gene or its fragment are shown in SEQ ID NO.13 and SEQ ID NO.14; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker SHOX gene or its fragment are shown in SEQ ID NO.16 and SEQ ID NO.17; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker NKX2-6 gene or its fragment are shown in SEQ ID NO.19 and SEQ ID NO.20; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker LOC728424 gene or its fragment are shown in SEQ ID NO.22 and SEQ ID NO.23; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker ALX3 gene or its fragment are shown in SEQ ID NO.25 and SEQ ID NO.26; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker EN1 gene or its fragment are shown in SEQ ID NO.28 and SEQ ID NO.29; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker PROX1-AS1 gene or its fragment are shown in SEQ ID NO.31 and SEQ ID NO.32; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker LHX4 gene or its fragment are shown in SEQ ID NO.34 and SEQ ID NO.35; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker DCHS2 gene or its fragment are shown in SEQ ID NO.37 and SEQ ID NO.38; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker FOXD3 gene or its fragment are shown in SEQ ID NO.40 and SEQ ID NO.41; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker TBX5-AS1 gene or its fragment are shown in SEQ ID NO.43 and SEQ ID NO.44; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker LOC100420879 gene or its fragment are shown in SEQ ID NO.46 and SEQ ID NO.47; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker CTNND2 gene or its fragment are shown in SEQ ID NO.49 and SEQ ID NO.50; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker ARHGEF4 gene or its fragment are shown in SEQ ID NO.52 and SEQ ID NO.53; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker ZNF536-1 gene or its fragment are shown in SEQ ID NO.55 and SEQ ID NO.56; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker LOC127274910 gene or its fragment are shown in SEQ ID NO.58 and SEQ ID NO.59; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker ST8SIA5 gene or its fragment are shown in SEQ ID NO.61 and SEQ ID NO.62; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker intergenic region 1 gene or its fragment are shown in SEQ ID NO.64 and SEQ ID NO.65; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker ZNF536-2 gene or its fragment are shown in SEQ ID NO.67 and SEQ ID NO.68; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker HTR1F gene or its fragment are shown in SEQ ID NO.70 and SEQ ID NO.71; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker PDE4B gene or its fragment are shown in SEQ ID NO.73 and SEQ ID NO.74; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker PROKR2 gene or its fragment are shown in SEQ ID NO.76 and SEQ ID NO.77; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker SFRP4 gene or its fragment are shown in SEQ ID NO.79 and SEQ ID NO.80; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker WEE1P1 gene or its fragment are shown in SEQ ID NO.82 and SEQ ID NO.83; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker VSX1 gene or its fragment are shown in SEQ ID NO.85 and SEQ ID NO.86; And / or, the nucleotide sequences of the primer pairs for detecting the methylation marker LINC01210 gene or its fragment are shown in SEQ ID NO.88 and SEQ ID NO.89; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker NOL4 gene or its fragment are shown in SEQ ID NO.91 and SEQ ID NO.92; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker ZNF610 gene or its fragment are shown in SEQ ID NO.94 and SEQ ID NO.95; And / or, the nucleotide sequences of the primer pair for detecting the methylation marker SLC30A10 gene or its fragment are shown in SEQ ID NO.97 and SEQ ID NO.

98.

12. The product according to claim 6, characterized in that The nucleotide sequence of the probe for detecting the methylation level of the methylation marker AMER2 gene or its fragment is shown in SEQ ID NO.3; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker TTC34 gene or its fragment is as shown in SEQ ID NO.6; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker NMNAT2 gene or its fragment is as shown in SEQ ID NO.9; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker SLC7A14 gene or its fragment is as shown in SEQ ID NO.12; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker VSTM2B gene or its fragment is as shown in SEQ ID NO.15; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker SHOX gene or its fragment is as shown in SEQ ID NO.18; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker NKX2-6 gene or its fragment is shown in SEQ ID NO.21; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker LOC728424 gene or its fragment is shown in SEQ ID NO.24; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ALX3 gene or its fragment is shown in SEQ ID NO.27; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker EN1 gene or its fragment is shown as SEQ ID NO.30; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker PROX1-AS1 gene or its fragment is as shown in SEQ ID NO.33; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker LHX4 gene or its fragment is as shown in SEQ ID NO.36; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker DCHS2 gene or its fragment is shown in SEQ ID NO.39; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker FOXD3 gene or its fragment is as shown in SEQ ID NO.42; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker TBX5-AS1 gene or its fragment is as shown in SEQ ID NO.45; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker LOC100420879 gene or its fragment is shown in SEQ ID NO.48; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker CTNND2 gene or its fragment is as shown in SEQ ID NO.51; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ARHGEF4 gene or its fragment is shown in SEQ ID NO.54; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ZNF536-1 gene or its fragment is as shown in SEQ ID NO.57; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker LOC127274910 gene or its fragment is shown in SEQ ID NO.60; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ST8SIA5 gene or its fragment is shown in SEQ ID NO.63; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker intergenic region 1 gene or its fragment is shown in SEQ ID NO.66; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ZNF536-2 gene or its fragment is as shown in SEQ ID NO.69; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker HTR1F gene or its fragment is shown in SEQ ID NO.72; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker PDE4B gene or its fragment is as shown in SEQ ID NO.75; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker PROKR2 gene or its fragment is as shown in SEQ ID NO.78; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker SFRP4 gene or its fragment is shown in SEQ ID NO.81; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker WEE1P1 gene or its fragment is shown in SEQ ID NO.84; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker VSX1 gene or its fragment is as shown in SEQ ID NO.87; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker LINC01210 gene or its fragment is shown in SEQ ID NO.90; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker NOL4 gene or its fragment is as shown in SEQ ID NO.93; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ZNF610 gene or its fragment is shown in SEQ ID NO.96; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker SLC30A10 gene or its fragment is as shown in SEQ ID NO.

99.

13. A device for detecting cervical cancer and / or cervical precancerous lesions, comprising the following modules: a data acquisition module for providing methylation level data of a target marker of a sample to be tested, wherein the target marker is a methylation marker in the application according to any one of claims 1 to 5; The judgment module is used to evaluate the cervical cancer and / or cervical precancerous lesions based on the methylation level data of the target markers of the sample to be tested.

14. The detection device according to claim 13, characterized in that: Based on the methylation level data of the target marker of the sample to be tested, the ΔCt value of the methylation marker is calculated. Preferably, the methylation level data is the Ct value of the methylation marker detected by quantitative methylation-specific PCR, and the ΔCt value is the difference between the Ct value of the target marker and the Ct value of the reference gene. More preferably, the reference gene is β-actin.

15. The detection device according to claim 14, characterized in that: The cervical cancer and / or cervical precancerous lesions are evaluated based on the ΔCt value and the preset ΔCt cutoff value. Preferably, the sample to be tested is judged to be negative or positive based on the ΔCt value and the preset ΔCt cutoff value. More preferably, the ΔCt cutoff value is the ΔCt value that maximizes the Youden Index.

16. The detection device according to claim 13, characterized in that: The sample to be tested includes any one or more of blood, serum, plasma, lymph, urine, cervical scraping cells or tissue, biopsy tissue, surgical tissue or cervical exfoliated cells. Preferably, the sample to be tested is cervical exfoliated cells.

17. The detection device according to claim 15, characterized in that: When the ΔCt value of the target marker of the sample to be tested is less than or equal to the cutoff value of ΔCt, the sample to be tested is determined to be positive; And / or, when the ΔCt value of the target marker of the sample to be tested is greater than the cutoff value of ΔCt, the sample to be tested is judged to be negative; and / or, the positive means that the individual corresponding to the sample to be tested is a patient with severe cervical precancerous lesions or cervical cancer, and the negative means that the individual corresponding to the sample to be tested is a healthy person, a patient with mild cervical precancerous lesions or a patient with moderate cervical precancerous lesions.

18. A computer-readable storage medium, characterized in that: The storage medium stores computer instructions, and when the computer instructions are executed by the processor, a method for detecting cervical cancer and / or cervical precancerous lesions is implemented, the method comprising: obtaining methylation level data of a target marker of a sample to be tested, wherein the target marker is a methylation marker in the application according to any one of claims 1 to 5; Based on the methylation level data of the target markers in the tested samples, the status of cervical cancer and / or cervical precancerous lesions is evaluated.

19. The computer-readable storage medium of claim 18, wherein: Based on the methylation level data of the target marker of the sample to be tested, the ΔCt value of the methylation marker is calculated. Preferably, the methylation level data is the Ct value of the methylation marker detected by quantitative methylation-specific PCR, and the ΔCt value is the difference between the Ct value of the target marker and the Ct value of the reference gene. More preferably, the reference gene is β-actin.

20. The computer-readable storage medium of claim 19, wherein: The cervical cancer and / or cervical precancerous lesions are evaluated based on the ΔCt value and the preset ΔCt cutoff value. Preferably, the sample to be tested is judged to be negative or positive based on the ΔCt value and the preset ΔCt cutoff value. More preferably, the ΔCt cutoff value is the ΔCt value that maximizes the Youden Index.

21. The computer-readable storage medium of claim 18, wherein: The sample to be tested includes any one or more of blood, serum, plasma, lymph, urine, cervical scraping cells or tissue, biopsy tissue, surgical tissue or cervical exfoliated cells. Preferably, the sample to be tested is cervical exfoliated cells.

22. The computer-readable storage medium of claim 20, wherein: When the ΔCt value of the target marker of the sample to be tested is less than or equal to the cutoff value of ΔCt, the sample to be tested is determined to be positive; And / or, when the ΔCt value of the target marker of the sample to be tested is greater than the cutoff value of ΔCt, the sample to be tested is judged to be negative; and / or, the positive means that the individual corresponding to the sample to be tested is a patient with severe cervical precancerous lesions or cervical cancer, and the negative means that the individual corresponding to the sample to be tested is a healthy person, a patient with mild cervical precancerous lesions or a patient with moderate cervical precancerous lesions.

23. An electronic terminal, characterized in that: The electronic terminal includes: a processor, a memory, an input / output interface and a communication port; the memory is used to store a computer program, and the processor is used to execute the computer program stored in the memory, so that the terminal executes the method for detecting cervical cancer and / or cervical precancerous lesions as described in the computer-readable storage medium as described in any one of claims 18 to 22.

24. A computer program product, comprising a computer program, which, when executed by a processor, implements the following steps: obtaining methylation level data of a target marker of a sample to be tested, and evaluating cervical cancer and / or cervical precancerous lesions based on the methylation level data of the target marker of the sample to be tested.

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