Use of a substance for detecting a methylation marker in the manufacture of a product for detecting cervical cancer and / or precancerous lesions of the cervix

Abstract

The application discloses methylation markers and application for detecting cervical cancer and / or cervical precancerous lesions, and particularly relates to the technical field of molecular biological medicine. The application studies the methylation differences between cervical cancer and / or cervical precancerous lesion patients and healthy people through cervical exfoliated cell DNA of Chinese population, screens out candidate methylation markers with obvious differences, and combines a LASSO-Logistic modeling, AUC ranking and an artificial screening method to first screen out 33 optimal methylation markers to establish a methylation risk prediction method for cervical cancer and / or cervical precancerous lesions of Chinese population, which is suitable for predicting the risk assessment of early cervical cancer and / or cervical precancerous lesions of Chinese population, and screening and diagnosing the cervical cancer and / or cervical precancerous lesions.

Description

TECHNICAL FIELD

[0001] The present specification relates to the field of molecular biomedical technology, in particular to the application of a substance for detecting methylation markers in the preparation of a product for detecting cervical cancer and / or precancerous lesions of the cervix. BACKGROUND

[0002] Cervical cancer threatens women's health. In global malignancies, the incidence and mortality of cervical cancer are both the fourth. In China, the new cases and deaths of cervical cancer account for nearly 20% of the total number in the world.

[0003] Only persistent infection of high-risk HPV (hrHPV) can lead to cervical lesions, through mild cervical precancerous lesions (cervical intraepithelial neoplasia, CIN), moderate CIN and severe CIN, and finally develop into cervical cancer. The current screening guidelines recommend HPV DNA detection as the preferred method, or combined with Thinprep cytologic test (TCT) detection. The hrHPV detection result is objective and has high repeatability, but it cannot distinguish between transient infection and transformed infection, which may lead to an increase in colposcopy referral and cause anxiety in positive women. Cell screening methods such as TCT have high specificity, but are subjective in diagnosis and have low sensitivity, which leads to missed diagnosis.

[0004] In recent years, studies have shown that early epigenetic changes are an important feature of tumor occurrence and development. DNA methylation detection has gradually become a new means of detecting cervical cancer and precancerous lesions of the cervix. A variety of methylation genes such as FAM19A4, Mir124-2, PAX1, ZNF582, SOX1, EPB41L3, etc. have been considered as biomarkers for cervical cancer screening, but the detection efficiency of cervical cancer and precancerous lesions of the cervix in Chinese population is generally poor. Therefore, it is of great clinical significance to explore and develop more effective and objective biomarkers suitable for Chinese population. SUMMARY

[0005] In view of the above-mentioned lack of methylation markers for Chinese population, the DNA of cervical exfoliated cells of Chinese population is subjected to RRBS (Reduced Representation Bisulfite Sequencing) preliminary screening and TBS (Targeted Bisulfite Sequencing) verification. Through the analysis of differential methylation regions of cervical cancer and precancerous lesions of the cervix and healthy people from high-throughput sequencing results, a model is constructed to provide a new idea and detection means for early diagnosis and treatment of cervical cancer and precancerous lesions of the cervix. The main contribution of the present 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 the present application.

[0006] The application provides an application 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, the methylation marker comprising 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.

[0007] The application also provides a product for detecting cervical cancer and / or precancerous lesions of the cervix, the product comprising a substance for detecting a methylation marker, the methylation marker comprising 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.

[0008] The application also provides a device for detecting cervical cancer and / or precancerous lesions of the cervix, comprising the following modules: a data acquisition module for providing methylation level data of a target marker of a sample to be detected, the target marker being a methylation marker in the application; and a judgment module for evaluating the cervical cancer and / or precancerous lesions of the cervix based on the methylation level data of the target marker of the sample to be detected.

[0009] The application also provides a computer-readable storage medium, the storage medium storing computer instructions, when the computer instructions are executed by a processor, a method for detecting cervical cancer and / or precancerous lesions of the cervix is implemented, the method comprising: acquiring methylation level data of a target marker of a sample to be detected, the target marker being a methylation marker in the application; and evaluating the cervical cancer and / or precancerous lesions of the cervix based on the methylation level data of the target marker of the sample to be detected.

[0010] The application also provides an electronic terminal, comprising 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 to enable the terminal to execute the method for detecting cervical cancer and / or precancerous lesions of the cervix described above.

[0011] The application also provides a computer program product comprising a computer program, which, when executed by a processor, implements the method of the following steps: obtaining methylation level data of a target marker of a sample to be tested, and evaluating the condition of cervical cancer and / or precancerous lesions of the cervix based on the methylation level data of the target marker of the sample to be tested.

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

[0013] The application will be further described in the form of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, in which:

[0014] Figure 1 The development process of screening methylation biomarkers for detecting the state of cervical cancer and precancerous lesions of the cervix using methylation sequencing data is shown.

[0015] Figure 2 The difference heat map of the methylation level of the methylation marker in the simplified genomic methylation sequencing between CIN1- and CIN3+ in Example 1 of the application is shown.

[0016] Figure 3 The receiver operating characteristic (ROC) curve of the screening of cervical cancer and precancerous lesions of the cervix by the combination of 20 methylation markers in Example 2 of the application is shown.

[0017] Figure 4 The receiver operating characteristic (ROC) curve of the screening of cervical cancer and precancerous lesions of the cervix by the AUC Top15 methylation markers in Example 2 of the application is shown.

[0018] Figure 5 The receiver operating characteristic (ROC) curve of the screening of cervical cancer and precancerous lesions of the cervix by the 3 methylation markers screened manually in Example 2 of the application is shown.

[0019] Figure 6 Module diagram for a cervical cancer and / or precancerous lesion detection device according to some embodiments of the present application.

[0020] Figure 7 Flowchart for a method of detecting cervical cancer and / or precancerous lesions according to some embodiments of the present application.

[0021] Figure 8 Architectural schematic of an electronic terminal 800 according to some embodiments of the present application. DETAILED DESCRIPTION

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present specification, the following will briefly introduce the drawings needed to be used in the embodiment description. Obviously, the drawings in the following description are only some examples or embodiments of the present specification, and for those skilled in the art, without paying creative labor, the present specification can also be applied to other similar scenarios according to these drawings. Unless the context clearly indicates otherwise or otherwise stated, the same reference numbers in the drawings represent the same structure or operation.

[0023] As shown in the specification and claims, unless the context clearly indicates otherwise or otherwise stated, "one", "a", "an", and / or "the" do not necessarily mean a single number, but can also include a plurality. Generally speaking, the terms "comprise" and "include" only indicate the inclusion of the steps and elements explicitly identified, and these steps and elements do not constitute an exclusive list, and the method or device can also include other steps or elements.

[0024] Flowcharts are used in the present specification to illustrate the operations performed by the system according to the embodiments of the present specification. It should be understood that the preceding or subsequent operations are not necessarily executed in sequence. On the contrary, each step can be processed in reverse order or simultaneously. At the same time, other operations can also be added to these processes, or one or more steps of operation can be removed from these processes.

[0025] The application provides application of a substance for detecting a methylation marker in preparation of a product for detecting cervical cancer and / or precancerous lesion of the cervix, the methylation marker comprising 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.

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

[0027] In some embodiments, the methylation marker can include 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, Chr2:119607088-119608622, 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, based on the sequence of human reference genome Hg19.

[0028] "Chr" followed by a number (1-20) indicates a specific chromosome, and the number after ":" indicates the base position on the chromosome. For example, "Chr1:220100662-220102096" indicates the base sequence from the 220100662th base to the 220102096th base on human chromosome 1.

[0029] In some embodiments, the cervical precancerous lesion can be a severe cervical precancerous lesion.

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

[0031] In some embodiments, the substance for detecting the methylation marker can 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 can include a primer pair and a probe.

[0032] In some embodiments, more preferably, the primer pairs and probes 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, Chr2:119607088-119608622, 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] "Corresponding" herein refers to the gene fragment after bisulfite treatment. After bisulfite treatment, the corresponding gene fragment will undergo different changes 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 primer pair nucleotide sequence for detecting the methylation marker AMER2 gene or a fragment thereof can be as shown in SEQ ID NO. 1 and SEQ ID NO. 2.

[0035] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker TTC34 gene or a fragment thereof can be as shown in SEQ ID NO. 4 and SEQ ID NO. 5.

[0036] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker NMNAT2 gene or a fragment thereof can be as shown in SEQ ID NO. 7 and SEQ ID NO. 8.

[0037] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker SLC7A14 gene or a fragment thereof can be as shown in SEQ ID NO. 10 and SEQ ID NO. 11.

[0038] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker VSTM2B gene or a fragment thereof can be as shown in SEQ ID NO. 13 and SEQ ID NO. 14.

[0039] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker SHOX gene or a fragment thereof can be as shown in SEQ ID NO. 16 and SEQ ID NO. 17.

[0040] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker NKX2-6 gene or a fragment thereof can be as shown in SEQ ID NO. 19 and SEQ ID NO. 20.

[0041] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker LOC728424 gene or a fragment thereof can be as shown in SEQ ID NO. 22 and SEQ ID NO. 23.

[0042] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker ALX3 gene or a fragment thereof can be as shown in SEQ ID NO. 25 and SEQ ID NO. 26.

[0043] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker EN1 gene or a fragment thereof can be as shown in SEQ ID NO. 28 and SEQ ID NO. 29.

[0044] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker PROX1-AS1 gene or a fragment thereof can be as set forth in SEQ ID NO. 31 and SEQ ID NO. 32.

[0045] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker LHX4 gene or a fragment thereof can be as set forth in SEQ ID NO. 34 and SEQ ID NO. 35.

[0046] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker DCHS2 gene or a fragment thereof can be as set forth in SEQ ID NO. 37 and SEQ ID NO. 38.

[0047] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker FOXD3 gene or a fragment thereof can be as set forth in SEQ ID NO. 40 and SEQ ID NO. 41.

[0048] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker TBX5-AS1 gene or a fragment thereof can be as set forth in SEQ ID NO. 43 and SEQ ID NO. 44.

[0049] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker LOC100420879 gene or a fragment thereof can be as set forth in SEQ ID NO. 46 and SEQ ID NO. 47.

[0050] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker CTNND2 gene or a fragment thereof can be as set forth in SEQ ID NO. 49 and SEQ ID NO. 50.

[0051] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker ARHGEF4 gene or a fragment thereof can be as set forth in SEQ ID NO. 52 and SEQ ID NO. 53.

[0052] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker ZNF536-1 gene or a fragment thereof can be as set forth in SEQ ID NO. 55 and SEQ ID NO. 56.

[0053] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker LOC127274910 gene or a fragment thereof can be as set forth in SEQ ID NO. 58 and SEQ ID NO. 59.

[0054] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker ST8SIA5 gene or a fragment thereof can be as set forth in SEQ ID NO. 61 and SEQ ID NO. 62.

[0055] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker intergenic 1 gene or a fragment thereof can be as set forth in SEQ ID NO. 64 and SEQ ID NO. 65.

[0056] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker ZNF536-2 gene or a fragment thereof can be as set forth in SEQ ID NO. 67 and SEQ ID NO. 68.

[0057] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker HTR1F gene or a fragment thereof can be as set forth in SEQ ID NO. 70 and SEQ ID NO. 71.

[0058] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker PDE4B gene or a fragment thereof can be as set forth in SEQ ID NO. 73 and SEQ ID NO. 74.

[0059] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker PROKR2 gene or a fragment thereof can be as set forth in SEQ ID NO. 76 and SEQ ID NO. 77.

[0060] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker SFRP4 gene or a fragment thereof can be as set forth in SEQ ID NO. 79 and SEQ ID NO. 80.

[0061] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker WEE1P1 gene or a fragment thereof can be as set forth in SEQ ID NO. 82 and SEQ ID NO. 83.

[0062] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker VSX1 gene or a fragment thereof can be as set forth in SEQ ID NO. 85 and SEQ ID NO. 86.

[0063] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker LINC01210 gene or a fragment thereof can be as set forth in SEQ ID NO. 88 and SEQ ID NO. 89.

[0064] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker NOL4 gene or a fragment thereof can be as set forth in SEQ ID NO. 91 and SEQ ID NO. 92.

[0065] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker ZNF610 gene or a fragment thereof can be as set forth in SEQ ID NO. 94 and SEQ ID NO. 95.

[0066] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker SLC30A10 gene or a fragment thereof can be as set forth in SEQ ID NO. 97 and SEQ ID NO. 98.

[0067] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker AMER2 gene or a fragment thereof can be as set forth in SEQ ID NO. 3.

[0068] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker TTC34 gene or a fragment thereof can be as set forth in SEQ ID NO. 6.

[0069] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker NMNAT2 gene or a fragment thereof can be as set forth in SEQ ID NO. 9.

[0070] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker SLC7A14 gene or a fragment thereof can be as set forth in SEQ ID NO. 12.

[0071] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker VSTM2B gene or a fragment thereof can be as set forth in SEQ ID NO. 15.

[0072] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker SHOX gene or a fragment thereof can be as set forth in SEQ ID NO. 18.

[0073] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker NKX2-6 gene or a fragment thereof can be as set forth in SEQ ID NO. 21.

[0074] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker LOC728424 gene or a fragment thereof can be as set forth in SEQ ID NO. 24.

[0075] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker ALX3 gene or a fragment thereof can be as set forth in SEQ ID NO. 27.

[0076] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker EN1 gene or a fragment thereof can be as set forth in SEQ ID NO. 30.

[0077] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker PROX1-AS1 gene or a fragment thereof can be as set forth in SEQ ID NO. 33.

[0078] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker LHX4 gene or a fragment thereof can be as set forth in SEQ ID NO. 36.

[0079] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker DCHS2 gene or a fragment thereof can be as set forth in SEQ ID NO. 39.

[0080] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker FOXD3 gene or a fragment thereof can be as set forth in SEQ ID NO. 42.

[0081] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker TBX5-AS1 gene or a fragment thereof can be as set forth in SEQ ID NO. 45.

[0082] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker LOC100420879 gene or a fragment thereof can be as set forth in SEQ ID NO. 48.

[0083] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker CTNND2 gene or a fragment thereof can be as set forth in SEQ ID NO. 51.

[0084] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker ARHGEF4 gene or a fragment thereof can be as set forth in SEQ ID NO. 54.

[0085] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker ZNF536-1 gene or a fragment thereof can be as set forth 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 a fragment thereof can be as represented 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 a fragment thereof can be as represented 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 1 gene or a fragment thereof can be as represented 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 a fragment thereof can be as represented 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 a fragment thereof can be as represented 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 a fragment thereof can be as represented 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 a fragment thereof can be as represented 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 a fragment thereof can be as represented 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 a fragment thereof can be as represented 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 a fragment thereof can be as represented 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 a fragment thereof can be as represented 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 a fragment thereof can be as set forth 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 a fragment thereof can be as set forth 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 a fragment thereof can be as set forth in SEQ ID NO. 99.

[0100] The present application also provides a product for detecting cervical cancer and / or precancerous lesions of the cervix, the product comprising a substance for detecting a methylation marker, the methylation marker comprising any one of the following genes or a fragment 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.

[0101] In some embodiments, the methylation markers can include any one of the following genes or fragments thereof: Chr 13:25744938-25746426, Chr 1:2705810-2707334, Chr 1:183385855-183387356, Chr 3:170302295-170303819, Chr 19:30018451-30019977, Chr X:590483-592172, Chr 8:23566680-23568179, Chr 15:30516816-30518367, Chr 1:110611042-110612605, Chr 2:119607088-119608622, Chr 1:214155611-214157113, Chr 1:180201754-180203218, Chr 4:155410591-155412092, Chr 1:63784781-63786348, Chr 12:114846786-114848325, Chr 1:243052908-243054404, Chr 5:11384181-11385604, Chr 2:131721013-131722519, Chr 19:30865153-30866677, Chr 2:137522897-137524244, Chr 18:44336420-44337856, Chr 10:23461814-23463239, Chr 19:30716060-30717060, Chr 3:87841226-87842641, Chr 1:66257794-66259310, Chr 20:5297005-5298538, Chr 7:37955552-37957019, Chr 4:107146-108577, Chr 20:25061403-25062879, Chr 3:137489322-137490790, Chr 18:31804106-31805604, Chr 19:52839052-52840463, Chr 1:220100662-220102096, based on the sequence of human reference genome Hgl9.

[0102] In some embodiments, the cervical precancerous lesion can be severe cervical precancerous lesion.

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

[0104] In some embodiments, the product can be any one of a kit, a chip, a strip, a protein array, a composition, or a detection system. In some embodiments, preferably, the product can include one or more of a DNA polymerase, a dNTP mixture, a buffer solution, a primer, a probe, sodium bisulfite, a positive control, or a negative control.

[0105] The term "kit" refers to a packaged set 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, or a desiccant.

[0106] The term "strip" refers to a diagnostic tool that utilizes specific biomolecular recognition principles to specifically bind to target substances in a sample by immobilizing biomolecules such as antigens or antibodies on a membrane, and qualitatively or quantitatively analyzes the target substances in the sample through visualization or other signal detection methods.

[0107] The term "chip" generally refers to a miniature device that integrates biosensors and microfluidic technology, which can perform sample preparation, reaction, detection, and other operations on a microscale 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 research of a large number of proteins. This technology allows rapid protein expression analysis, protein-protein interaction, protein-small molecule binding, and other research by orderly arranging thousands of different protein or protein interaction probes on a solid surface.

[0109] In some embodiments, the substance for detecting the methylation marker can 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 can include a primer pair and a probe.

[0110] In some embodiments, more preferably, the primer pairs and probes can be specific primer pairs and probes corresponding to any one of the following genes or fragments thereof: Chr 13:25744938-25746426, Chr 1:2705810-2707334, Chr 1:183385855-183387356, Chr 3:170302295-170303819, Chr 19:30018451-30019977, Chr X:590483-592172, Chr 8:23566680-23568179, Chr 15:30516816-30518367, Chr 1:110611042-110612605, Chr 2:119607088-119608622, Chr 1:214155611-214157113, Chr 1:180201754-180203218, Chr 4:155410591-155412092, Chr 1:63784781-63786348, Chr 12:114846786-114848325, Chr 1:243052908-243054404, Chr 5:11384181-11385604, Chr 2:131721013-131722519, Chr 19:30865153-30866677, Chr 2:137522897-137524244, Chr 18:44336420-44337856, Chr 10:23461814-23463239, Chr 19:30716060-30717060, Chr 3:87841226-87842641, Chr 1:66257794-66259310, Chr 20:5297005-5298538, Chr 7:37955552-37957019, Chr 4:107146-108577, Chr 20:25061403-25062879, Chr 3:137489322-137490790, Chr 18:31804106-31805604, Chr 19:52839052-52840463, Chr 1:220100662-220102096.

[0111] The term "primer" as used herein refers to an oligonucleotide (e.g., restriction fragment) that is either naturally occurring full-length or generated synthetically, which is capable of acting as a point-mutated initiation point for the synthesis of a primer extension product that is complementary to a nucleic acid strand (template or target sequence) when placed 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- or RNA-dependent polymerase). Typically, a primer set will consist of at least two primers, an "upstream primer" and a "downstream primer," which together define an amplicon (the sequence that will be amplified using the primers).

[0112] The term "probe" refers to any molecule that is capable of selectively binding 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 fluorescent group and a quenching 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] Antibody refers to a protein produced by the body in response to stimulation by an antigen, which has a protective effect. In some embodiments, the antibody can be used as a detection reagent to detect the expression of a gene.

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

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

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

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

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

[0119] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker SHOX gene or a fragment thereof can be as shown in SEQ ID NO. 16 and SEQ ID NO. 17.

[0120] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker NKX2-6 gene or a fragment thereof can be as shown in SEQ ID NO. 19 and SEQ ID NO. 20.

[0121] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker LOC728424 gene or a fragment thereof can be as shown in SEQ ID NO. 22 and SEQ ID NO. 23.

[0122] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker ALX3 gene or a fragment thereof can be as shown in SEQ ID NO. 25 and SEQ ID NO. 26.

[0123] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker EN1 gene or a fragment thereof can be as shown in SEQ ID NO. 28 and SEQ ID NO. 29.

[0124] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker PROX1-AS1 gene or a fragment thereof can be as shown in SEQ ID NO. 31 and SEQ ID NO. 32.

[0125] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker LHX4 gene or a fragment thereof can be as shown in SEQ ID NO. 34 and SEQ ID NO. 35.

[0126] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker DCHS2 gene or a fragment thereof can be as shown in SEQ ID NO. 37 and SEQ ID NO. 38.

[0127] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker FOXD3 gene or a fragment thereof can be as shown in SEQ ID NO. 40 and SEQ ID NO. 41.

[0128] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker TBX5-AS1 gene or a fragment thereof can be as shown in SEQ ID NO. 43 and SEQ ID NO. 44.

[0129] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker gene LOC100420879 or a fragment thereof can be as set forth in SEQ ID NO. 46 and SEQ ID NO. 47.

[0130] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker gene CTNND2 or a fragment thereof can be as set forth in SEQ ID NO. 49 and SEQ ID NO. 50.

[0131] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker gene ARHGEF4 or a fragment thereof can be as set forth in SEQ ID NO. 52 and SEQ ID NO. 53.

[0132] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker gene ZNF536-1 or a fragment thereof can be as set forth in SEQ ID NO. 55 and SEQ ID NO. 56.

[0133] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker gene LOC127274910 or a fragment thereof can be as set forth in SEQ ID NO. 58 and SEQ ID NO. 59.

[0134] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker gene ST8SIA5 or a fragment thereof can be as set forth in SEQ ID NO. 61 and SEQ ID NO. 62.

[0135] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker gene Intergenic 1 or a fragment thereof can be as set forth in SEQ ID NO. 64 and SEQ ID NO. 65.

[0136] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker gene ZNF536-2 or a fragment thereof can be as set forth in SEQ ID NO. 67 and SEQ ID NO. 68.

[0137] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker gene HTR1F or a fragment thereof can be as set forth in SEQ ID NO. 70 and SEQ ID NO. 71.

[0138] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker gene PDE4B or a fragment thereof can be as set forth in SEQ ID NO. 73 and SEQ ID NO. 74.

[0139] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker PROKR2 gene or a fragment thereof can be as set forth in SEQ ID NO. 76 and SEQ ID NO. 77.

[0140] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker SFRP4 gene or a fragment thereof can be as set forth in SEQ ID NO. 79 and SEQ ID NO. 80.

[0141] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker WEE1P1 gene or a fragment thereof can be as set forth in SEQ ID NO. 82 and SEQ ID NO. 83.

[0142] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker VSX1 gene or a fragment thereof can be as set forth in SEQ ID NO. 85 and SEQ ID NO. 86.

[0143] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker LINC01210 gene or a fragment thereof can be as set forth in SEQ ID NO. 88 and SEQ ID NO. 89.

[0144] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker NOL4 gene or a fragment thereof can be as set forth in SEQ ID NO. 91 and SEQ ID NO. 92.

[0145] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker ZNF610 gene or a fragment thereof can be as set forth in SEQ ID NO. 94 and SEQ ID NO. 95.

[0146] In some embodiments, the primer pair nucleotide sequence for detecting the methylation marker SLC30A10 gene or a fragment thereof can be as set forth in SEQ ID NO. 97 and SEQ ID NO. 98.

[0147] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker AMER2 gene or a fragment thereof can be as set forth in SEQ ID NO. 3.

[0148] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker TTC34 gene or a fragment thereof can be as set forth in SEQ ID NO. 6.

[0149] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker NMNAT2 gene or a fragment thereof can be as set forth in SEQ ID NO. 9.

[0150] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker SLC7A14 gene or a fragment thereof can 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 methylation marker VSTM2B gene or a fragment thereof can 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 methylation marker SHOX gene or a fragment thereof can 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 methylation marker NKX2-6 gene or a fragment thereof can 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 methylation marker LOC728424 gene or a fragment thereof can 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 methylation marker ALX3 gene or a fragment thereof can 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 methylation marker EN1 gene or a fragment thereof can 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 methylation marker PROX1-AS1 gene or a fragment thereof can 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 methylation marker LHX4 gene or a fragment thereof can 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 methylation marker DCHS2 gene or a fragment thereof can 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 methylation marker FOXD3 gene or a fragment thereof can be as shown in SEQ ID NO. 42.

[0161] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker TBX5-AS1 gene or a fragment thereof can be as set forth in SEQ ID NO. 45.

[0162] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker LOC100420879 gene or a fragment thereof can be as set forth in SEQ ID NO. 48.

[0163] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker CTNND2 gene or a fragment thereof can be as set forth in SEQ ID NO. 51.

[0164] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker ARHGEF4 gene or a fragment thereof can be as set forth in SEQ ID NO. 54.

[0165] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker ZNF536-1 gene or a fragment thereof can be as set forth in SEQ ID NO. 57.

[0166] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker LOC127274910 gene or a fragment thereof can be as set forth in SEQ ID NO. 60.

[0167] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker ST8SIA5 gene or a fragment thereof can be as set forth in SEQ ID NO. 63.

[0168] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker intergenic 1 gene or a fragment thereof can be as set forth in SEQ ID NO. 66.

[0169] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker ZNF536-2 gene or a fragment thereof can be as set forth in SEQ ID NO. 69.

[0170] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker HTR1F gene or a fragment thereof can be as set forth in SEQ ID NO. 72.

[0171] In some embodiments, the nucleotide sequence of the probe for detecting the methylation marker PDE4B gene or a fragment thereof can be as set forth 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 a fragment thereof can be as set forth 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 a fragment thereof can be as set forth 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 a fragment thereof can be as set forth 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 a fragment thereof can be as set forth 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 a fragment thereof can be as set forth 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 a fragment thereof can be as set forth 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 a fragment thereof can be as set forth 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 a fragment thereof can be as set forth in SEQ ID NO. 99.

[0180] The application also provides a method for diagnosing human cervical cancer and / or precancerous lesions of the cervix, which utilizes the above product to detect the methylation level of the 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 gene or fragment thereof in the sample to be tested relative to that in a healthy person.

[0181] In some embodiments, the method can comprise the following steps: (1) collecting the sample to be tested, extracting the sample genomic DNA, performing bisulfite treatment to obtain the converted DNA; (2) preparing a reaction system comprising the sample genomic converted DNA, DNA polymerase, a mixture of deoxynucleotides (dNTPs), a buffer solution, primers, and probes; (3) performing an amplification reaction; (4) analyzing the results to obtain the methylation level of the 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 gene or fragment thereof in the sample to be tested; and (5) determining whether the corresponding human of the sample to be tested has cervical cancer and / or precancerous lesions of the cervix.

[0182] In some embodiments, the amplification reaction can be quantitative methylation-specific PCR (qMSP). The ΔCt value of the methylation marker is calculated 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 the sensitivity (true positive rate) as the vertical coordinate and the 1-specificity (false positive rate) as the horizontal coordinate. The Youden index = sensitivity - (1-specificity), and the optimal critical point (cut-off value of ΔCt) was determined according to the maximum value of the Youden index.

[0184] In some embodiments, when the ΔCt value of the AMER2 gene or a fragment thereof in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt 10.80, it can be judged that the person corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of the cervix.

[0185] In some embodiments, when the ΔCt value of the TTC34 gene or a fragment thereof in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt 7.96, it can be judged that the person corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of the cervix.

[0186] In some embodiments, when the ΔCt value of the NMNAT2 gene or a fragment thereof in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt 12.17, it can be judged that the person corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of the cervix.

[0187] In some embodiments, when the ΔCt value of the SLC7A14 gene or a fragment thereof in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt 6.37, it can be judged that the person corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of the cervix.

[0188] In some embodiments, when the ΔCt value of the VSTM2B gene or a fragment thereof in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt 8.96, it can be judged that the person corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of the cervix.

[0189] In some embodiments, when the ΔCt value of the SHOX gene or a fragment thereof in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt 8.92, it can be judged that the person corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of the cervix.

[0190] In some embodiments, when the ΔCt value of the NKX2-6 gene or a fragment thereof in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt 7.62, it can be judged that the person corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of the cervix.

[0191] In some embodiments, when the ΔCt value of the LOC728424 gene or a fragment thereof in the sample to be tested is lower than or significantly lower than the cut-off value of ΔCt 6.48, it can be judged that the person corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of the cervix.

[0192] In some embodiments, when the ΔCt value of the ALX3 gene or a fragment thereof of the sample to be tested is lower than or significantly lower than the ΔCt cutoff value 11.45, it can be judged that the human corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of the cervix.

[0193] In some embodiments, when the ΔCt value of the EN1 gene or a fragment thereof of the sample to be tested is lower than or significantly lower than the ΔCt cutoff value 6.83, it can be judged that the human corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of the cervix.

[0194] In some embodiments, when the ΔCt value of the PROX1-AS1 gene or a fragment thereof of the sample to be tested is lower than or significantly lower than the ΔCt cutoff value 7.25, it can be judged that the human corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of the cervix.

[0195] In some embodiments, when the ΔCt value of the LHX4 gene or a fragment thereof of the sample to be tested is lower than or significantly lower than the ΔCt cutoff value 9.56, it can be judged that the human corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of the cervix.

[0196] In some embodiments, when the ΔCt value of the DCHS2 gene or a fragment thereof of the sample to be tested is lower than or significantly lower than the ΔCt cutoff value 16.68, it can be judged that the human corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of the cervix.

[0197] In some embodiments, when the ΔCt value of the FOXD3 gene or a fragment thereof of the sample to be tested is lower than or significantly lower than the ΔCt cutoff value 15.33, it can be judged that the human corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of the cervix.

[0198] In some embodiments, when the ΔCt value of the TBX5-AS1 gene or a fragment thereof of the sample to be tested is lower than or significantly lower than the ΔCt cutoff value 6.33, it can be judged that the human corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of the cervix.

[0199] In some embodiments, when the ΔCt value of the LOC100420879 gene or a fragment thereof of the sample to be tested is lower than or significantly lower than the ΔCt cutoff value 5.27, it can be judged that the human corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of the cervix.

[0200] In some embodiments, when the ΔCt value of the CTNND2 gene or a fragment thereof of the sample to be tested is lower than or significantly lower than the ΔCt cutoff value 15.37, it can be judged that the human corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of the cervix.

[0201] In some embodiments, when the ΔCt value of the ARHGEF4 gene or a fragment thereof of the sample to be tested is lower than or significantly lower than the ΔCt cutoff value 7.44, it can be judged that the human corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of cervix.

[0202] In some embodiments, when the ΔCt value of the ZNF536-1 gene or a fragment thereof of the sample to be tested is lower than or significantly lower than the ΔCt cutoff value 10.93, it can be judged that the human corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of cervix.

[0203] In some embodiments, when the ΔCt value of the LOC127274910 gene or a fragment thereof of the sample to be tested is lower than or significantly lower than the ΔCt cutoff value 15.35, it can be judged that the human corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of cervix.

[0204] In some embodiments, when the ΔCt value of the ST8SIA5 gene or a fragment thereof of the sample to be tested is lower than or significantly lower than the ΔCt cutoff value 8.72, it can be judged that the human corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of cervix.

[0205] In some embodiments, when the ΔCt value of the intergenic 1 gene or a fragment thereof of the sample to be tested is lower than or significantly lower than the ΔCt cutoff value 9.50, it can be judged that the human corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of cervix.

[0206] In some embodiments, when the ΔCt value of the ZNF536-2 gene or a fragment thereof of the sample to be tested is lower than or significantly lower than the ΔCt cutoff value 8.83, it can be judged that the human corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of cervix.

[0207] In some embodiments, when the ΔCt value of the HTR1F gene or a fragment thereof of the sample to be tested is lower than or significantly lower than the ΔCt cutoff value 7.32, it can be judged that the human corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of cervix.

[0208] In some embodiments, when the ΔCt value of the PDE4B gene or a fragment thereof of the sample to be tested is lower than or significantly lower than the ΔCt cutoff value 9.67, it can be judged that the human corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of cervix.

[0209] In some embodiments, when the ΔCt value of the PROKR2 gene or a fragment thereof of the sample to be tested is lower than or significantly lower than the ΔCt cutoff value 12.04, it can be judged that the human corresponding to the sample to be tested suffers from cervical cancer and / or precancerous lesion of cervix.

[0210] In some embodiments, when the ΔCt value of the SFRP4 gene or its fragment in the test sample is lower than or significantly lower than the cutoff value of 7.91 for ΔCt, it can be determined that the person corresponding to the test sample has cervical cancer and / or cervical precancerous lesions.

[0211] In some embodiments, when the ΔCt value of the WEE1P1 gene or its fragment in the test sample is lower than or significantly lower than the cutoff value of 6.68 for ΔCt, it can be determined that the person corresponding to the test sample has cervical cancer and / or cervical precancerous lesions.

[0212] In some embodiments, when the ΔCt value of the VSX1 gene or its fragment in the test sample is lower than or significantly lower than the cutoff value of 4.85 for ΔCt, it can be determined that the person corresponding to the test sample has cervical cancer and / or cervical precancerous lesions.

[0213] In some embodiments, when the ΔCt value of the LINC01210 gene or its fragment in the test sample is lower than or significantly lower than the cutoff value of ΔCt 3.97, it can be determined that the person corresponding to the test sample has cervical cancer and / or cervical precancerous lesions.

[0214] In some embodiments, when the ΔCt value of the NOL4 gene or its fragment in the test sample is lower than or significantly lower than the cutoff value of 7.15 for ΔCt, it can be determined that the person corresponding to the test sample has cervical cancer and / or cervical precancerous lesions.

[0215] In some embodiments, when the ΔCt value of the ZNF610 gene or its fragment in the test sample is lower than or significantly lower than the cutoff value of 9.48 for ΔCt, it can be determined that the person corresponding to the test sample has cervical cancer and / or cervical precancerous lesions.

[0216] In some embodiments, when the ΔCt value of the SLC30A10 gene or its fragment in the test sample is lower than or significantly lower than the cutoff value of ΔCt 7.67, it can be determined that the person corresponding to the test sample has cervical cancer and / or cervical precancerous lesions.

[0217] This application also provides a device for detecting cervical cancer and / or precancerous cervical lesions, such as Figure 6 As shown, it includes the following modules: data acquisition module 610 and judgment module 620.

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

[0219] In some embodiments, a 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 a Ct value of the methylation marker detected by quantitative methylation-specific PCR. In some embodiments, preferably, the ΔCt value can be a difference between a Ct value of the target marker and a Ct value of a reference gene. In some embodiments, more preferably, the reference gene can be β-actin.

[0220] The judging module 620 is configured to evaluate the cervical cancer and / or cervical precancerous lesion based on the methylation level data of the target marker of the sample to be tested. In some embodiments, the cervical cancer and / or cervical precancerous lesion can be evaluated based on the ΔCt value and a preset ΔCt cutoff value. In some embodiments, preferably, the sample to be tested can be determined as negative or positive based on the ΔCt value and the preset ΔCt cutoff value. In some embodiments, more preferably, the ΔCt cutoff value can be a 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 ΔCt cutoff value, the sample to be tested can be determined as positive. In some embodiments, when the ΔCt value of the target marker of the sample to be tested is greater than the ΔCt cutoff value, the sample to be tested can be determined as negative.

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

[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 tissue, biopsy tissue, surgical tissue 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 storing computer instructions, which, when executed by a processor, implement a method for detecting cervical cancer and / or cervical precancerous lesion, a flowchart of the method being shown in Figure 7 .

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

[0226] In some embodiments, a 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 a Ct value of the methylation marker detected by quantitative methylation-specific PCR. In some embodiments, preferably, the ΔCt value can be a difference between a Ct value of the target marker and a Ct value of a reference gene. In some embodiments, more preferably, the reference gene can be β-actin.

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

[0228] In some embodiments, a cervical cancer and / or cervical precancerous lesion condition can be evaluated based on the ΔCt value and a preset ΔCt cutoff value. In some embodiments, preferably, the ΔCt value and the preset ΔCt cutoff value can be used to determine whether the sample to be tested is negative or positive. In some embodiments, more preferably, the ΔCt cutoff value can be a 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 ΔCt cutoff value, 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 ΔCt cutoff value, the sample to be tested can be determined to be negative.

[0230] In some embodiments, the positive refers to an individual corresponding to the sample to be tested being a severe cervical precancerous lesion patient or a cervical cancer patient, and the negative refers to an individual corresponding to the sample to be tested being a healthy person, a mild cervical precancerous lesion patient, or a moderate cervical precancerous lesion patient.

[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 tissue, biopsy tissue, surgical tissue, 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 comprises a processor 810, a memory 820, an input / output interface 830, and a communication port 840; the memory 820 is configured to store a computer program, and the processor 810 is configured 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 lesion described above.

[0233] The processor 810 can execute computing instructions (program code) and perform the functions of the detection device described in the present application. The computing instructions can include programs, objects, components, data structures, procedures, modules, and functions (functions refer to specific functions described in the present application). For example, the processor 810 can process instructions for evaluating the condition of cervical cancer and / or precancerous lesions of the cervix in the cervical cancer and / or precancerous lesion detection device. In some embodiments, the processor 810 can 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 physical 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 executing one or more functions, etc., or any combination thereof. For the sake of illustration only, Figure 8 Only one processor 810 is described in the present application, but it should be noted that the present application can include multiple processors.

[0234] The memory 820 can store data / information obtained from any component of the cervical cancer and / or precancerous lesion detection device. In some embodiments, the memory 820 can include a mass storage, a removable storage, a volatile read and write memory, and a read only memory (ROM), etc., or any combination thereof. The exemplary mass storage can include a magnetic disk, an optical disk, and a solid state drive, etc. The removable storage can include a flash drive, a floppy disk, an optical disk, a memory card, a U disk, a compact disk, and a mobile hard disk, etc. The volatile read and write memory can include a random access memory (RAM). The RAM can include a dynamic RAM (DRAM), a double rate synchronous dynamic RAM (DDR SDRAM), a static RAM (SRAM), a thyristor RAM (T-RAM), and a zero-capacitor (Z-RAM), etc. The ROM can include a mask ROM (MROM), a programmable ROM (PROM), an erasable programmable ROM (PEROM), an electrically erasable programmable ROM (EEPROM), an optical disk ROM (CD-ROM), and a digital versatile disk ROM, etc.

[0235] The input / output interface 830 can be used for inputting or outputting signals, data, or information. In some embodiments, the input / output interface 830 can be used to implement the interaction behavior of a user (e.g., a person corresponding to the sample to be tested, a user of the cervical cancer and / or precancerous lesion detection device, etc.) with the processor 710. In some embodiments, the user can input the feature 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. The wired connection can include a cable, an optical cable, or a telephone line, etc., or any combination thereof. The wireless connection 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] The present application also provides a computer program product comprising a computer program which, when executed by a processor, implements the method of the following steps: obtaining methylation level data of a target marker of a sample to be tested, and evaluating the cervical cancer and / or precancerous lesion based on the methylation level data of the target marker of the sample to be tested.

[0238] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent companies unless otherwise specified. In the following examples, three repeated experiments were set for the quantitative test, and the average value was taken as the result.

[0239] The normal population (Normal), mild cervical precancerous lesion (CIN1), moderate cervical precancerous lesion (CIN2), severe cervical precancerous lesion (CIN3), and cervical cancer patients (Cancer) involved in the examples all meet the diagnostic criteria. The diagnostic criteria refer to the fifth edition of the World Health Organization (WHO) classification of female reproductive organ tumors.

[0240] The cervical exfoliative cell specimens used in the examples, the reagent materials, and their sources are as follows:

[0241] 1. Specimen

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

[0243] 2. Main reagent materials

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

[0245] The analysis flowchart of using methylation sequencing data to screen methylation biomarkers for detecting cervical cancer and cervical precancerous lesions is shown in Figure 1 .

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

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

[0248] Take 100-300 ng of qualified genomic DNA, first use MspI (methylation-insensitive restriction enzyme) for enzyme treatment. The DNA fragments after enzyme treatment were end repaired, adapter connected. After gel cutting for fragment selection, EZ DNA Methylation Gold Kit was used for bisulfite (Bisulfite, BS) treatment of DNA, and DNA library was obtained by PCR amplification. After library quality inspection, different libraries were pooled according to the effective concentration and target number of machine requirements for sequencing. The quality of the sequencing data was evaluated, and the sequencing adapter and low-quality data of the sequencing data were cut off by Trimming to obtain clean data for subsequent analysis. Bsmap software was used for methylation data alignment analysis to the reference genome. Use metilene software (Ver 0.2-7) for differentially methylated region (differentially methylated regions, DMR) analysis. Use metilene software (Ver 0.2-7) for DMR analysis.

[0249] The samples were grouped according to CIN1- and CIN3+, and the negative population, CIN1 and CIN2 patients were defined as CIN2-, and CIN3 and cervical cancer patients were defined as CIN3+ patients. The hypermethylated DMRs were screened according to the following conditions: ① annotated region; ② the average methylation difference between CIN1- and CIN3+ groups is Top500; ③ the average methylation value difference between CIN1- and CIN3+ groups is Top500; ④ the methylation level of CIN1- group is <0.2, a total of 572 DMRs were screened for subsequent verification, see Figure 2 .

[0250] Example 2: Screening of cervical cancer and cervical precancerous lesion methylation markers based on targeted methylation sequencing (TBS).

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

[0252] According to p < 0.05, and the difference between the mean methylation levels of the CIN3+ vs CIN2- groups is greater than 0.15, a total of 1103 hypermethylated sites are screened. Then through ① negative group, the mean methylation level < 0.1; ② CIN3 vs negative group, p < 0.05; ③ CIN3 vs CIN2 group, p < 0.10; a total of 125 DMCs are screened for subsequent analysis. LASSO regression analysis is performed using R language, the pathological grade of the patient is defined as the result variable (CIN2- is defined as 0, CIN3+ is defined as 1), and 125 DMCs are the independent variables of the model. A total of 20 DMCs are screened out after LASSO regression analysis. The 20 differential methylation markers (Table 1) screened are modeled using SPSS statistic 21 software, and the formula is:

[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*DCHS2 + 2.253*FOXD3-1 + 1.212*FOXD3-2 + 2.177*TBX5-AS1 + 1.766*LOC100420879.

[0254] Table 1 20 methylation markers of LASSO-Logistic modeling

[0255] Methylation marker Chromosome location AMER2 Chr 13:25744938-25746426 TTC34 Chr 1:2705810-2707334 NMNAT2 Chr 1:183385855-183387356 SLC7A14 Chr 3:170302295-170303819 VSTM2B Chr 19:30018451-30019977 SHOX Chr X:590483-592172 NKX2-6-1 Chr 8:23566680-23568179 NKX2-6-2 Chr 8:23566680-23568179 LOC728424 Chr 15:30516816-30518367 ALX3-1 Chr 1:110611042-110612605 ALX3-2 Chr 1:110611042-110612605 EN1 Chr 2:119607088-119608622 PROX1-AS1-1 Chr 1:214155611-214157113 PROX1-AS1-2 Chr 1:214155611-214157113 LHX4 Chr 1:180201754-180203218 DCHS2 Chr 4:155410591-155412092 FOXD3-1 Chr 1:63784781-63786348 FOXD3-2 Chr 1:63784781-63786348 TBX5-AS1 Chr 12:114846786-114848325 LOC100420879 Chr 1:243052908-243054404

[0256] The receiver operating characteristic (ROC) curve of the 20 methylation markers combination was obtained using SPSS statistic 21 software, with sensitivity (true positive rate) as the vertical coordinate and 1-specificity (false positive rate) as the horizontal coordinate. The 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 predicting the risk of cervical cancer and precancerous lesions of the cervix is shown, with a predicted sensitivity of 86.67%, a specificity of 94.87%, and an AUC of 0.979.

[0257] The ROC and AUC of 1103 candidate methylation markers were obtained using SPSS statistic 21 software to distinguish CIN2- and CIN3+, and the AUC of 1103 high methylation sites was sorted to screen the top 15 high AUC sites (Table 2), Figure 4 The ROC of the Top 15 methylation markers predicting the risk of cervical cancer and precancerous lesions of the cervix is shown.

[0258] Table 2 AUC of Top 15 methylation markers and screening CIN3+

[0259] Methylation marker Chromosome location AUC TTC34 Chr 1:2705810-2707334 0.870 CTNND2 Chr 5:11384181-11385604 0.867 ARHGEF4 Chr 2:131721013-131722519 0.866 ZNF536-1 Chr 19:30865153-30866677 0.866 LOC127274910 Chr 2:137522897-137524244 0.864 ST8SIA5 Chr 18:44336420-44337856 0.863 Intergenic 1 Chr 10:23461814-23463239 0.857 ZNF536-2 Chr 19:30716060-30717060 0.853 HTR1F Chr 3:87841226-87842641 0.851 PDE4B Chr 1:66257794-66259310 0.847 PROKR2 Chr 20:5297005-5298538 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] The 36 cases of negative, 35 cases of CIN1, 46 cases of CIN2, 33 cases of CIN3 and 42 cases of cervical cancer samples, i.e. 117 cases of CIN2- and 75 cases of CIN3+ samples. Artificial analysis was performed on 1103 sites of candidate methylation markers corresponding to 117 cases of CIN2- and 75 cases of CIN3+ samples, to screen methylation markers that can detect the missed CIN3+ samples of the first 35 methylation markers, a total of 3 supplementary methylation markers, as shown in Table 3. Figure 5 The ROC of the supplementary methylation markers for predicting 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 position 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) verification of the screened methylation markers for cervical cancer and precancerous lesions of the cervix.

[0264] Genomic DNA samples obtained from 19 cases of negative, 13 cases of CIN1, 37 cases of CIN2, 33 cases of CIN3 and 44 cases of cervical cancer exfoliated cells were used for qMSP detection to further verify the performance of the screened methylation markers, and a total of 33 methylation marker detection systems were successfully established. The specific method is as follows: after the sample detection is qualified, 1.5 μL of genomic DNA is taken, and EZ DNA Methylation Gold Kit (Zymo Research) is used for Bisulfite treatment. The product after transformation is taken for PCR reaction, and the total volume of PCR reaction solution is 20 μL, which is composed of the following: 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 product after transformation (1.5 μL). The primer sequences and probe sequences are shown in Tables 4 and 5, respectively. The PCR reaction is carried out according to the following reaction program: first 95°C for 5 min, then 95°C for 15 sec and 60°C for 30 sec for a total of 50 cycles. According to the qMSP results, the ΔCt value of the methylation marker is calculated. 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 vertical coordinate and the 1-specificity (false positive rate) as the horizontal coordinate. The Youden index = sensitivity - (1-specificity), and the optimal critical point (cut-off value of ΔCt) was determined according to the maximum value of the Youden index. Table 6 shows the performance of each methylation marker in detecting cervical cancer and precancerous lesions of the cervix by qMSP.

[0266] Table 4 primer sequences

[0267]

[0268]

[0269]

[0270] Table 5 probe sequences

[0271]

[0272]

[0273] Table 6 qMSP method validation of the sensitivity and specificity of methylation markers in detecting CIN3+

[0274]

[0275]

[0276] The above results show that candidate methylation markers for patients with cervical cancer and / or precancerous lesions of the cervix and healthy people are screened out from the DNA of cervical exfoliated cells of the Chinese population, and combined with LASSO-Logistic modeling, AUC ranking and artificial screening methods, 33 methylation markers for screening cervical cancer and / or precancerous lesions of the cervix in the Chinese population are first screened out, and a methylation risk prediction model for cervical cancer and / or precancerous lesions of the cervix in the Chinese population is established, which is suitable for predicting the risk assessment of early cervical cancer and / or precancerous lesions of the cervix in the Chinese population, as well as screening and diagnosis of cervical cancer and / or precancerous lesions of the cervix, providing a fast, effective and accurate new way for early screening, auxiliary diagnosis and evaluation of cervical cancer and / or precancerous lesions of the cervix.

[0277] The foregoing merely illustrates the principles of the application. It will thus be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the application and are thus within its spirit and scope.

[0278] Also, the description uses specific terminology when describing embodiments of the present description. Use of such specific terminology is for the benefit of the reader. For example, the phrases "one embodiment," "an embodiment," and / or "some embodiments" do not necessarily refer to the same embodiment, although they can. Thus, use of these phrases in this description is not intended to necessarily limit the scope of the embodiments described herein. Furthermore, the description is not intended to be limited to the embodiments described herein. Accordingly, the description is not intended to be limited to the embodiments described herein. The description is merely intended to illustrate principles of the description.

[0279] Some embodiments use numerical terms to describe quantities of ingredients, attributes, and the like. It is understood that such numerical terms used in embodiments descriptions are approximations. Unless otherwise specified, "approximately," "about," or "substantially" shall mean ±20% of the value of the measured, calculated, or obtained quantity. Accordingly, numerical parameters used in the description and claims are approximations. Although these numerical parameters are approximations, the disclosed embodiments are not to be limited to the specific values set forth. Other embodiments can employ different numerical parameters depending on the particular embodiments. It is noted that, as used in this description, "exemplary" shall mean "serving as an example," "example," and "exemplary embodiment" shall mean "an example embodiment that serves as illustration of the description." It will be apparent to those skilled in the art that various modifications and variations can be made to the present embodiments without departing from the spirit or scope of the description. Thus, it is intended that the description cover the modifications and variations of this description provided they come within the scope of the appended claims and their equivalents.

[0280] Finally, it should be understood that the embodiments described herein are merely exemplary of the principles of the description. Other embodiments can be devised which fall within the scope of the description. Accordingly, the description is not intended to be limited to the embodiments described herein.

Claims

1. Use of a substance for detecting the methylation level of a methylation marker in the preparation of a product for detecting cervical cancer and severe cervical precancer, the methylation marker being a TTC34 gene fragment, and the chromosomal location of the TTC34 gene fragment being Chr1: 2705810-2707334 based on the sequence of human reference genome Hg19.

2. Use according to claim 1, wherein The sample detected by the product is cervical exfoliated cells.

3. The use according to claim 1, wherein The substance for detecting the methylation level of the methylation marker comprises a primer pair and a probe.

4. The use according to claim 3, wherein the compound is ###0002### The nucleotide sequences of the primer pair are shown in SEQ ID NO. 4 and SEQ ID NO.

5.

5. The use according to claim 3, wherein the compound is ###0002### The nucleotide sequence of the probe is shown in SEQ ID NO.

6.

6. A device for detecting cervical cancer and severe cervical precancer, comprising the following modules: a data acquisition module for providing the methylation level data of a target marker in a sample to be tested, the target marker being the methylation marker in the use of claim 1; a judgment module for evaluating the condition of cervical cancer and severe cervical precancer based on the methylation level data of the target marker in the sample to be tested.

7. The detection device of claim 6, wherein, Based on the methylation level data of the target marker in the sample to be tested, the ΔCt value of the methylation marker is calculated.

8. The detection device of claim 7, wherein, 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.

9. The detection device of claim 8, wherein, The reference gene is β-actin.

10. The detection device of claim 9, wherein, The condition of cervical cancer and severe cervical precancer is evaluated based on the ΔCt value and the preset ΔCt cutoff value.

11. The detection device of claim 10, wherein, Based on the ΔCt value and the preset ΔCt cutoff value, the sample to be tested is determined to be negative or positive.

12. The detection device of claim 11, wherein, The ΔCt cutoff value is the ΔCt value that maximizes the Youden index.

13. The detection device of claim 6, wherein, The sample to be tested is cervical exfoliated cells.

14. The detection device of claim 12, wherein, When the ΔCt value of the target marker in the sample to be tested is less than or equal to the ΔCt cutoff value, the sample to be tested is determined to be positive; When the ΔCt value of the target marker in the sample to be tested is greater than the ΔCt cutoff value, the sample to be tested is determined to be negative; The positive refers to the individual corresponding to the sample to be tested being a severe cervical precancer patient or a cervical cancer patient, and the negative refers to the individual corresponding to the sample to be tested being a healthy person, a mild cervical precancer patient, or a moderate cervical precancer patient.

15. 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 severe cervical precancer is implemented, the method comprising: acquiring the methylation level data of a target marker in a sample to be tested, the target marker being the methylation marker in the use of claim 1; evaluating the condition of cervical cancer and severe cervical precancer based on the methylation level data of the target marker in the sample to be tested.

16. The computer-readable storage medium of claim 15, wherein, Based on the methylation level data of the target marker in the sample to be tested, the ΔCt value of the methylation marker is calculated.

17. The computer-readable storage medium of claim 16, wherein, 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.

18. The computer-readable storage medium of claim 17, wherein, The reference gene is β-actin.

19. The computer-readable storage medium of claim 18, wherein, Based on the ΔCt value and a preset ΔCt cutoff value, the cervical cancer and severe cervical precancerous lesion are evaluated.

20. The computer-readable storage medium of claim 19, wherein, Based on the ΔCt value and a preset ΔCt cutoff value, the sample to be tested is determined as negative or positive.

21. The computer-readable storage medium of claim 20, wherein, The ΔCt cutoff value is a ΔCt value that maximizes the Youden index.

22. The computer-readable storage medium of claim 15, wherein, The sample to be tested is cervical exfoliated cells.

23. The computer-readable storage medium of claim 21, wherein, When the ΔCt value of the target marker of the sample to be tested is less than or equal to the ΔCt cutoff value, the sample to be tested is determined as positive. When the ΔCt value of the target marker of the sample to be tested is greater than the ΔCt cutoff value, the sample to be tested is determined as negative. The positive refers to that the individual corresponding to the sample to be tested is a severe cervical precancerous lesion patient or a cervical cancer patient, and the negative refers to that the individual corresponding to the sample to be tested is a healthy person, a mild cervical precancerous lesion patient or a moderate cervical precancerous lesion patient.

24. An electronic terminal, characterized in that The electronic terminal comprises a processor, a memory, an input / output interface and a communication port; the memory is used for storing a computer program, and the processor is used for executing the computer program stored in the memory to enable the terminal to execute the method for detecting cervical cancer and severe cervical precancerous lesion in the computer readable storage medium according to any one of claims 15-23.

25. A computer program product comprising a computer program, which, when executed by a processor, implements the method for detecting cervical cancer and severe cervical precancerous lesion in the computer readable storage medium according to any one of claims 15-23.

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