Methylation gene detection kit for early detection of cervical cancer

Abstract

The invention relates to the field of medicine, in particular to a methylation gene detection kit for early detection of cervical cancer, compared with a published cervical cancer methylation marker, the AUC corresponding to cervical cancer lesion detection of the marker can reach 0.986, and when the specificity is 100%, the sensitivity can reach 90.9%. According to the results, the detection sensitivity and the specificity of the kit are greatly improved compared with those of HPV (human papillomavirus) detection. In addition, in an HPV positive sample, the sensitivity and the specificity can be further improved through methylation joint detection. Therefore, the kit can better detect cervical cancer to a great extent, meanwhile, the phenomena of excessive diagnosis and excessive anxiety caused by HPV detection are well solved, and the kit has very high social value.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and more particularly, to a methylation gene detection kit for early detection of cervical cancer. Background Art

[0002] Early detection, early diagnosis, and early treatment of cervical cancer are of great significance. Regular cervical cancer screening is the best way to prevent cervical cancer. There is an urgent need in this field to develop early detection techniques for cervical cancer with advantages such as rapidity, simplicity, high sensitivity, and high specificity. The present invention can perform early detection of cervical cancer through the detection of circulating free DNA in plasma, exfoliated cells in cervical secretions, and free DNA.

[0003] DNA methylation is the most common epigenetic modification, which can regulate cell proliferation, apoptosis, and differentiation, and its level is closely related to the biological characteristics of tumors. Therefore, the detection of DNA methylation indicators can be used as an important biomarker for cancer diagnosis, early screening, and prognosis. Methylation fluorescence quantitative method is widely used in the detection of DNA methylation due to its high throughput and high sensitivity, and the need for post-PCR electrophoresis, hybridization and other operations is avoided, reducing contamination and operation errors.

[0004] The prior art CN115896289A discloses a detection primer, probe, kit and method for methylation of cervical cancer-related genes, which uses the multiplex Q-MSP method to detect the methylation degree of 8 genes related to cervical cancer, namely ADCYAP1, AJAP1, CDKN2A, GATA4, LHX8, PAX1, SOX1 and MIR214, with high sensitivity and strong specificity, and is suitable for large-scale population screening and routine molecular diagnosis.

[0005] The prior art CN113502327A discloses a biomarker molecule for cancer, a screening test kit thereof, and a detection method thereof, which uses the methylation regions of the target genes PAX1, ZNF582, SOX1, and NKX6-1 in the analyzed sample to design several oligonucleotide primers or probes specific to the target genes, and uses the oligonucleotide probes to detect the presence or absence of methylation of the target genes, and further judges the possibility of cancer occurrence.

[0006] The prior art CN116463422A discloses an integrated detection model for human papillomavirus (HPV) genotyping and methylation of cervical cancer-related genes and its construction method, wherein the cervical cancer-related genes are: CADM1, CCNA2, CDH1, CDH13, CDH6, CDKN2A, DAPK1, DLX1, EPB41L3, FAM19A4, FANCI, FHIT, GATA4, GFRA1, HS3ST2, JAM3, KCNIP4, LHX8, LMX1A, MAL, MIR124-1, MIR124-2, MIR124-3, PAX1, PCDHA13, PCDHA4, POU4F3, RARB, RNASEH2A, RUBCNL, RXFP3, SLIT2, SOX1, SOX17, ST6GALNAC5, TERT, TIMP3, WIF1, ZNF671 and ZSCAN1; the sensitivity is 79.59%, the specificity is 67.74%, and the AUC value is 0.74.

[0007] The prior art by Zhou Zijin discloses the selection of C13ORF18, TERT and SLIT1 as detection markers for cervical cancer, and its sensitivity and specificity are 78% and 96.9% (see Zhou Zijin, "Methylation levels and clinical significance of C13ORF18, TERT and SLIT1 genes in cervical lesions and cervical cancer", Master's thesis of Tianjin Medical University).

[0008] However, the sensitivities and specificities of the above prior arts are uneven. Therefore, there is an urgent need to provide a set of methylation gene markers and detection reagents with higher sensitivity and specificity. Accordingly, the present invention provides a primer-probe composition for detecting gene methylation of cervical cancer, which has high sensitivity, strong specificity, can perform methylation-specific amplification detection, and uses methylation fluorescence quantitative method for the screening and diagnosis of cervical cancer. Summary of the Invention

[0009] To solve the above problems, the present invention first provides a methylation gene for early detection of cervical cancer, and the methylation gene includes one or more of PCDHA4, C13ORF18, SOX1, PAX1, TERT, and JAM3.

[0010] In certain embodiments, the methylation gene is one or more of PCDHA4, C13ORF18, SOX1, PAX1, TERT, and JAM3.

[0011] In certain embodiments, the methylation gene is PCDHA4, C13ORF18, SOX1, PAX1, TERT and JAM3.

[0012] The present invention also provides an application of the above-mentioned methylated gene in the preparation of early diagnosis products for cervical cancer.

[0013] In some embodiments, the product comprises primers, probes or a kit.

[0014] The present invention also provides an application of the above-mentioned methylated gene in the early diagnosis of cervical cancer, and the application is for non-diagnostic or non-therapeutic purposes;

[0015] The application includes the following:

[0016] (a) Diagnosing the progression of cervical cancer;

[0017] (b) Screening drugs for the treatment of cervical cancer;

[0018] (c) Assisting in constructing a cell or animal model of cervical cancer;

[0019] Optionally, the application is for scientific research purposes.

[0020] The present invention also provides a primer-probe set for detecting the above-mentioned methylated gene, and the sequences of the primer-probe set are shown as SEQ ID NO: 1 - SEQ ID NO: 21.

[0021] The present invention also provides a kit for the early diagnosis of cervical cancer, and the kit comprises the above-mentioned primer-probe set.

[0022] The present invention also provides an application of the above-mentioned primer set in the preparation of a kit for the early diagnosis of cervical cancer.

[0023] Finally, the present invention provides a method for the early diagnosis of cervical cancer, which comprises the step of using the above-mentioned primers or the above-mentioned kit to detect the above-mentioned methylated gene in a sample to be tested, and the application is for non-diagnostic purposes. Optionally, the application is for scientific research purposes; optionally, the sample is an ex vivo sample.

[0024] In some embodiments, the methylation modification status of cytosine sites in the CpG dinucleotide sequence within the detection target region in the above DNA sample is detected by using methylation conversion technology and methylation detection technology; the methylation conversion technology includes but is not limited to bisulfite conversion, conversion based on enzymes such as TET, APOBEC, etc., conversion based on borane, and preferably bisulfite conversion;

[0025] In some embodiments, the methylation detection techniques include, but are not limited to, methylation-specific PCR, quantitative methylation-specific PCR, digital PCR, bisulfite sequencing, methylation-specific microarray, whole-genome methylation sequencing, pyrosequencing, methylation-specific high performance liquid chromatography, methylation-specific high-resolution melting curve, methylation-sensitive restriction endonuclease or fluorescence quantification, preferably quantitative methylation-specific PCR or digital PCR method.

[0026] Compared with the prior art, the present invention has at least the following beneficial effects:

[0027] Compared with the published cervical cancer methylation markers, the target genes detected by the composition for detecting cervical cancer provided by the present invention have high sensitivity and specificity, because the target genes are screened after collecting and processing a large amount of cervical cancer-related data. The AUC of the marker of the present invention for detecting cervical cancer lesions can reach 0.984375, and when the specificity is 100%, the sensitivity can reach 90.9%. Compared with cytological detection, HPV DNA detection can type and quantitatively detect high-risk and low-risk types. However, more than 80% of women will be infected with HPV in their lifetime, and the virus in more than 90% of the infected people will be automatically cleared within two years. HPV DNA detection produces a large number of false positives (low specificity), which will cause over-treatment clinically and increase the burden on patients. From the above results, it can be seen that this kit has a great improvement in detection sensitivity and specificity compared with HPV detection. In addition, in HPV-positive samples, the combined detection of methylation can further improve the sensitivity and specificity. Therefore, to a great extent, this kit can better detect cervical cancer, and at the same time, it can well solve the phenomenon of over-diagnosis and over-anxiety caused by HPV detection, and has high social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a schematic diagram of the amplification curves and result judgment of positive samples of the target genes SOX1 gene and PAX1 gene.

[0029] Figure 2 is a schematic diagram of the amplification curves and result judgment of positive samples of the target genes TERT gene and C13ORF18 gene.

[0030] Figure 3 is a schematic diagram of the amplification curves and result judgment of positive samples of the target genes JAM3 gene and PCDHA4 gene.

[0031] Figure 4 is the ROC curve obtained by six-gene combined diagnosis of cervical cancer after obtaining the calculation formula through a logistic regression model. DETAILED DESCRIPTION OF THE INVENTION

[0032] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0033] Term Explanation:

[0034] QMSP, Quantitative Methylation-Specific PCR, quantitative methylation-specific PCR;

[0035] AUC, Area Under Curve, the area enclosed by the ROC curve and the coordinate axes;

[0036] ROC, Receiver Operating Characteristic curve, is a tool for evaluating the performance of classification models;

[0037] PCDHA4, protocadherin alpha4, a gene encoding a transmembrane cell adhesion protein, mainly distributed on the surface of neurons in the central nervous system;

[0038] C13ORF18, chromosome 13 open reading frame 18

[0039] JAM3, Junctional Adhesion Molecule 3, is considered a tumor suppressor gene;

[0040] PAX1, Paired Box 1, is an important transcription factor and belongs to the PAX1 gene family;

[0041] SOX1, SRY (sex determining region Y)-box 1, a similar protein regulates the γ-crystallin gene;

[0042] TERT, Telomerase Reverse Transcriptase, a gene encoding the catalytic subunit of telomerase, i.e., telomerase reverse transcriptase.

[0043] Example 1, Whole Genome Methylation Chip Screening

[0044] Ten cervical cancer exfoliated cell samples and ten normal female cervical exfoliated cell samples were collected. The Illumina Infinium MethylationEPIC Bead Chip was used to screen methylation sites. According to the method reported in Comprehensive analysis of DNA methylation data with RnBeads (Nat Methods. 2014), differential DNA methylation sites were screened, and the candidate genes JAM3, PCDHA4, C13OEF18, PAX1, SOX1, and TERT were obtained.

[0045] Example 2

[0046] The present invention is a kit for detecting gene methylation in cervical cancer, including primer pairs and probes for detecting the methylation of JAM3, PCDHA4, C13OEF18, PAX1, SOX1, and TERT genes; primer pairs and probes for the internal reference ACTB gene, and the sequences are shown in Table 1;

[0047] Table 1. Primer and probe sequences

[0048]

[0049]

[0050] Real-time fluorescence quantitative PCR reaction solution; it also includes a lysis solution, a bisulfite conversion reagent, and a washing solution. The reaction procedure is shown in Table 2. The reaction system is shown in Tables 3 - 5.

[0051] Table 2. Reaction procedure

[0052]

[0053] Table 3. Reaction system

[0054] Primer and Probe Name Volume Added (μL) PAX1-F 0.25 PAX1-Probe(ROX) 0.15 PAX1-R 0.25 SOX1-F 0.25 SOX1-Probe(FAM) 0.15 SOX1-R 0.25 ACTB-F 0.25 ACTB-R 0.25 ACTB-Probe(CY5) 0.15 Bis-DNA 2 2×TaqMan qPCR Mix 12.5 Rnase-Free Water 8.55 Total Volume 25

[0055] Table 4. Reaction system

[0056]

[0057]

[0058] Table 5. Reaction system

[0059] Primer and Probe Name Volume Added (μL) PCDH4-F 0.25 PCDH4-Probe(FAM) 0.15 PCDH4-R 0.25 JAM3-F 0.25 JAM3-Probe(ROX) 0.15 JAM3-R 0.25 ACTB-F 0.25 ACTB-Probe(CY5) 0.15 ACTB-R 0.25 Bis-DNA 2 2×TaqMan qPCR Mix 12.5 Rnase-Free Water 8.55 Total Volume 25

[0060] 1) Collect samples (the samples include any one of plasma, serum, and cervical secretions), and the clinical samples include: 22 exfoliated cell samples of cervical cancer, and 20 exfoliated cell samples of the cervix of normal women.

[0061] Among them, the samples numbered 1-22 are exfoliated cell samples of cervical cancer, and the samples numbered 23-42 are exfoliated cell samples of the cervix of normal women, and free DNA is extracted from the said samples;

[0062] 2) Perform methylation conversion on the said free DNA with a bisulfite conversion reagent to obtain bis-DNA;

[0063] 3) Perform a real-time fluorescence quantitative PCR amplification reaction on the bis-DNA, detect the fluorescence signal and determine the result, determine the positive and negative of the target gene according to the CT value obtained for each marker and the CT value of ACTB, and then determine the positive and negative of the sample according to the logical model.

[0064] Judgment criteria for positive samples

[0065] Judgment of methylation detection results

[0066] (1) The CT value of the internal reference ≤ 35; the CT value of the target gene ≤ 35, and this target gene is determined to be positive, scoring one point;

[0067] (2) The CT value of the internal reference ≤ 35; the CT value of the target gene > 35, and this target gene is determined to be negative, scoring 0 points;

[0068] (3) The CT value of the internal reference > 35 or the amplification curve has no obvious logarithmic growth phase, and it is determined that the sample is unqualified.

[0069] Determine the positive and negative of the sample according to the logical model. The sum of the scores of each target gene ≥ 3 is counted as a positive sample. When the sample to be tested is determined to be positive, it indicates a high risk of high-grade cervical lesions and / or progressive cervical cancer; on the contrary, when the sample to be tested is determined to be negative, it indicates a low risk of high-grade cervical lesions and / or progressive cervical cancer. It is applicable to the early detection and auxiliary diagnosis of cervical cancer in women.

[0070] The results are shown in Table 6, Figures 1 - 4 as follows.

[0071] Table 6 Results of the combined diagnosis of six genes for cervical cancer

[0072]

[0073]

[0074]

[0075] Simultaneously perform multiplex detection on the above genes, obtain the calculation formula through a logistic regression model, and then conduct six-gene combined diagnosis of cervical cancer, and prepare the corresponding ROC curve. The results are as Figure 4 shown. The AUC of the biomarker of the present invention for detecting cervical cancer lesions can reach 0.986, and when the specificity is 100%, the sensitivity can reach 90.9%.

[0076] The above is the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle described in the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A methylation gene for early detection of cervical cancer, characterized in that: The methylation genes include one or more of PCDHA4, C13ORF18, SOX1, PAX1, TERT, and JAM3.

2. The methylation gene according to claim 1, characterized in that The methylation gene is one or more of PCDHA4, C13ORF18, SOX1, PAX1, TERT, and JAM3.

3. The methylation gene according to claim 1, characterized in that The methylated genes are PCDHA4, C13ORF18, SOX1, PAX1, TERT and JAM3.

4. Use of the methylation gene as described in any one of claims 1 to 3 in preparing a product for early diagnosis of cervical cancer.

5. The use according to claim 4, characterized in that The product comprises a primer, a probe or a kit.

6. A use of the methylation gene according to claim 1 in diagnosing cervical cancer, wherein the use is for non-diagnostic or non-therapeutic purposes; The application includes the following: (a) Diagnosis of cervical cancer; (b) Screening of drugs for the treatment of cervical cancer; (c) Assist in the construction of cell or animal models of cervical cancer; Optionally, the use is for the purpose of scientific research.

7. A primer probe set for detecting the methylated gene according to claim 1, characterized in that: The primer pair sequences are shown in SEQ ID NO: 1-SEQ ID NO:

21.

8. A methylation gene detection kit for early detection of cervical cancer, characterized in that: The kit comprises the primer-probe set according to claim 7.

9. Use of the primer set according to claim 7 in preparing a kit for early diagnosis of cervical cancer.

10. A method for early diagnosis of cervical cancer, characterized in that: It comprises the step of using the primers described in claim 7 or the kit described in claim 8 to detect the methylated gene described in any one of claims 1 to 3 in the sample to be tested, and the application is for non-diagnostic purposes. Optionally, the application is for the purpose of scientific research; optionally, the sample is an in vitro sample.

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