A DNA methylation detection kit for cervical cancer diagnosis

Abstract

This invention discloses a DNA methylation detection kit for cervical cancer diagnosis, belonging to the field of molecular diagnostic technology. This invention achieves highly sensitive and specific diagnosis of cervical cancer by jointly detecting the methylation status of specific sites in the human ZNF671 and PAX1 genes. This invention provides a specific primer and probe combination and an optimized detection system, which can accurately quantify the methylation status of the above genes through bisulfite treatment and real-time quantitative PCR technology. Results show that this method has a 100% detection rate in 27 cervical cancer samples and no false positives in 25 healthy control samples, with 100% specificity. Particularly noteworthy is its extremely high detection sensitivity, stably detecting samples with a methylation frequency as low as 0.5%. This invention solves the problems of insufficient sensitivity or low specificity in existing cervical cancer screening methods, providing a powerful tool for early diagnosis, risk stratification, and prognostic monitoring of cervical cancer, and has significant clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostic technology, specifically a DNA methylation detection kit for cervical cancer diagnosis. It is suitable for early screening, diagnosis and risk assessment of cervical cancer, and can quickly and accurately identify the methylation status of specific genes in cervical cells, providing reliable detection basis for clinical practice. Background Technology

[0002] Cervical cancer is one of the most common malignant tumors in women, and early screening is crucial for improving cure rates and reducing mortality. Current screening methods have the following shortcomings: 1. Cytological examination: low sensitivity, high false negative rate, and subjective interpretation of results. 2. HR-HPV testing: while highly sensitive, its specificity is relatively low; most HPV infections are transient and do not lead to precancerous lesions or cancer, resulting in many false positives, causing unnecessary psychological anxiety and over-treatment (such as colposcopy and biopsy). Therefore, developing a highly sensitive and highly specific auxiliary diagnostic method for cervical cancer is of significant clinical importance.

[0003] DNA methylation is an important form of epigenetic modification, playing a crucial role in gene expression regulation. Tumor development and progression are often accompanied by hypomethylation of oncogenes and hypermethylation of tumor suppressor genes. Studies have found that the methylation status of certain genes is closely related to the progression of cervical intraepithelial neoplasia (CIN) and cervical cancer, and can serve as potential biomarkers. Methylation detection, as an important means of early diagnosis of cervical cancer, relies on the effective extraction, methylation treatment, and accurate detection of genomic DNA from exfoliated cervical cells.

[0004] PAX1 (Parasyte Box Family 1) is an important transcription factor involved in embryonic development. Its hypermethylation in cervical cancer tissue has been reported in the literature, and its abnormal silencing is significantly associated with cervical cancer development. ZNF671 (Zinc Finger Protein 671) is a zinc finger protein that normally participates in gene transcriptional regulation. In the development and progression of cervical cancer, the promoter region of the ZNF671 gene undergoes abnormal hypermethylation. This epigenetic alteration leads to gene expression silencing, causing it to lose its function as a tumor suppressor gene, thereby promoting the malignant transformation of cervical epithelial cells.

[0005] Existing technologies have reported the association between methylation of single genes (such as PAX1, SOX1, ZNF671, etc.) and cervical cancer. However, the detection efficacy (sensitivity and specificity) of single gene markers is sometimes insufficient to meet the clinical demand for high-precision screening. How to screen for the optimal gene combination and develop detection products with higher clinical value remains a technical problem that needs to be solved in this field. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, the present invention aims to provide a highly sensitive DNA methylation detection kit for cervical cancer diagnosis. This invention designs primer and probe combinations based on two target genes, ZNF671 and PAX1, and the internal reference gene β-actin, for non-invasive screening and triage of cervical cancer. This invention solves the problems of insufficient sensitivity or low specificity in existing cervical cancer screening methods, providing a powerful tool for early diagnosis, risk stratification, and prognostic monitoring of cervical cancer, and has significant clinical application value.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a molecular marker combination for early screening of cervical cancer, the molecular marker combination being composed of a methylated ZNF671 gene and a methylated PAX1 gene.

[0009] Secondly, the present invention provides a primer and probe combination for high-sensitivity DNA methylation detection for cervical cancer diagnosis, wherein the primer and probe combination is used to detect the methylated ZNF671 gene and the methylated PAX1 gene after bisulfite treatment; the primer and probe combination includes ZNF671 forward primer F, ZNF671 reverse primer R, ZNF671 probe P, PAX1 forward primer F, PAX1 reverse primer R and PAX1 probe P.

[0010] Based on the above technical solution, further, the nucleotide sequence of ZNF671 forward primer F is shown in SEQ ID NO.1, the nucleotide sequence of ZNF671 reverse primer R is shown in SEQ ID NO.2, the nucleotide sequence of ZNF671 probe P is shown in SEQ ID NO.3, the nucleotide sequence of PAX1 forward primer F is shown in SEQ ID NO.4, the nucleotide sequence of PAX1 reverse primer R is shown in SEQ ID NO.5, and the nucleotide sequence of PAX1 probe P is shown in SEQ ID NO.6.

[0011] Based on the above technical solution, further, the 5' end of the ZNF671 probe P is marked with ROX and the 3' end is marked with BHQ1 or BHQ2; the 5' end of the PAX1 probe P is marked with VIC and the 3' end is marked with BHQ1 or BHQ2.

[0012] Thirdly, the present invention provides a kit for cervical cancer diagnosis, the kit comprising the above-mentioned primer and probe combination for cervical cancer methylation detection.

[0013] Based on the above technical solution, the kit further includes a forward primer F, a reverse primer R, and a detection probe for detecting the internal reference gene β-actin. The nucleotide sequence of the forward primer F is shown in SEQ ID NO.7, the nucleotide sequence of the reverse primer R is shown in SEQ ID NO.8, and the nucleotide sequence of the detection probe is shown in SEQ ID NO.9.

[0014] Based on the above technical solution, further, the 5' end of the detection probe is marked with ROX, and the 3' end is marked with BHQ1 or BHQ2.

[0015] Based on the above technical solution, the kit further includes DNA polymerase and Mg 2+ The PCR reaction solution consists of dNTPs, nuclease-free water, and Taq enzyme.

[0016] Fourthly, the present invention provides a method for using the above-mentioned reagent kit, comprising the following steps: (1) Extract genomic DNA from the cervical exfoliated cells of the subjects; (2) Take a portion of the genomic DNA obtained in step (1) and process it with a methylation detection kit; (3) Using the DNA processed in step (2) as a template, add the above primer and probe combination and PCR reaction solution to perform real-time fluorescence quantitative PCR amplification.

[0017] Based on the above technical solution, the procedure for real-time quantitative PCR amplification in step (3) is as follows: Stage 1: pre-denaturation at 94~96℃ for 4~6 minutes; Stage 2: reaction at 94~96℃ for 8~15 seconds, reaction at 58~62℃ for 25~35 seconds; 3 cycles; Stage 3: reaction at 94~96℃ for 8~15 seconds, reaction at 58~62℃ for 25~35 seconds, collect fluorescence signal; 45 cycles.

[0018] Based on the above technical solution, further, after step (3) is completed, the amplification curve and Ct value are analyzed, and the methylation status of the sample is determined according to the difference in CT values ​​between the two target genes and the internal reference gene: Determination of valid sample results: If the Ct of β-actin is ≤35, the sample is valid; if the Ct of the β-actin gene is >35 or there is no amplification, the test is considered invalid. Positive result judgment: If the CT value of ZNF671 and the Ct value of β-actin are △Ct≦8, or the CT value of PAX1 and the Ct value of β-actin are △Ct≦8, then it is a positive sample; otherwise, it is a negative sample.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. High sensitivity and high specificity: ZNF671 and PAX1 genes exhibit highly specific methylation in cervical cancer tissues, while almost no methylation is observed in normal tissues; combined detection of the two can produce a synergistic effect, and its sensitivity and specificity for detecting high-grade lesions (CIN2+) are significantly better than single gene detection and HPV detection, effectively reducing false positive and false negative results.

[0020] 2. Objective Quantification: The qMSP method is used, and the results are output in the form of Ct values, which can realize the absolute and relative quantification of methylation levels, making the interpretation objective and avoiding the subjectivity of cytological examination.

[0021] 3. Samples are readily available and non-invasive: The test sample is cervical exfoliated cells, which are obtained in the same way as TCT / HPV tests, making them easy to accept and convenient for large-scale population screening.

[0022] 4. Clear triage value: For HPV-positive individuals, the detection method of this invention can effectively distinguish between transient infection and potential high-grade lesions, providing an important basis for decision-making on whether colposcopy is necessary and avoiding over-medicalization. Attached Figure Description

[0023] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0024] Figure 1 This is a fluorescence detection result of ZNF671 gene methylation in one cervical cancer patient in Example 1.

[0025] Figure 2 This is a graph showing the fluorescence detection results of PAX1 gene methylation in a cervical cancer patient in Example 1.

[0026] Figure 3 This is a graph showing the fluorescence detection results of β-actin gene methylation in one cervical cancer patient in Example 1. Detailed Implementation

[0027] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0028] Example 1: 1. Experimental Materials Cervical exfoliated cells from 27 patients with cervical cancer and 25 patients with normal pathology, nucleic acid extraction kit, ZYMO methylation kit (EZ DNA Methylation-Lightning™ Kit), fluorescence detection reagent, and quantitative real-time PCR instrument were used.

[0029] 2. Experimental Procedure 2.1. DNA Extraction: Genomic DNA was extracted from the 52 cervical exfoliated cell samples using a nucleic acid extraction kit. The DNA concentration and OD260 of each sample were measured. nm / 280 nm The values ​​were set to ensure that the quality of the extracted samples was up to standard (the DNA 260 / 280 values ​​of the 52 samples extracted by the nucleic acid extraction kit were all within the range of 1.8~2.0, and the concentration met the requirements for subsequent methylation treatment and fluorescence detection).

[0030] 2.2 Methylation treatment: The extracted DNA samples were processed using the ZYMO methylation detection kit, and the reaction conditions were strictly controlled according to the kit's operating instructions.

[0031] 2.3 Primer Design The risk genes ZNF671 and PAX1 associated with cervical cancer were identified, and methylation-specific primer and probe combinations were designed for the target regions. The specific primer and probe combinations are shown in Table 1.

[0032] Table 1 Primers and probes for ZNF671 and PAX1

[0033] 2.4 Preparation of fluorescence detection reagents: Prepare PCR reaction solution according to Table 2-4. Three reaction systems should be prepared for each sample. Add 2µL of the above-treated methylated DNA sample to each reaction system.

[0034] Table 2 Reaction System 1

[0035] Table 3 Reaction System 2

[0036] Table 4 Reaction System 3

[0037] 2.5. Fluorescence Detection: Using a quantitative real-time PCR instrument, set up the PCR reaction program according to the following procedure: Stage 1: Pre-denaturation at 95℃ for 5 minutes; Phase 2: Reaction at 95℃ for 10 seconds, reaction at 60℃ for 30 seconds; 3 cycles; Phase 3: React at 95℃ for 10 seconds, then at 60℃ for 30 seconds, and collect fluorescence signals; 45 cycles; 2.6 Results Statistics and Comparison: Using β-actin as the internal reference gene and ZNF671 and PAX1 as target genes, the Ct values ​​of ZNF671, PAX1, and β-actin genes for each sample were recorded. The criteria for valid sample results were: Ct value of the β-actin gene Ct ≤ 35; if the Ct value of the β-actin gene > 35 or there was no amplification, the test was considered invalid. If the difference between the CT value of the ZNF671 gene and the CT value of the internal reference gene β-actin, ΔCt ≤ 8, or the difference between the CT value of the PAX1 gene and the CT value of the internal reference gene β-actin, ΔCt ≤ 8, the sample was considered positive; otherwise, it was considered negative.

[0038] 3. Experimental Results Detection results and comparison: Among the 52 samples to be tested, samples 1-27 were known pathological positive samples for cervical cancer, and samples 28-52 were negative control samples. The detection results of the above method of the present invention are shown in Table 5, and all of them are consistent with the pathological results.

[0039] Table 5 Comparison of test results and pathological results in this application

[0040] Example 2: 1. Experimental Materials Samples with methylation rates of 0%, 0.5%, 1%, 2.5%, 5%, 10%, 25%, 50%, and 100% were simulated using standards (Human Methylated & Non-methylated DNA kit standards), ZYMO methylation kit (EZ DNA Methylation-Lightning™ Kit), fluorescent detection reagents, and a real-time PCR instrument.

[0041] 2. Experimental Procedure 2.1 Methylation treatment: Samples with simulated methylation rates of 0%, 0.5%, 1%, 2.5%, 5%, 10%, 25%, 50%, and 100% were treated with the ZYMO methylation detection kit, and the reaction conditions were strictly controlled according to the kit's operating instructions.

[0042] 2.3 Preparation of fluorescence detection reagents: Using the primers and probes designed in Table 1 of Example 1, prepare PCR reaction solutions according to Tables 2-4 of Example 1. Three reaction systems need to be prepared for each sample. Add 2µL of the above-treated methylated DNA sample to each reaction system.

[0043] 2.4. Fluorescence Detection: Using a quantitative real-time PCR instrument, set up the PCR reaction program according to the following procedure: Stage 1: Pre-denaturation at 95℃ for 5 minutes; Phase 2: Reaction at 95℃ for 10 seconds, reaction at 60℃ for 30 seconds; 3 cycles; Phase 3: React at 95℃ for 10 seconds, then at 60℃ for 30 seconds; collect fluorescence signals; 45 cycles; 2.5 Results Statistics and Comparison: Using β-actin as the internal reference gene and ZNF671 and PAX1 as target genes, the Ct values ​​of ZNF671, PAX1, and β-actin genes for each sample were recorded. The criteria for valid sample results were: Ct value of the β-actin gene Ct ≤ 35; if the Ct value of the β-actin gene > 35 or there was no amplification, the test was considered invalid. If the difference between the CT value of the ZNF671 gene and the CT value of the internal reference gene β-actin, ΔCt ≤ 8, or the difference between the CT value of the PAX1 gene and the CT value of the internal reference gene β-actin, ΔCt ≤ 8, the sample was considered positive; otherwise, it was considered negative.

[0044] 3. Experimental results: Repeated detection was performed using standards with simulated methylation rates of 0%, 0.5%, 1%, 2.5%, 5%, 10%, 25%, 50%, and 100%. The results are shown in Table 6. The results show that the standards with simulated methylation rates ≥0.5% can be detected stably and effectively, proving that the detection sensitivity of this method reaches 0.5%.

[0045] Table 6 Comparison of test results and pathological results in this application

[0046] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. The protection scope of the present invention shall be determined by the claims.

Claims

1. A primer and probe combination for DNA methylation detection in cervical cancer diagnosis, characterized in that, The primer and probe combination is used to detect the methylated ZNF671 gene and the methylated PAX1 gene after bisulfite treatment; the primer and probe combination includes ZNF671 forward primer F, ZNF671 reverse primer R, ZNF671 probe P, PAX1 forward primer F, PAX1 reverse primer R and PAX1 probe P.

2. The primer and probe combination according to claim 1, characterized in that, The nucleotide sequence of ZNF671 forward primer F is shown in SEQ ID NO.1, the nucleotide sequence of ZNF671 reverse primer R is shown in SEQ ID NO.2, the nucleotide sequence of ZNF671 probe P is shown in SEQ ID NO.3, the nucleotide sequence of PAX1 forward primer F is shown in SEQ ID NO.4, the nucleotide sequence of PAX1 reverse primer R is shown in SEQ ID NO.5, and the nucleotide sequence of PAX1 probe P is shown in SEQ ID NO.

6.

3. The primer and probe combination according to claim 2, characterized in that, The 5' end of the ZNF671 probe P is marked with ROX, and the 3' end is marked with BHQ1 or BHQ2; the 5' end of the PAX1 probe P is marked with VIC, and the 3' end is marked with BHQ1 or BHQ2.

4. A reagent kit for cervical cancer diagnosis, characterized in that, The kit includes the primer and probe combination for cervical cancer methylation detection as described in any one of claims 1-3.

5. The reagent kit according to claim 4, characterized in that, The kit also includes a forward primer F, a reverse primer R, and a detection probe for detecting the internal reference gene β-actin. The nucleotide sequence of the forward primer F is shown in SEQ ID NO.7, the nucleotide sequence of the reverse primer R is shown in SEQ ID NO.8, and the nucleotide sequence of the detection probe is shown in SEQ ID NO.

9.

6. The reagent kit according to claim 5, characterized in that, The 5' end of the detection probe is marked with ROX, and the 3' end is marked with BHQ1 or BHQ2.

7. The reagent kit according to claim 4, characterized in that, The kit also contains DNA polymerase, Mg 2+ The PCR reaction solution consists of dNTPs, nuclease-free water, and Taq enzyme.

8. The method of using the reagent kit according to any one of claims 4-7, characterized in that, Includes the following steps: (1) Extract genomic DNA from the cervical exfoliated cells of the subjects; (2) Take a portion of the genomic DNA obtained in step (1) and process it with a methylation detection kit; (3) Using the DNA processed in step (2) as a template, add the primer and probe combination and PCR reaction solution to perform real-time quantitative PCR amplification.

9. The method of use according to claim 8, characterized in that, The procedure for real-time quantitative PCR amplification described in step (3) is as follows: Stage 1: pre-denaturation at 94~96℃ for 4~6 minutes; Stage 2: reaction at 94~96℃ for 8~15 seconds, reaction at 58~62℃ for 25~35 seconds; 3 cycles; Stage 3: reaction at 94~96℃ for 8~15 seconds, reaction at 58~62℃ for 25~35 seconds, collect fluorescence signal; 45 cycles.

10. The method of use according to claim 8, characterized in that, After step (3) is completed, the amplification curve and Ct value are analyzed, and the methylation status of the sample is determined based on the difference in CT values ​​between the two target genes and the internal reference gene: Determination of valid sample results: If the Ct of β-actin is ≤35, the sample is valid; if the Ct of the β-actin gene is >35 or there is no amplification, the test is considered invalid. Positive result judgment: If the CT value of ZNF671 and the Ct value of β-actin are △Ct≦8, or the CT value of PAX1 and the Ct value of β-actin are △Ct≦8, then it is a positive sample; otherwise, it is a negative sample.

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