阅读说明：本技术 一种***相关基因甲基化检测方法 (Methylation detection method for cervical cancer related gene ) 是由 刘文兰 徐迹 李赟 徐勇 余志英 黄建林 林穆奇 于 2020-07-30 设计创作，主要内容包括：本发明提供一种宫颈癌相关基因甲基化检测方法。述检测方法包括以下步骤：(1)样本DNA的提取：从宫颈脱落细胞样本中提取得到样本DNA；(2)样本DNA的转化纯化：将提取得到的样本DNA进行重硫酸盐转化反应后纯化,得到Bis DNA；(3)Bis DNA的PCR扩增：采用PCR扩增液对Bis DNA进行探针荧光定量PCR扩增；(4)根据探针荧光定量PCR扩增的结果确定待检测样本的甲基化值。所述检测方法具有快速、高通量、灵敏及特异性好的特点。(The invention provides a methylation detection method of a gene related to cervical cancer. The detection method comprises the following steps: (1) extraction of sample DNA: extracting sample DNA from a cervical exfoliated cell sample; (2) transformation and purification of sample DNA: performing a bisulfate conversion reaction on the extracted sample DNA, and then purifying to obtain Bis DNA; (3) PCR amplification of Bis DNA: performing probe fluorescent quantitative PCR amplification on the Bis DNA by using a PCR amplification solution; (4) and determining the methylation value of the sample to be detected according to the result of the fluorescent quantitative PCR amplification of the probe. The detection method has the characteristics of high speed, high flux, sensitivity and good specificity.)

1. A methylation detection method of a gene related to cervical cancer, which is characterized by comprising the following steps:

(1) extraction of sample DNA: extracting sample DNA from a cervical exfoliated cell sample;

(2) transformation and purification of sample DNA: performing a bisulfate conversion reaction on the extracted sample DNA, and then purifying to obtain Bis DNA;

(3) PCR amplification of Bis DNA: performing probe fluorescent quantitative PCR amplification on Bis DNA by using a PCR amplification solution, wherein the PCR amplification solution comprises: a PAX1 forward primer, a PAX1 reverse primer, a PAX1 detection probe, an HPV16 forward primer, an HPV16 reverse primer, an HPV16 detection probe, an HPV18 forward primer, an HPV18 reverse primer, an HPV18 detection probe, an ACTB forward primer, an ACTB reverse primer, an ACTB detection probe, deoxyribonucleoside triphosphates, and nuclease-free water;

(4) and determining the methylation value of the sample to be detected according to the result of the fluorescent quantitative PCR amplification of the probe.

2. The method for detecting methylation of a gene associated with cervical cancer according to claim 1, wherein the extracting in step (1) specifically comprises the steps of: centrifuging a cervical exfoliated cell sample, and removing supernatant to obtain a sample precipitate; mixing the sample precipitate, proteinase K and lysis solution ABL, and incubating after vortexing to obtain sample DNA;

preferably, the time of the centrifugation is 1-2min, and the rotation speed of the centrifugation is 10000-15000 rpm;

preferably, the volume ratio of the cervical exfoliated cell sample, the proteinase K and the lysis solution ABL is (100-;

preferably, the incubation time is 20-40min and the incubation temperature is 60-70 ℃.

3. The method for detecting methylation of a gene associated with cervical cancer according to claim 1 or 2, wherein the step (2) comprises the following steps: mixing the sample DNA extracted in the step (1), a sodium bisulfite solution, a DNA gel sample adding buffer solution and water, heating to 90-100 ℃ for reaction for 5-15min, then cooling to 60-65 ℃ for reaction for 2-3h, and finally keeping at 0-10 ℃ for 4-5 h;

preferably, the volume ratio of the sample DNA, the sodium bisulfite solution, the DNA gel loading buffer solution and the water is 1 (80-90): 30-40): 1;

preferably, the mass concentration of the sodium bisulfite solution is 40-50 wt%.

4. The methylation detection method of a cervical cancer-associated gene according to any one of claims 1 to 3, wherein the purification in step (2) comprises the following specific steps: purifying the reaction liquid obtained by the heavy sulfate conversion reaction by using a UNIQ-10 column, washing by using a washing buffer solution, desulfonating the desulfonation liquid, and finally eluting by using TE eluent to obtain Bis DNA;

preferably, the washing buffer comprises, in terms of the concentration of the substance: 1-3mM Tris, 1-3mM NaCl and 0.1-0.5mM disodium EDTA, wherein the solvent of the washing buffer solution is ethanol water solution;

preferably, the sulfonation liquid comprises, by mass concentration: 40-60mM Tris, 50-150mM NaCl and 150-250mM NaOH, wherein the solvent of the sulfonation solution is ethanol water solution.

5. The method for detecting methylation of a gene related to cervical cancer according to any one of claims 1 to 4, wherein the sequence of the PAX1 forward primer in step (2) is 5'-TTAGGGGGTAGTTGAGTAAGTT-3', the sequence of the PAX1 reverse primer is 5'-AAAATAACCTATAAATCCCCAAACAA-3', and the sequence of the PAX1 detection probe is 5'-AAATAAAACGCGACGCATTAT-3'.

6. The method for detecting methylation of a gene related to cervical cancer according to any one of claims 1 to 5, wherein the sequence of the HPV16 forward primer is 5'-TGACACAGAAAATGCTAGTGCT-3', the sequence of the HPV16 reverse primer is 5'-TCACCTGGATTTACTGCAACAT-3', and the sequence of the HPV16 detection probe is 5'-AGGGGAACACTGGGGCAAAGGATCCC-3'.

7. The method for detecting methylation of a gene related to cervical cancer according to any one of claims 1 to 6, wherein the sequence of the HPV18 forward primer is 5'-AATGTCTGCAGATCCTTATGGG-3', the sequence of the HPV18 reverse primer is 5'-CTTGGAGAGGGAGAATACACAC-3', and the sequence of the HPV18 detection probe is 5'-CAGGTACTATGGGTGACACTGTGCCTCA-3'.

8. The method for detecting methylation of a gene associated with cervical cancer according to any one of claims 1 to 7, wherein the sequence of the ACTB forward primer is 5'-GGTTAGGAAGGAGGTTGTTTGTTTT-3'; the sequence of the ACTB reverse primer is 5'-ATCATCTTTCCCACCAAACTATAACC-3'; the sequence of the ACTB detection probe is 5'-CCCATTAACTAAACACAACCT-3'.

9. The method for detecting methylation of a gene associated with cervical cancer according to any one of claims 1 to 8, wherein the PCR amplification solution in the step (2) comprises, in terms of the concentration of the substance: 0.1-0.3 mu M PAX1 forward primer, 0.1-0.3 mu M PAX1 reverse primer, 0.2-0.5 mu M PAX1 detection probe, 0.1-0.3 mu M HPV16 forward primer, 0.1-0.3 mu M HPV16 reverse primer, 0.2-0.5 mu M HPV16 detection probe, 0.1-0.3 mu M HPV18 forward primer, 0.1-0.3 mu M HPV18 reverse primer, 0.2-0.5 mu M HPV18 detection probe, 0.1-0.3 mu M ACTB forward primer, 0.1-0.3 mu M ACTB reverse primer, 0.2-0.5 mu M ACTB detection probe, 0.2-0.5 mu M deoxynucleoside triphosphate solution, and PCR solution without ribonuclease.

10. The method for detecting methylation of a gene associated with cervical cancer according to any one of claims 1 to 9, wherein the volume ratio of the PCR amplification solution to the Bis DNA in step (2) is (2-4): 1;

preferably, the reaction conditions for the amplification in step (2) are: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, annealing and extension at 60 ℃ for 40s, and 50 cycles; storing at 4 ℃.

Technical Field

The invention belongs to the technical field of in-vitro detection, and particularly relates to a methylation detection method of a gene related to cervical cancer.

Background

Persistent infection by high-risk human papillomavirus (HR-HPV) is the cause of cervical cancer. The process of cervical epithelial carcinogenesis also involves many genetic and epigenetic changes. DNA methylation is the most common epigenetic change, often occurring in the promoter region of tumor suppressor genes, causing transcriptional inactivation of the gene, thereby promoting tumorigenesis. DNA methylation can be detected in cervical cancer reported in the existing literature, and can be detected in 30-80% of cervical cancer prophase lesions. It has been shown that detection of methylation of specific genes in exfoliated cervical cells may serve as a potential molecular marker for early detection of cervical cancer.

The conventional cervical cancer screening technology adopted at present is HPV detection, wherein HPV-DNA detection: the technology has high sensitivity, low specificity and high flux of the detection method, is suitable for large-scale clinical screening, but has high false positive rate and can only perform early warning but not early detection. HPV-E6E7mRNA detection: the technology has high sensitivity and general specificity, the detection method has a complex typing system and unstable RNA, viruses but not individual genes are detected, and early warning but early detection is only carried out. On the other hand, conventionally, methylation state differences between the CDH1, p16, SOX1 and RASSFA1 genes in cervical cancer precursor lesions and invasive cervical cancer were found by a Methylation Specific PCR (MSP) method. Conventional methylation detection methods include: methylation specific PCR and bisulfite sequencing. However, these methods have the disadvantages of complicated operation, low accuracy and low sensitivity, which limits their wide application in clinical laboratories.

Therefore, there is a need to provide a method for detecting methylation of cervical cancer genes to overcome the above-mentioned drawbacks.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide a methylation detection method of a gene related to cervical cancer. The detection method designs a scientific and reasonable PCR reaction system by designing a primer and a probe with high specificity, and has the characteristics of high speed, high flux, sensitivity and good specificity.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a method for detecting methylation of a gene associated with cervical cancer, the method comprising the steps of:

(1) extraction of sample DNA: extracting sample DNA from a cervical exfoliated cell sample;

(2) transformation and purification of sample DNA: performing a bisulfate conversion reaction on the extracted sample DNA, and then purifying to obtain Bis DNA;

(3) PCR amplification of Bis DNA: performing probe fluorescent quantitative PCR amplification on Bis DNA by using a PCR amplification solution, wherein the PCR amplification solution comprises: a PAX1 forward primer, a PAX1 reverse primer, a PAX1 detection probe, an HPV16 forward primer, an HPV16 reverse primer, an HPV16 detection probe, an HPV18 forward primer, an HPV18 reverse primer, an HPV18 detection probe, an ACTB forward primer, an ACTB reverse primer, an ACTB detection probe, deoxyribonucleoside triphosphates, and nuclease-free water;

(4) and determining the methylation value of the sample to be detected according to the result of the fluorescent quantitative PCR amplification of the probe.

In the invention, a pair of specific primers and probes for methylation detection are respectively designed in promoter regions of a PAX1 gene and an internal standard gene ACTB (b-actin) in a human genome. Then, the primers and the probes are used for amplifying sample DNA converted by sulfite, the methylation value of a sample to be detected is determined according to the relative fluorescence CT value of the PCR amplification result of the PAX1 and ACTB genes, and the risk of cervical canceration is judged according to the methylation value. On the basis, the HPV16 and the HPV18 are combined to be used as a biomarker for assisting the PAX1 gene in detecting the cervical cancer, so that the influence of non-methylated DNA is avoided, and the kit has the advantages of high speed, high sensitivity, high specificity and the like.

Preferably, the extraction in step (1) specifically comprises the following steps: centrifuging a cervical exfoliated cell sample, and removing supernatant to obtain a sample precipitate; and mixing the sample precipitate, proteinase K and lysis solution ABL, and incubating after vortexing to obtain sample DNA.

Preferably, the centrifugation time is 1-2min, such as 1min, 1.2min, 1.4min, 1.6min, 1.8min, 2min, etc., and the centrifugation rotation speed is 10000-15000rpm, such as 10000rpm, 11000rpm, 12000rpm, 13000rpm, 14000rpm, 15000rpm, etc.

Preferably, the volume ratio of the cervical exfoliated cell sample, the proteinase K and the lysis solution ABL is (100-;

wherein "100-" 200 "may be 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, etc.;

wherein "1-3" can be 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, etc.;

wherein "20-30" may be 20, 22, 24, 26, 28, 30, etc.

Preferably, the incubation time is 20-40min, such as 20min, 22min, 24min, 26min, 28min, 30min, 32min, 34min, 36min, 38min, 40min, etc., and the incubation temperature is 60-70 deg.C, such as 60 deg.C, 62 deg.C, 64 deg.C, 66 deg.C, 68 deg.C, 70 deg.C, etc.

Preferably, the specific steps of the bisulfate conversion reaction in step (2) are: mixing the sample DNA extracted in step (1), a sodium bisulfite solution, a DNA gel loading buffer solution and water, heating to 90-100 deg.C (e.g. 90 deg.C, 92 deg.C, 94 deg.C, 96 deg.C, 98 deg.C, 100 deg.C), reacting for 5-15min (e.g. 5min, 6min, 8min, 10min, 12min, 15min, etc.), cooling to 60-65 deg.C (e.g. 60 deg.C, 61 deg.C, 62 deg.C, 63 deg.C, 64 deg.C, 65 deg.C, etc.), reacting for 2-3h (e.g. 2h, 2.2h, 2.4h, 2.6h, 2.8h, 3h, etc.), and keeping for 4-5h (e.g. 4h, 4.2h, 4.4h, 4.6h, 4.8h, 5h, etc.) at 0-10 deg.C (e.g. 0 deg.C, 2 deg.C.

Preferably, the volume ratio of the sample DNA, the sodium bisulfite solution, the DNA gel loading buffer solution and the water is 1 (80-90): 30-40): 1;

wherein "80-90" may be, for example, 80, 82, 84, 86, 88, 90, etc.;

the "30 to 40" may be, for example, 30, 32, 34, 36, 38, 40, or the like.

Preferably, the sodium bisulfite solution has a mass concentration of 40 to 50 wt%, and may be, for example, 40 wt%, 42 wt%, 44 wt%, 46 wt%, 48 wt%, 50 wt%, or the like.

Preferably, the specific steps of the purification in step (2) are: and purifying the reaction liquid obtained by the heavy sulfate conversion reaction by using a UNIQ-10 column, washing by using a washing buffer solution, desulfonating the desulfonation liquid, and finally eluting by using TE eluent to obtain the Bis DNA.

Preferably, the washing buffer comprises, in terms of the concentration of the substance: tris at 1-3mM (for example, 1mM, 1.5mM, 2mM, 2.5mM, 3 mM), NaCl at 1-3mM (for example, 1mM, 1.5mM, 2mM, 2.5mM, 3 mM), and disodium EDTA at 0.1-0.5mM (for example, 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5 mM), wherein the solvent of the washing buffer is ethanol aqueous solution.

Preferably, the sulfonation liquid comprises, by mass concentration: 40-60mM (for example, 40mM, 45mM, 50mM, 55mM, 60mM, etc.) Tris, 50-150mM (for example, 50mM, 70mM, 90mM, 110mM, 130mM, 150mM, etc.) NaCl, 150-250mM (for example, 150mM, 170mM, 190mM, 210mM, 230mM, 250mM, etc.) NaOH, wherein the solvent of the sulfonation solution is ethanol water solution.

Preferably, in the step (2), the sequence of the PAX1 forward primer is 5'-TTAGGGGGTAGTTGAGTAAGTT-3', the sequence of the PAX1 reverse primer is 5'-AAAATAACCTATAAATCCCCAAACAA-3', and the sequence of the PAX1 detection probe is 5'-AAATAAAACGCGACGCATTAT-3'.

Preferably, the sequence of the HPV16 forward primer is 5'-TGACACAGAAAATGCTAGTGCT-3', the sequence of the HPV16 reverse primer is 5'-TCACCTGGATTTACTGCAACAT-3', and the sequence of the HPV16 detection probe is 5'-AGGGGAACACTGGGGCAAAGGATCCC-3'.

Preferably, the sequence of the HPV18 forward primer is 5'-AATGTCTGCAGATCCTTATGGG-3', the sequence of the HPV18 reverse primer is 5'-CTTGGAGAGGGAGAATACACAC-3', and the sequence of the HPV18 detection probe is 5'-CAGGTACTATGGGTGACACTGTGCCTCA-3'.

Preferably, the sequence of the ACTB forward primer is 5'-GGTTAGGAAGGAGGTTGTTTGTTTT-3'; the sequence of the ACTB reverse primer is 5'-ATCATCTTTCCCACCAAACTATAACC-3'; the sequence of the ACTB detection probe is 5'-CCCATTAACTAAACACAACCT-3'.

The 5 'end of the sequence of all the primers and the probe detection probes is marked with a fluorescence reporter group, and the 3' end is marked with a fluorescence quenching group.

Preferably, the PCR amplification solution in step (2) comprises, in terms of the concentration of the substance: a PAX1 forward primer of 0.1-0.3. mu.M (e.g., 0.1. mu.M, 0.15. mu.M, 0.2. mu.M, 0.25. mu.M, 0.3. mu.M, etc.), a PAX1 reverse primer of 0.1-0.3. mu.M (e.g., 0.1. mu.M, 0.15. mu.M, 0.2. mu.M, 0.25. mu.M, 0.3. mu.M, etc.), a PAX1 detection probe of 0.2. mu.M, 0.25. mu.M, 0.3. mu.M, 0.35. mu.M, 0.4. mu.M, 0.45. mu.M, 0.5. mu.M, etc.), a HPV 54 forward primer of 0.1-0.3. mu.M (e.1. mu.M, 0.15. mu.M, 0.2. mu.M, 0.25. mu.M, 0.3. mu.M, 16. mu.M, a HPV 3. forward primer of 0.M, 0.25. mu.25. mu.M, 0.5. mu.25. mu.M, a HPV 3. mu.M forward primer of HPV 3. mu.25. mu.M, a HPV 3. mu.25. mu.M (e), a 3. mu.M, 0.0.25. mu.M, 0.M, 0.7. mu.25. mu.7. mu.M, 0.0.M, 0., 0.1-0.3. mu.M (e.g., 0.1. mu.M, 0.15. mu.M, 0.2. mu.M, 0.25. mu.M, 0.3. mu.M, etc.) HPV18 forward primer, 0.1-0.3. mu.M (e.g., 0.1. mu.M, 0.15. mu.M, 0.2. mu.M, 0.25. mu.M, 0.3. mu.M, etc.) HPV18 reverse primer, 0.2-0.5. mu.M (e.g., 0.2. mu.M, 0.25. mu.M, 0.3. mu.M, 0.35. mu.M, 0.4. mu.M, 0.45. mu.M, 0.5. mu.M, etc.) HPV18 detection probe, 0.1-0.3. mu.M (e.1. mu.M, 0.15. mu.M, 0.2. mu.M, 0.25. mu.M, 0.3. mu.M, etc.), ACTB forward primer, 0.1-0.5. mu.M (e.0.1-0.5. mu.M, 0.M, 0.25. mu.25. mu.5. mu.M, 0.M, 0.5. mu.M, etc.), ACTB reverse primer, etc.) detection probe, etc. (e.g., ACTB), ACTB, etc.) 0.2 to 0.5. mu.M (for example, 0.2. mu.M, 0.25. mu.M, 0.3. mu.M, 0.35. mu.M, 0.4. mu.M, 0.45. mu.M, 0.5. mu.M and the like) of deoxyribonucleoside triphosphates, and the solvent of the PCR amplification solution is nuclease-free water.

Preferably, the volume ratio of the PCR amplification solution to the Bis DNA in step (2) is (2-4):1, and may be, for example, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, or the like.

Preferably, the reaction conditions for the amplification in step (2) are: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, annealing and extension at 60 ℃ for 40s, and 50 cycles; storing at 4 ℃.

Compared with the prior art, the invention has the following beneficial effects:

the methylation detection method of the cervical cancer related gene has the advantages of high speed, high sensitivity and specificity and the like.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Outpatients who find cervical abnormality and see doctor's office in the second people hospital in Shenzhen city are selected as study objects, and patients with pregnancy, cervix resection history and malignant tumor history are excluded. Cervical precancerous lesions (cervical intraepithelial neoplasia CIN I, CIN II, CIN III). The materials are taken in non-menstrual period, sexual life is prohibited within three days before examination, and repeated cleaning and medication in vagina are prohibited. Collecting cervical orifice and cervical tube sloughed cell samples by using a cervical tube brush, and taking a small amount of cervical tissues to carry out pathological biopsy by using a pair of forceps under a colposcope. The samples were stored at-20 ℃ until use.

Proteinase K purchased from Beijing Solebao technologies, lysis Buffer ABL purchased from Beijing Biyan blue Biotech, DNA gel Loading Buffer (10 XDNA Loading Buffer) purchased from Beijing Legen biotech, TE eluent purchased from Shanghai research biotech, deoxyribonucleoside triphosphate Saint Biopsis, and nuclease-free Water purchased from Shanghai Limmai bioengineering, Inc