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Cervical cancer gene methylation detection primer probe combination and kit and application thereof

A technology of detection kits and primer probes, applied in the biological field, can solve the problems of high experimental cost, limited diagnostic effect, and reduced specificity, and achieve the effect of reducing labor costs, reducing consumable costs, and reducing operating steps

Pending Publication Date: 2022-05-31
广州达健生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection accuracy of a single gene in the detection of cervical cancer DNA methylation based on fluorescence quantitative method of methylation is not ideal, and the diagnostic effect is limited. Researchers often combine the joint detection of multiple genes to improve the detection sensitivity, but many Gene joint detection may lead to a decrease in specificity. At the same time, if a single-tube single-gene test is used for detection, it needs to consume more reagents, increase the operation of the experimenter, and the cost of the experiment is high.

Method used

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  • Cervical cancer gene methylation detection primer probe combination and kit and application thereof
  • Cervical cancer gene methylation detection primer probe combination and kit and application thereof
  • Cervical cancer gene methylation detection primer probe combination and kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: Sample DNA Extraction and Bisulfite Conversion

[0067] 1. Processing of cervical exfoliated cell samples and DNA extraction

[0068] 1) Sample collection:

[0069] The operation of collecting cervical exfoliated cell samples is as follows: the medical staff first exposes the cervix with a speculum, and wipes off the excessive secretion of the cervix with a cotton swab. Place the cervical brush on the cervix and rotate it 5 times in one direction to obtain a sufficient sample of epithelial cells, then put the head of the cervical brush into the sample tube containing the cell preservation solution, and place the cervical brush handle along the crease of the brush handle. Break off, leave the brush head in the sample tube, tighten the tube cap, and mark the sample

[0070] 2) DNA extraction:

[0071] Extract the DNA of cervical exfoliated cells by using the nucleic acid extraction or purification reagent (general type) produced by Anhui Dajian Medical Techn...

Embodiment 2

[0091] Example 2: Screening of cervical cancer hypermethylation candidate genes and specific primers and probes

[0092] 1. Screening of candidate genes for hypermethylation in cervical exfoliated cells of patients with cervical cancer

[0093] Comprehensive analysis of literature research results, TCGA methylation chip database and transcriptome sequencing expression profiles, screening of methylation sites with significant differences, and through multiple data filtering analysis, final screening and determination of SOX1, AJAP1, and ZNF671 as candidates for cervical cancer hypermethylation Gene.

[0094] 2. Screening of primer-probe combinations for cervical cancer methylation detection

[0095] 1) Screening of specific primers and probes:

[0096] According to the nucleic acid sequences of SOX1, AJAP1, and ZNF671 mentioned above, methylation primers and probes were designed on the Methyl primer Express v1.0 software. After repeated design and deliberation by the applican...

Embodiment 3

[0101] Example 3: Methylation fluorescence quantitative PCR amplification detection of cervical cancer hypermethylation candidate genes

[0102] 1. The reaction system of methylation fluorescent quantitative PCR is as follows: 7.5 μL of 2×PCR reaction master mix, 0.1 μL of 10 μM GAPDH primers and probes, 0.5 μL of each primer and 0.2 μL of probes in the 10 μM above primer-probe combination , 3 μL Bis-DNA, and rehydrate to 15 μL.

[0103] 2. Reaction conditions for methylation fluorescent quantitative PCR

[0104]

[0105] 3. Interpretation of detection results of methylation fluorescent quantitative PCR

[0106] 1) Threshold value setting: It can be output automatically according to the instrument, or manually adjust the baseline according to the instrument’s instructions, set the threshold value at the linear part of the logarithmic graph of the fluorescence value, export the data from the software and read the CT value.

[0107] 2) Judgment of the effectiveness of the k...

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Abstract

The invention provides a cervical cancer gene methylation detection primer probe combination and a kit and application thereof, a cervical cancer nucleic acid sample to be detected is subjected to hydrosulphite conversion by adopting a hydrosulphite modification method, a fluorescent quantitative PCR technology is combined, a literature research result, a TCGA methylation chip database and a transcriptome sequencing expression profile are comprehensively analyzed, and the detection sensitivity of the cervical cancer gene methylation detection primer probe combination is improved. The method comprises the following steps: screening cervical cancer hypermethylation candidate genes through multiple data filtration analysis, designing specific gene methylation detection primers and probes aiming at multiple methylation detection sites on the cervical cancer hypermethylation candidate genes, covering more than 10 methylation CpG sites, amplifying a to-be-detected DNA sample modified by hydrosulfite through a multiple PCR amplification technology, and detecting the methylation of the to-be-detected DNA sample. The methylation condition of a target gene in a sample to be detected is determined according to a PCR amplification result, the sensitivity and specificity of the kit are improved through multiple ways, and early screening and diagnosis of cervical cancer are achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a combination of primers and probes for detection of cervical cancer gene methylation, a kit and application thereof. Background technique [0002] Cervical cancer is one of the most common reproductive system malignancies in women, ranking second among female malignancies in the world, and its morbidity and mortality are second only to breast cancer, seriously threatening women's physical and mental health. The incidence of cervical cancer is almost always related to high-risk human papillomavirus (HR-HPV) infection, and the age of onset is the most between 40 and 50 years old, and the incidence tends to be younger in recent years. Cervical cancer occurs in the cervix, most of which are squamous cell carcinoma, followed by adenocarcinoma and adenosquamous carcinoma, and rare types include small cell carcinoma and clear cell carcinoma. The occurrence and development of c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/154C12Q2600/166C12Q2600/16C12Q2600/118C12Q2531/113C12Q2537/143C12Q2545/101C12Q2563/107C12Q2523/125
Inventor 邵琦
Owner 广州达健生物科技有限公司
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