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Composition for detecting cervical cancer as well as kit and application thereof

A detection kit and technology for cervical cancer, applied in biochemical equipment and methods, recombinant DNA technology, microbiological determination/inspection, etc., can solve the problems of high experimental cost, limited diagnostic effect, and reduced specificity, and reduce operations steps, reduce the cost of consumables, and reduce the effect of labor costs

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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  • Composition for detecting cervical cancer as well as kit and application thereof
  • Composition for detecting cervical cancer as well as kit and application thereof
  • Composition for detecting cervical cancer as well as 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 collection operation of cervical exfoliated cell samples is as follows: the medical staff first exposes the cervix with a speculum, and wipes off the excessive secretions from the cervix with a cotton swab. Place the cervical brush on the cervix, rotate it 5 times in one direction to obtain a sufficient amount of epithelial cell samples, 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 nucleic acid extraction or purification reagents (universal) 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 hypermethylated candidate genes in cervical exfoliated cells from patients with cervical cancer

[0093] Comprehensive analysis of literature research results, TCGA methylation chip database, and transcriptome sequencing expression profiles were used to screen methylation sites with significant differences. Through multiple data filtering analysis, PCDH10, LMX1A, and ZNF582 were finally screened to identify cervical cancer hypermethylation candidates. Gene.

[0094] 2. Primer-probe combination screening for cervical cancer methylation detection

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

[0096] According to the nucleic acid sequences of PCDH10, LMX1A and ZNF582 mentioned above, the methylation primers and probes were designed on the Methyl primerExpress v1.0 software. After repeated design and deliberation by the applicant, the ...

Embodiment 3

[0102] Example 3: Detection of methylation fluorescence quantitative PCR amplification of hypermethylated candidate genes of cervical cancer

[0103] 1. The reaction system of methylation fluorescence quantitative PCR is as follows: 7.5 μL of 2× PCR reaction master mix, 0.1 μL of 10 μM GAPDH primer and probe each, 0.5 μL of each primer and 0.2 μL of each probe in the 10 μM primer-probe combination above , 3 μL Bis-DNA, and make up to 15 μL with water.

[0104] 2. Reaction conditions of methylation fluorescence quantitative PCR

[0105]

[0106] 3. Interpretation of the detection results of methylation fluorescence quantitative PCR

[0107] 1) Threshold setting: It can be automatically output by the instrument, or the baseline can be manually adjusted according to the instrument's instructions, set the threshold in the linear part of the logarithmic graph of the fluorescence value, export the data from the software and read the CT value.

[0108] 2) Determination of the va...

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Abstract

According to the composition for detecting the cervical cancer and the kit and application thereof, a to-be-detected cervical cancer nucleic acid sample is subjected to hydrosulphite conversion through a hydrosulphite modification method, a fluorescent quantitative PCR technology is combined, literature research results, a TCGA methylation chip database and a transcriptome sequencing expression profile are comprehensively analyzed, and the detection result of the cervical cancer is obtained. 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 particularly relates to a composition for detecting cervical cancer, a kit and application thereof. Background technique [0002] Cervical cancer (cervical cancer) is one of the most common female reproductive system malignant tumors, ranking second in the global female malignant tumor, its morbidity and mortality are second only to breast cancer, a serious threat to 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 40 to 50 years old. 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. There are generally no symptoms in the early stage of cervical cancer. As the disease progresses, symptoms such as contact bleeding and abn...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6886C12Q1/6851C12Q2600/154C12Q2600/118C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2545/101C12Q2563/107C12Q2523/125Y02A50/30
Inventor 邵琦
Owner 广州达健生物科技有限公司
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