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Composition for detecting bladder cancer and kit and application thereof

A detection kit and technology for bladder cancer, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of high experimental cost, reduced specificity, multiple reagents, etc., and improve detection sensitivity , reduce the operation steps, reduce the effect of reagent consumption

Pending Publication Date: 2022-01-18
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 bladder cancer DNA methylation based on the 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 bladder cancer and kit and application thereof
  • Composition for detecting bladder cancer and kit and application thereof
  • Composition for detecting bladder cancer and kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: Sample DNA Extraction and Bisulfite Conversion

[0068]1. Sample processing and DNA extraction

[0069] 1) Sample pretreatment:

[0070] Tissue and cell samples do not need to be pretreated, and the pretreatment of urine samples is as follows: take 80mL of morning urine in a 100mL centrifuge tube, centrifuge at 4200rpm for 10min, discard the supernatant, add 10mL of PBS to mix and wash the cell sediment, centrifuge at 4200rpm for 5min, discard part of the supernatant Only keep about 1mL supernatant, mix well and transfer to a 1.5ml sterile centrifuge tube.

[0071] 2) DNA extraction:

[0072] Extract bladder exfoliated cell DNA by using nucleic acid extraction or purification reagent (general type) produced by Anhui Dajian Medical Technology Co., Ltd. The specific steps are as follows:

[0073] (a) Add 200 μL Lysis Solution I and 30 μL Proteinase K to the sample to be tested, mix thoroughly, and incubate at 60°C for 2 hours until the sample is completely l...

Embodiment 2

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

[0092] 1. Screening of candidate genes for urinary hypermethylation in patients with bladder 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, the final screening determined that ECRG4, TMEFF2, and TWIST1 are candidates for bladder cancer hypermethylation Gene.

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

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

[0096] According to the nucleic acid sequences of ECRG4, TMEFF2, and TWIST1 mentioned above, methylation primers and probes were designed on the Methyl primer Expressv1.0 software. After repeated design and deliberation by the applicant, PCR probes and p...

Embodiment 3

[0102] Example 3: Methylation fluorescence quantitative PCR amplification detection of bladder cancer hypermethylation candidate genes

[0103] 1. The reaction system of methylation fluorescent quantitative PCR is as follows: 10 μL of 2×methylation PCR reaction master mix, 0.1 μL of 10 μM GAPDH primers and probes, 0.5 μL of each primer in the 10 μM primer-probe combination, and 0.5 μL of each probe. 0.2 μL, 5 μL Bis-DNA, and rehydrate to 20 μL.

[0104] 2. Reaction conditions for methylation fluorescent quantitative PCR

[0105]

[0106]

[0107] 3. Interpretation of detection results of methylation fluorescent quantitative PCR

[0108] 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.

[0109] 2) Judgment o...

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Abstract

The invention provides a composition for detecting the bladder cancer and a kit and application thereof. A bladder cancer nucleic acid sample to be detected is subjected to hydrosulphite conversion by adopting a hydrosulphite modification method, by combining a fluorescent quantitative PCR technology, a literature research result, a TCGA methylation chip database and a transcriptome sequencing expression profile are comprehensively analyzed, a bladder cancer hypermethylation candidate gene is screened through multiple data filtering analysis, specific gene methylation detection primers and probes are designed aiming at a plurality of methylation detection sites on the bladder cancer hypermethylation candidate gene, more than 10 methylation CpG sites are covered, a to-be-detected DNA sample modified by hydrosulfite is amplified through a multiple PCR amplification technology, the methylation condition of a target gene in the 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 bladder cancer are achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a composition and its use in disease detection, in particular to a composition for detecting bladder cancer and its corresponding kit and application. Background technique [0002] Bladder cancer (BC) refers to malignant tumors that occur on the bladder mucosa. It is a common urinary system tumor and one of the top ten common tumors in the whole body. It is the second leading cause of death in patients with urological tumors. Bladder cancer can occur at any age, mostly over the age of 50, and the incidence rate increases with age, and the incidence rate of men is 3-4 times that of women. Bladder cancer can be divided into non-muscle marsh bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC) according to different treatment modes and disease prognosis, and can be divided into bladder urothelial carcinoma, bladder squamous cell carcinoma, Bladder adenocarcinoma, among which ...

Claims

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Application Information

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