Kit for BRAFV600E gene mutation and detection method

A kit and gene technology, applied in the fields of biotechnology and medicine, can solve the problem of high cost

Inactive Publication Date: 2017-09-19
安徽安龙基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to overcome the deficiency of the high cost of the test kit for detecting BRAF V600E gene mutation in the prior...

Method used

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  • Kit for BRAFV600E gene mutation and detection method
  • Kit for BRAFV600E gene mutation and detection method
  • Kit for BRAFV600E gene mutation and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The method for detecting 7 kinds of gene mutations of BRAF V600E by real-time fluorescent PCR of the present invention comprises the following steps:

[0035] 1. Nucleic acid extraction

[0036] Taking paraffin-embedded tissue as an example, extract genomic DNA from paraffin-embedded tissue according to the recommended QIAamp DNA FFPE Tissue Kit instructions.

[0037] 2. Preparation of PCR reaction system (25ul)

[0038] Reagent name

Amount added

PCR reaction solution

16ul

Enzyme 1

2ul

Primer Probe Mix

2ul

template DNA

5ul

[0039] The template DNA includes sample DNA and negative and positive control DNA.

[0040] 3. PCR amplification

[0041] Real-time PCR amplification conditions are: 50°C for 2min, 1 cycle; 95°C for 5min; 95°C for 10s, 55°C for 15s, 72°C for 30s, 5 cycles; 95°C for 10s, 60°C for 45s (collect FAM and HEX fluorescence signals) , 40 cycles.

[0042] 4. Result judgment

[0043] 1) In additi...

Embodiment 2

[0051] The invention was used to detect 10 cases of tumor tissue samples from patients with non-small cell lung cancer, and compared with the sequencing results, the result: 2 cases were detected positive for V600E, and the coincidence rate with the sequencing method was 100%.

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Abstract

The invention discloses a kit for BRAFV600E gene mutation. The kit comprises a PCR (Polymerase Chain Reaction) buffer solution, primer probe mixed liquor, an enzyme system 1, positive control and negative control. The primer probe mixed liquor comprises a mutation site gene and internal control primer probe, a quality control gene and internal control detection primer probe, a mutation and quality control probe 5' terminal marking FAM fluorescein, a 3' terminal marking MGB fluorescein, an internal control 5' terminal marking HEX fluorescein, and a 3' terminal marking BHQ1 fluorescein. Quality control gene selects a relatively conservative section of a human BRAF V600E gene, internal control selects a human beta-aCtin conservative gene, and a double control system is jointly used for monitoring a plasma sample DNA quality and PCR reaction process. Compared with the prior art, the kit utilizing the primer, the probes and an amplification system provided by the invention is high in detection sensitivity, and can be used for detecting a sample with the mutation content being 0.1 percent; the detection accuracy is high, a double-control detection system is adopted, and the reliability of a detection result is ensured.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular to a kit and a detection method for BRAF V600E gene mutation. Background technique [0002] At present, lung cancer has become the leading cause of cancer death in the world, with 1.5 million new cases each year worldwide, seriously threatening human health. Among lung cancer patients, non-small cell lung cancer (NSCLC) accounts for about 80%, and 80% of them have lost the best chance of surgery or radiotherapy at the time of treatment, resulting in low long-term survival rate and worrying efficacy. The 2-year survival rate of stage IIIB / IV non-small cell lung cancer is only 10%-20%, and the median survival time is only 10-12 months. Therefore, people need to continuously study new treatment methods to improve the effectiveness of lung cancer treatment. Among them, new treatment methods represented by molecular targeted therapy have brought new hope for the treatment of a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/156C12Q2600/166
Inventor 韦玉军李航王娟苏军吴远航
Owner 安徽安龙基因科技有限公司
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