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Human b-raf gene v600 mutation detection kit

A detection kit and b-raf technology, which are applied in the fields of biotechnology and clinical molecular diagnosis, can solve the problems of long B-raf gene mutation detection cycle, complicated result interpretation, cumbersome operation process, etc., and achieve clear and intuitive result interpretation and acquisition. The effect of low quality requirements and reduced chance of contamination

Active Publication Date: 2016-03-09
武汉海吉力生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a fast, accurate, and effective contamination-preventing human B-raf gene V600 mutation detection kit, which solves the problem of long B-raf gene mutation detection cycle, cumbersome operation process, and complicated result interpretation in the prior art. Low sensitivity, easy contamination, etc., especially to provide non-invasive detection of plasma or other body fluids except tumor tissue

Method used

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  • Human b-raf gene v600 mutation detection kit
  • Human b-raf gene v600 mutation detection kit
  • Human b-raf gene v600 mutation detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1: Extraction of clinical sample DNA

[0056] In this example, DNA is extracted from paraffin-embedded tissue sections of papillary thyroid cancer patients, and is quantified as a template for PCR detection. The QIAamp paraffin-embedded tissue extraction kit from Qiagen was used, as detailed below.

[0057] 1. Preparation before experiment

[0058] (1) Adjust the water bath to 56°C.

[0059] (2) Place BufferATL and BufferAL in the QIAamp kit at 56°C until the precipitate dissolves.

[0060] 2. Sample processing

[0061] (1) Cut off the excess paraffin on the paraffin section, cut the section into an appropriate size, and install it on a paraffin microtome.

[0062] (2) Adjust the caliper to 10 μm, and then cut the wax block until a uniform tissue can be clearly seen on the cut section.

[0063] (3) Remove the first 5 pieces, and start from the 6th piece, put them into 1.5ml EP tubes, 5 pieces per tube, and then number them.

[0064] 3. DNA extraction ...

Embodiment 2

[0082] Example 2: Real-time fluorescent PCR method to amplify clinical sample DNA

[0083] The human B-raf gene V600 mutation detection kit contains 2 PCR reaction systems, which are the detection reaction system and the quantitative reaction system, and the even-numbered wells of the 8-way PCR reaction strip are the detection reaction system amplification mutation templates, which are used to detect the mutation status of the samples to be tested ; The odd-numbered wells of the 8-link PCR reaction strip are quantitative reaction systems, which are used to control the quality of the samples to be tested. In this example, the primers and probes shown in Table 1 were used to amplify the DNA sample extracted in Example 1.

[0084] Table 1: Primer and probe sequences provided in the kit

[0085]

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Abstract

The invention discloses a human B-raf gene V600 mutation detection kit (fluorescent PCR method), the detection kit includes a peptide nucleic acid PNA probe for blocking wild-type B-raf gene amplification, for A set of primer pairs and detection probes for specifically amplifying the V600 mutant B-raf gene sequence, a set of primer pairs and detection probes as the internal quality control of the kit. The kit of the invention is suitable for the selection of various clinical sample types, and has significant advantages such as strong specificity, high sensitivity, short experiment period, simple operation, safety and non-toxicity, and low cost.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and clinical molecular diagnosis, in particular to a human B-raf gene V600 mutation detection kit. technical background [0002] The B-raf gene is a member of the RAF family and encodes a serine-threonine protein kinase in the MAPK pathway, which transduces signals from ras to MEK1 / 2, thereby participating in the regulation of various biological activities in cells. Mutations in the B-raf gene cause changes in the encoded amino acids, resulting in the continuous activation of the protein encoded by the B-raf gene, which changes the activity of phosphokinases in the MAPK signaling pathway, causing abnormal cell proliferation and differentiation, and eventually forming tumors. [0003] B-raf gene missense mutations account for 8% of the incidence of human solid tumors. In a variety of human malignant tumors, such as colorectal cancer, melanoma, thyroid cancer, lung cancer, etc., there are di...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q2525/107C12Q2537/163C12Q2561/101
Inventor 王秀娟梁惠喻德华
Owner 武汉海吉力生物科技有限公司
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