BRAF gene mutation detection kit and application thereof

A detection kit and the technology of the kit, which are applied in the fields of biotechnology and medicine, can solve the problems of long operation time, low sensitivity and low cost, and achieve the effect of fast detection speed and high sensitivity

Inactive Publication Date: 2016-11-23
上海济远生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The direct sequencing method has always been considered the gold standard for detecting gene mutations. This method is relatively cheap, but it takes a long time to operate, and it has two obvious disadvantages: one is low sensitivity, and generally requires the abundance of mutated genes to reach more than 20%. in order to accurately detect
Second, due to the subsequent need to process the PCR product, contamination is

Method used

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  • BRAF gene mutation detection kit and application thereof
  • BRAF gene mutation detection kit and application thereof
  • BRAF gene mutation detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: The kit provided by the invention detects BRAF gene artificial plasmid

[0044] 1. Artificial plasmid DNA purification

[0045] Purify the BRAF wild-type and V600E mutant artificial plasmids with the QIAprep Spin Miniprep (Qiagen) kit as follows:

[0046] Take 1.5mL of bacterial solution, centrifuge at 8000rpm for 3min to collect the bacterial cells, add 250μL of P1 buffer solution containing RNase A enzyme and mix well, then add the same volume of P2 buffer solution, mix up and down until the solution becomes clear (do not allow the reaction time to exceed 5min ), add 350μL N3 buffer, mix immediately, centrifuge at 13000rpm for 10min, transfer the supernatant to a QIAprep spin tube, centrifuge at 13000rpm for 1min, discard the liquid; wash once with 0.5mL PB, then add PE buffer containing absolute ethanol 0.75 Wash twice with mL, transfer the QIAprep spin to a new 1.5 mL centrifuge tube, add 50 μL sterile water, let stand for 1 min, centrifuge for 1 min,...

Embodiment 2

[0054] Embodiment 2: The kit provided by the invention detects fresh tissue samples

[0055] 1. Purification of genomic DNA from tissue samples

[0056] Genomic DNA (confirmed negative for KRAS mutations) from fresh tissue specimens was purified using the QIAamp DNA Mini (Qiagen) kit as follows:

[0057] Take less than 25 mg of fresh tissue and use scissors to cut the sample into small pieces, put it in a 1.5 mL centrifuge tube, add 180 μL ATL, 20 μL proteinase K, mix well, incubate at 56 °C for 1-3 hours until the sample dissolves, shake 2-3 times per hour ; Add 200 μL AL, shake for 15 seconds, and incubate at 70°C for 10 minutes; then add 200 μL of absolute ethanol, shake for 15 seconds, carefully transfer the liquid (including the precipitate) to the column provided in the kit without polluting the tube mouth, centrifuge at 8000 rpm for 1 minute, Carefully add 500 μL of AW1 buffer to the column in a new 2 mL centrifuge tube, centrifuge at 8000 rpm for 1 min, discard the fi...

Embodiment 3

[0066] Embodiment 3: The kit provided by the present invention detects plasma samples

[0067] 1. Purification of genomic DNA from plasma samples

[0068] Collect 80 samples of peripheral blood from colorectal cancer patients and 20 samples from healthy people, centrifuge at 2500 rpm for 10 minutes, and absorb 2 mL of supernatant for purification of circulating free DNA.

[0069] Use the QIAampDNABloodMidi (Qiagen) kit to purify circulating cell-free DNA in plasma samples as follows:

[0070] Add 200μL PK and 2mL AL to 2mL serum (or whole blood), mix thoroughly for 15 seconds, incubate at 56°C for 10min; add 2mL absolute ethanol, mix thoroughly for 15 seconds, transfer the liquid to the QIAamp Midi Spin Column, and centrifuge at 8000rpm for 2min , discard the filtrate; centrifuge again, transfer the column to another collection tube, add 5mLAW1 buffer, centrifuge at 8000rpm for 1min, discard the filtrate, add 5mLAW2 buffer, centrifuge at 13000rpm for 1min, discard the filtrat...

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Abstract

The invention discloses a detection kit and detection method for BRAF gene mutations, and belongs to the technical field of B2D10 currently first developed high-tech industrialization important field guide/biology/novel medical precise diagnosis and treatment equipment. The detection kit is characterized by comprising two pairs of specifically amplified primers for BRAF gene mutation loci, an efficient blocking probe of a wild sequence and a BRAF gene specific TaqMan fluorescent probe. The kit can detect specimens of which the number of the mutant copies is as low as 5 to 10, and the mutation content is as low as 0.1%. The detection kit can detect five gene mutations of a BRAF gene simultaneously, the sensitivity is high, operation is easy, detection is low in price, the clinical application range is wide, samples can adopt fresh pathological tissue or paraffin-embedded tissue or a pleural fluid or serum or plasma, the detection speed is high, and only 90 minutes are needed for completing the detection process.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular to a rapid detection of BRAF gene mutation related to the selection and diagnosis of tumor medication. Background technique [0002] The World Health Organization published the "World Cancer Report" on the occasion of World Cancer Day in 2014, stating that cancer has become the leading cause of death in the world, and both morbidity and mortality are on the rise. The global cancer incidence rate has increased by 11% in four years, and by 2012 there were approximately 14.1 million cases and 8.2 million deaths due to cancer. Half of the new cases of cancer worldwide in 2012 occurred in Asia, most of them in mainland China. And predicted that in the next 20 years, the world's cancer cases will increase by 75%, reaching nearly 25 million. In early 2013, the China Cancer Registry Center released the "2012 China Cancer Registry Annual Report", which shows that there are about ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 李瑶景奉香鲍升林刘明华曹婕王晓娟
Owner 上海济远生物科技有限公司
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