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KRAS 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, inaccurate results, etc., and achieve the effects of ensuring detection stability, fast detection speed and high stability rate.

Active 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 prone to occur and the results are inaccurate
[0011] However, due to the low content of circulating DNA in peripheral blood, the content of tumor-specific mutant DNA is even less, especially in early cancer patients. In the same amplification system, a large number of wild-type templates are dominantly amplified, resulting in the inhibition of the amplification of a small amount of mutant templates and cannot be detected

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1: The kit provided by the invention detects KRAS gene artificial plasmid

[0057] 1. Artificial plasmid DNA purification

[0058] KRAS wild-type and mutant artificial plasmids were purified using the QIAprep Spin Miniprep (Qiagen) kit as follows:

[0059] 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 the QIAprep spin tube, centrifuge at 13000rpm for 1min, discard the liquid; wash once with 0.5mL PB, then add PE buffer containing absolute ethanol Wash twice with 0.75 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

[0066] Embodiment 2: The kit provided by the invention detects fresh tissue samples

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

[0068] Genomic DNA was purified from fresh tissue specimens using the QIAamp DNA Mini (Qiagen) kit as follows:

[0069] 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 filtrate; carefully add 500 μL of AW2 buff...

Embodiment 3

[0078] Embodiment 3: The kit provided by the present invention detects plasma samples

[0079] 1. Purification of genomic DNA from plasma samples

[0080] Collect 5 mL of peripheral blood samples from 80 patients with colorectal cancer and 20 healthy controls, centrifuge at 2500 rpm for 10 minutes, and absorb 2 mL of supernatant for purification of circulating free DNA.

[0081] Circulating cell-free DNA in plasma samples was purified using the QIAamp DNA Blood Midi (Qiagen) kit as follows:

[0082] 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 5mL AW1 buffer, centrifuge at 8000rpm for 1min, discard the filtrate, add 5mL AW2 buffer, centrifuge at 13000rpm for 1min, discard th...

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Abstract

The invention discloses a detection kit and detection method for KRAS 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 eight pairs of specifically amplified primers for KRAS gene mutation loci, two efficient blocking probes of a wild sequence and two KRAS gene specific TaqMan fluorescent probes. The kit can detects 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 KRAS 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 KRAS gene mutations related to 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 3.1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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