Kit for detecting EGFR gene mutation and application of kit

A detection kit and kit technology, applied in the fields of biotechnology and medicine, can solve problems such as easy pollution, inaccurate results, and low sensitivity

Active Publication Date: 2015-09-30
SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI +1
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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
[0008] 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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  • Kit for detecting EGFR gene mutation and application of kit
  • Kit for detecting EGFR gene mutation and application of kit
  • Kit for detecting EGFR gene mutation and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1: The kit provided by the invention detects EGFR gene artificial plasmid

[0066] 1. Artificial plasmid DNA purification

[0067] EGFR wild-type and mutant artificial plasmids were purified using the QIAprep Spin Miniprep (Qiagen) kit as follows:

[0068] 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 Wash 0.75ml twice, transfer the QIAprep spin to a new 1.5ml centrifuge tube, add 50μL sterile water, let stand for 1min, centrifuge for 1min, and collect...

Embodiment 2

[0076] Embodiment 2: The kit provided by the invention detects fresh tissue samples

[0077] 1. Purification of genomic DNA from tissue samples

[0078] Genomic DNA was purified from fresh tissue specimens using the QIAamp DNA Mini (Qiagen) kit as follows:

[0079] Collect 40 fresh tissue samples from patients with non-small cell lung cancer, cut each sample within 25 mg with scissors to small pieces, put them in a 1.5 mL centrifuge tube, add 180 μL ATL, 20 μL proteinase K, mix well, and incubate at 56 °C for 1 ~3h until the sample is dissolved, shake 2-3 times per hour; add 200μL AL, shake for 15 seconds, incubate at 70°C for 10min; then add 200μL absolute ethanol, shake for 15 seconds, carefully transfer the liquid (including the precipitate) to the kit provided In the column, do not pollute the nozzle. After centrifuging at 8000rpm for 1min, carefully add 500μL AW1 buffer to a new 2ml centrifuge tube, centrifuge at 8000rpm for 1min, discard the filtrate; carefully add 500μ...

Embodiment 3

[0088] Embodiment 3: The kit provided by the present invention detects plasma samples

[0089] 1. Purification of genomic DNA from plasma samples

[0090] Collect 5 mL of peripheral blood samples from 80 non-small cell lung cancer patients, 10 small cell lung cancer patients and 26 healthy people, centrifuge at 2500 rpm for 10 minutes, and absorb 2 mL of the supernatant for the purification of circulating free DNA.

[0091] Circulating cell-free DNA in plasma samples was purified using the QIAamp DNA Blood Midi (Qiagen) kit as follows:

[0092] 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 Medi 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 buffe...

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Abstract

The invention discloses a kit for detecting EGFR gene mutation and an application of the kit. The kit is characterized by containing 20 specific amplification primers for an EGFR gene mutation site, 5 efficient blocking probes for wild sequences, and 4 EGFR gene specific TaqMan fluorescent probes. The kit can be used for detecting samples of which the mutation copy number is as low as 5-10 copies and the mutation content is as low as 0.1%. The kit disclosed by the invention can be used for simultaneously detecting 5 types of gene mutations of an EGFR gene, is high in sensitivity, simple to operate, low-cost in detection and wide in clinical application range, the samples can be fresh pathological tissues, paraffin embedded tissues, pleural fluid, serum or plasma, the detection speed is high, and the detection process can be finished in only 90 minutes.

Description

technical field [0001] The invention relates to an EGFR gene mutation detection kit and application thereof, in particular to a rapid detection of EGFR gene mutation related to selection and diagnosis of tumor medication. Belongs to the field of biotechnology and medicine. 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 Chi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 景奉香鲍升林张冀申贾春平金庆辉赵建龙
Owner SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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