Primers, probes, detection systems and kits for one-time detection of multiple gene mutations in colorectal cancer

A kit and probe technology, applied in the direction of recombinant DNA technology, DNA / RNA fragments, etc., can solve the problems of low detection throughput and long time consumption, and achieve the effect of consistent detection reaction system and cost saving

Active Publication Date: 2018-04-27
AMOY DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
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Problems solved by technology

Therefore, it is very important to detect the genetic variation of KRAS / NRAS / PIK3CA / BRAF gene in colorectal cancer. However, most of the current clinical practice can only detect one mutation or a mutation of a gene at a time, and the detection throughput is low and time-consuming. A kit capable of simultaneously and one-time detection of the four gene mutations

Method used

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  • Primers, probes, detection systems and kits for one-time detection of multiple gene mutations in colorectal cancer
  • Primers, probes, detection systems and kits for one-time detection of multiple gene mutations in colorectal cancer
  • Primers, probes, detection systems and kits for one-time detection of multiple gene mutations in colorectal cancer

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Experimental program
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Embodiment 1

[0046] According to the wild-type gene sequences of human KRAS, NRAS, BRAF and PIK3CA published by Cosmic data, specific primers and new probes were designed based on the driver mutation sites of KRAS, NRAS, BRAF and PIK3CA (see Table 1 ).

[0047] In this example, KRAS-M1, KRAS-M5, KRAS-M7, KRAS-M 15, KRAS-M 22, NRAS-M10, NRAS-M 7, NRAS-M 1, NRAS-M 12, PIK3CA-M1, The BRAF-M1 mutation is taken as an example to illustrate the detection of the above intestinal cancer mutation gene by fluorescent PCR of the present invention.

[0048] The experiment uses plasmid templates containing the above mutations respectively, and its fluorescent PCR detection includes the following steps:

[0049] (1) Plasmid treatment and extraction:

[0050] The extraction of each plasmid was performed using a plasmid extraction kit, and the specific extraction steps were operated according to the instructions of the kit. The extracted DNA was dissolved in Tris-HCl (10mmol / L, PH 8.0), and the quality ...

Embodiment 2

[0070] In August 2013, 4 positive samples and 16 negative samples of paraffin-embedded tissue samples of clinical intestinal cancer confirmed by direct sequencing method were sent to our company for one-time multiple detection of intestinal cancer gene mutations.

[0071] 1. Extraction of test sample DNA: DNA extraction can use Xiamen Aide Biomedical Technology Co., Ltd. FFPE sample DNA isolation kit, Cat No. ) or QIAGEN Paraffin Tissue DNA Extraction Kit, Cat NO.56404; follow the instructions of the extraction kit. The extracted DNA was dissolved in Tris-HCL (10mmol / L, pH 8.0). The quality of the extraction was detected by a UV spectrophotometer to determine its concentration OD 260 / OD 280 1.8 to 2.0, then adjust the DNA concentration to 2ng / μL with Tris-HCL (10mmol / L, pH 8.0) solution as a PCR template. The DNA extracted above was used as a template for fluorescent PCR amplification of intestinal cancer mutant genes.

[0072] 2. Carry out PCR amplification of each DNA m...

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Abstract

The invention discloses primers, probes, a detection system and a kit for one-time detection of intestinal cancer multiple mutation genes, including primers, probes and distribution methods for detecting intestinal cancer KRAS, NRAS, PIK3CA and BRAF gene mutations. The detection kit of the present invention adopts the design of 12 PCR reaction strips, and each 12 PCR strips detects a sample, and the 1-11 tubes of the 12 strips are equipped with 37 kinds of mutation detection and internal control of corresponding KRAS / NRAS / PIK3CA / BRAF genes Reagent, mutation is indicated by FAM signal, internal control is indicated by HEX (or VIC) signal; tube No. 12 is used as an external control detection tube for DNA extraction quality, and is also indicated by FAM signal. The invention can realize one-time detection of 37 mutations of KRAS / NRAS / PIK3CA / BRAF genes in DNA, greatly shortens the detection time, has high sensitivity, strong specificity, simple and fast operation, and can be used for clinicians to select tumor targets for intestinal cancer patients Provides a reference to drug therapy.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to primers, probes, detection kits and systems for lung cancer multiple gene mutations. Background technique [0002] Colorectal cancer is the third most common malignant tumor in the world, and its mortality rate is the second highest, with about 1.2 million new patients every year. In China, among all malignant tumors, colorectal cancer has become the third most fatal malignant tumor. The treatment of colorectal cancer is an active comprehensive treatment based on surgery, which can remove the primary tumor and relieve the obstructive symptoms caused by the tumor. Postoperative chemotherapy has developed rapidly in recent years, supplemented by immunotherapy, traditional Chinese medicine therapy, gene-targeted therapy, especially targeted therapy is increasingly used in clinical practice. The occurrence of colorectal cancer is multi-stage, multi-step, involving complex processes suc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12N15/11
Inventor 江风阁林清华曹樱陈婷宋庆涛阮力郑立谋
Owner AMOY DIAGNOSTICS CO LTD
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