Primers, probes, detection system and kit for one time detection of intestinal cancer multiple gene mutation

A kit and technology for colorectal cancer, applied in biochemical equipment and methods, recombinant DNA technology, microbial assay/inspection, etc., can solve the problems of low detection throughput and long time consumption, achieve consistent detection reaction system and save costs Effect

Active Publication Date: 2015-08-05
AMOY DIAGNOSTICS CO LTD
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  • 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

Method used

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  • Primers, probes, detection system and kit for one time detection of intestinal cancer multiple gene mutation
  • Primers, probes, detection system and kit for one time detection of intestinal cancer multiple gene mutation
  • Primers, probes, detection system and kit for one time detection of intestinal cancer multiple gene mutation

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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 embodiment, the mutations of KRAS-M1, KRAS-M5, KRAS-M7, KRAS-M15, KRAS-M22, NRAS-M10, NRAS-M7, NRAS-M1, NRAS-M12, PIK3CA-M1, and BRAF-M1 are respectively An example is used 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, PH8.0)...

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, pH8.0). The quality of the extraction was detected by a UV spectrophotometer to determine its concentration OD 260 / OD 280 1.8 ~ 2.0, then use Tris-HCL (10mmol / L, pH8.0) solution to adjust the DNA concentration to 2ng / μL 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 me...

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Abstract

The present invention discloses primers, probes, a detection system and a kit for one time detection of intestinal cancer multiple gene mutation, wherein the primers, the probes, and the distribution way for detecting gene mutations of intestinal cancer genes such as KRAS, NRAS, PIK3CA, and BRAF are provided. According to the present invention, the detection kit adopts the 12 linking PCR reaction strip design, each 12 linking PCR strip detects multiple genes of a sample, the corresponding detection reagents for the 37 mutations of KRAS/NRAS/PIK3CA/BRAF and the internal control reagents are filled in the pipes 1-11 of the 12 linking PCR strip, the mutation is indicated by the FAM signal, and the internal control is indicated by the HEX (or VIC) signal; the pipe 12 is adopted as the DNA extraction quality external control detection pipe and is indicated by the FAM; and with the primers, the probes, the detection system and the kit, the one-time detection of the 37 mutations of the KRAS/NRAS/PIK3CA/BRAF gene can be achieved, such that the detection time is substantially shortened, the sensitivity is high, the specificity is strong, the operation is simple and rapid, and the reference for selection of tumor targeting drug therapy on intestinal cancer patients can be provided for clinician.

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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IPC IPC(8): C12Q1/68C12N15/11
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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