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Primer probe composition, kit and detection method for detecting 7 kinds of hotspot mutations in human kras gene

A primer-probe and composition technology, applied in the field of molecular biology, can solve the problems of low cost, inaccurate results, low sensitivity and the like

Active Publication Date: 2017-12-05
SHANDONG VIGENE BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most common method is the direct sequencing method, which is relatively cheap, but takes a long time to operate, and it has two obvious disadvantages: one is low sensitivity, and generally requires the abundance of mutant genes to reach more than 10% to be accurately detected
Second, due to the subsequent need to process the PCR product, contamination is prone to occur and the results are inaccurate

Method used

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  • Primer probe composition, kit and detection method for detecting 7 kinds of hotspot mutations in human kras gene
  • Primer probe composition, kit and detection method for detecting 7 kinds of hotspot mutations in human kras gene
  • Primer probe composition, kit and detection method for detecting 7 kinds of hotspot mutations in human kras gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] 1. According to the wild-type gene sequence of codons 12 and 13 of exon 2 of KRAS gene and 7 kinds of mutant gene sequences published by Cosmic data, design ARMS primers, general downstream primers, specific probes, quality control primers and quality control primers. Control probe, designed primers and probes can be artificially synthesized according to existing methods.

[0075] Specifically, the primers and probes are as follows:

[0076]

[0077] Among them, ARMS primer 1 and universal downstream primer, ARMS primer 2 and universal downstream primer, ARMS primer 3 and universal downstream primer, ARMS primer 4 and universal downstream primer, ARMS primer 5 and universal downstream primer, ARMS primer 6 and universal downstream primer, ARMS primer 7 and universal downstream primers consist of 7 pairs of specific primers, which can amplify 7 hotspot mutations of KRAS gene respectively.

[0078] 2. In addition, it also includes the internal control primers and internal contr...

Embodiment 2

[0085] Using the kit of Example 1 of the present invention to detect KRAS gene mutations includes the following steps:

[0086] (1) Sample processing and template extraction to be tested;

[0087] Take fresh frozen or paraffin-embedded human tumor tissue, extract DNA with extraction kit, and quantitatively dilute it to 5ng / μL as a template for PCR detection.

[0088] (2) Using the kit of the present invention, take 1 quality control tube and 7 mutation detection tubes, and add the sample DNA template, PCR mixed reaction solution, specific probes and internal control primers to the 7 mutation detection tubes respectively. Probe, each mutation detection tube is also added with a pair of specific primers composed of corresponding ARMS primers and universal downstream primers; 1 quality control tube is added with the DNA template of the test sample, PCR mixed reaction solution, and 1 pair of quality control tubes. Control primers and quality control probes for ARMS-qPCR detection of KRA...

Embodiment 3

[0111] Example 3 Practical application of the detection method

[0112] The kit and detection method of the present invention are used to detect whether there are mutations in codons 12 and 13 of exon 2 of KRAS gene in tumor patients, and the method is as follows:

[0113] 1. Sample acquisition and template extraction

[0114] 63 patients with non-small cell lung cancer and colorectal cancer were selected, and paraffin-embedded tissue sections from lungs of patients with non-small cell lung cancer and colorectal cancer were taken as test samples, using Qiagen’s QIAamp paraffin-embedded tissue The extraction kit (Cat No.56404) extracts DNA and quantitatively dilutes it to 5ng / μL as a template for PCR detection.

[0115] 2. Perform real-time fluorescence quantitative PCR amplification

[0116] The above-mentioned 63 samples were tested by PCR. The method was as follows: each sample DNA was tested with 7 mutation detection tubes and 1 quality control tube. The 7 mutation detection tubes w...

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Abstract

The invention discloses a primer probe composition for detecting 7 kinds of hot spot mutations in the human KRAS gene, including 7 pairs of specific primers and a specific probe, and the 7 pairs of specific primers are composed of 7 kinds of ARMS primers respectively. and a universal downstream primer. The invention also discloses a kit comprising the composition and a mutation detection method using the kit. The primer-probe composition of the present invention has high sensitivity and strong specificity. The detection process using the kit is a closed-tube reaction, which significantly reduces pollution. In addition, the UNG enzyme anti-pollution system and internal control system are added, which can more accurately and stably test samples. The typing test ensures that the results are authentic and reliable, and the detection method is fast, and the test can be completed within 100 minutes. The result interpretation is simple and objective, and it is convenient for analysis.

Description

Technical field [0001] The invention relates to a primer probe composition for detecting 7 hot mutations (ie 7 common mutations) of human KRAS gene, a kit and a method for detecting mutations, belonging to the field of molecular biology. Background technique [0002] KRAS gene is a kind of RAS gene family, located on human chromosome 12, is a kind of proto-oncogene, which has a great influence on human cancer. KRAS gene encodes ras protein with a molecular weight of 21kD, also known as p21 protein. The p21 protein is located on the inner surface of the cell membrane. It has GTPase activity and participates in intracellular signal transduction. It is activated when combined with GTP and inactive when combined with GDP. The KRAS gene is like a molecular switch in the body. It plays an important regulatory role in the signal transduction pathways of tumor cell growth and angiogenesis. The normal KRAS gene can inhibit the growth of tumor cells, but when abnormal, it will induce perma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 陈英孙秀莲孙利王春苗张玉丽
Owner SHANDONG VIGENE BIOSCI
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