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Primer-probe composition, kit and detection method for detecting seven kinds of hot-spot mutation of KRAS gene of humans

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

Active Publication Date: 2015-07-29
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 seven kinds of hot-spot mutation of KRAS gene of humans
  • Primer-probe composition, kit and detection method for detecting seven kinds of hot-spot mutation of KRAS gene of humans
  • Primer-probe composition, kit and detection method for detecting seven kinds of hot-spot mutation of KRAS gene of humans

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0076]

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

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

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 freshly frozen or paraffin-embedded human tumor tissue, use an extraction kit to extract DNA, 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, add the sample DNA template to be tested, PCR mixed reaction solution, specific probes, internal control primers and internal control Probes, a pair of specific primers consisting of corresponding ARMS primers and universal downstream primers were added to each mutation detection tube; DNA template of the sample to be tested, PCR mixed reaction solution, Control primers and quality control probes for ARMS-qPCR detection of KRAS gene mutation typing, the model used can be Bio-Rad CFX96.

[0089] The cont...

Embodiment 3

[0111] The practical application of embodiment 3 detection method

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

[0113] 1. Sample acquisition and template extraction

[0114] Select 63 patients with non-small cell lung cancer and colorectal cancer, and take paraffin-embedded tissue sections from the lungs of non-small cell lung cancer patients and intestines of colorectal cancer patients as test samples, and use Qiagen's QIAamp paraffin-embedded tissue The extraction kit (Cat No.56404) extracts DNA, and quantitatively dilutes it to 5ng / μL, which is used as a template for PCR detection.

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

[0116] The above-mentioned 63 samples were subjected to PCR detection, the method was as follows: each sample DNA was tested with 7 mutation detection tubes and 1 quality co...

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Abstract

The invention discloses a primer-probe composition for detecting seven kinds of hot-spot mutation of KRAS gene of humans. The primer-probe composition comprises 7 pairs of specific primers and one kind of specific probes, wherein the 7 pairs of specific primers are respectively prepared from 7 kinds of ARMS primers and one kind of downstream primer. The invention further discloses a kit containing the primer-probe composition, and a method for detecting mutation by using the kit. The primer-probe composition disclosed by the invention is high in sensitivity and specificity; when the kit is used, the detection processes are all reactions carried out in closed tubes, so that the pollution is significantly reduced, and besides, a UNG enzyme anti-pollution system and an internal control system are added, so that more accurate and stable typing detection can be performed on samples, and the results are guaranteed to be genuine and credible; the detection method is fast, the detection can be completed within 100 minutes, the result judgment is simple and objective, and the analysis is convenient.

Description

technical field [0001] The invention relates to a primer probe composition, a kit and a mutation detection method for detecting seven hotspot mutations (ie, seven common mutations) of human KRAS gene, belonging to the field of molecular biology. Background technique [0002] KRAS gene is one of the RAS gene family, located on human chromosome 12, is a proto-oncogene, which has a great impact on human cancer. The KRAS gene encodes a 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, has GTPase activity, and participates in intracellular signal transduction. It is active 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 pathway of tumor cell growth and angiogenesis. The normal KRAS gene can inhibit the growth of tumor cells, but it will induce permanent activa...

Claims

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Application Information

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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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