Method for rapidly detecting mutation of KRAS gene

A rapid and genomic technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of inaccurate detection and low sensitivity, and achieve the effect of simple result analysis, high sensitivity, and optimized amplification effect

Active Publication Date: 2012-11-28
陕西佰美医学检验有限公司
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Problems solved by technology

[0005] In order to overcome the shortcomings of low sensitivity and inaccurate detection of traditional probe melting curve method, the present invention proposes a method for rap...

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  • Method for rapidly detecting mutation of KRAS gene
  • Method for rapidly detecting mutation of KRAS gene
  • Method for rapidly detecting mutation of KRAS gene

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Embodiment Construction

[0032] The present invention combines molecular beacon probes with extension-blocking primers, and utilizes a polymerase without 5'→3' exonuclease activity to rapidly detect KRAS mutations in vitro, which not only saves consumables, but also has high sensitivity and high specificity The characteristics make the test results more reliable.

[0033] Extension-Refractory primers (Extension-Refractory primers) are primers that can form a "neck loop" structure with their own extension products; in addition to the normal primer sequence, the 5' end of the extension-refractory primer contains an additional base sequence, This sequence can be complementary to a base sequence including the mutation site in the single strand of the product obtained by its own extension, forming a "neck loop" structure; due to the difference in sequence between the wild-type and mutant templates, the formed "neck loop" There is a significant difference in the strength (Tm value) of the "ring" structure. ...

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Abstract

The invention provides a method for rapidly detecting the mutation of KRAS gene, to overcome the disadvantages of low sensitivity and inaccuracy in conventional probe melting curve assay. The method for rapidly detecting the mutation of KRAS gene comprises the following steps: (1) designing and synthesizing molecular beacon probe and template amplification primers; (2) extracting the KRAS genome DNA of a detected sample; (3) preparing a reaction system; (4) performing PCR reaction; performing melting curve analysis after the PCR reaction is finished, and determining if a KRAS mutation occurs in the detected sample through the Tm value of a melting peak. The method combines the molecular beacon probe and extension retardance primers, and uses the DNA polymerase without 5'->3' exonuclease activity to detect KRAS mutation rapidly in vitro, thereby saving consumables simultaneously having the characteristics of high sensitivity and specificity which ensure a more reliable detection result.

Description

technical field [0001] The invention relates to a method for detecting KRAS gene mutation. Background technique [0002] Gene mutation refers to the change of base composition or arrangement sequence in the structure and function of genomic DNA molecules, mainly including base substitution and fragment insertion and deletion, and is one of the important causes of genotype diseases. Gene mutation analysis plays a very important role in biomedical research, especially in genotypic disease diagnosis and pathological research. The protein encoded by the KRAS gene is involved in the cell signal transduction pathway mediated by the epidermal growth factor receptor (EGFR), affecting cell proliferation, growth and metastasis; the protein encoded by the normal KRAS gene is activated after receiving upstream signal activation, and The inactivated state will be restored after the signal is transmitted to the downstream cytokines; while the protein encoded by the mutated KRAS gene is a...

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 陈超刘金辉戴鹏高王浩王刚
Owner 陕西佰美医学检验有限公司
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