Fluorescent quantitation PCR detection kit and detection method for PIK3CA (phosphatidylinositol3-kinase catalytic alpha) gene mutations

Abstract

The invention relates to the field of molecular biology, in particular to a method for detecting gene mutations by adopting fluorescent quantitation PCR, as well as a fluorescent quantitation PCR detection kit and a detection method for PIK3CA (phosphatidylinositol3-kinase catalytic alpha) gene mutations. The method comprises the following steps: designing a specific primer and a probe; preparing a fluorescent quantitation PCR reaction system and designing reaction conditions; amplifying a mutant gene target sequence in a sample by utilizing the specific primer; detecting the fluorescence signal strengths of FAM, HEX and Cy5 in the reaction system, and taking the fluorescence signal strengths as judgement standards of results. The kit provided by the invention can detect 8 types of mutations of a PIK3CA gene simultaneously; the specificity and the selection capability are high; a PIK3CA mutated molecule, which is as low as 5 copy, can be detected in a clinical sample of which the mutation content is only 0.1 percent; the detection speed is high, that is, the detection can be fulfilled within 90 minutes.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for detecting gene mutations by fluorescent quantitative PCR, in particular to a fluorescent quantitative PCR detection kit and detection method for PIK3CA gene mutations, which can be detected in clinical samples with only 0.1% mutation content As few as 5 copies of PIK3CA mutant molecules were detected. Background technique [0002] Phosphatidylinositol-3-kinase (PI3K) is an important signal transduction pathway molecule. It consists of a catalytic subunit p110 and a regulatory subunit p85. According to the structural characteristics of p110 and different substrate molecules, PI3K can be divided into three subtypes: type I, type II and type III. PI3K-mediated signal transduction pathway plays an important role in cell proliferation, transformation, apoptosis and tumorigenesis, and regulates the proliferation and survival of tumor cells. Studies have found that dysre...

AI Technical Summary

Problems solved by technology

The detection sensitivity of direct DNA sequencing method is only 20-30%, that is, tumor samples must contain 20-30% mutation-positive cells to be detected; for the detection of small samples of pathological tissue, the detection accuracy of direct sequencing method is limited, it is difficult to Meet the needs of practical applications

At the same time, this method is easy to cause pollution and false positives; the operation is complicated and time-consuming (usually it takes 2 to 3 days to get the test result)

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