A gene mutation analysis and judgment method based on real-time fluorescent quantitative PCR platform

A technology of real-time fluorescence quantification and determination method, which is applied in the fields of biochemical equipment and methods, and the determination/inspection of microorganisms. small effect

Active Publication Date: 2017-01-04
JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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Problems solved by technology

[0006] The detection sensitivity of the above methods is low, subjectivity is strong, and it is easy to cause misjudgment

Method used

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  • A gene mutation analysis and judgment method based on real-time fluorescent quantitative PCR platform
  • A gene mutation analysis and judgment method based on real-time fluorescent quantitative PCR platform
  • A gene mutation analysis and judgment method based on real-time fluorescent quantitative PCR platform

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Embodiment 1

[0042] Example 1, taking the detection and analysis of EGFR gene mutation as an example

[0043] Materials: Plasma samples to be tested, positive quality control products, and negative quality control products.

[0044] Instruments: Lightcycler480, nanodrop1000, high-speed centrifuge, water bath, vortex oscillator, refrigerator, oven, sterilizer.

[0045] Reagents: DNA polymerase (Roche), 10×PCR Buffer (Roche), MgCl2 (Roche), dNTP (TaKaRa), purified water.

[0046] Primers: The purity of all primers should reach electrophoresis grade (PAGE) or HPLC grade, free of impurities. Provide the quality inspection certificate of the synthetic product issued by the synthesis institution, such as PAGE electrophoresis results or HPLC analysis patterns, which prove that there should be obvious single-peak PAGE or HPLC purification patterns after purification by PAGE or HPLC, and the concentration is 10ng / μl for use.

[0047] 1. PCR reaction system and reaction conditions

[0048] The fina...

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Abstract

The invention discloses a gene mutation analysis and judgment method based on a real-time fluorescent quantitative PCR platform, which uses the fluorescent quantitative PCR amplification result of the target gene to analyze and judge, and the steps of the analysis and judgment method are as follows: the first step, after the PCR experiment ends Afterwards, calculate the CT value of the experimental results and judge the CT value to determine whether to continue to analyze the experimental situation; the second step is to run the internal control, and determine whether the DNA sample is qualified according to the internal control results; the third step is to run the positive quality control product, according to The CT value of the positive quality control product determines whether the experimental system is qualified; the fourth step, if the CT value is qualified by the above-mentioned steps, run the Tm calling program, and judge whether there is a mutation in the sample through melting curve analysis and CT value analysis. The invention has a clear and objective interpretation of the PCR results, completes the interpretation of the gene type of the sample according to the amplification data and the melting curve, and has a low misjudgment rate.

Description

technical field [0001] The invention relates to the field of gene mutation analysis, in particular to a gene mutation analysis and determination method based on a real-time fluorescent quantitative PCR platform. Background technique [0002] At present, the existing methods for identifying genetic mutations mainly include the following: [0003] (1) High resolution melting curve (high resolution melting, HRM). The high-resolution melting curve is based on the physical properties of nucleic acid, through the binding of saturated dyes to PCR amplification products, and monitoring the changes in the melting curve of the product to analyze gene mutations. The detection sensitivity is about 5%, and the specific location of the positive result cannot be determined, and it needs to be confirmed by sequencing. [0004] (2) Probe amplification retardation mutation method (amplification refractory mutation system, ARMS). Using the principle that the base at the 3' end of the PCR pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 王弢李宗飞张利清张丽乐飚徐维琛
Owner JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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