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ARMS-based method for detecting botryis cinerea SdhB gene H272Y mutation

A technology for detection of Botrytis cinerea and mutation, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of low sensitivity of low copy number templates and influence of detection results, etc., and achieves cost reduction and accuracy rate. The effect of improving and reducing workload

Inactive Publication Date: 2015-07-08
BEIJING UNIV OF TECH +1
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  • Summary
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  • Application Information

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Problems solved by technology

However, precisely because of its very high detection sensitivity, the primer dimers produced in the PCR process will have a great impact on the detection results, and the false positive results are very serious
This makes the method less sensitive for detecting low copy number templates

Method used

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  • ARMS-based method for detecting botryis cinerea SdhB gene H272Y mutation
  • ARMS-based method for detecting botryis cinerea SdhB gene H272Y mutation
  • ARMS-based method for detecting botryis cinerea SdhB gene H272Y mutation

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Experimental program
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Effect test

Embodiment example 1

[0033] Implementation case 1, detection of wild-type strains without mutations in the SdhB gene

[0034] The UNIQ-10 Column Fungal Genomic DNA Extraction Kit was purchased from Sangon Bioengineering (Shanghai) Co., Ltd. to extract the genomic DNA of the samples to be tested. Three sets of PCR reactions were performed, the first of which used a universal primer pair to amplify the entire SdhB gene. The mixed solution of reaction is prepared in the following way:

[0035]

[0036] Mix the upstream primer of the reaction, SEQ ID NO:1, at a concentration of 5 mM, in 0.5 μL, with sucrose solution, with a mass fraction of 20%, 0.5 μL, and SYBR GreenⅠ, at a concentration of 25×, in 1 μL, and add it to the bottom of the PCR tube. Add 5 μL of DNA template to the upper layer of the primers, then add the PCR reaction mixture, and add 30 μL of mineral oil to the top layer to seal. Do not shake the PCR tube during this process to keep it layered.

[0037] PCR reaction conditions: 94°...

Embodiment example 2

[0047] Implementation case 2, detection of strains with mutations in the SdhB gene

[0048] The UNIQ-10 Column Fungal Genomic DNA Extraction Kit was purchased from Sangon Bioengineering (Shanghai) Co., Ltd. to extract the genomic DNA of the samples to be tested. Three sets of PCR reactions were performed, the first of which used a universal primer pair to amplify the entire SdhB gene. The mixed solution of reaction is prepared in the following way:

[0049]

[0050] Mix the upstream primer of the reaction, SEQ ID NO:1, at a concentration of 5 mM, in 0.5 μL, with sucrose solution, with a mass fraction of 20%, 0.5 μL, and SYBR GreenⅠ, at a concentration of 25×, in 1 μL, and add it to the bottom of the PCR tube. Add 5 μL of DNA template to the upper layer of the primers, then add the PCR reaction mixture, and add 30 μL of mineral oil to the top layer to seal. Do not shake the PCR tube during this process to keep it layered.

[0051] PCR reaction conditions: 94°C for 3 minut...

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Abstract

An ARMS-based method for detecting botryis cinerea SdhB gene H272Y mutation belongs to a real-time fluorescent quantitative detection method of gene mutation, integrates the real-time PCR technology and the ARMS technology, and greatly reduces the interference of a primer dimmer on the detection process through special primer design, so as to improve the detection sensitivity and accuracy. Moreover, the SYBR Green I dye method is adopted to reduce the detection cost.

Description

technical field [0001] The invention relates to the field of molecular biology, and specifically relates to an ARMS-based method for detecting the H272Y mutation of the Botrytis cinerea SdhB gene. Background technique [0002] Botrytis cinerea is a common and difficult to control fungal disease of crops in open fields and protected areas. It is a low-temperature and high-humidity disease. Botrytis cinerea is caused by the infection of Botrytis cinerea, a fungus belonging to the genus Botrytis cinerea. There are as many as 230 species of hosts for this pathogen. In addition to infecting tomato fruits, it can also infect tomato stems, leaves, and flowers, resulting in severe yield reduction or even failure. The disease occurs early, spreads quickly, causes serious damage, and causes large losses, seriously affecting vegetable production. At present, for the gray mold of crops, chemical control is the main method of production. Traditional fungicides mainly include benzimida...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6858C12Q2531/113C12Q2527/107C12Q2563/107
Inventor 谢飞吕宝北马雪梅江洪刘珊珊
Owner BEIJING UNIV OF TECH
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