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Method for amplifying four genes of Botrytis cinerea and multiplex PCR (polymerase chain reaction) primer set thereof

A botrytis cinerea and genome technology, applied in the field of molecular biology, can solve the problems of ineffective bactericides and point mutations, and achieve the effect of less non-specific amplification and reduced workload

Active Publication Date: 2015-12-16
BEIJING UNIV OF TECH
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  • Abstract
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  • Claims
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Problems solved by technology

The main reason for the resistance to fungicides is the point mutation of the target site gene of drug action, which makes the fungicides unable to exert their original effect

Method used

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  • Method for amplifying four genes of Botrytis cinerea and multiplex PCR (polymerase chain reaction) primer set thereof
  • Method for amplifying four genes of Botrytis cinerea and multiplex PCR (polymerase chain reaction) primer set thereof
  • Method for amplifying four genes of Botrytis cinerea and multiplex PCR (polymerase chain reaction) primer set thereof

Examples

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Effect test

Embodiment 1

[0033] Example 1. Multiplex PCR amplification of strain samples

[0034] Using UNIQ-10 column type fungal genomic DNA extraction kit, purchased from Sangon Bioengineering (Shanghai) Co., Ltd., the genomic DNA of 5 strains to be tested was extracted. PCR reaction components, purchased from Invitrogen, including Taq enzyme, dNTPs, PCR buffer (without Mg 2+ ), MgCl 2 .

[0035] Carry out the PCR reaction of the experimental group and the control group, wherein the PCR reaction mixture of the experimental group contains the extracted genomic DNA of the sample to be tested as the template of the PCR reaction, and the reaction system is as follows,

[0036]

[0037]

[0038] An equal amount of double distilled water (ddH) was added to the PCR reaction mixture of the control group. 2 0) As the template of PCR reaction, the reaction system is as follows:

[0039]

[0040] PCR reaction program: 94°C for 5min; 94°C for 30sec, 59°C for 30sec, 72°C for 20sec; 72°C for 10min. ...

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Abstract

The invention provides a method for amplifying four genes of Botrytis cinerea and a multiplex PCR (polymerase chain reaction) primer set thereof, relating to the field of molecular biology. On the basis of drug-resistant mutant sites of known Botrytis cinerea p-benzimidazole, boscalid, iprodione, fenhexamid and many other bactericides, a PCR primer set for simultaneously amplifying four gene segments beta-tub, BsOS1, erg27 and SdhB is established. The extracted Botrytis cinerea genome can be directly used as a template and subjected to amplification by using the designed primer set to obtain the enriched four gene segments. The multiplex PCR process is used for the first time to simultaneously amplify the four drug-resistant mutant genes of Botrytis cinerea, thereby greatly reducing the workload for drug-resistant site detection in the later period. By optimizing the PCR reaction system, the four gene segments are amplified in one PCR process.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for simultaneously amplifying Botrytis cinerea and a primer set for resistance mutant genes of four fungicides. Background technique [0002] Botrytis cinerea is a fungal disease that is common in open fields and protected fields and is very difficult to control. The host range of Botrytis cinerea is very wide, including more than 200 kinds of plants, such as tomatoes, cucumbers, grapes and other common vegetables and fruits in life, which are extremely harmful in agricultural production. At present, the control methods for Botrytis cinerea include agricultural control, biological control and chemical control, among which chemical control is still the main one. Commonly used fungicides include benzimidazoles, dicarboximides, N-phenylcarbamates, anilinopyrimidines, and the like. [0003] Botrytis cinerea has the characteristics of rapid reproduction, large genetic varia...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/11C12Q1/68
Inventor 马雪梅张鑫吕宝北赵鹏翔谢飞
Owner BEIJING UNIV OF TECH
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