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Specific primer used for rapid detection of kiwi fruit soft rot pathogenic bacterium Botryosphaeria dothidea, and detection method of Botryosphaeria dothidea

A technology for kiwifruit and pathogenic bacteria, which is applied in the field of detection of plant pathogenic fungi, can solve the problem of inability to quickly and accurately detect whether there are kiwifruit soft rot pathogenic bacteria in kiwifruit, and achieve the effects of high specificity, simple operation and accurate detection.

Inactive Publication Date: 2014-01-15
SICHUAN AGRI UNIV
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Problems solved by technology

[0006] The object of the present invention is to provide a method for rapid detection of kiwifruit soft rot pathogenic bacteria Botryosphaeria dothidea The specific primers and detection methods thereof mainly solve the problem that the existing technical means cannot quickly and accurately detect the presence of kiwifruit soft rot pathogens in kiwifruit

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  • Specific primer used for rapid detection of kiwi fruit soft rot pathogenic bacterium Botryosphaeria dothidea, and detection method of Botryosphaeria dothidea
  • Specific primer used for rapid detection of kiwi fruit soft rot pathogenic bacterium Botryosphaeria dothidea, and detection method of Botryosphaeria dothidea

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Embodiment

[0043] The present invention is mainly aimed at kiwi fruit soft rot pathogenic bacteria Botryosphaeria dothidea To detect, the specific method is as follows.

[0044] First, the present invention designs a set of primers for PCR amplification, this set of primers includes BZY-1 and BZY-2, wherein BZY-1 is a forward primer, BZY-2 is a reverse primer, the two The sequences are as follows:

[0045] BZY-1: 5'-CCATCAAACTCCAGTCAGTAAACG-3';

[0046] BZY-2: 5'-CTGCGCTCCGAAGCGAGATGTATG-3.

[0047] Then extract the DNA of the fruit sample to be detected. In the present invention, the DNA of the fruit sample to be detected is extracted by the CTAB method, and its extraction process comprises the following steps:

[0048] (1) Weigh 1 g of kiwi fruit sample frozen at -60°C, put it into a mortar and add liquid nitrogen to grind it into a fine powder, and quickly transfer the fine powder to a 10mL centrifuge tube , add 3 mL of 2×CTAB preheated to 65°C and 10 μL of 2-mercaptoethanol at th...

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Abstract

The invention discloses a specific primer used for rapid detection of a kiwi fruit soft rot pathogenic bacterium, and a detection method of the kiwi fruit soft rot pathogenic bacteriaum. A problem that existing technology is incapable of realizing accurate and rapid detection of the kiwi fruit soft rot pathogenic bacterium is solved. The specific primer comprises BZY-1 and BZY-2, the sequence of BZY-1 is 5'-CCATCAAACTCCAGTCAGTAAACG-3', and the sequence of BZY-2 is 5'-CTGCGCTCCGAAGCGAGATGTATG-3. The detection method of the kiwi fruit soft rot pathogenic bacterium comprises following steps: (1) DNA of samples are extracted; (2) the sample DNA is taken as a template, and primer BZY-1, primer BZY-2, 2*TaqPCRMasterMix and sterile ultrapure water are added for uniform mixing, and then PCR amplification is performed; and (3) an amplification product is subjected to agarose gel electrophoresis analysis, and the existence of the kiwi fruit soft rot pathogenic bacterium is determined based on the existence of amplification bands. Specificity of the primer is extremely high, only the kiwi fruit soft rot pathogenic bacterium band can be obtained by amplification, so that it is just needed to extract genome DNA from kiwi fruit tissue to be detected for PCR amplification, and the existence of the kiwi fruit soft rot pathogenic bacterium is determined based on the existence of amplification bands in electrophoretograms.

Description

technical field [0001] The invention relates to a method for detecting plant pathogenic fungi, in particular to a method for rapidly detecting kiwifruit soft rot pathogens Botryosphaeria dothidea The specific primers and detection method thereof are used to quickly and accurately detect whether there is kiwifruit soft rot pathogen in kiwifruit fruit. Background technique [0002] Post-harvest fruit rot is a global problem, and more than 25% to 30% of fresh fruits worldwide cannot be used due to rot and deterioration during storage. Postharvest fruit rot is mainly caused by pathogen infection during postharvest processing, transportation, storage and sales of fruits. With the development of my country's fruit industry and the expansion of planting area, postharvest fruit rot has gradually been paid attention to. However, due to incomplete preharvest disease control and backward storage and transportation technology, the situation of postharvest fruit rot is increasing d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/645
CPCC12Q1/04C12Q1/686C12Q2531/113
Inventor 龚国淑何书婷周游徐文俊张敏陈华保唐贵婷叶梦萍彭丹丹
Owner SICHUAN AGRI UNIV
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