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Polymerase chain reaction (PCR) detection method of specificity of botrytis cinerea

A detection method and technology for Botrytis cinerea, which is applied in the field of microbial detection, can solve the problems of long PCR reaction time and limited to microgram level, etc., and achieve the effects of simple result determination, short detection time and high sensitivity

Inactive Publication Date: 2013-06-12
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0006] Although some scholars have detected Botrytis cinerea by designing specific PCR primers, the required PCR reaction time is long, and only specific PCR primers are provided to amplify three species of cucumber downy mildew, cucumber powdery mildew, and capsicum phytophthora. The detection results of fungi, among them, downy mildew of cucumber and Phytophthora capsici belong to flagellate fungus, while powdery mildew of cucumber belongs to Ascomycota Sclerotinia fungus, these three fungi are far from Botrytis and easy to distinguish
Other species of Botrytis and related genera have not been specifically detected, and the sensitivity of specific PCR primers is limited to the microgram level

Method used

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  • Polymerase chain reaction (PCR) detection method of specificity of botrytis cinerea
  • Polymerase chain reaction (PCR) detection method of specificity of botrytis cinerea
  • Polymerase chain reaction (PCR) detection method of specificity of botrytis cinerea

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Embodiment 1

[0032] The present invention designs a kind of Botrytis cinerea specific PCR detection method, comprises the steps:

[0033] Step 1, designing primer pairs for specific amplification of Botrytis cinerea

[0034] By searching the literature and referring to the RAPD (randomly amplified polymorphic DNA fragment) primers provided by Shanghai Sangon Bioengineering Co., Ltd., 23 random primers were selected (see Table 1, the sequences were synthesized by Beijing Aoke Dingsheng Biotechnology Co., Ltd.) , amplified using the genomic DNA of Botrytis cinerea and its relatives (see Table 3) as a template.

[0035] 50μL RAPD reaction system: sample genomic DNA 1μL (40ng); 10×PCR buffer 5μL; 25mM MgCl 2 2 μL; 2.5mM dNTP Mixture (TAKARA Biotechnology Co., Dalian) 1.6 μL; 5U / μL rTaq enzyme (TAKARA Biotechnology Co., Dalian) 0.5 μL; 20 μM RAPD random primer 3 μL; ddH 2 O36.9 μL. RAPD reaction program: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 36°C for...

Embodiment 2

[0053] Specificity evaluation experiment of botrytis cinerea PCR detection method

[0054] Step 1, DNA template preparation

[0055] According to step 2 of Example 1, the genomic DNAs of 16 strains (see Table 3) of other fungi of the genus Botrytis and their related genera were respectively extracted.

[0056] The strains used in the present invention and their identification methods are the fungi that have been identified in the reference literature (Zhang Jing. Studies on Botrytis cinerea flora and Botrytis cinerea diversity in Hubei Province. Huazhong Agricultural University, doctoral dissertation, 2010). All experimental strains came from the research group of Phytopathogenic Fungus Li Guoqing of the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University. At the same time, the present invention amplifies part of the eukaryotic ITS1-5.8S-ITS2 (eukaryotic ribosome transcribed spacer) region based on the universal primer pair ITS1 and ITS4 to ver...

Embodiment 3

[0066] Amplification of different concentrations of Botrytis cinerea DNA to evaluate the sensitivity of Botrytis cinerea PCR detection method

[0067] Step 1, DNA template preparation

[0068] According to step 2 of Example 1, extract Botrytis cinerea DNA, ddH 2 O was dissolved and diluted, and the concentration detected by the ultraviolet spectrophotometer was 40ng / μL, 20ng / μL, 4ng / μL, 0.8ng / μL, 0.4ng / μL, and 0.04ng / μL.

[0069] Step 2: Sensitivity evaluation test of Botrytis cinerea PCR detection method

[0070] Take 1 μL of each concentration sample and add it into the PCR reaction system, and perform PCR amplification detection on the DNA template to be tested according to the method in Step 3 of Example 1.

[0071] Step 3, result judgment

[0072] Take 10 μL of the PCR amplification product, detect it by 1.5% agarose gel electrophoresis, stain with EB, and observe under ultraviolet light. If there is a single amplification band at 327 bp, it means that the sample conta...

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Abstract

The invention discloses a polymerase chain reaction (PCR) detection method of specificity of botrytis cinerea. The PCR detection method of the specificity of the botrytis cinerea includes the following steps of obtaining specificity fragment of the botrytis cinerea according to randomly amplified polymorphic DNA (RAPD) marks, designing amplification primer pair sequences, B1SE11f and B1SE11r, extracting sample deoxyribonucleic acid (DNA), conducting amplification through the PCR method, detecting an amplification product through gel electrophoresis, and judging whether the sample contains botrytis cinerea, wherein the judging is conducted according to the fact that the sample contains the botrytis cinerea when the electrophoresis result appears an amplification band on 327bp. The PCR detection method of the specificity of the botrytis cinerea can detect DNA of 0.4-nanogram botrytis cinerea, distinguish the botrytis cinerea from other fungus and relative genus fungus of the botrytis cinerea, and be directly applied to field detection. The PCR detection method of the specificity of the botrytis cinerea is short in detection time, low in cost, distinctive in detection result and simple in result judgment.

Description

technical field [0001] The invention relates to the field of microorganism detection. In particular, it relates to a botrytis cinerea-specific PCR detection method, which has short detection time, low cost, specific result, simple result determination and high sensitivity. Background technique [0002] According to the latest classification system, Botrytis cinerea belongs to the genus Deuteromycetes in its anamorphic form, Hyphophyceae, Hyphospora, Phyllosporaceae, and Botrytis genus. Sexuality belongs to the Ascomycota, Panmycetes, Mollicolales, Sclerotiniaceae, Botrytis genus. [0003] Botrytis cinerea is a facultative parasite, which can infect the aboveground parts of plants at any stage of plant growth and produce a large number of conidia, which can be spread by wind, water and agricultural operations to re-infect. Saprophytic strong, the disease caused by Botrytis cinerea is a kind of intractable disease. Among the reported fungi of the genus Botrytis, Botrytis ci...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/645
Inventor 张静范璇李国庆杨龙
Owner HUAZHONG AGRI UNIV
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