High throughput real-time detection technology for drug resistance gene frequency of Sclerotinia sclerotiorum population

A technology for gene frequency and real-time detection, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., to achieve the effect of improving specificity, improving amplification efficiency, and simple detection and monitoring

Inactive Publication Date: 2009-07-08
NANJING AGRICULTURAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

On the basis of research on the mechanism of drug resistance, according to the principle of gene point mutation, the application of conventional nucleic acid technology such as ASO-PCR technology to detect the resistance of pathogenic fungi to benzimidazole fungicides such as carbendazim can

Method used

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  • High throughput real-time detection technology for drug resistance gene frequency of Sclerotinia sclerotiorum population
  • High throughput real-time detection technology for drug resistance gene frequency of Sclerotinia sclerotiorum population
  • High throughput real-time detection technology for drug resistance gene frequency of Sclerotinia sclerotiorum population

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0066] Example 1. Suitable for real-time quantitative PCR (Real-time quantitative PCR) specific primer screening.

[0067] The mutation of amino acid at position 198 of the β-tubulin gene from glutamic acid (Glu) to alanine (Ala) is the main reason that causes S. sclerotiorum to develop resistance to carbendazim in the field, and its corresponding code From GAG to GCG, the upstream ASO specific primer 5'-TGGTCGAGAACTCTGACGC-3', which can amplify the characteristic band of resistant sclerotinia, was designed based on this mutation. This primer cannot be used for real-time quantitative PCR detection. Because there is a secondary structure in the amplified product; because SYBR Green I is a fluorescent dye that only binds to DNA double-strands, but it cannot select a specific DNA template, this method requires that the primers cannot form dimers for quantification. No mismatches. In addition, in the present invention, the drug-resistant strain has only one base change (A→C) in the β-...

Example Embodiment

[0095] Example 2. Optimization of SYBR Green I fluorescent dye quantitative PCR system

[0096] 2.1 Annealing temperature In order to ensure the stability and reliability of the test, the experiment selected Bao Biological Engineering (Dalian) Co., Ltd. Premix Ex Taq TM (Perfect Real Time) DRR041S kit. Enzymes, Mg in the kit 2+ , DNTP, etc. have been optimized, you only need to add your own template and primers. Therefore, the experiment only optimizes the annealing temperature and primer concentration.

[0097] The annealing temperature Tm is an important guarantee for the specificity of the reaction. If the annealing temperature is too low, non-specific amplification will occur. Therefore, choosing a higher annealing temperature can greatly reduce the non-specific binding between the primer and the template and improve the specificity of the PCR reaction. However, a too high annealing temperature will reduce the amplification efficiency, so it needs to be optimized. In the expe...

Example Embodiment

[0100] Example 3 Sensitivity of SYBR Green I fluorescent dye quantitative PCR

[0101] Dilute the standard genomic DNA of strain JSJ6 into 10 gradients: 140ng / μL, 14ng / μL, 1.4ng / μL, 1.4×10 -1 ng / μL, 1.4×10 -2 ng / μL, 1.4×10 -3 ng / μL, 1.4×10 -4 ng / μL, 1.4×10 -5 ng / μL, 1.4×10 -6 ng / μL, 1.4×10 -7 ng / μL, quantitatively amplified with two pairs of primers respectively; the obtained amplification profile ( image 3 ), when the template concentration is less than 1.4×10 -3 After ng / μL, the fluorescence value of the amplification curve is lower than the threshold; while the product is detected by 1.5% agarose gel electrophoresis, when the template concentration is less than 1.4×10 -2 No electrophoresis band after ng / μL ( Figure 4 ). It can be seen that the lowest sample concentration that can be detected by SYBR Green I fluorescent dye quantitative PCR is 1.4×10 -3 ng / μL (2.8×10 per 25μL system -3 ng, 3.17×10 3 copy), the sensitivity is at least 10-100 times that of ordinary PCR detection...

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Abstract

The present invention belongs to a real-time detection method of the frequency of drug resistance genes in Sclerotinia sclerotiorum syngen, which can be used for drug resistance monitoring and popularity warning of carbendazim and other benzimidazole fungicide resistance in sclerotinia sclerotium disease of cole and the like crops. The detection method is divided into three major steps, the first step: respectively extracting the nuclear genome DNA of known drug-resistant strains, sensitive strains and sample to be measured; the second step: using the specific detection primers of drug-resistant strains and Sclerotinia sclerotiorum strains for real-time quantitative PCR reactions, respectively establishing the standard curves of the drug-resistant strains and Sclerotinia sclerotiorum strains detection; the third step: sample to be measured respectively using the two specific primers for real-time quantitative PCR reactions, contradistinguishing with the established standard curves, obtaining the corresponding numbers of the drug-resistant strains and sensitive strains and the proportion of the drug-resistant strains in the total number of the Sclerotinia sclerotiorum. The use of real-time quantitative PCR technology can perform high-throughput, rapid and accurate quantitative detection of the number of field drug-resistant Sclerotinia sclerotiorum and its proportion in the pathogen syngens, which has high-throughput characteristic. Compared with the conventional biometrics and general PCR detection of drug-resistant strains, the invention is time-saving, cost-saving and fast. The whole process from directly extracting the genome DNA from the sclerotia, disease spots and spores collected to real-time quantitative-PCR detection is only 6h needed, and the detection accuracy rate is more than 96%.

Description

1. Technical field [0001] The invention belongs to a real-time detection method for the frequency of drug-resistant genes in sclerotinia populations, which can be used to monitor the development dynamics of benzimidazole fungicide-resistant sclerotinia populations and to predict the prevalence of drug-resistant rapeseed sclerotinia. 2. Technical background [0002] Sclerotinia sclerotiorum is a worldwide disease caused by Sclerotinia sclerotiorum (Lib.) de Bary, which seriously affects the yield and quality of rapeseed. For a long time, benzimidazole fungicides or compound agents based on such agents have been used for chemical control. Benzimidazole fungicides are used in production as a class of high-efficiency, broad-spectrum systemic fungicides, which solve the environmental toxicity problem of protective fungicides and improve the ability of humans to control the disease. Benzimidazole fungicides include carbendazim, benomyl, thiabendazole, thiophanate, etc. These fun...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06
Inventor 周明国陈长军王建新
Owner NANJING AGRICULTURAL UNIVERSITY
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