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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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  • Abstract
  • Description
  • Claims
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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 be quickly and accurately detected. samples, but a PCR reaction system can only detect the sensitivity of one strain to carbendazim fungicide, and the sensitivity of a single strain to carbendazim can only be detected by electrophoresis after the completion of the entire PCR reaction system

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
Effect test

Embodiment 1

[0066] Example 1. Screening of specific primers suitable for real-time quantitative PCR (Real-time quantitative PCR).

[0067] The mutation of amino acid 198 of β-tubulin gene from glutamic acid (Glu) to alanine (Ala) is the main cause of field resistance of S. sclerotiorum to carbendazim, and the corresponding code According to this mutation, the upstream ASO-specific primer 5'-TGGTCGAGAACTCTGACGC-3', which can amplify the characteristic band of resistant S. sclerotiorum, was designed. This primer cannot be used for real-time quantitative PCR detection. Because there is a secondary structure in the amplification product; since SYBR Green I is a fluorescent dye that only binds to the DNA double strand, but it cannot select a specific DNA template, the quantification of this method requires that the primers cannot form dimers. No mismatch occurs. In addition, in the present invention, compared with the sensitive strain, only one base has changed in the β-tubulin gene sequence ...

Embodiment 2

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

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

[0097] The annealing temperature Tm value 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, too high annealing temperature will reduce the amplification efficiency, so it needs to be optimized. ...

Embodiment 3

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

[0101] Standards of strain JSJ6 genomic DNA were serially diluted 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 pattern ( 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 value; 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 detectable 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 to 100 times...

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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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