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Test kit for detecting drug resistance of botrytis cinerea to benzimidazole fungicides

A technology for benzimidazoles and Botrytis cinerea, which is applied in the field of drug-resistant primer combinations and kits thereof, can solve the problems of limited number of assay samples, high technical requirements, large workload and the like, and achieves convenient and sensitive detection, The detection speed is simple and the method is simple and easy to learn.

Inactive Publication Date: 2018-05-11
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The traditional method for determining the susceptibility of mycelium to pharmaceuticals usually takes several weeks, with heavy workload, limited sample quantity, strict aseptic operation, and it is difficult to detect the existence of drug-resistant strains at an early stage
The detection method based on PCR technology requires expensive instruments and requires high technical requirements for operators, so it is not suitable for promotion in the field or in agricultural departments such as local cities and counties.

Method used

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  • Test kit for detecting drug resistance of botrytis cinerea to benzimidazole fungicides
  • Test kit for detecting drug resistance of botrytis cinerea to benzimidazole fungicides
  • Test kit for detecting drug resistance of botrytis cinerea to benzimidazole fungicides

Examples

Experimental program
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Effect test

Embodiment 1

[0066] (1) Pick the mycelium of Botrytis cinerea to be tested in 50 μL of 10×TE lysate, boil it in boiling water for 2 minutes, and obtain the DNA template;

[0067] (2) Prepare the solution according to the reaction system, the reaction system (25 μL): 4U Bst DNA polymerase, 1×ThermoPol Buffer, 4mM MgSO 4 , 1mM dNTPs, 1.2mM internal primers, 0.4mM external primers, 0.8M betaine, 1μL DNA template in step (1); reaction conditions: firstly at 63°C for 60min and then at 85°C for 10min ;

[0068] (3) Add 0.2 μL of 10000×SYBR Green I dye to the obtained amplification product, observe the color reaction, and obtain the result.

[0069] The above method was used to detect and analyze 16 botrytis cinerea to be tested, numbered 1-16 in turn, and number 17 was distilled water control. Wherein, the primer group III used in this method, that is, the inner primers are Tub-FIP-E198K and Tub-BIP, each 1.2 mM, and the outer primers are Tub-F3 and Tub-B3, each 0.4 mM. The result is as figu...

Embodiment 2

[0074] (1) Pick the mycelium of Botrytis cinerea to be tested in 50 μL of 10×TE lysate, boil it in boiling water for 2 minutes, and obtain the DNA template;

[0075] (2) Prepare the solution according to the reaction system, the reaction system (25 μL): 4U Bst DNA polymerase, 1×ThermoPol Buffer, 4mM MgSO 4 , 1mM dNTPs, 1.2mM Tub-BIP, 1.2mM Tub-FIP-E198A or Tub-FIP-E198V or Tub-FIP-E198K, 0.4mM Tub-F3, 0.4mM Tub-B3, 0.8M betaine, 1μL step ( 1) Medium DNA template; reaction conditions: first keep the temperature at 63°C for 60 minutes, then keep the temperature at 85°C for 10 minutes;

[0076] (3) Perform agarose gel electrophoresis on the amplified product obtained in step (2), and observe whether there is an amplified product.

[0077] The above method was used to detect and analyze 16 botrytis cinerea to be tested, numbered 1-16 in turn, wherein number 17 was the distilled water control, and M represented Marker. Wherein, the primer group III used in this method, that is, t...

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Abstract

The invention provides a test kit for detecting drug resistance of botrytis cinerea to benzimidazole fungicides. The kit comprises a primer combination. The primer combination comprises a primer set I, a primer set II, and / or a primer set III; the primer set I consists of Tub-F3, Tub-B3, Tub-FIP-E198A and Tub-BIP; the primer set II consists of Tub-F3, Tub-B3, Tub-FIP-E198V, and Tub-BIP; the primerset III consists of Tub-F3, Tub-B3, Tub-FIP-E198K, and Tub-BIP. The invention provides a convenient and flexible method for detecting the drug resistance of the botrytis cinerea with direct judgmentresults and capable of being operated in the field based on a molecular level, and can ensure a primary technician to obtain objective and reliable detection results in the field within two hours.

Description

technical field [0001] The invention relates to the field of detection of drug resistance of plant pathogenic bacteria, in particular to a combination of primers and a kit for detecting the resistance of Botrytis cinerea to benzimidazole fungicides. Background technique [0002] Gray mold caused by Botrytis cinerea is a very common disease on fruits and vegetables, and the damage is very serious, which has caused huge losses to fruit and vegetable production. At present, the control of this disease still mainly depends on fungicides. Benzimidazole fungicides are the most widely used fungicides in production. However, due to its extensive use, Botrytis cinerea has developed resistance to it, and the control effect of the drug has also declined significantly. Research on resistant strains found that the 198th codon of the β-tubulin gene of the resistant strains had a point mutation, so that the original glutamic acid was replaced by alanine, valine or lysine (E198A / V / K). ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2600/156C12Q2531/119C12Q2563/107
Inventor 罗朝喜范飞林杨阴伟晓
Owner HUAZHONG AGRI UNIV
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