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Device for rapidly detecting benzimidazole fungicide-resistant botrytis cinerea Pers. based on LAMP

A technology for benzimidazoles and Botrytis cinerea, which is applied in the fields of plant disease detection, control and early warning of drug-resistant pathogens, biology, and identification, can solve the problems of high detection cost, cumbersome experimental process, long detection time, etc. The effect of high cost, simple reaction process and long detection time

Active Publication Date: 2017-01-25
ZHEJIANG FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional method of detecting or monitoring fungicide resistance is mainly to isolate and cultivate pathogenic bacteria, and then culture them on the drug-containing medium, and identify whether they are drug-resistant strains according to the inhibitory effect of the drug on the growth of mycelium. This method has a long detection cycle. It takes one week or even several weeks from isolation to identification, and there are often bacteria contaminations during the cultivation of pathogenic bacteria, which brings errors to the test results; at the same time, it also requires a lot of human resources to increase the cost of testing
Polymerase chain reaction (Polymerase Chain Reaction, PCR) is also the most commonly used method to rapidly amplify specific genes or DNA sequences in vitro to detect drug-resistant mutants. It has high sensitivity and specificity, but detection requires expensive experimental instruments and cumbersome experiments. process, and the detection time is long and the detection cost is high, which cannot meet the needs of economical, efficient and rapid detection

Method used

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  • Device for rapidly detecting benzimidazole fungicide-resistant botrytis cinerea Pers. based on LAMP
  • Device for rapidly detecting benzimidazole fungicide-resistant botrytis cinerea Pers. based on LAMP
  • Device for rapidly detecting benzimidazole fungicide-resistant botrytis cinerea Pers. based on LAMP

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1, a kind of LAMP kit that is used to detect the Botrytis cinerea of ​​resistance benzimidazoles fungicide E198A genotype:

[0063] The kit contains a detection solution composed of a loop-mediated isothermal amplification primer mixture and a loop-mediated isothermal amplification reaction master mix. The concentration of the primer combination mixture is: 1.6 μM forward internal primer FIP, 1.6 μM reverse internal primer BIP, 0.2 μM forward outer primer F3, 0.2 μM reverse outer primer B3; loop-mediated isothermal amplification reaction master mix: 10×ThermoPol Buffer, 1mM dNTPs, 4mM MgCl 2 , 0.6M betaine, 150μM hydroxynaphthol blue (HNB), 8U / μL Bst DNA polymerase, ddH 2 O.

[0064]A total of 24 μL of the detection solution was added, and 1 μL of the DNA template to be tested was added to form a 25 μL detection reaction system. The system contains 8U / μL Bst DNA polymerase 0.5μL (4U), 10×ThermoPol Buffer 2.5μL, 20μM FIP 2.0μL, 20μM BIP2.0μL, 10μM F3 0.5μL, ...

Embodiment 2

[0065] Embodiment 2, the universality investigation of kit of the present invention

[0066] Select the E198A mutant Botrytis cinerea strain, and the sensitive Botrytis cinerea strain DNA template as a negative control, ddH 2 O As a blank control, take 1 μL of DNA solution (concentration: 1ng / μL), add 24 μL of the detection solution in Example 1 to carry out LAMP reaction, the reaction program is: 65°C, 60min; 80°C inactivation for 10min. Observe the color change of the reaction solution after the reaction. Under normal light, the sample containing the E198A mutant gray mold strain should turn into sky blue under normal light. The negative control of the sensitive gray mold strain sample and ddH 2 The reaction solution of the O blank control is blue-purple in average range, and the results are shown in figure 2 . It shows that the primer composition of the present invention and the kit prepared therefrom have good versatility.

Embodiment 3

[0067] Embodiment 3, kit sensitivity detection of the present invention

[0068] In order to test the sensitivity of this kit for detecting Botrytis cinerea of ​​the E198A genotype resistant to benzimidazole fungicides, the DNA template of the E198A mutant Botrytis cinerea strain was diluted into different concentration gradients, respectively: 10ng / μL, 1ng / μL, 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL, 10fg / μL, 1fg / μL, 0.1fg / μL, 0.01fg / μL, 10 -3 fg / μL, 10 -4 fg / μL, 10 -5 fg / μL. Take 1 μL of E198A mutant DNA at different concentrations as a template, and add 24 μL of the detection solution in Example 1 to carry out LAMP reaction. The reaction program is: 65°C, 60min; 80°C inactivation for 10min. After the reaction, observe the color change of the reaction solution, the results are shown in Figure 5 . When the template concentration is as low as 1fg / μL, this kit can turn the reaction solution from blue-purple to sky blue, and gel electrophoresis can detect ladder-shaped bands, ...

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Abstract

The invention discloses a loop-mediated isothermal amplification primer composition for detecting benzimidazole fungicide E198A genotype-resistant botrytis cinerea Pers.. The LAMP primer composition consists of the following four primers: an F3, a B3, an FIP and a BIP. The primer composition consists of primer groups designed by mismatching a basic group in a mutation region containing a 198 locus of a botrytis cinerea Pers. beta-tubulin gene; loop-mediated isothermal amplification reaction is used for detecting the benzimidazole fungicide E198A genotype-resistant botrytis cinerea Pers.. The invention further provides a loop-mediated isothermal amplification method for detecting the benzimidazole fungicide E198A genotype-resistant botrytis cinerea Pers. and a kit used thereby.

Description

technical field [0001] The invention belongs to the field of biological technology, relates to a primer set and a method for using a loop-mediated isothermal amplification technique (LAMP) for detecting the E198A genotype Botrytis cinerea resistant to benzimidazole fungicides, and belongs to the detection, identification, prevention and control of plant diseases. The technical field of early warning of medicinal pathogens. Background technique [0002] Gray mold (Botrytis cinerea Pers.) belongs to the genus Botrytis of the genus Hyphophyceae Hyphospora, which has strong saprophytic properties, low host specialization, and a wide range of hosts. It can infect more than 230 kinds of plants. Botrytis cinerea can cross-infect different host plants. It belongs to necrotic plant pathogenic fungi and is the earliest described fungus. Botrytis cinerea mainly destroys mature and aging tissues of double-leaved plants, but it can also invade host tissues in the early stage of host gro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12Q1/04
Inventor 张传清胡小然戴德江时浩杰
Owner ZHEJIANG FORESTRY UNIVERSITY
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