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Method for rapidly evaluating resistance risk of botrytis cinerea for QoIs bactericide

A botrytis cinerea, positive technology, applied in the field of plant disease detection, biology, control and risk early warning of pathogenic bacteria resistance, identification, can solve the problems of high workload and long time, and achieve low cost, high specificity and high sensitivity Effect

Inactive Publication Date: 2017-05-31
ZHEJIANG FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ordinary bioassay techniques or molecular biology techniques such as PCR cannot meet this requirement. Bioassay techniques require a long time (at least more than 1 week) and heavy workload; molecular biology techniques such as PCR not only require expensive instruments The equipment also requires skilled senior technical operators

Method used

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  • Method for rapidly evaluating resistance risk of botrytis cinerea for QoIs bactericide
  • Method for rapidly evaluating resistance risk of botrytis cinerea for QoIs bactericide
  • Method for rapidly evaluating resistance risk of botrytis cinerea for QoIs bactericide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1, the optimal LAMP reaction primer group screening of the present invention:

[0059] According to Botrytis cinerea cytb gene (NW_001814458.1), BCbi143 / 144 intron 1205bp sequence, design LAMP primers, reasonably set the CG content of each set of primers to 50-60%, hairpin structure formation tendency, self-pairing tendency, etc. Indicators, select a more optimized combination for design, adjust the annealing temperature of each primer: Flc and Blc (64-66°C), F2 and B2 (59-61°C), F3 and B3 (55-60°C), so as to determine the correct Outward primer FIP, reverse inner primer BIP, forward outer primer F3, and reverse outer primer B3, 5 sets of primers were screened and designed, and the DNA of Botrytis cinerea strain containing BCbi143 / 144 intron was used as a template for LAMP test , to screen out the combination of LAMP primers that can specifically detect Botrytis cinerea strains containing BCbi143 / 144 introns.

[0060] The sequences of each primer are as foll...

Embodiment 2

[0065] Embodiment 2, reaction system of the present invention and detection kit system optimization:

[0066]In order to determine the optimal composition of each component in the reaction detection system, set Bst DNA polemerase (0.08 / 0.16 / 0.24 / 0.32 / 0.40U / μL), dNTPs (0.4 / 0.6 / 0.8 / 1.0 / 1.2mM), betaine (0.6 / 0.8 / 1.0 / 1.2 / 1.4M), F3 / B3 primers (0.1 / 0.2 / 0.3 / 0.4 / 0.5μM) and FIP / BIP primers (0.4 / 0.8 / 1.2 / 1.6 / 2.0μM), finalize the reaction 25 μL system: 1.6 μM forward inner primer FIP, 1.6 μM reverse inner 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, 5mM MgCl 2 , 0.8M betaine, 150μM hydroxynaphthol blue (HNB), 8U / μL Bst DNA polymerase, ddH 2 O.

Embodiment 3

[0067] Embodiment 3, a kind of LAMP kit that is used to detect the Botrytis cinerea containing BCbi143 / 144 intron:

[0068] 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, 5mM MgCl 2 , 0.8M betaine, 150μM hydroxynaphthol blue (HNB), 8U / μL Bst DNA polymerase, ddH 2 O.

[0069] And the LAMP primer set sequence is as follows:

[0070] F3: 5'-CCTAATCAAATGGCTAAACGTATT-3'

[0071] B3: 5'-CGTACAGTAACCATGGGATA-3'

[0072] FIP: 5'-TGAGAATCACCTAAGAGTGAACCATGCTTTTAAACGAATAGGACCG-3'

[0073] BIP: 5'-CCGATTACATGGAAACGGAACTCAAAAGTCATGCAGTCACAAT-3'.

[0074] A total of 24 μL of the det...

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Abstract

The invention discloses a loop-mediated isothermal amplification primer for detecting botrytis cinerea containing BCbil43 / 144 intron. The invention further simultaneously discloses a loop-mediated isothermal amplification kit for detecting the botrytis cinerea containing the BCbil43 / 144 intron. Besides the LAMP (loop-mediated isothermal amplification) primer composition, the kit further comprises 10*ThermoPol Buffer, dNTPs, MgCl2, betaine, hydroxynaphthol blue, 8U / mu L Bst DNA polymerase and ddH2O. By adopting the loop-mediated isothermal amplification primer and the loop-mediated isothermal amplification kit which are disclosed by the invention, rapid detection on the botrytis cinerea containing the BCbil43 / 144 intron can be implemented in the field, so that scientific medicine application for preventing and treating the botrytis cinerea is timely guided.

Description

technical field [0001] The invention belongs to the field of biological technology, relates to a loop-mediated isothermal amplification (LAMP) primer set and a method for using a loop-mediated isothermal amplification technique for detecting Botrytis cinerea containing BCbi143 / 144 introns, and belongs to the detection, identification, and use of plant diseases. The technical field of prevention and control and risk early warning of pathogenic bacteria resistance. Background technique [0002] Gray mold (Botrytis cinerea Pers.) belongs to the genus Botrytis of the half-known fungus Hyphophyceae Hyphospora. It is an opportunistic pathogenic fungus with strong saprophytic properties. It is a necrotic plant pathogenic fungus and has the characteristics of latent infection. longer. The host of Botrytis cinerea is not very specific and has a wide range of hosts, which can infect more than 230 kinds of plants. Botrytis cinerea can infect cross-infection among different host plants...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6844C12Q1/6895C12Q2531/119C12Q2537/1376C12Q2563/107
Inventor 张传清胡小然时浩杰戴德江
Owner ZHEJIANG FORESTRY UNIVERSITY
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