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Fast detection method for high-resistance botrytis cinerea bacterial strains of carbendazim based on LAMP (loop-mediated isothermal amplification) technology

A technology of Botrytis cinerea and carbendazim, which is applied in the field of rapid detection of carbendazim high resistance to Botrytis cinerea strains based on LAMP technology, and achieves the effects of high sensitivity, reduced drug use and strong specificity

Active Publication Date: 2015-01-21
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are currently no relevant reports on the rapid molecular detection of LAMP against carbendazim-resistant strains of Botrytis cinerea at home and abroad.

Method used

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  • Fast detection method for high-resistance botrytis cinerea bacterial strains of carbendazim based on LAMP (loop-mediated isothermal amplification) technology
  • Fast detection method for high-resistance botrytis cinerea bacterial strains of carbendazim based on LAMP (loop-mediated isothermal amplification) technology
  • Fast detection method for high-resistance botrytis cinerea bacterial strains of carbendazim based on LAMP (loop-mediated isothermal amplification) technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 LAMP reaction system optimization

[0036] In order to save detection cost and ensure the stability and reliability of the detection method, Bst DNA polymerase (8U / μL) (0.8-4.0U), Mg 2+ (25mM) concentration (0.6-2.0μL), primer FIP / BIP (40μM) and F3 / B3 (10μM) concentration (0.1-0.5μL), betaine (8M) concentration (0.4-2.0μL), HNB (2.5mM ) concentration (0.4-1.2μL) was optimized, and the best reaction system was determined to be: Bst DNA polymerase (8U / μL) 0.3μL, 10×ThermoPol 1μL, MgCl 2 (25mM) 1.4μL, dNTP (10mM) 1.0μL, FIP (40μM) 0.3μL, BIP (40μM) 0.3μL, F3 (10μM) 0.2μL, B3 (10μM) 0.2μL, betaine (8M) 0.8μL, HNB (2.5mM) 0.8μL, Genomic DNA 0.4μL, sterile ddH 2 O 3.3 μL.

Embodiment 2

[0037] Embodiment 2LAMP reaction condition optimization

[0038] In order to obtain the most suitable reaction temperature and time and ensure the high efficiency of the detection method, the reaction temperature and time in the reaction parameters were optimized, and the reaction temperature and time were respectively 58-65°C ( figure 1 ) and 45-90min ( figure 2) conditions, DNA bands were amplified, and DNA bands were more abundant at 62°C and 60 min, so the optimal reaction temperature and time were 62°C and 60 min, respectively.

Embodiment 3

[0039] Example 3 LAMP reaction sensitivity detection

[0040] In order to determine the lower limit of detection of the LAMP reaction, this experiment PCR amplified the DNA fragment containing the 198-position mutation site, cloned the fragment into the vector pMD-18, transformed Escherichia coli, picked positive transformants, extracted the plasmid, and measured the concentration. , using 10-fold serial dilution as a template for LAMP and PCR amplification, respectively. Finally, the lower limit of detection of LAMP is 10 times of the lower limit of detection of ordinary PCR ( image 3 ).

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Abstract

The invention discloses a fast detection method for high-resistance botrytis cinerea bacterial strains of carbendazim. The method can be used for performing dynamic monitoring and early warning on botrytis cinerea bacteria causing gray mold of crops for the resistance groups of the carbendazol. In the method disclosed by the invention, a molecular detection technology having the advantages of fastness, convenience, strong specificity, high sensitivity and low cost is established on the basis of a loop-mediated isothermal amplification technology (LAMP). The detection method comprises the following steps: designing 2 pairs of specific primers on beta tubulin of high-resistance bacterial strains of the botrytis cinerea bacteria for the carbendazol, performing LAMP amplification, and judging whether products are the high-resistance bacterial strains of the botrytis cinerea bacteria for the carbendazol or not according to the colors of reaction products. If the LAMP amplification products appear to be azure, electrophoresis patterns present ladder-shaped strips, and the product amplification is generated, the products are identified as the high-resistance bacterial strains of the botrytis cinerea bacteria for the carbendazol; if the LAMP amplification products present violet, the electrophoresis patterns do not have strips, and the product amplification is not generated, then the products are identified as non high-resistance bacterial strains of the botrytis cinerea bacteria for the carbendazol. The method is simple, convenient, fast, and low in cost and has important actual significance in the resistance risk assessment of the gray mold of the crops and reasonable medication.

Description

technical field [0001] The present invention is based on loop-mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) rapid molecular detection method for Botrytis cinerea to carbendazim-resistant strains, which can be used for Botrytis cinerea to carbendazim The dynamic monitoring of the development of resistant populations and the assessment of resistance risk provide an important theoretical basis for the control of resistance, epidemic early warning and rational drug use guidance of Botrytis cinerea. Background technique [0002] Loop-mediated constant temperature amplification reaction (LAMP) is a novel constant temperature nucleic acid in vitro amplification technology invented by Japanese scholar Notomi et al. in 2000. It is widely used in gene diagnosis of diseases such as animals and plants. The principle of this technology is: using a set (4 types) of specific primers to cause a self-circulating strand displacement reaction under the action...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6844C12Q2531/119
Inventor 段亚冰周明国杨莹张晓柯王建新曹君红
Owner NANJING AGRICULTURAL UNIVERSITY
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