Molecular detection method for rapidly identifying carbendazim-resistant genotype-F200Y botrytis cinerea strain

A technology for Botrytis cinerea and molecular detection, which is applied in the detection field of molecular biology to achieve the effects of high accuracy, strong specificity and improved detection efficiency

Active Publication Date: 2015-01-28
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no relevant reports on the rapid molecular identification of LAMP for the carbendazim-resistant genotype F200Y of Botrytis cinerea at home and abroad.

Method used

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  • Molecular detection method for rapidly identifying carbendazim-resistant genotype-F200Y botrytis cinerea strain
  • Molecular detection method for rapidly identifying carbendazim-resistant genotype-F200Y botrytis cinerea strain
  • Molecular detection method for rapidly identifying carbendazim-resistant genotype-F200Y botrytis cinerea strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 LAMP reaction system optimization

[0031] In order to save the identification cost and ensure the stability and reliability of the identification method, BstDNA 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: BstDNA polymerase (8U / μL) 0.25μL, 10×ThermoPol 1μL, MgCl 2 (25mM) 1.6μL, dNTP (10mM) 0.8μL, FIP (40μM) 0.4μL, BIP (40μM) 0.4μL, F3 (10μM) 0.4μL, B3 (10μM) 0.4μL, betaine (8M) 1.0μL, HNB (2.5mM) 0.8μL, Genomic DNA 0.4μL, sterile ddH 2 O 2.55 μL.

Embodiment 2

[0032] Example 2 LAMP reaction parameter optimization

[0033] In order to obtain the optimum 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 optimum reaction temperature and time were obtained to be 60°C and 45min, respectively.

Embodiment 3

[0034] Embodiment 3 LAMP reaction sensitivity test

[0035] In order to determine the lower limit of detection of the LAMP reaction, clone the DNA fragment containing the 200-position mutation site of Botrytis cinerea to the carbendazim-resistant genotype F200Y strain into the vector pEASY-T3, transform Escherichia coli, and pick positive transformants, After the plasmid was extracted, the 10-fold serial dilution was used as a template for LAMP and PCR amplification respectively. Finally, it was concluded that the lowest detection limit of LAMP was 100 times that of ordinary PCR.

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Abstract

The invention discloses a molecular detection method for rapidly identifying a carbendazim-resistant genotype-F200Y botrytis cinerea strain. The molecular detection method is a molecular detection technology, which is established based on a LAMP (loop-mediated isothermal amplification) technology and is rapid, convenient, high in specificity and sensitivity and low in cost. The detection method comprises the following steps of designing two pairs of specific primers on a beta tubulin of a detected sample, performing LAMP, and judging whether the detected sample is a carbendazim-resistant genotype-F200Y botrytis cinerea strain or not according to the color of a reaction product; if the LAMP product is sky-blue and an electrophoretogram is a ladder band, determining that the product amplification exists and that the detected sample is the carbendazim-resistant genotype-F200Y botrytis cinerea strain; if the LAMP product is purple and the electrophoretogram is free of band, determining that the product amplification does not exist and that the detected sample is a carbendazim-nonresistant genotype-F200Y botrytis cinerea strain. The method is convenient, rapid, low in cost and significant for theoretically guiding the resistance risk evaluation and reasonable medication of botrytis of crops.

Description

technical field [0001] The present invention is a rapid molecular identification method for the carbendazim-resistant genotype F200Y strain of Botrytis cinerea based on loop-mediated isothermal amplification technology (loop-mediated isothermal amplification, LAMP), and belongs to the field of molecular biology in the field of biology. Detection method. Background technique [0002] Botrytis cinerea is a kind of plant fungal disease caused by Botrytis cinerea, which can harm more than 200 kinds of important economic crops such as vegetables, fruit trees and ornamental plants. The occurrence of this disease can cause plant seedlings, fruits and storage Organ damping, fallen leaves, flower rot, rotten fruit and rotten kiln, causing serious economic losses. In recent years, with the development of vegetable production in protected areas, the occurrence and prevalence of Botrytis cinerea have increased. At present, due to the lack of varieties with high resistance to Botrytis ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/645
CPCC12Q1/6844C12Q1/689C12Q2531/119
Inventor 段亚冰周明国杨莹张晓柯王建新
Owner NANJING AGRICULTURAL UNIVERSITY
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