A method for rapid detection of Fusarium graminearum strains with moderate resistance to carbendazim

A technology of Fusarium graminearum and carbendazim, which is applied in the directions of biochemical equipment and methods, microbial determination/inspection, etc., to achieve the effects of high accuracy, strong specificity, and simple and fast methods

Active Publication Date: 2014-10-29
NANJING AGRICULTURAL UNIVERSITY
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  • Description
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Problems solved by technology

However, there are no relevant reports on the rapid molecular detection of LAMP for carbendazim-resistant strains of Fusarium graminearum at home and abroad.

Method used

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  • A method for rapid detection of Fusarium graminearum strains with moderate resistance to carbendazim
  • A method for rapid detection of Fusarium graminearum strains with moderate resistance to carbendazim
  • A method for rapid detection of Fusarium graminearum strains with moderate resistance to carbendazim

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Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1 LAMP reaction system optimization

[0033] In order to save detection cost and ensure the stability and reliability of the detection method, Bst DNA polymerase (8U / μL) (0.8U-4U), Mg 2+ (25mM) concentration (0.4-2.4μL), primer FIP / BIP (40μM) and F3 / B3 (20μM) concentration (0.1-0.5μL), betaine (8M) concentration (0.4-2.4μL), HNB (2.5mM ) concentration (0.2-1μL) was optimized, and the best reaction system was determined to be: Bst DNA polymerase (8U / μL) 0.3μL, 10×ThermoPol 1μL, MgCl 2 (25mM) 1.6μL, dNTP (10mM) 1.0μL, FIP (40μM) 0.4μL, BIP (40μM) 0.4μL, F3 (10μM) 0.2μL, B3 (10μM) 0.2μL, betaine (8M) 1.2uL, HNB (2.5mM) 0.6μL, Genomic DNA 0.5μL, dH 2 O (sterilized distilled water) 2.6 μL.

Embodiment 2

[0034] Embodiment 2LAMP reaction condition optimization

[0035] In order to obtain the optimum reaction temperature and time and ensure the high efficiency of the detection method, the reaction temperature (60-65°C) and time (15-90min) in the reaction parameters were optimized, and finally the optimum reaction temperature and The time is 63°C and 60 min, respectively.

Embodiment 3

[0036] Example 3 LAMP reaction sensitivity detection

[0037] In order to determine the lower limit of detection of the LAMP reaction, genomic DNA was used as a template in this experiment and diluted in a 10-fold gradient. Using the above-mentioned diluted genomic DNA as a template, LAMP and PCR amplification were performed respectively. Finally, it was concluded that the lowest detection limit of LAMP was 10 times that of ordinary PCR.

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Abstract

The invention discloses a method for rapidly detecting moderately-resistant strain in fusarium graminearum to carbendazim, which can be used for dynamical monitoring and epidemic warning of resistant groups of the fusarium graminearum causing wheat scab to the carbendazim. The method is a molecular technology for rapidly detecting the moderately-resistant strain in the fusarium graminearum to the carbendazim and is established on the basis of the loop-mediated isothermal amplification technology (LAMP). The method comprises the following steps of: designing two pairs of specific primers on beta2 tubulin of the moderately-resistant strain (F167Y) of the fusarium graminearum to the carbendazim, carrying out LAMP amplification, and judging whether to be the moderately-resistant strain of the fusarium graminearum to the carbendazim according to the color of a reaction product, wherein if the color is sky-blue (with an amplification product), the strain is the moderately-resistant strain in the fusarium graminearum to the carbendazim, and if the color is purple (without the amplification product), the strain is the sensitive strain of the fusarium graminearum. The method disclosed by the invention is simple, fast and low in cost and has important practical significance for risk evaluation on resistance of the wheat scab and the reasonable pesticide use.

Description

technical field [0001] The present invention is based on loop-mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) rapid molecular detection method for Fusarium graminearum carbendazim medium-resistant strains, which can be used for Fusarium graminearum for multi- Dynamic monitoring and resistance risk assessment of the development of wartazim-resistant populations, forecasting the prevalence of resistant wheat scab, and providing medication guidance for the control of wheat scab. 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 of a highly active s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 周明国段亚冰葛常艳张晓柯
Owner NANJING AGRICULTURAL UNIVERSITY
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