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LAMP (loop-mediated isothermal amplification)-based method for rapid detection of sclerotinia sclerotiorum strain with high resistance to carbendazim

A technology of carbendazim and sclerotinia, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, to achieve the effects of improving detection efficiency, guiding scientific medication, and improving detection accuracy

Active Publication Date: 2014-05-28
NANJING AGRICULTURAL UNIVERSITY
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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 S. sclerotiorum at home and abroad.

Method used

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  • LAMP (loop-mediated isothermal amplification)-based method for rapid detection of sclerotinia sclerotiorum strain with high resistance to carbendazim
  • LAMP (loop-mediated isothermal amplification)-based method for rapid detection of sclerotinia sclerotiorum strain with high resistance to carbendazim
  • LAMP (loop-mediated isothermal amplification)-based method for rapid detection of sclerotinia sclerotiorum strain with high resistance to carbendazim

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Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1 LAMP reaction system optimization

[0032] In order to save detection cost and ensure the stability and reliability of the detection method, Bst DNA polymerase (8U / μL) (2U-10U), Mg 2+ (25mM) concentration (1-6μL), primer FIP / BIP (40μM) and F3 / B3 (20μM) concentration (0.25-1.25μL), betaine (8M) concentration (1-6μL), HNB (2.5mM) concentration (0.5-2.5μL) was optimized, and the optimal reaction system was determined to be: BstDNA polymerase (8U / μL) 0.4μL, 10×ThermoPol 1.0μL, MgCl2 (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 0.4μL, dH 2 O (sterilized distilled water) 2.6 μL.

Embodiment 2

[0033] Embodiment 2LAMP reaction condition optimization

[0034] 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

[0035] Embodiment 3 LAMP primer specific detection

[0036] Genomic DNA of carbendazim-resistant S. sclerotiorum strain (NT36) and sensitive strain (HA61S) were used as templates for LAMP amplification respectively, and this technique has good specificity. When the template is a highly antibacterial strain, the color of the reaction product is sky blue, and the electrophoresis spectrum forms a ladder-like band (Figure 1); when the template is a sensitive strain, the color of the reaction product is purple, and there is no band in the electrophoresis (Figure 1).

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Abstract

The invention discloses an LAMP (loop-mediated isothermal amplification)-based method for rapid detection of a sclerotinia sclerotiorum strain with high resistance to carbendazim. The method can be used for dynamic monitoring and epidemic warning of field sclerotinia sclerotiorum for a carbendazim resistance group, and is a molecular technique established based on LAMP and used for rapid detection of the sclerotinia sclerotiorum strain with high resistance to the carbendazim. According to the detection method, two pairs of LAMP specific primers are designed on beta-tubulin (comprising 198 amino acid mutation sites) of the sclerotinia sclerotiorum strain with high resistance to the carbendazim, and LAMP is performed; whether the strain is the sclerotinia sclerotiorum strain with high resistance to the carbendazim is determined according to the color of a reaction product; if the color is sky blue (an amplification product exists), the strain is the sclerotinia sclerotiorum strain with high resistance to the carbendazim; and if the color is purple (the amplification product does not exist), the strain is the sclerotinia sclerotiorum strain sensitive to the carbendazim. The method is simple, convenient, fast and low in cost, and has important practical significance for sclerotinia sclerotiorum resistance monitoring and rational drug use.

Description

technical field [0001] The present invention is based on loop-mediated isothermal amplification (1oop-mediated isothermal amplification, LAMP) rapid molecular detection of carbendazim-resistant Sclerotinia sclerotiorum strains, and can be used for the dynamics of the development of sclerotinia sclerotinia to carbendazim-resistant populations Monitoring and resistance risk assessment, early warning of the prevalence of drug-resistant rapeseed sclerotinia, and providing medication guidance for the prevention and treatment of rapeseed sclerotinia. 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 ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/645
CPCC12Q1/04C12Q1/6844C12Q2531/119
Inventor 周明国段亚冰葛常艳张晓柯
Owner NANJING AGRICULTURAL UNIVERSITY
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