LAMP technology based rapid Botrytis cinerea detection method

A rapid technology for Botrytis cinerea, applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of rapid molecular detection of LAMP of Botrytis cinerea, so as to timely understand the development of pathogens and reduce environmental pollution , The effect of reducing the probability of false positive occurrence

Inactive Publication Date: 2013-09-04
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 Botrytis cinerea at home and abroad after searching

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  • LAMP technology based rapid Botrytis cinerea detection method
  • LAMP technology based rapid Botrytis cinerea detection method
  • LAMP technology based rapid Botrytis cinerea detection method

Examples

Experimental program
Comparison scheme
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, the experiment was performed on Bst DNA polymerase (8U / μL) (1U-12U), Mg 2+ (25mM) concentration (1-8μL), primer FIP / BIP (40μM) and F3 / B3 (20μM) concentration (0.25-2μL), betaine (8M) concentration (0.125-4μL), HNB (2.5mM) concentration ( 0.25-2μL) was optimized, and the best reaction system was determined to be: Bst DNA polymerase (8U / μL) 1μL, 10×ThermoPol 2.5μL, MgCl 2 (25mM) 3.0μL, dNTP (10mM) 2.5μL, FIP (40μM) 0.75μL, BIP (40μM) 0.75μL, F3 (10μM) 0.5μL, B3 (10μM) 0.5μL, betaine (8M) 2.0uL, HNB (2.5mM) 1.5μL, Genomic DNA 1.0μL, dH 2 O (sterilized distilled water) 9.0 μL.

Embodiment 2

[0034] The optimization of embodiment 2 LAMP reaction conditions

[0035] In order to obtain the most suitable reaction temperature and time and ensure the high efficiency of the detection method, the reaction μL in the reaction parameters of the experiment, F3 (10μM) 0.5μL, B3 (10μM) 0.5μL, betaine (8M) 2.0uL, HNB (2.5mM) 1.5μL, Genomic DNA 1.0μL, dH 2 O (sterilized distilled water) 9.0 μL.

[0036] Embodiment 2 LAMP reaction condition optimization

[0037] In order to obtain the optimum reaction temperature and time and ensure the high efficiency of the detection method, the experiment optimized the reaction temperature (60-65°C) and time (15-90min) in the reaction parameters, and finally determined the optimum reaction temperature and time are 63°C and 45min, respectively.

Embodiment 3

[0038] Example 3 Detection of LAMP reaction sensitivity

[0039] In order to determine the lower limit of detection of the LAMP reaction, in this experiment, the kit was used to extract and purify genomic DNA as a template, which was diluted in a 10-fold gradient. Using the above-mentioned diluted genomic DNA as a template, LAMP and PCR amplification were performed respectively. From figure 2 and image 3 It can be known that the lowest detection limit of LAMP technology is 1pg, while the detection limit of ordinary PCR is only 10pg.

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Abstract

The invention discloses a rapid Botrytis cinerea detection method. The method can be used for the rapid diagnosis of botrytis and the dynamic monitoring of the development of pathogenies, and is of great practical significance for the early warning of the botrytis circulation and the rational drug use guidance. The method is a rapid and simple Botrytis cinerea molecule detection method established based on an LAMP (loop-mediated isothermal amplification) technology. The detection method comprises the following steps: 1, respectively extracting genome DNA of samples to be detected; 2, carrying out an LAMP reaction; and 3, determining whether there is Botrytis cinerea or not according to the color of a reaction product, determining there is Botrytis cinerea if the color is sky-blue, and determining there is no Botrytis cinerea if the color is purple (there is no amplification product). The Botrytis cinerea detection method provided for the scientific researches and the production practices has the advantages of simplicity, rapidness and low cost, and is of practical and far-reaching significance to increasing ecologic, social and economic benefits.

Description

technical field [0001] The invention is a rapid molecular detection method for Botrytis cinerea based on loop-mediated isothermal amplification technology (Loop-mediated isothermal amplification, LAMP), which can be used for the diagnosis of Botrytis cinerea in various host plants and the development trend of pathogen groups monitoring, forecasting and early warning 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. This method uses a set (4 kinds) of specific primers to identify six a specific area. The principle of this technology is: under the action of Bst large fragment polymerase, a self-circulating strand displacement reaction is caused, and a large amount of target DNA is synthesized within 60 minutes at 60-65°C, accompanied by the by-product—white magnesium pyrophosphate p...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 周明国葛常艳段亚冰张晓柯王建新陈长军
Owner NANJING AGRICULTURAL UNIVERSITY
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