Fluorescent recombinase-mediated isothermal amplification detection kit for Clavibacter michiganensis subsp. nebraskensis

Abstract

The invention belongs to the technical field of detection, and discloses a fluorescent recombinase-mediated isothermal amplification detection kit for Clavibacter michiganensis subsp. nebraskensis. The kit comprises a forward primer as shown in SEQ ID NO: 1, a reverse primer as shown in SEQ ID NO: 2 and a probe as shown in SEQ ID NO: 3. A construction method of the kit comprises the following steps: activating strains by using an NA culture medium, collecting thalli by using an inoculating loop scraper, extracting bacterial DNA, verifying an extraction effect by using a nucleic acid tester, and storing the bacterial DNA at -20 DEG C for later use; designing and synthesizing the primers and the probe, screening the primers, establishing a system and conducting verifying; verifying the specificity of a fluorescent RAA detection method; and verifying the detection effects of a simulated sample and a real sample to obtain the fluorescent recombinase-mediated isothermal amplification detection kit for Clavibacter michiganensis subsp. nebraskensis. The kit is rapid in reaction, only needs 20 min, is high in specificity and sensitivity, and passes detection and verification based on actual samples and simulated samples.

Description

technical field [0001] The invention belongs to the technical field of detection, and in particular relates to a fluorescent recombinase-mediated isothermal amplification detection kit for Neizhou wilt pathogen of corn. Background technique [0002] At present, Corynebacterium michiganii subsp. Nebraska causes Nebraska wilt disease in corn, which can lead to a loss of up to 44% of corn. It is an important bacterial pathogen on corn and a quarantine pathogen of great concern in my country. . Cmn was first discovered in the central part of Nebraska, USA, and then gradually expanded to many states in the United States and parts of Canada. It has not been reported in my country. Cmn mainly spreads long-distance through seed belts, and the GRAP model prediction shows that it is suitable for colonization in most corn planting areas in my country. Although my country's corn production has been stable at more than 200 million tons for many years, with the surge in domestic industri...

AI Technical Summary

Problems solved by technology

[0004] Through the above analysis, the existing problems and defects of the existing technology are as follows: the separation and culture and serological detection take a long time, the operation is complicated and the sensitivity is low; the traditional molecular biology detection method requires strict temperature control, needs specific instrument support, and consumes It takes a long time; the product of the LAMP detection method is a series of bands of different sizes, and the results cannot be further verified by cloning and sequencing.

Images