Multiplex PCR primer group and next-generation sequencing database building method for MLST (multilocus sequence typing) tracing of vibrio parahaemolyticus

Abstract

The invention provides a multiplex PCR primer group and next-generation sequencing database building method for MLST (multilocus sequence typing) tracing of vibrio parahaemolyticus, and belongs to thetechnical field of microbiological detection. The multiplex PCR primer group for the MLST tracing of the vibrio parahaemolyticus comprises a primer for gyrB gene amplification, a primer for dnaE geneamplification, a primer for dtdS gene amplification, a primer for pyrC gene amplification, a primer for tnaA gene amplification, a primer for pntA gene amplification and one of a primer for recA-1 gene amplification and a primer for recA-2 gene amplification. The next-generation sequencing based database building method is established by multiplex PCR amplification for the seven genes, amplification and sequencing of the two types of RecA genes can be realized, and a sequencing result is relatively consistent with a first-generation sequencing result; and meanwhile, amplification of all genescan be realized by one-time PCR, the method can be directly applied to database building, and the operation is simple.

Description

technical field [0001] The invention belongs to the technical field of microbial detection, and in particular relates to a multiplex PCR primer set for MLST typing and traceability of Vibrio parahaemolyticus and a method for building a library by next-generation sequencing. Background technique [0002] With the development of traffic and trade, the flow of people and goods has become more frequent, and a large number of cross-border organisms have been intercepted and detected at ports, including a large number of food-borne pathogenic bacteria, among which Vibrio parahaemolyticus is a common diarrheal bacterium . [0003] Vibrio parahaemolyticus, Vibrio family Vibrio, is commonly found in coastal seawater, seabed sediments, seafood and salted foods; it is more common in Japan, Southeast Asia, the United States and Chinese Taipei, and is an important pathogen that causes foodborne diseases One; food poisoning caused by this bacterium accounts for 40-60% of bacterial food p...

AI Technical Summary

Problems solved by technology

[0010] The currently promulgated industry standard "SNT 4525.3-2016 Molecular Typing of Pathogenic Bacteria in Food for Export MLST Method Part 3: Vibrio parahaemolyticus" specifies 7 housekeeping genes for MLST typing of Vibrio parahaemolyticus , but for the amplification of these 7 genes, the ordinary PCR method is used, and the sequencing method is used for the first-generation sequencing. The first-generation sequencing needs to be amplified multiple times for the same bacterium, and the sequencing needs to be sent to a third-party company. The operation is complicated and the workload is heavy.

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