Molecular typing method for distinguishing different strains of cronobacter through performing single enzyme digestion on gluA gene based on RsaI

Abstract

The invention relates to a typing method for distinguishing different strains of cronobacter through performing single enzyme digestion on gluA gene based on RsaI, and belongs to the technical field of microbiological examination of food. Operation steps of the method disclosed by the invention are divided into two stages: 1, in the first stage, specific primer amplification on the cronobacter isperformed, so that gluA gene fragments of six different strains of the cronobacter are obtained; and 2, in the second stage, the specific gluA gene fragments of the cronobacter, which is amplified inthe first stage, are subjected to RsaI enzyme digestion, so that differential PCR-RFLP fingerprint graphs of the six different strains of the cronobacter are obtained, and the different strains of thecronobacter are further distinguished. A large quantity of examples of strains of the cronobacter verify that by adopting the molecular typing method, the differential enzyme digestion polymorphic graphs of the different strains can be effectively obtained, and the six different strains of the cronobacter can be quickly and accurately distinguished. The molecular typing method disclosed by the invention is used for distinguishing the strains of the cronobacter, and has an important practical meaning on accurately tracing pollution of the cronobacter in the food.

Description

technical field [0001] The invention belongs to the technical field of food microbiological testing, and in particular relates to a molecular typing method for distinguishing different species of Cronobacter based on RsaI single-enzyme cutting gluA gene. Background technique [0002] Cronobacter ( Cronobacter ), are foodborne pathogens that can cause serious infections. The survey of food contamination data shows that infant formula milk powder is the main transmission medium of its infection. Currently, Cronobacter typing techniques mainly include antibiotic typing, phage typing, Enterobabcterial Repetitive Intergenic Consensus-PCR (ERIC-PCR), pulsed field gel electrophoresis (Pulsed Field Gel Electrophoresis, PFGE), random amplification Polymorphism (Random Applied Polymorphic DNA-PCR) and other technologies, these methods cannot effectively distinguish different species of Cronobacter genus, while multilocus sequence analysis (Multil℃us Sequencing Typing, MLST) can be u...

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Problems solved by technology

Currently, Cronobacter typing techniques mainly include antibiotic typing, phage typing, Enterobabcterial Repetitive Intergenic Consensus-PCR (ERIC-PCR), pulsed field gel electrophoresis (Pulsed Field Gel Electrophoresis, PFGE), random amplification Polymorphism (Random Applied Polymorphic DNA-PCR) and other technologies, these methods cannot effectively distinguish different species of Cronobacter genus, while multilocus sequence analysis (Multil℃us Sequencing Typing, MLST) can be used for Cronobacter Different species of the genus can be distinguished, but the operation is cumbersome and requires high technical operations (nucleic acid purification, sequencing and sequence comparison analysis), which takes a long time (at least 2 days for ordinary laboratories), which is not conducive to the contamination of the bacteria in the food processing process Rapid identification of source

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