A method for typing Vibrio parahaemolyticus based on real-time fluorescent PCR

Abstract

The invention provides a real-time fluorescent PCR-based typing method of vibrio parahaemolyticus. The method comprises the following steps: extracting genome DNA of the vibrio parahaemolyticus, carrying out fluorescent quantitative PCR detection on the extracted genome DNA, using "1" to represent a condition that a melting curve peak exists in a result of the fluorescent quantitative PCR detection, and using "0" to represent a condition that no melting curve peak exists in the result of the fluorescent quantitative PCR detection; and processing binary data composed of "0" and "1" in order to obtain a typing result. The fluorescence change of a sample to be detected is detected in real time mainly based on the fluorescent quantitative PCR in order to complete the detection of the sample. The method has the advantages of high resolution, no tedious operating step of electrophoretic analysis, simplicity in operation, and high automation degree.

Description

technical field [0001] The invention belongs to the technical field of microbial identification, in particular to a typing method for vibrio parahaemolyticus, more specifically to a typing method for vibrio parahaemolyticus based on real-time fluorescent PCR. Background technique [0002] Vibrio parahaemolyticus (Vibrioparahaemolyticus) is a halophilic Gram-negative Vibrio. Vibrio parahaemolyticus widely exists in seawater and seafood. Eating food contaminated by this bacteria will cause food poisoning. In coastal countries, such as China, the United States, and Japan, V. parahaemolyticus has become an important foodborne pathogen. In the coastal provinces of China, most bacterial food poisoning is caused by V. parahaemolyticus of. With the increasing number of food poisoning cases reported by Vibrio parahaemolyticus, the epidemiological investigation of this bacteria has attracted more and more attention. Bacterial typing is an important means of epidemiological research. ...

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Problems solved by technology

Compared with phenotyping methods, these methods have advantages such as speed and high specificity, but they are still relatively laborious due to the step of electrophoretic analysis required

[0004] In addition, although the ERIC-PCR method is used to amplify bacterial genomic DNA, DNA fingerprints can be generated, and the size and number of DNA migration bands within a certain range represent the distance and copy of the repeat sequence. According to the ERIC fingerprint of genomic DNA The classification and identification of pathogenic bacteria can be achieved, but the ERIC sequence also exists in highly homologous sequences of these sequences in Salmonella and other intestinal bacteria, so there is specificity in the molecular analysis of Vibrio parahaemolyticus based on the ERIC sequence Lesser deficiencies, may not be able to distinguish between Vibrio parahaemolyticus and microorganisms within its genus

[0005] In addition, Xiao Xiao et al. reported a new genotyping method for Vibrio parahaemolyticus. Although a gene cluster LVPC consisting of 18 genes was disclosed, the loci of the LVPC gene cluster were selected for typing analysis, but The resolution of the selected gene loci and their primers is limited, and it is necessary to analyze the LVPC gene cluster and the VNTRE gene cluster together to obtain the analysis results

The joint analysis of two sets of gene clusters is not only cumbersome and long-term, but also causes a large detection workload. Therefore, it cannot meet the requirements of rapid typing

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