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Abstract
The serotyping of Vibrio parahaemolyticus, which is crucial to the surveillance and detection of outbreaks of vibriosis infection, has been widely used in many countries. In this study, we developed a molecular assay, named multiplex ligation reaction based on probe melting curve analysis (MLMA), for simultaneous identification of V. parahaemolyticus 57 K-serogroups. Based on the previous genomes of 418 strains including 39 K-serogroups and the 18 K-serogroups sequences from public databases, we obtained 57 K-serogroups specific gene sequences for designing primers and probes. The developed MLMA assay for identifying the V. parahaemolyticus 57 K-serogroups showed high reproducibility, with the intra- and inter-assay standard deviations and coefficients of variation of no more than 1°C and 1%, respectively. The limit of detection for all gene targets ranged from 0.1 to 1.0 ng/µl. We validated the MLMA assay with a double-blind test identifying 595 V. parahaemolyticus isolates using conventional serotyping methods for comparison. The results showed the kappa value between the MLMA assay and the traditional serological method was 0.936 and that there was a 96.97% consistency rate with conventional serotyping methods for all detected isolates. Additionally, five rare K-serogroups were identified using the MLMA assay, as well as 18 strains that could not be identified using the traditional serotyping method. Thus, the MLMA assay provides a rapid, robust, and promising tool for the molecular serotyping of V. parahaemolyticus K-serogroups and has the potential application to the detection of outbreaks and surveillance of V. parahaemolyticus infection.
Highlights
Vibrio parahaemolyticus is a Gram-negative, motile bacterium commonly found in marine and estuarine environments worldwide (Letchumanan et al, 2014; Wang et al, 2015)
There were 3,826 strains stored that were isolated from the stool specimens of infectious diarrheal patients and food samples over 15 years (2003–2018) in Shenzhen Center for Disease Control and Prevention, 3,590 strains were K-serogroups typeable and 236 strains were K-serogroups untypeable (KUT)
18 rare K-serogroups were incorporated into the assay using Escherichia coli TOP10 strains (n=18, Table S1) containing serogroupspecific genes cloned into a pUC57 vector
Summary
Vibrio parahaemolyticus is a Gram-negative, motile bacterium commonly found in marine and estuarine environments worldwide (Letchumanan et al, 2014; Wang et al, 2015). Between 2011 and 2016, 790 outbreaks of foodborne diseases were reported in more than 703 hospitals of China, causing 13,013 individuals to become ill. Of these cases, V. parahaemolyticus was the most common pathogen being responsible for foodborne disease outbreaks that caused most cases reported (42.3%). A newly emerging shrimp disease, acute hepatopancreatic necrosis disease (AHPND), is known to be caused by strains of V. parahaemolyticus that contain a unique virulence plasmid. It has caused significant economic loss in global shrimp industry (Lai et al, 2015). V. parahaemolyticus has become a large public health issue on a global scale, especially in coastal regions (Han et al, 2016)
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