Simultaneous Identification of Clinically Common Vibrio parahaemolyticus Serotypes Using Probe Melting Curve Analysis

Abstract

The dynamic nature of Vibrio parahaemolyticus epidemiology has presented a unique challenge for disease intervention strategies. Despite the continued rise of disease incidence and outbreaks of vibriosis, as well as the global emergence of pandemic clones and serovariants with enhanced virulence, there is a paucity of molecular methods for the serotyping of V. parahaemolyticus strains to improve disease surveillance and outbreak investigations. We describe the development of a multiplex ligation reaction based on probe melting curve analysis (MLMA) for the simultaneous identification of 11 clinically most common V. parahaemolyticus serotypes spanning a 10-year period. Through extensive sequence analyses using 418 genomes, specific primers and probes were designed for a total of 22 antigen gene targets for the O- and K- serogroups. Additionally, the toxR gene was incorporated into the assay for the confirmation of V. parahaemolyticus. All gene targets were detected by the assay and gave expected Tm values, without any cross reactions between the 11 clinically common serotypes or with 38 other serotypes. The limit of identification for all gene targets ranged from 0.1 to 1 ng/μL. The intra- and inter-assay standard deviations and the coefficients of variation were no more than 1°C and <1% respectively, indicating a highly reproducible assay. A multicenter double-blind clinical study was conducted using the traditional V. parahaemolyticus identification workflow and the MLMA assay workflow in parallel. From consecutive diarrheal stool specimens (n = 6118) collected over a year at 10 sentinel hospitals, a total of 153 V. parahaemolyticus isolates (2.5%) were identified by both workflows. A total agreement (kappa = 1.0) between the serotypes identified by the MLMA assay and conventional serological method was demonstrated. This is the first molecular assay to simultaneously identify multiple clinically important V. parahaemolyticus serotypes, which satisfies the acute need for a practical, rapid and robust identification of V. parahaemolyticus serotypes to facilitate the timely detection of vibriosis outbreaks and surveillance.

A Novel Molecular Method for Simultaneous Identification of Vibrio parahaemolyticus 57 K-Serogroups Using Probe Melting Curve Analysis

“…There were 3,826 strains stored that were isolated from the stool specimens of infectious diarrheal patients and food samples over 15 years (2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015)(2016)(2017)(2018) in Shenzhen Center for Disease Control and Prevention, 3,590 strains were K-serogroups typeable and 236 strains were K-serogroups untypeable (KUT). Among 3,826 strains, 418 strains were previously sequenced (Li et al, 2019;Bian et al, 2021). Among 418 strains, 338 strains were selected to develop the MLMA assay (Table 1), which included 328 strains representing 39 K-serogroups and 10 strains of untyped Kserogroups for detection of cross-reactions.…”

“…In this study, the primers were designed as previously described (Li et al, 2019). Primers designed for development of the MLMA assay included two parts ( Table 2).…”

“…Primers designed for development of the MLMA assay included two parts ( Table 2). (1) The whole genome sequences of 418 V. parahaemolyticus strains were sequenced in the previous study (Li et al, 2019;Bian et al, 2021) and 39 pairs of primers of different K-serogroups were designed based on specific serogroup antigen genes sequences of the 418 genomes which the bioproject numbers could be obtained from the GenBank database (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA677930). Among 39 pairs of primers, the eight pairs of primers were previously reported (Li et al, 2019).…”

“…(1) The whole genome sequences of 418 V. parahaemolyticus strains were sequenced in the previous study (Li et al, 2019;Bian et al, 2021) and 39 pairs of primers of different K-serogroups were designed based on specific serogroup antigen genes sequences of the 418 genomes which the bioproject numbers could be obtained from the GenBank database (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA677930). Among 39 pairs of primers, the eight pairs of primers were previously reported (Li et al, 2019). 2Additionally, 18 pairs of primers targeting 18 rare K-serogroup gene sequences were designed based on the wzy or wzx gene as previously described (Pang et al, 2019) using sequence information obtained from the GenBank database (https://www.ncbi.nlm.nih.gov/).…”

“…In MLMA, a fluorescence signal and melting temperature (Tm) are combined and used as a virtual 2D label that enables homogenous detection of one order of magnitude more targets (Liao et al, 2013). Previously, we presented a molecular assay based on the principles of MLMA to simultaneously identify the 11 clinically most common V. parahaemolyticus serotypes (Li et al, 2019). In this study, to further increase the number of target K-serogroups in multiplexity, we established a three-tube system to serotype V. parahaemolyticus 57 K-serogroups that could be applied as an alternative for the surveillance and control of vibriosis infections.…”