Multiplex PCR assays for simultaneous detection and characterization of Vibrio vulnificus strains

Abstract

Aims: Vibrio vulnificus is a major cause of seafood-related deaths in the United States. Several biomarkers, e.g. the virulence-correlated gene (vcg), 16S rRNA, and the capsular polysaccharide operon (CPS) have been used to differentiate virulent- from nonvirulent-type V. vulnificus strains. In this study, we combined the use of these biomarkers with a species-specific V. vulnificus cytolysin/haemolysin gene (vvhA) to develop two pairs of multiplex PCR assays that simultaneously detect and characterize V. vulnificus strains.

Methods and results: The first multiplex PCR pair amplified four genes (vvhA, vcg, 16S rRNA, and CPS), with one for virulent-type and the other one for nonvirulent-type V. vulnificus strains, while the second pair targeted three of those genes excluding CPS. Primer concentration and annealing temperature were optimized for the four multiplex PCR assays. When testing ten V. vulnificus reference strains and 80 field oyster isolates, results from each multiplex PCR matched 100% with known strain characteristics for these target genes.

Conclusions: The optimized multiplex PCR assays were capable of simultaneously detecting and characterizing V. vulnificus with high specificity and speed.

Significance and impact of the study: Multiplex PCR assays designed in this study are valuable tools for microbial ecology and epidemiology studies. They may facilitate better control of V. vulnificus risks in oysters, thereby reducing the number of illnesses and deaths because of V. vulnificus in the long run.