Triplex-PCR Assay for the Simultaneous Detection and Differentiation of Vibrio parahaemolyticus in Farmed Shrimp

Abstract

Vibrio parahaemolyticus is an important bacterial pathogen affecting both human health and aquatic animal health. Initially, a multiplex PCR employing four sets of primers targeting Vibrio genus-specific regions and V. parahaemolyticus species-specific
regions of the genome was optimized using a gradient PCR. The amplicons sizes for Vibrio genus detection were 663 bp (16S rRNA) and 689 bp (genus specific region of genome), while the amplicon sizes for V. parahaemolyticus species detection were 450 bp (tlh gene) and 297 bp (species specific region of the genome). PCR conditions were first optimized using a V. parahaemolyticus type culture (ATCC 17802) and later used for testing Vibrio isolates from apparently healthy Penaeus vannamei shrimp and water samples from shrimp ponds. However, the use of only one set of primers targeting 16S rRNA was sufficient for detecting the Vibrio at the genus level, hence the multiplex-PCR was modified into a triplex PCR (663 bp, 450 bp and 297 bp amplicons). This triplex PCR finds application not only in screening shrimp samples, both during the aquaculture phase and in processed shrimp products, but also in screening aquaculture environment samples for the presence of V. parahaemolyticus.