Vibrio harveyi, V. parahaemolyticus, and V. vulnificus detection in Thai shellfishes by the triplex PCR method

Abstract

Context: Shellfish sold in Southeast Asian markets are highly contaminated. Uncooked seafood samples were collected from markets in Bangkok, Thailand, which were contaminated with Vibrio species (27%) and in which antibiotic resistance was relatively high. Aims: To simultaneously detect V. harveyi, V. parahaemolyticus, and V. vulnificus in shellfish samples including life mussel (Perna viridis), blood clam (Tegillarca granosa), and baby clam (Paphia undulata) in the local markets in Bangkok. The sensitivity, accuracy, and specificity of triplex PCR method were also evaluated. Materials and Methods: The 150 frozen shellfishes were purchased from five local markets in Bangkok. Each sample was homogenized, enriched, and prepared for colony counting and isolation. Three Vibrio species were identified according to biochemical tests and then later confirmed by triplex PCR. The accuracy and specificity of triplex PCR were evaluated and compared with conventional tests. The sensitivity of triplex PCR was explained as total count (CFU/mL) of Vibrio species. Results: Using the biochemical test and triplex PCR method in the three Vibrio species of marine shellfishes, 14 and 15 isolates of V. harveyi, 8 isolates of V. parahaemolyticus, and undetectable and 1 isolate for V. vulnificus were identified, respectively. The accuracy of triplex PCR was higher than the conventional method. Triplex PCR was shown to be sensitive as total bacterial count for three Vibrio detection in marine shellfishes was 1.2 × 107-2 × 106 CFU/mL. Conclusions: Triplex PCR assay was higher in sensitivity, accuracy, and specificity, which proved to be convenient, simple, and effective method for Vibrio detection and identification in shellfishes.