Species-specific detection of Vibrio alginolyticus in shellfish and shrimp by real-time PCR using the groEL gene

Vibrio alginolyticus is an important opportunistic pathogen in humans and marine animals. Culture-based methods and the traditional polymerase chain reaction (PCR) cannot quantify the pathogen with sufficient sensitivity. Thus, reliable, rapid, and accurate detection and quantification methods are essential to prevent and control V. alginolyticus. We developed a real-time PCR assay using SYBR Green I targeting the groEL gene to detect and quantify V. alginolyticus. A species-specific primer was designed based on the groEL gene. Primer specificity was confirmed using 3 V. alginolyticus strains and 32 other Vibrio and non-Vibrio strains. Only the V. alginolyticus strain showed a positive result in the specificity test. A melting curve analysis showed a specific peak with a melting temperature of 85.80 +/- 0.15 degrees C. A standard curve was produced to permit quantification of the target organism. Detection sensitivity was 0.14 pg of genomic DNA (equivalent to 10 cells per ml) for a pure culture of V. alginolyticus. V. alginolyticus was also quantified in artificially inoculated shellfish and living shrimp. The results indicated that SYBR Green I-based quantitative real-time polymerase chain reaction targeting the groEL gene enabled accurate, sensitive, and rapid quantitative detection of V. alginolyticus in seawater, shellfish, and shrimp.