SYBR Green I-based real-time PCR assay targeting groEL gene for the detection and quantification of Vibrio alginolyticus from shellfish and shrimp

Abstract

V. alginolyticus is an important opportunistic pathogen for humans and marine animals. Culture-based methods and the traditional polymerase chain reaction (PCR) can not quantify the pathogen with sufficient sensitivity. Thus, reliable, rapid, and accurate detection and quantification methods are essential to prevent and control V. alginolyticus. A real-time PCR assay was developed by using SYBR Green I targeting groEL gene to detect and quantify V. alginolyticus. A species-specific primer was designed based on groEL gene. Specificity of the primer was confirmed by using three 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 ± 0.15ºC. 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/ml) for a pure culture of V. alginolyticus. V. alginolyticus was also quantified in artificially inoculated shellfish and shrimp. The results indicated that SYBR Green I-based quantitative real-time polymerase chain reaction (qRT-PCR) targeting the groEL gene enabled accurate, sensitive, and rapid quantitative detection of V. alginolyticus in shellfish, shrimp and seawater.