Detection of Vibrio parahaemolyticus in shellfish using polymerase chain reaction–enzyme‐linked immunosorbent assay

Aims: This study evaluated the application of polymerase chain reaction–enzyme‐linked immunosorbent assay (PCR‐ELISA) for the detection of Vibrio parahaemolyticus in shellfish. Methods and Results: The PCRs were selected to amplify a species‐specific sequence region. In particular, internal tl biotin‐labelled oligonucleotide probe was used to capture the DIG‐labelled PCR products. Next, the probe PCR product hybrids, immobilized on a streptavidin‐coated microtiter plate, were detected with peroxidase‐conjugated anti‐digoxigenin antibody (anti‐DIG‐POD) and the colorimetric peroxidase substrate ABTS [2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulphonic acid)] using an ELISA plate reader. Conclusions: The PCR‐ELISA system described is a feasible, sensitive method for the direct and specific detection of V. parahaemolyticus in shellfish samples. Compared with gel‐based detection methods, PCR‐ELISA in this study increased sensitivity by 100‐fold for V. parahaemolyticus. Significance and Impact of the Study: The PCR‐ELISA described may be used for potential rapid detection in routine shellfish analysis for the seafood industry. The sector requires simultaneous large‐scale sample screenings to monitor contamination levels in processing plants and evaluate the performance of the hazard analysis and critical control point (HACCP) system. PCR‐ELISA also proved to be economical, with a cost of about 9 Euros per sample, and the quick assay taking 8 h to complete starting from DNA extraction.