Detection ofVibrio parahaemolyticusin shellfish using polymerase chain reaction–enzyme-linked immunosorbent assay


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Abstract

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.

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