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Abstract
Vibrio parahaemolyticus is one of the most important foodborne pathogens that cause various life-threatening diseases in human and animals. A selective method for detection of viable V. parahaemolyticus in codfish was created by combining propidium monoazide Polymerase Chain Reaction, and nucleic acid lateral-flow test strip. The VP1332 gene of V. parahaemolyticus was used as the detection target in PCR, and the 5′-end of the primers were labeled with biotin and digoxygenin, respectively. The PMA-PCR-LFS assay was further applied for detection of viable V. parahaemolyticus cells in codfish. The results showed that the PCR assay exhibited superior specificity (100%) on 70V. parahaemolyticus strains and 37 non-V. parahaemolyticus strains examined in the inclusivity and exclusivity tests; and the limit of detection of the PCR-LFS reached 50 CFU/mL. The optimized conditions for PMA treatment were 40 µM PMA, with light exposure at 40% for 3 min. Furthermore, the PMA-PCR-LFS assay accurately detected as low as 50 CFU/g of viable V. parahaemolyticus in the presence of a large number of non-viable cells in spiked codfish after a 2-hour enrichment, which provides a selective, simple and economically method for field detection of viable V. parahaemolyticus in codfish.
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