A polymerase chain reaction based lateral flow test strip with propidium monoazide for detection of viable Vibrio parahaemolyticus in codfish

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.