Evaluation of outer membrane protein U (OmpU) as a novel capture target of Vibrio parahaemolyticus and rapid detection of acute hepatopancreatic necrosis disease (AHPND) using PCR combined with immunomagnetic separation

A polymerase chain reaction (PCR) assay combined with immunomagnetic separation (IMS) was established to detect a virulence plasmid harbored by the bacterium Vibrio parahaemolyticus which can cause acute hepatopancreatic necrosis disease (AHPND). The outer membrane protein OmpU, a conserved antigen among Vibrio species, was evaluated as a novel capture target for the immunomagnetic separation to capture and concentrate V. parahaemolyticus from environmental sources. A PCR assay targeting pirA- (284 bp) and pirB- (392 bp) like genes was used for screening AHPND V. parahaemolyticus. Immunomagnetic beads (IMBs), coated with anti-VpOmpU antibodies, presented high capture efficiency (> 90%) for V. parahaemolyticus within 60 min. When PCR assay was combined with IMS (IMS-PCR), the detection limit was 101 CFU/mL in pure cultures and was unaffected by the presence of 105 CFU/mL of competing microflora. When applied in artificially contaminated environmental samples, the IMS-PCR could also detect as few as 101 CFU/mL AHPND V. parahaemolyticus within 4-5 h. These results suggest that the IMS-PCR approach could be a rapid and effective method for the detection of AHPND V. parahaemolyticus in shrimp and water samples from environmental sources.