QPCR assay for detecting and quantifying a virulence plasmid in acute hepatopancreatic necrosis disease (AHPND) due to pathogenic Vibrio parahaemolyticus

A quantitative polymerase chain reaction (qPCR) assay was developed, based on a TaqMan probe, to detect and quantify a virulence plasmid harbored by the bacterium Vibrio parahaemolyticus which can cause acute hepatopancreatic necrosis disease (AHPND). The assay uses a pair of PCR primers, which amplify a 135-bp DNA fragment, and a TaqMan probe selected from the plasmid pirA-like gene. This qPCR assay reacted with AHPND-pathogenic isolates of V. parahaemolyticus collected from Vietnam and Mexico, but not with non-pathogenic strains of Vibrio spp. For quantification, a plasmid (pVpPirA-1) containing the target pirA-like gene was constructed, purified and serially diluted to be used as a standard. With this standard, the qPCR assay was then used to quantify the virulence plasmid in shrimp samples collected from different farms. Up to 5.8×10⁵ copy per mg tissue were detected in AHPND-affected shrimp collected from Vietnam. Lower quantities, up to 1.5×10⁴ copies per mg of tissues were detected in affected shrimp collected from a Chinese farm. In the laboratory bioassays, similar plasmid quantities, 1.8×10³ to 4.7×10⁶ copies of plasmid per mg of tissues were found in the moribund/dead shrimp, 3.5×10² to 2.2×10⁶ copies of plasmid per mL were detected in the water samples. This assay is specific with high sensitivity (10 copies of virulence plasmid) and can be used to detect AHPND-pathogenic V. parahaemolyticus in shrimp and water samples.