A Duplex PCR Assay for Rapid Detection of and in Large Yellow Croaker Fish.

Both and cause an increasing number of diseases in fish, resulting in great economic losses in aquaculture. In addition, the disease infected with or exhibited the similar clinical symptoms in aquatic animals. However, there is no effective means for the simultaneous detection of co-infection and discrimination them for these two pathogens. Here, we developed a duplex polymerase chain reaction (PCR) method based on the outer membrane protein A () gene of and . The specificity and validity of the designed primers were confirmed experimentally using simplex PCR. The expected amplicons for and had a size of 663 and 1404 bp, respectively. The optimal condition for duplex PCR were determined to encompass a primer concentration of 0.5 μM and annealing temperature of 57°C. This method was analytical specific with no amplification being observed from the genomic DNA of , , , and . The limit of detection was estimated to be 20 fg of genomic DNA for and 200 fg for , or 100 colony-forming units (CFU) of bacterial cells in both cases. The duplex PCR was capable of simultaneously amplifying target fragments from genomic DNA extracted from the bacteria and fish liver. For practical validation of the method, 20 diseased fish were collected from farms, among which 4 samples were PCR-positive for and . The duplex PCR method developed here is time-saving, specific, convenient, and may prove to be an invaluable tool for molecular detection and epidemiological investigation of and in the field of aquaculture.