Innovative Multiplex PCR Assay for Detection of , , and Genes in with Reference to the U.S. FDA's ().

is an important foodborne bacterium that causes severe gastroenteritis following the consumption of contaminated seafood. To identify and determine its pathogenicity, the U.S. Food and Drug Administration (FDA)'s () recommends a multiplex polymerase chain reaction (PCR) protocol to simultaneously detect the species-specific thermolabile hemolysin () gene and the pathogenic thermostable-related hemolysin () and thermostable-direct hemolysin ( genes. However, this assay has shown two limitations: difficulty in separating the amplicons of the (486 bp) and (450 bp) genes due to their highly similar sizes, and the weaker band exhibited by the gene amplicon (270 bp). The present study aimed to improve the 's multiplex PCR assay by separating the three amplicons with similar intensity. A new primer set was applied for the gene (369 bp) alongside the existing primers for the and genes. The amplicons for the three genes were effectively separated by electrophoresis on a 2% tris-borate-EDTA (TBE) agarose gel within 45 min. Primer concentrations of 0.25 µM for three genes produced a significant amount of amplicons among various combinations of primer concentrations with 35 PCR cycles. This assay exhibited a detection limit of 10 pg of bacterial DNA, demonstrating its high sensitivity. It did not display amplicons from nine species known to be human pathogens or from 18 well-documented foodborne pathogens. Therefore, the present multiplex PCR protocol could help overcome the limitations of existing assays and provide a more reliable method for detecting the three genes of .