Multiplex PCR assay to identify clinically important <i>Aeromonas</i> species

The genus <i>Aeromonas</i> is increasingly implicated in human infections. However, accurate species-level identification remains challenging, particularly in clinical microbiology laboratories. This study aimed to develop a multiplex polymerase chain reaction (PCR) assay to identify four <i>Aeromonas</i> species-<i>Aeromonas hydrophila</i>, <i>Aeromonas caviae</i>, <i>Aeromonas veronii</i>, and <i>Aeromonas dhakensis-</i>most frequently associated with human infectious diseases. A total of 788 whole genome sequencing (WGS) data sets from 31 <i>Aeromonas</i> species were analyzed to identify open reading frames (ORFs) specifically present in <i>A. hydrophila</i>, <i>A. caviae</i>, <i>A. veronii</i>, and <i>A. dhakensis</i>. Primer sets were designed based on sequences of ORFs specific to each species to develop a multiplex PCR assay. To validate the efficacy of the assay, 256 clinical <i>Aeromonas</i> isolates were tested, and the results were compared with taxonomic affiliation inferred by WGS data, along with 19 type strains. The multiplex PCR successfully identified all strains of the four target species and produced no amplification in non-target species strains except the band for internal control. The multiplex PCR enables rapid and reliable identification of four <i>Aeromonas</i> spp. commonly involved in human infectious diseases.IMPORTANCEThe multiplex PCR assay facilitates accurate identification of clinically important <i>Aeromonas</i> spp. in clinical microbiology laboratories, providing crucial information to guide appropriate antimicrobial therapy and advance understanding of the epidemiology of <i>Aeromonas</i> spp.