Application of newly developed PCR detection and sequencing methods for the lafA gene to Aeromonas isolates

The novel polymerase chain reaction (PCR) and sequencing assays were developed to reveal the prevalence and nucleotide sequence of the lateral flagellar A gene (lafA) in clinical and environmental strains of Aeromonas spp. and were applied to both origins of the Aeromonas species. We designed a total of ten primers for PCR detection and sequencing of the lafA gene and used them on 63 clinical and 110 environmental strains of Aeromonas spp. The lafA1 and/or lafA2 (lafA1/A2) genes of approximately 800 bp size and the lafA1-A2 genes of approximately 1800 bp size from the lafA1 to lafA2 genes could be detected specifically by the PCR assay. The overall detection rate of lafA1/A2 genes was significantly higher in environmental than clinical strains, and strains bearing the lafA1-A2 genes appeared in several species. The sequencing assay was able to easily sequence the lafA1 gene of 68 strains. However, there was little specific clustering that can distinguish clinical and environmental strains in the phylogenetic tree. New PCR detection and sequencing methods for the lafA gene of Aeromonas spp. were successfully applied to clinical and environmental strains. Since the genetic testing methods developed in this study could specifically detect the lafA1/A2 and lafA1-A2 genes and easily sequence the lafA1 gene, they may be useful for public health and environmental monitoring of Aeromonas spp.