Development of a PCR Assay Based on a Single–Base Pair Substitution for the Detection of Aeromonas caviae by Targeting the gyrB Gene

Aeromonas caviae is a bacterial pathogen that causes various infectious diseases in both humans and animals. To facilitate its detection, we developed species‐specific primer sets targeting polymorphisms in the gyrB gene for use in a PCR assay. The technique was able to detect 100% (29/29) of the A. caviae strains tested using either of two sets of primers (designated ACF1‐ACR and ACF3‐ACR), which produced 293‐bp and 206‐bp amplicons, respectively. Another set of primers (designated ACF2‐ACR) yielded a 237‐bp amplicon and exhibited 90% (26/29) positive results with respect to A. caviae . None of the primer sets exhibited cross‐reactivity with 12 non– A . caviae isolates and 52 other non‐ Aeromonas bacteria. The detection limit using the ACF2‐ACR and ACF3‐ACR primer sets in pure culture was 1.6 × 10 3 CFU/mL, or 6 CFU per reaction, whereas that of the ACF1‐ACR primer set was 1.6 × 10 4 CFU/mL, or 60 CFU per reaction. In the case of spiked Nile Tilapia Oreochromis niloticus, the sensitivity of all primer sets without enrichment was 1.8 × 10 4 CFU/g, or 30 CFU per reaction. Primer set ACF3‐ACR was the best for a PCR assay targeting the gyrB gene, and the PCR technique developed was rapid, specific, and sensitive for the identification of A. caviae . Received December 2, 2014; accepted April 9, 2015