title: Development of a PCR Assay Based on a Single-Base Pair Substitution for the Detection of Aeromonas caviae by Targeting the gyrB Gene.
abstract: 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.
authors.affiliation: a Department of Biology, Faculty of Science, Srinakharinwirot University , Bangkok , 10110 , Thailand.
authors.forename: Penpan
authors.initials: P
authors.lastname: Payattikul
authors.forename: Siwaporn
authors.initials: S
authors.lastname: Longyant
authors.forename: Paisarn
authors.initials: P
authors.lastname: Sithigorngul
authors.forename: Parin
authors.initials: P
authors.lastname: Chaivisuthangkura
mesh_terms: D057585:Aeromonas caviae / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D023681:Cichlids; D027081:DNA Gyrase / Q000235:genetics / Q000378:metabolism*; D005393:Fish Diseases / Q000382:microbiology; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D016905:Gram-Negative Bacterial Infections / Q000382:microbiology / Q000662:veterinary; D016133:Polymerase Chain Reaction / Q000379:methods*; D013045:Species Specificity
chemical_list: D001426:Bacterial Proteins; D027081:DNA Gyrase