Rapid identification of Pseudomonas aeruginosa from ocular isolates by PCRusing exotoxin A-specific primers

The purpose of this research was to evaluate the use of PCR for the identif ication of ocular isolates of Pseudomonas aeruginosa by using primers speci fic to the exotoxin A gene of the bacteria. Genomic DNA was obtained from o cular microbial isolates of keratitis patients. Primers were designed based on the published sequence of the exotoxin A gene of P. aeruginosa. Using t he primers designed, PCR reactions were performed on the DNA samples. The P CR was also examined for its specificity and sensitivity. In addition, a di rect PCR using heating method was attempted on P. aeruginosa with no separa te DNA extraction step. ATCC strains of P. aeruginosa were included as posi tive controls. The rest of the bacteria other than P. aeruginosa served as negative controls. A single band was obtained when analysed on agarose gel electrophoresis only from samples that contained genomic DNA of P. aerugino sa. The direct PCR method was also successful with the same band produced f rom the amplification. The whole process was completed within 4 h. The dire ct PCR amplification targeting at the exotoxin A gene of P. aeruginosa is p otentially a rapid, specific, sensitive and relatively simple method for th e identification of ocular isolates of P. aeruginosa. (C) 2000 Academic Pre ss.