The purpose of this research was to evaluate the use of PCR for the identification of ocular isolates of Pseudomonas aeruginosa by using primers specific to the exotoxin A gene of the bacteria. Genomic DNA was obtained from ocular microbial isolates of keratitis patients. Primers were designed based on the published sequence of the exotoxin A gene of P. aeruginosa. Using the primers designed, PCR reactions were performed on the DNA samples. The PCR was also examined for its specificity and sensitivity. In addition, a direct PCR using heating method was attempted on P. aeruginosa with no separate DNA extraction step. ATCC strains of P. aeruginosa were included as positive 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. aeruginosa. The direct PCR method was also successful with the same band produced from the amplification. The whole process was completed within 4 h. The direct PCR amplification targeting at the exotoxin A gene of P. aeruginosa is potentially a rapid, specific, sensitive and relatively simple method for the identification of ocular isolates of P. aeruginosa.
Copyright 2000 Academic Press.