DETECTION OF PSEUDOMONAS-AERUGINOSA FROM OVINE FLEECE WASHINGS BY PCRAMPLIFICATION OF 16S RIBOSOMAL-RNA

Two oligonucleotides were selected from the variable regions of the 16 S rRNA gene of P. aeruginosa and used as PCR primers for the detection of P. aeruginosa. The specificity of the primers was tested against t he following bacterial species; Pseudomonas putida, Pseudomonas cepaci a, Xanthamonas maltophilia, Pseudomonas mendocina, Pseudomonas stutzer i, Pseudomonas fluouescens, Pseudomonas alcaligenes and Pseudomonas di minuta. These primers had a sensitivity of detection of 1 pg of chromo somal DNA or 1 X 10(5) cfu/mu l and were species-specific. The sensiti vity of detection was increased to 1 fg or less than 10 cfu/mu l using a non-radioactively labelled probe. Using these PCR primers it was po ssible to detect the presence of P. aeruginosa from fleece washings co llected from a flock of 100 sheep. Correlation between single PCR and bacteriological isolation showed agreement in 89% of fleece samples te sted, 2% of the samples contained organic PCR inhibitors in the fleece washings, 3% were below the level of sensitivity of the test and the remaining 6% were culture negative for P. aeruginosa but PCR positive. Use of nested PCR did not increase the sensitivity of detection over single round PCR combined with the use of a non-radiactively labelled probe.