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

Two oligonucleotides were selected from the variable regions of the 16S rRNA gene of P. aeruginosa and used as PCR primers for the detection of P. aeruginosa. The specificity of the primers was tested against the following bacterial species; Pseudomonas putida, Pseudomonas cepacia, Xanthamonas maltophilia, Pseudomonas mendocina, Pseudomonas stutzeri, Pseudomonas fluouescens, Pseudomonas alcaligenes and Pseudomonas diminuta. These primers had a sensitivity of detection of 1 pg of chromosomal DNA or 1 X 10(5) cfu/mu l and were species-specific. The sensitivity 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 possible to detect the presence of P. aeruginosa from fleece washings collected from a flock of 100 sheep. Correlation between single PCR and bacteriological isolation showed agreement in 89% of fleece samples tested, 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.