A COLONY BASED CONFIRMATION ASSAY FOR LEGIONELLA AND LEGIONELLA-PNEUMOPHILA EMPLOYING THE ENVIROAMP(TM) LEGIONELLA SYSTEM AND SEROAGGLUTINATION

Modifications to the EnviroAmp(TM) Legionella detection System are des cribed which permit the rapid analysis of bacterial colonies taken fro m Legionella selective media. Capillary PCR permitted twice the number of samples to be analysed with a single kit; When PCR was positive fo r Leg. pneumophila, this result was confirmed by seroagglutination. Th e reverse dot blot hybridization assay was only used where PCR indicat ed a Legionella sp. other than Leg. pneumophila, permitting further sa vings on detection system components. This technique and standard conf irmation procedures were applied to 133 isolates arising from 63 water samples plated to Legionella isolation media. Results agreed except f or two isolates which gave a positive result for Legionella spp. by PC R and hybridization but were negative using standard procedures. Raisi ng the annealing/extension temperature of the PCR by 2 degrees C elimi nated the false positive result with these two isolates but did not ad versely effect the sensitivity of the assay, as determined by re-testi ng of 68 environmental isolates and testing of 69 new environmental is olates and 12 Legionella reference species. The modified technique pro vides a convenient and cost effective alternative to standard confirma tion procedures.