MODIFICATION OF REAGENTS IN THE ENVIROAMP(TM) KIT TO INCREASE RECOVERY OF LEGIONELLA ORGANISMS IN WATER

Organisms of the bacterial genus Legionella, commonly found in aqueous reservoirs, have been associated with Legionnaires' disease (legionel la pneumonia, caused by Legionella pneumophila) and Pontiac fever (non pneumonic legionellosis). EnviroAmp(TM) Legionella sample preparation, polymerase chain reaction amplification, and detection kits (Perkin-E lmer Corp.) were developed for rapid detection of DNA from organisms o f the genus Legionella and the species L. pneumophila from environment al water samples. The kits are based on molecular techniques incorpora ting polymerase chain reaction amplification and detection by reverse dot blot hybridization to particular genus and species probes. The man ufacturer states that the EnviroAmp(TM) Legionella sample preparation, polymerase chain reaction amplification, and detection kits can detec t approximately 100 Legionella organisms/mL (10 000 organisms/100 mt) in the original water sample. The sensitivity of the kits was increase d to 0.1 colony-forming units/mL (10 colony-forming units/100mL), at l east for cultured organisms, by modifying the EnviroAmp(TM) Legionella sample preparation kit protocol. Data obtained in this study indicate d that sample volume could be increased from 100 to 1000 mt (in the ab sence of interfering substances such as humic acid) and DNA extraction volume could be decreased from 2 to 0.5 mt to increase the ability of the kit to detect lower numbers of Legionella spp. or L. pneumophila per volume.