Modification of reagents in the EnviroAmp™ kit to increase recovery of Legionella organisms in water

Abstract

Organisms of the bacterial genus Legionella, commonly found in aqueous reservoirs, have been associated with Legionnaires' disease (legionella pneumonia, caused by Legionella pneumophila) and Pontiac fever (nonpneumonic legionellosis). EnviroAmp™ Legionella sample preparation, polymerase chain reaction amplification, and detection kits (Perkin-Elmer Corp.) were developed for rapid detection of DNA from organisms of the genus Legionella and the species L. pneumophila from environmental water samples. The kits are based on molecular techniques incorporating polymerase chain reaction amplification and detection by reverse dot blot hybridization to particular genus and species probes. The manufacturer states that the EnviroAmp™ Legionella sample preparation, polymerase chain reaction amplification, and detection kits can detect approximately 100 Legionella organisms/mL (10 000 organisms/100 mL) in the original water sample. The sensitivity of the kits was increased to 0.1 colony-forming units/mL (10 colony-forming units/100 mL), at least for cultured organisms, by modifying the EnviroAmp™ Legionella sample preparation kit protocol. Data obtained in this study indicated that sample volume could be increased from 100 to 1000 mL (in the absence of interfering substances such as humic acid) and DNA extraction volume could be decreased from 2 to 0.5 mL to increase the ability of the kit to detect lower numbers of Legionella spp. or L. pneumophila per volume.Key words: Legionella, environment, water, EnviroAmp™ kit.