Nested polymerase chain reaction for detection of Legionella pneumophila in water

Abstract

A highly sensitive nested polymerase chain reaction (PCR) method was evaluated for detection of Legionella pneumophila in water. Two sets of primers homologous to the coding region of the L. pneumophila macrophage infectivity potentiator ( mip) gene were used. Even when starting from minute amounts of L. pneumophila DNA, the double PCR products were readily detected by direct visualization in ethidium-bromide-stained agarose gels. The method was tested on 34 potable water samples from a hospital building and compared with standard culture isolation. L. pneumophila was isolated in only twelve samples, whereas, by nested PCR, 19 samples were positive, 12 of them coincidental with culturing. These results indicate that nested PCR permits detection of L. pneumophila in samples where culturing fails, with the advantage of a rapid turnaround time, simplicity and the ability to detect non-culturable cells, fulfilling the requirements of sensitivity and specificity for routine use in an environmental laboratory.