Detection of viable Legionella pneumophila in water by polymerase chain reaction and gene probe methods

Abstract

Methods using polymerase chain reaction (PCR) and gene probes to detect viable Legionella pneumophila were investigated with cells exposed to biocide or elevated temperature. Exposure to hypochlorite caused viable nonculturable cells to form. Culturable and viable nonculturable cells showed positive PCR amplification, whereas nonviable cells did not. Viable cells were also specifically detected with mip mRNA as the target, reverse transcription (to form cDNA), and PCR amplification. After exposure to elevated temperature, only viable culturable cells were detected, which corresponded with positive PCR amplification.

Development of a simplified NASBA protocol for detecting viable cells of the citrus pathogen Xanthomonas citri subsp. citri under different treatments

“…PCR-based analyses are highly sensitive, rapid and specific, although both live and dead cells may contribute to positive signals (Josephson et al, 1993;Masters et al, 1994), and DNA may persist in a detectable form long after all viable organisms have been killed (Masters et al, 1994;Hellyer et al, 1999a). For these reasons, RNA has been proposed as a more representative target for assessing bacterial viability (Bej et al, 1991). Although rRNA has been employed in several works as a target for viability testing (McKillip et al, 1998;Villarino et al, 2000;Aellen et al, 2006), the long half-life of rRNA species makes this target a less accurate indicator than messenger RNA (Tolker-Nielsen et al, 1997;Rodriguez-Lá zaro et al, 2006).…”

An RNA transcription-based amplification technique (NASBA) for the detection of viable Salmonella enterica

“…Several types of RNA are produced in cells and one, messenger RNA (mRNA), functions as an intermediary in protein synthesis, and is thus found only in viable cells. The use of mRNA as a diagnostic of cell viability has been proposed (Bej et al 1991), but approaches to its detection have not produced unambiguous results. Generally, RT-PCR has been employed (Sheridan et al 1998;Vaitilingom et al 1998).…”

Effect of stress treatments on the detection of Listeria monocytogenes and enterotoxigenic Escherichia coli by the polymerase chain reaction

“…Although PCR gene probe methods can indicate the presence of a particular organism, they may not demonstrate unequivocally whether the cells are alive or dead. It has been claimed, however, that PCR can indicate the pres- ence of viable but non-culturable (VNC) cells of Legionella pneumophila (Bej et al 1991) and Shigella dysenteriae (Islam et al 1993).…”