Comparison of conventional culture and real-time quantitative PCR using SYBR Green for detection of <i>Llegionella pneumophila</i> in water samples

Abstract

The genus Legionella comprises more than 40 species and 64 serogroups with approximately half of those species associated with human diseases. Legionella pneumophila Serogroup 1 is the most common pathogenic species and is responsible for up to 80% of legionellosis cases in the world. Legionella levels in water are assessed routinely by culture on a selective medium, but its slow growth is a serious drawback, given that at least 10 days are required to obtain results. In an attempt to provide a simple screening method for Legionella pneumophila in water systems samples a real time PCR assay using SYBR Green was developed. A total of 50 samples from cooling towers and hot tap water systems were analysed by DNA amplification using 2 pairs of primers targeting the mip and dot genes. Legionella pneumophila Serogroup 1 (NCTC12821) was used as a reference strain and to evaluate real-time PCR performance. The assays were successful with both primer sets; good and similar amplification efficiencies were achieved. In addition, high sensitivity was obtained; the method proved to allow for the detection of fewer than 10 gene copies per reaction. Results of real-time PCR were compared to conventional analysis based on culture. Although no strong correlation was observed between both methods and consequently real-time PCR could not substitute for the reference method, it represents a powerful screening tool. The inexpensive, sensitive and rapid real-time PCR based in SYBR Green method is of interest in monitoring Legionella pneumophila contamination, especially in environmental samples, and should be economical for large-scale routine tests.

Fate of bacterial indicators and Salmonella in biofilm developed on ultrafiltration membranes treating secondary effluents of domestic wastewater

“…4). Higher qrt-PCR counts compared to plate counts have been reported in other studies 32–34 and were attributed to the higher sensitivity of the qrt-PCR for quantification of bacteria in the VBNC state 33–35 . Higher qrt-PCR counts may also result from overestimation of the E. coli numbers on the membrane surface.…”

Legionella Detection in Environmental Samples as an Example for Successful Implementation of qPCR

“…Another difficulty in the comparison of qPCR and culture is the difference between GU and CFU, where a conversion equation for Legionella has not been established yet. Elevated detection of L. pneumophila by qPCR is also displayed in the low PPV values [18][19][20][21][22]. The sample type also seems to determine the ability to compare qPCR and culture results.…”

Viability qPCR, a new tool for Legionella risk management

“…This has been one of the drawbacks, but not the main one, for the implementation of molecular methods in risk management in water systems. Previous studies conducted with qPCR methods demonstrate a bias of several orders of magnitude between qPCR and culture (Fittipladi et al, 2010), and one additional persistent reality is that the qPCR also detects DNA from dead cells (Whiley, 2016). Although the bias can be managed by applying correction factors, the detection of DNA from dead cells introduces considerable uncertainty.…”