Comparison of conventional culture and real-time quantitative PCR using SYBR Green for detection of Llegionella pneumophila 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.

Keywords

: Legionella pneumophila , real time PCR, SYBR Green