Quantitative Detection ofLegionella pneumophilain Water Samples by Immunomagnetic Purification and Real-Time PCR Amplification of thedotAGene

Abstract

A new real-time PCR assay was developed and validated in combination with an immunomagnetic separation system for the quantitative determination of Legionella pneumophila in water samples. Primers that amplify simultaneously an 80-bp fragment of the dotA gene from L. pneumophila and a recombinant fragment including a specific sequence of the gyrB gene from Aeromonas hydrophila, added as an internal positive control, were used. The specificity, limit of detection, limit of quantification, repetitivity, reproducibility, and accuracy of the method were calculated, and the values obtained confirmed the applicability of the method for the quantitative detection of L. pneumophila. Moreover, the efficiency of immunomagnetic separation in the recovery of L. pneumophila from different kinds of water was evaluated. The recovery rates decreased as the water contamination increased (ranging from 59.9% for distilled water to 36% for cooling tower water), and the reproducibility also decreased in parallel to water complexity. The feasibility of the method was evaluated by cell culture and real-time PCR analysis of 60 samples in parallel. All the samples found to be positive by cell culture were also positive by real-time PCR, while only eight samples were found to be positive only by PCR. Finally, the correlation of both methods showed that the number of cells calculated by PCR was 20-fold higher than the culture values. In conclusion, the real-time PCR method combined with immunomagnetic separation provides a sensitive, specific, and accurate method for the rapid quantification of L. pneumophila in water samples. However, the recovery efficiency of immunomagnetic separation should be considered in complex samples.

Quantitative Real-Time Legionella PCR for Environmental Water Samples: Data Interpretation

“…We observed, as in other studies (24,27), higher concentrations of legionellae by quantitative PCR than by conventional culture, both for hot water system and cooling tower samples (Fig. 2).…”

Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations

“…Commercial and inhouse-developed NAATs are used in both clinical and environmental laboratories; however, the list of Legionella genes amplified is limited. The most common targets include a conserved segment of the rRNA genes for the 5S and 16S subunits, the 16S-23S spacer, and/or the macrophage inhibitor protein mip, found primarily in the genus Legionella and highly conserved in all L. pneumophila isolates (226,(332)(333)(334)(349)(350)(351)(352)(353)(354)(355)(356)(357)(358). Several studies have also examined alternative chromosomal targets, such as dotA, gyrB, dnaJ, wzm, and wzt (340).…”

Surveillance of parasiticLegionellain surface waters by using immunomagnetic separation and amoebae enrichment

“…It is widely applied for separating pathogens in foods, clinical specimens and environmental samples. [19][20][21] Several studies have independently confirmed the efficiency of IMS in recovering Legionella spp. from different aquatic environments.…”

“…from different aquatic environments. [20][21][22] In this study, we utilised IMS to pinpoint extracellular Legionella or to remove most free organisms to facilitate detecting parasitic Legionella after selective amoebae enrichment. By comparing the detection rates of different approaches, we determined the presence and species of parasitic Legionella.…”