Detection of Legionella pneumophila in wastewater by nested polymerase chain reaction

Catalán, Vicente; García, Fernando; Moreno, Carmen; Vila, M.J.; Apráiz, D.

doi:10.1016/s0923-2508(97)81902-x

Cited by 44 publications

(30 citation statements)

References 25 publications

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“…The NRCL anti-L. pneumophila sg 1 polyclonal antibody does not cross-react with other serogroups (data not shown). The Accurate Chemical & Scientific anti-L. pneumophila polyclonal antibody was found to be specific for sgs 1,6,7, and 12, as demonstrated by tests on L. pneumophila type strains (data not shown). The specificity of the MAbs from Dresden has already been reported for the different serogroups (10).…”

Section: Resultsmentioning

confidence: 92%

“…However, these techniques cannot be applied to the detection of rare events (15). Alternatively, PCR-based assays have been developed for Legionella but remain limited mainly because of (i) the potential presence of PCR inhibitors, (ii) the lack of information on the viability of cells, and (iii) the low sensitivity for the quantification of cells (6,28). The recent development of solid-phase cytometry (ChemScanRDI; Chemunex, Ivry-sur-Seine, France) has been seen as a great stride forward in the field of quality control, since it represents a rapid and sensitive device for the enumeration of low concentrations of fluorescence-labeled cells in water samples (15).…”

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confidence: 99%

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Rapid Detection and Enumeration of Legionella pneumophila in Hot Water Systems by Solid-Phase Cytometry

Aurell

Catala

Farge

et al. 2004

Appl Environ Microbiol

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A new method for the rapid and sensitive detection of Legionella pneumophila in hot water systems has been developed. The method is based on an IF assay combined with detection by solid-phase cytometry. This method allowed the enumeration of L. pneumophila serogroup 1 and L. pneumophila serogroups 2 to 6, 8 to 10, and 12 to 15 in tap water samples within 3 to 4 h. The sensitivity of the method was between 10 and 100 bacteria per liter and was principally limited by the filtration capacity of membranes. The specificity of the antibody was evaluated against 15 non-Legionella strains, and no cross-reactivity was observed. When the method was applied to natural waters, direct counts of L. pneumophila were compared with the number of CFU obtained by the standard culture method. Direct counts were always higher than culturable counts, and the ratio between the two methods ranged from 1.4 to 325. Solid-phase cytometry offers a fast and sensitive alternative to the culture method for L. pneumophila screening in hot water systems.

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Section: Resultsmentioning

confidence: 92%

mentioning

confidence: 99%

Rapid Detection and Enumeration of Legionella pneumophila in Hot Water Systems by Solid-Phase Cytometry

Aurell

Catala

Farge

et al. 2004

Appl Environ Microbiol

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“…Present in soil and natural aquatic environments (12), legionellae sometimes survives as an intracellular parasite of amoebae and ciliates (7). Legionellae have also found a niche in several man-made aquatic environments such as potable water systems, cooling towers, and wastewater systems (9,10). Consequently, the possible presence of legionellae in bioaerosols generated from soil or aquatic environments poses a significant hazard for human health (34).…”

mentioning

confidence: 99%

“…To avoid these problems, nucleic acid amplification techniques, mainly PCR, have been described as useful tools for the detection of L. pneumophila in clinical and environmental samples. Several PCR-based methods for the detection of L. pneumophila DNA have been described, but most of them are based on the amplification of the macrophage infectivity potentiator (mip) gene (4,9) or the 16S or 5S rRNA gene (17,36,45).…”

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confidence: 99%

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

Yáñez¹,

Carrasco-Serrano²,

Barberá³

et al. 2005

Appl Environ Microbiol

101

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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.

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“…Therefore, PCR is becoming an important and powerful tool for detection of pathogenic micro-organisms in the environment [16][17][18] . Selective enrichment followed by PCR is a rapid, sensitive, specifi c and viable method for screening of a large number of samples.…”

Section: Resultsmentioning

confidence: 99%

Semi-nested polymerase chain reaction for detection of toxigenic Vibrio cholerae from environmental water samples

et al. 2007

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A rapid and sensitive direct cell semi-nested PCR assay was developed for the detection of viable toxigenic V. cholerae in environmental water samples. The semi-nested PCR assay amplifi ed cholera toxin (ctxA2B) gene present in the toxigenic V. cholerae. The detection sensitivity of direct cell semi-nested PCR was 2 x 10 3 CFU of V. cholerae whereas direct cell single-step PCR could detect 2 x 10 4 CFU of V. cholerae. The performance of the assay was evaluated using environmental water samples after spiking with known number of Vibrio cholerae O1. The spiked water samples were fi ltered through a 0.22 micrometer membrane and the bacteria retained on fi lters were enriched in alkaline peptone water and then used directly in the PCR assay. The semi-nested PCR procedure coupled with enrichment could detect less than 1 CFU/ml in ground water and sea water whereas 2 CFU/ml and 20 CFU/ml could be detected in pond water and tap water, respectively. The proposed method is simple, faster than the conventional detection assays and can be used for screening of drinking water or environmental water samples for the presence of toxigenic V. cholerae.

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Detection of Legionella pneumophila in wastewater by nested polymerase chain reaction

Cited by 44 publications

References 25 publications

Rapid Detection and Enumeration of Legionella pneumophila in Hot Water Systems by Solid-Phase Cytometry

Rapid Detection and Enumeration of Legionella pneumophila in Hot Water Systems by Solid-Phase Cytometry

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

Semi-nested polymerase chain reaction for detection of toxigenic Vibrio cholerae from environmental water samples

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