Evaluation of nested PCR assays for the detection of Legionella pneumophila in a wide range of aquatic samples.
OBJECTIVE
To compare the sensitivities of two nested PCR assays for the detection of Legionella pneumophila to each other and to the plate counting technique (ISO 11731) in a wide range of aquatic samples.


RESULTS
The nested PCR assay with the primer set LEG 225-LEG 858 revealed 56% of the 46 analysed aquatic samples as being positive for Legionella spp., while the primer set JFP-JRP yielded 98% positive samples. The detection was confirmed by sequencing the PCR products. These results are considerably higher than the result obtained with the plate counting technique (41%), indicating the higher sensitivity of PCR-based diagnostic methods. As the PCR assay with the LEG 225-LEG 858 primer set resulted in a lower number of positive samples, it is considered not sensitive enough for aquatic samples. Similar results for the respective primer sets were obtained for the detection of the species L. pneumophila, responsible for 90% of all human Legionella infections, in the aquatic samples analysed. Both microbial community analysis by PCR-denaturing gradient gel electrophoresis and the analysis of biotic and abiotic water quality parameters revealed no relation between L. pneumophila-positive and -negative samples and the physico-chemical and bacteriological characteristics of the aquatic samples.


CONCLUSIONS
The results show the additional value of the PCR assay with the JFP-JRP primer set compared with the plate counting technique, as well as its applicability in a wide range of aquatic samples.


CONCLUSIONS
This study shows the importance of comparing different primer sets for nested PCR assays for the detection of L. pneumophila in aquatic samples, as well as the lower sensitivity of the widely accepted plate counting technique (ISO 11731).
Devos
L
L
Laboratory of Microbial Ecology and Technology (LabMET), Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
Clymans
K
K
Boon
N
N
Verstraete
W
W
D001483:Base Sequence; D015169:Colony Count, Microbial / Q000379:methods; D003470:Culture Media; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D004586:Electrophoresis / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D016952:Legionella pneumophila / Q000302:isolation & purification*; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D014871:Water Microbiology
D003470:Culture Media; D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal