A novel triplex quantitative PCR strategy for quantification of toxigenic and nontoxigenic Vibrio cholerae in aquatic environments.
Vibrio cholerae is a severe human pathogen and a frequent member of aquatic ecosystems. Quantification of V. cholerae in environmental water samples is therefore fundamental for ecological studies and health risk assessment. Beside time-consuming cultivation techniques, quantitative PCR (qPCR) has the potential to provide reliable quantitative data and offers the opportunity to quantify multiple targets simultaneously. A novel triplex qPCR strategy was developed in order to simultaneously quantify toxigenic and nontoxigenic V. cholerae in environmental water samples. To obtain quality-controlled PCR results, an internal amplification control was included. The qPCR assay was specific, highly sensitive, and quantitative across the tested 5-log dynamic range down to a method detection limit of 5 copies per reaction. Repeatability and reproducibility were high for all three tested target genes. For environmental application, global DNA recovery (GR) rates were assessed for drinking water, river water, and water from different lakes. GR rates ranged from 1.6% to 76.4% and were dependent on the environmental background. Uncorrected and GR-corrected V. cholerae abundances were determined in two lakes with extremely high turbidity. Uncorrected abundances ranged from 4.6×10(2) to 2.3×10(4) cell equivalents liter(-1), whereas GR-corrected abundances ranged from 4.7×10(3) to 1.6×10(6) cell equivalents liter(-1). GR-corrected qPCR results were in good agreement with an independent cell-based direct detection method but were up to 1.6 log higher than cultivation-based abundances. We recommend the newly developed triplex qPCR strategy as a powerful tool to simultaneously quantify toxigenic and nontoxigenic V. cholerae in various aquatic environments for ecological studies as well as for risk assessment programs.
Institute for Hygiene and Applied Immunology, Water Hygiene, Medical University of Vienna, Vienna, Austria Armament and Defence Technology Agency, NBC & Environmental Protection Technology Division, Vienna, Austria.
Rupert
R
Bliem
Institute for Hygiene and Applied Immunology, Water Hygiene, Medical University of Vienna, Vienna, Austria.
Sonja
S
Schauer
Armament and Defence Technology Agency, NBC & Environmental Protection Technology Division, Vienna, Austria.
Helga
H
Plicka
Federal Ministry of Defence and Sports, Division of Science, Research and Development, Vienna, Austria.
Adelheid
A
Obwaller
Institute for Hygiene and Applied Immunology, Water Hygiene, Medical University of Vienna, Vienna, Austria Interuniversity Cooperation Centre Water and Health (ICC Water & Health), Vienna, Austria.
Regina
R
Sommer
Institute for Veterinary Disease Control Mödling, Dept. for Molecular Biology, Austrian Agency for Health and Food Safety (AGES), Mödling, Austria.
Adolf
A
Steinrigl
Enteric & Food Microbiology Laboratory, Centre for Food and Waterborne Diseases (CFWD) International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), Dhaka, Bangladesh.
Munirul
M
Alam
Institute of Chemical Engineering, Research Group Environmental Microbiology and Molecular Ecology, Vienna University of Technology, Vienna, Austria Interuniversity Cooperation Centre Water and Health (ICC Water & Health), Vienna, Austria.
Georg H
GH
Reischer
Institute of Chemical Engineering, Research Group Environmental Microbiology and Molecular Ecology, Vienna University of Technology, Vienna, Austria Interuniversity Cooperation Centre Water and Health (ICC Water & Health), Vienna, Austria.
Andreas H
AH
Farnleitner
Institute for Hygiene and Applied Immunology, Water Hygiene, Medical University of Vienna, Vienna, Austria Interuniversity Cooperation Centre Water and Health (ICC Water & Health), Vienna, Austria alexander.kirschner@meduniwien.ac.at.
Alexander
A
Kirschner
D001431:Bacteriological Techniques / Q000379:methods* / Q000592:standards; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014734:Vibrio cholerae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D014871:Water Microbiology
D004269:DNA, Bacterial