title: Evaluation of Various Campylobacter-Specific Quantitative PCR (qPCR) Assays for Detection and Enumeration of Campylobacteraceae in Irrigation Water and Wastewater via a Miniaturized Most-Probable-Number-qPCR Assay.
abstract: Campylobacter spp. are the leading cause of bacterial gastroenteritis worldwide, and water is increasingly seen as a risk factor in transmission. Here we describe a most-probable-number (MPN)-quantitative PCR (qPCR) assay in which water samples are centrifuged and aliquoted into microtiter plates and the bacteria are enumerated by qPCR. We observed that commonly used Campylobacter molecular assays produced vastly different detection rates. In irrigation water samples, detection rates varied depending upon the PCR assay and culture method used, as follows: 0% by the de Boer Lv1-16S qPCR assay, 2.5% by the Van Dyke 16S and Jensen glyA qPCR assays, and 75% by the Linton 16S endpoint PCR when cultured at 37°C. Primer/probe specificity was the major confounder, with Arcobacter spp. routinely yielding false-positive results. The primers and PCR conditions described by Van Dyke et al. (M. I. Van Dyke, V. K. Morton, N. L. McLellan, and P. M. Huck, J Appl Microbiol 109:1053-1066, 2010, http://dx.doi.org/10.1111/j.1365-2672.2010.04730.x) proved to be the most sensitive and specific for Campylobacter detection in water. Campylobacter occurrence in irrigation water was found to be very low (<2 MPN/300 ml) when this Campylobacter-specific qPCR was used, with the most commonly detected species being C. jejuni, C. coli, and C. lari Campylobacters in raw sewage were present at ∼10(2)/100 ml, with incubation at 42°C required for reducing microbial growth competition from arcobacters. Overall, when Campylobacter prevalence and/or concentration in water is reported using molecular methods, considerable validation is recommended when adapting methods largely developed for clinical applications. Furthermore, combining MPN methods with molecular biology-based detection algorithms allows for the detection and quantification of Campylobacter spp. in environmental samples and is potentially suited to quantitative microbial risk assessment for improved public health disease prevention related to food and water exposures.
  
  
  
  The results of this study demonstrate the importance of assay validation upon data interpretation of environmental monitoring for Campylobacter when using molecular biology-based assays. Previous studies describing Campylobacter prevalence in Canada utilized primers that we have determined to be nonspecific due to their cross-amplification of Arcobacter spp. As such, Campylobacter prevalence may have been vastly overestimated in other studies. Additionally, the development of a quantitative assay described in this study will allow accurate determination of Campylobacter concentrations in environmental water samples, allowing more informed decisions to be made about water usage based on quantitative microbial risk assessment.
authors.affiliation: School of Public Health, University of Alberta, Edmonton, Alberta, Canada.|Environmental Microbiology Program, Provincial Laboratory for Public Health, Edmonton, Alberta, Canada.
authors.forename: Graham S
authors.initials: GS
authors.lastname: Banting
authors.affiliation: School of Public Health, University of Alberta, Edmonton, Alberta, Canada.|Environmental Microbiology Program, Provincial Laboratory for Public Health, Edmonton, Alberta, Canada.
authors.forename: Shannon
authors.initials: S
authors.lastname: Braithwaite
authors.affiliation: School of Public Health, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Candis
authors.initials: C
authors.lastname: Scott
authors.affiliation: School of Public Health, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Jinyong
authors.initials: J
authors.lastname: Kim
authors.affiliation: School of Public Health, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Byeonghwa
authors.initials: B
authors.lastname: Jeon
authors.affiliation: School of Public Health, University of Alberta, Edmonton, Alberta, Canada.|Environmental Microbiology Program, Provincial Laboratory for Public Health, Edmonton, Alberta, Canada.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Ashbolt
authors.affiliation: Water Quality Services, Calgary, Alberta, Canada.
authors.forename: Norma
authors.initials: N
authors.lastname: Ruecker
authors.affiliation: Irrigation and Farm Water Division, Alberta Agriculture and Forestry, Lethbridge, Alberta, Canada.
authors.forename: Lisa
authors.initials: L
authors.lastname: Tymensen
authors.affiliation: Irrigation and Farm Water Division, Alberta Agriculture and Forestry, Lethbridge, Alberta, Canada.
authors.forename: Jollin
authors.initials: J
authors.lastname: Charest
authors.affiliation: Infectious Disease Prevention and Control Branch, Public Health Agency of Canada, Ottawa, Ontario, Canada.
authors.forename: Katarina
authors.initials: K
authors.lastname: Pintar
authors.affiliation: Environmental Microbiology Program, Provincial Laboratory for Public Health, Edmonton, Alberta, Canada.|Department of Ecosystem and Public Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Checkley
authors.affiliation: School of Public Health, University of Alberta, Edmonton, Alberta, Canada nfneuman@ualberta.ca.|Environmental Microbiology Program, Provincial Laboratory for Public Health, Edmonton, Alberta, Canada.
authors.forename: Norman F
authors.initials: NF
authors.lastname: Neumann
mesh_terms: D057914:Agricultural Irrigation; D002167:Campylobacter / Q000145:classification / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification*; D005618:Fresh Water / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D013045:Species Specificity; D062065:Wastewater / Q000382:microbiology*
chemical_list: D062065:Wastewater