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
Authors: Banting Graham S. , Braithwaite Shannon , Scott Candis , Kim Jinyong , Jeon Byeonghwa , Ashbolt Nicholas , Ruecker Norma , Tymensen Lisa , Charest Jollin , Pintar Katarina , Checkley Sylvia , Neumann Norman F. 
Published: May 2016

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.

IMPORTANCE
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.