title: Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces.
abstract: Cattle are considered to be the main reservoir for Shiga toxin-producing Escherichia coli (STEC) and are often the direct or indirect source of STEC outbreaks in humans. Accurate measurement of the concentration of shed STEC in cattle feces could be a key answer to questions concerning transmission of STEC, contamination sources and efficiency of treatments at farm level. Infected animals can be identified and the contamination level quantified by real-time quantitative PCR (qPCR), which has its specific limitations. Droplet digital PCR (ddPCR) has been proposed as a method to overcome many of the drawbacks of qPCR. This end-point amplification PCR is capable of absolute quantification independent from any reference material and is less prone to PCR inhibition than qPCR. In this study, the qPCR-based protocol described by Verstraete et al. (2014) for Shiga toxin genes stx1 and stx2 and the intimin gene eae quantification was optimized for ddPCR analysis. The properties of ddPCR and qPCR using two different mastermixes (EMM: TaqMan(®) Environmental Master Mix 2.0; UMM: TaqMan(®) Universal PCR Master Mix) were evaluated, using standard curves and both artificial and natural contaminated cattle fecal samples. In addition, the susceptibility of these assays to PCR-inhibitors was investigated. Evaluation of the standard curves and both artificial and natural contaminated cattle fecal samples suggested a very good agreement between qPCR using EMM and ddPCR. Furthermore, similar sensitivities and no PCR inhibition were recorded for both assays. On the other hand, qPCR using UMM was clearly prone to PCR inhibition. In conclusion, the ddPCR technique shows potential for the accurate absolute quantification of STEC on the farms, without relying on standardized reference material.
authors.affiliation: Institute of Agriculture and Fishery Research (ILVO), Technology and Food Science Unit, Brusselsesteenweg 370, Melle 9090, Belgium. bavo.verhaegen@ilvo.vlaanderen.be.|Department of Veterinary Public Health and Food Safety, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, Merelbeke 9820, Belgium. bavo.verhaegen@ilvo.vlaanderen.be.
authors.forename: Bavo
authors.initials: B
authors.lastname: Verhaegen
authors.affiliation: Institute of Agriculture and Fishery Research (ILVO), Technology and Food Science Unit, Brusselsesteenweg 370, Melle 9090, Belgium. koen.dereu@ilvo.vlaanderen.be.
authors.forename: Koen
authors.initials: K
authors.lastname: De Reu
authors.affiliation: Department of Veterinary Public Health and Food Safety, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, Merelbeke 9820, Belgium. lieven.dezutter@ugent.be.
authors.forename: Lieven
authors.initials: L
authors.lastname: De Zutter
authors.affiliation: Institute of Agriculture and Fishery Research (ILVO), Technology and Food Science Unit, Brusselsesteenweg 370, Melle 9090, Belgium. karen.verstraete@ilvo.vlaanderen.be.
authors.forename: Karen
authors.initials: K
authors.lastname: Verstraete
authors.affiliation: Institute of Agriculture and Fishery Research (ILVO), Technology and Food Science Unit, Brusselsesteenweg 370, Melle 9090, Belgium. marc.heyndrickx@ilvo.vlaanderen.be.|Department of Pathology, Faculty of Veterinary Medicine, Bacteriology and Poultry Diseases, Ghent University; Salisburylaan 133, Merelbeke 9820, Belgium. marc.heyndrickx@ilvo.vlaanderen.be.
authors.forename: Marc
authors.initials: M
authors.lastname: Heyndrickx
authors.affiliation: Institute of Agriculture and Fishery Research (ILVO), Technology and Food Science Unit, Brusselsesteenweg 370, Melle 9090, Belgium. els.vancoillie@ilvo.vlaanderen.be.
authors.forename: Els
authors.initials: E
authors.lastname: Van Coillie
mesh_terms: D000818:Animals; D002417:Cattle; D005243:Feces / Q000382:microbiology*; D005798:Genes, Bacterial; D016133:Polymerase Chain Reaction / Q000379:methods*; D054323:Shiga-Toxigenic Escherichia coli / Q000235:genetics / Q000302:isolation & purification*; D014774:Virulence / Q000235:genetics
keywords: PCR inhibition; Shiga Toxin-producing Escherichia coli; cattle; droplet digital PCR; real-time qualitative PCR