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Toxins 2016, 8(5), 157; doi:10.3390/toxins8050157

Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces

1
Institute of Agriculture and Fishery Research (ILVO), Technology and Food Science Unit, Brusselsesteenweg 370, Melle 9090, Belgium
2
Department of Veterinary Public Health and Food Safety, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, Merelbeke 9820, Belgium
3
Department of Pathology, Faculty of Veterinary Medicine, Bacteriology and Poultry Diseases, Ghent University; Salisburylaan 133, Merelbeke 9820, Belgium
*
Author to whom correspondence should be addressed.
Academic Editors: Gerald B. Koudelka and Steven A. Mauro
Received: 29 March 2016 / Revised: 21 April 2016 / Accepted: 10 May 2016 / Published: 18 May 2016
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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. View Full-Text
Keywords: Shiga Toxin-producing Escherichia coli; real-time qualitative PCR; droplet digital PCR; PCR inhibition; cattle Shiga Toxin-producing Escherichia coli; real-time qualitative PCR; droplet digital PCR; PCR inhibition; cattle
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Verhaegen, B.; De Reu, K.; De Zutter, L.; Verstraete, K.; Heyndrickx, M.; Van Coillie, E. Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces. Toxins 2016, 8, 157.

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