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Comparison of droplet digital PCR and qPCR for the quantification of Shiga toxin-producing Escherichia coli in bovine feces

Bavo Verhaegen, Koen De Reu, Lieven De Zutter UGent, Karen Verstraete, Marc Heyndrickx UGent and Els Van Coillie (2016) TOXINS. 8(5).
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((R)) Environmental Master Mix 2.0; UMM: TaqMan((R)) 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.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
Shiga Toxin-producing Escherichia coli, REAL-TIME PCR, cattle, PCR inhibition, real-time qualitative PCR, droplet digital PCR, POLYMERASE-CHAIN-REACTION, QUANTITATIVE PCR, CATTLE FECES, O157, STEC, ASSAY, TRANSMISSION, SEROGROUPS, STRAINS
journal title
TOXINS
Toxins
volume
8
issue
5
article number
157
pages
11 pages
Web of Science type
Article
Web of Science id
000377428300037
JCR category
TOXICOLOGY
JCR impact factor
3.03 (2016)
JCR rank
30/92 (2016)
JCR quartile
2 (2016)
ISSN
2072-6651
DOI
10.3390/toxins8050157
language
English
UGent publication?
yes
classification
A1
copyright statement
I have retained and own the full copyright for this publication
id
8029151
handle
http://hdl.handle.net/1854/LU-8029151
date created
2016-07-07 11:15:25
date last changed
2016-12-21 15:42:10
@article{8029151,
  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((R)) Environmental Master Mix 2.0; UMM: TaqMan((R)) 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.},
  articleno    = {157},
  author       = {Verhaegen, Bavo and De Reu, Koen and De Zutter, Lieven and Verstraete, Karen and Heyndrickx, Marc and Van Coillie, Els},
  issn         = {2072-6651},
  journal      = {TOXINS},
  keyword      = {Shiga Toxin-producing Escherichia coli,REAL-TIME PCR,cattle,PCR inhibition,real-time qualitative PCR,droplet digital PCR,POLYMERASE-CHAIN-REACTION,QUANTITATIVE PCR,CATTLE FECES,O157,STEC,ASSAY,TRANSMISSION,SEROGROUPS,STRAINS},
  language     = {eng},
  number       = {5},
  pages        = {11},
  title        = {Comparison of droplet digital PCR and qPCR for the quantification of Shiga toxin-producing Escherichia coli in bovine feces},
  url          = {http://dx.doi.org/10.3390/toxins8050157},
  volume       = {8},
  year         = {2016},
}

Chicago
Verhaegen, Bavo, Koen De Reu, Lieven De Zutter, Karen Verstraete, Marc Heyndrickx, and Els Van Coillie. 2016. “Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-producing Escherichia Coli in Bovine Feces.” Toxins 8 (5).
APA
Verhaegen, Bavo, De Reu, K., De Zutter, L., Verstraete, K., Heyndrickx, M., & Van Coillie, E. (2016). Comparison of droplet digital PCR and qPCR for the quantification of Shiga toxin-producing Escherichia coli in bovine feces. TOXINS, 8(5).
Vancouver
1.
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(5).
MLA
Verhaegen, Bavo, Koen De Reu, Lieven De Zutter, et al. “Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-producing Escherichia Coli in Bovine Feces.” TOXINS 8.5 (2016): n. pag. Print.