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

(2016) TOXINS. 8(5).
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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((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.
Keywords
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

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Citation

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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.
@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},
}

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