Post by flounder9 on Apr 16, 2012 17:00:18 GMT -5
Highly Efficient Amplification of Chronic Wasting Disease Agent by Protein Misfolding Cyclic Amplification with Beads (PMCAb)
Chad J. Johnson1, Judd M. Aiken2, Debbie McKenzie2, Michael D. Samuel3, Joel A. Pedersen1*
1 Environmental Chemistry and Technology Program and Department of Soil Science, University of Wisconsin, Madison, Wisconsin, United States of America, 2 Center for Prions and Protein Misfolding Diseases, University of Alberta, Edmonton, Alberta, Canada, 3 U.S. Geological Survey, Wisconsin Cooperative Wildlife Research Unit, University of Wisconsin, Madison, Wisconsin, United States of America
Abstract Top
Protein misfolding cyclic amplification (PMCA) has emerged as an important technique for detecting low levels of pathogenic prion protein in biological samples. The method exploits the ability of the pathogenic prion protein to convert the normal prion protein to a proteinase K-resistant conformation. Inclusion of Teflon® beads in the PMCA reaction (PMCAb) has been previously shown to increase the sensitivity and robustness of detection for the 263 K and SSLOW strains of hamster-adapted prions. Here, we demonstrate that PMCAb with saponin dramatically increases the sensitivity of detection for chronic wasting disease (CWD) agent without compromising the specificity of the assay (i.e., no false positive results). Addition of Teflon® beads increased the robustness of the PMCA reaction, resulting in a decrease in the variability of PMCA results. Three rounds of serial PMCAb allowed detection of CWD agent from a 6.7×10−13 dilution of 10% brain homogenate (1.3 fg of source brain). Titration of the same brain homogenate in transgenic mice expressing cervid prion protein (Tg(CerPrP)1536+/− mice) allowed detection of CWD agent from the 10−6 dilution of 10% brain homogenate. PMCAb is, thus, more sensitive than bioassay in transgenic mice by a factor exceeding 105. Additionally, we are able to amplify CWD agent from brain tissue and lymph nodes of CWD-positive white-tailed deer having Prnp alleles associated with reduced disease susceptibility.
SNIP...
Our study extends the utility of PMCAb to a new prion strain and demonstrates that this method is substantially more sensitive than animal bioassay. The higher sensitivity and increased robustness of PMCAb is expected to allow early stages of disease to be delineated (i.e., PrPTSE trafficking during initial exposure), determination of efficacies of decontamination methods, and detection of CWD agent in naturally contaminated environmental samples. Enabling the identification of environmental reservoirs of infectivity would lead to better understanding of CWD epizootics.
Citation: Johnson CJ, Aiken JM, McKenzie D, Samuel MD, Pedersen JA (2012) Highly Efficient Amplification of Chronic Wasting Disease Agent by Protein Misfolding Cyclic Amplification with Beads (PMCAb). PLoS ONE 7(4): e35383. doi:10.1371/journal.pone.0035383
Editor: Ilia V. Baskakov, University of Maryland, United States of America
Received: December 18, 2011; Accepted: March 15, 2012; Published: April 13, 2012
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported by NSF grants DEB-0914484 (MDS) and CBET-0547484 (JAP), NIH grant R01 NS060034 (JMA/JAP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: joelpedersen@wisc.edu
www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035383
chronic-wasting-disease.blogspot.com/
transmissiblespongiformencephalopathy.blogspot.com/
TSS
Chad J. Johnson1, Judd M. Aiken2, Debbie McKenzie2, Michael D. Samuel3, Joel A. Pedersen1*
1 Environmental Chemistry and Technology Program and Department of Soil Science, University of Wisconsin, Madison, Wisconsin, United States of America, 2 Center for Prions and Protein Misfolding Diseases, University of Alberta, Edmonton, Alberta, Canada, 3 U.S. Geological Survey, Wisconsin Cooperative Wildlife Research Unit, University of Wisconsin, Madison, Wisconsin, United States of America
Abstract Top
Protein misfolding cyclic amplification (PMCA) has emerged as an important technique for detecting low levels of pathogenic prion protein in biological samples. The method exploits the ability of the pathogenic prion protein to convert the normal prion protein to a proteinase K-resistant conformation. Inclusion of Teflon® beads in the PMCA reaction (PMCAb) has been previously shown to increase the sensitivity and robustness of detection for the 263 K and SSLOW strains of hamster-adapted prions. Here, we demonstrate that PMCAb with saponin dramatically increases the sensitivity of detection for chronic wasting disease (CWD) agent without compromising the specificity of the assay (i.e., no false positive results). Addition of Teflon® beads increased the robustness of the PMCA reaction, resulting in a decrease in the variability of PMCA results. Three rounds of serial PMCAb allowed detection of CWD agent from a 6.7×10−13 dilution of 10% brain homogenate (1.3 fg of source brain). Titration of the same brain homogenate in transgenic mice expressing cervid prion protein (Tg(CerPrP)1536+/− mice) allowed detection of CWD agent from the 10−6 dilution of 10% brain homogenate. PMCAb is, thus, more sensitive than bioassay in transgenic mice by a factor exceeding 105. Additionally, we are able to amplify CWD agent from brain tissue and lymph nodes of CWD-positive white-tailed deer having Prnp alleles associated with reduced disease susceptibility.
SNIP...
Our study extends the utility of PMCAb to a new prion strain and demonstrates that this method is substantially more sensitive than animal bioassay. The higher sensitivity and increased robustness of PMCAb is expected to allow early stages of disease to be delineated (i.e., PrPTSE trafficking during initial exposure), determination of efficacies of decontamination methods, and detection of CWD agent in naturally contaminated environmental samples. Enabling the identification of environmental reservoirs of infectivity would lead to better understanding of CWD epizootics.
Citation: Johnson CJ, Aiken JM, McKenzie D, Samuel MD, Pedersen JA (2012) Highly Efficient Amplification of Chronic Wasting Disease Agent by Protein Misfolding Cyclic Amplification with Beads (PMCAb). PLoS ONE 7(4): e35383. doi:10.1371/journal.pone.0035383
Editor: Ilia V. Baskakov, University of Maryland, United States of America
Received: December 18, 2011; Accepted: March 15, 2012; Published: April 13, 2012
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported by NSF grants DEB-0914484 (MDS) and CBET-0547484 (JAP), NIH grant R01 NS060034 (JMA/JAP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: joelpedersen@wisc.edu
www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035383
chronic-wasting-disease.blogspot.com/
transmissiblespongiformencephalopathy.blogspot.com/
TSS