Highly Efficient Amplification of Chronic Wasting Disease Agent by Protein Misfolding Cyclic Amplification with Beads (PMCAb)
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{"title"=>"Highly efficient amplification of chronic wasting disease agent by protein misfolding cyclic amplification with beads (PMCAB)", "type"=>"journal", "authors"=>[{"first_name"=>"Chad J.", "last_name"=>"Johnson", "scopus_author_id"=>"26027904200"}, {"first_name"=>"Judd M.", "last_name"=>"Aiken", "scopus_author_id"=>"7101788870"}, {"first_name"=>"Debbie", "last_name"=>"McKenzie", "scopus_author_id"=>"7202305081"}, {"first_name"=>"Michael D.", "last_name"=>"Samuel", "scopus_author_id"=>"7102728127"}, {"first_name"=>"Joel A.", "last_name"=>"Pedersen", "scopus_author_id"=>"7401515158"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pmid"=>"22514738", "doi"=>"10.1371/journal.pone.0035383", "pui"=>"364617613", "issn"=>"19326203", "sgr"=>"84859729426", "scopus"=>"2-s2.0-84859729426"}, "id"=>"520ffbc3-e67e-37d0-bd5d-cab65f6124fc", "abstract"=>"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 10(5). 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.", "link"=>"http://www.mendeley.com/research/highly-efficient-amplification-chronic-wasting-disease-agent-protein-misfolding-cyclic-amplification", "reader_count"=>21, "reader_count_by_academic_status"=>{"Student > Doctoral Student"=>2, "Researcher"=>7, "Student > Ph. D. Student"=>5, "Other"=>1, "Student > Bachelor"=>3, "Professor"=>2, "Unspecified"=>1}, "reader_count_by_user_role"=>{"Student > Doctoral Student"=>2, "Researcher"=>7, "Student > Ph. D. Student"=>5, "Other"=>1, "Student > Bachelor"=>3, "Professor"=>2, "Unspecified"=>1}, "reader_count_by_subject_area"=>{"Engineering"=>1, "Unspecified"=>2, "Environmental Science"=>2, "Biochemistry, Genetics and Molecular Biology"=>6, "Agricultural and Biological Sciences"=>5, "Medicine and Dentistry"=>1, "Neuroscience"=>1, "Veterinary Science and Veterinary Medicine"=>3}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Neuroscience"=>{"Neuroscience"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>5}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>6}, "Unspecified"=>{"Unspecified"=>2}, "Environmental Science"=>{"Environmental Science"=>2}, "Veterinary Science and Veterinary Medicine"=>{"Veterinary Science and Veterinary Medicine"=>3}}, "reader_count_by_country"=>{"Italy"=>1}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/653775"], "description"=>"<p>CWD-positive brain homogenate (BH; 10% w/v) was diluted using serial five-fold dilutions in NBH and subjected to one round of 96-cycle PMCAb in the presence of absence of 0.045% saponin. BH, brain homogenate; MWM, molecular weight marker.</p>", "links"=>[], "tags"=>["increases", "pmcab"], "article_id"=>324264, "categories"=>["Physics", "Virology", "Infectious Diseases", "Biophysics"], "users"=>["Chad J. Johnson", "Judd M. Aiken", "Debbie McKenzie", "Michael D. Samuel", "Joel A. Pedersen"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0035383.g002", "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Saponin_increases_the_sensitivity_of_PMCAb_for_PrP_CWD_detection_/324264", "title"=>"Saponin increases the sensitivity of PMCAb for PrP<sup>CWD</sup> detection.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-13 01:11:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/653963"], "description"=>"<p>Four 10 µL samples of 4×10<sup>−3</sup> or 1.6×10<sup>−4</sup> dilutions of CWD-positive BH (10%) were added to 90 µL NBH containing 0.05% saponin and subjected to PMCAb for the indicated number of cycles.</p>", "links"=>[], "tags"=>["cwd", "amplification"], "article_id"=>324454, "categories"=>["Physics", "Virology", "Infectious Diseases", "Biophysics"], "users"=>["Chad J. Johnson", "Judd M. Aiken", "Debbie McKenzie", "Michael D. Samuel", "Joel A. Pedersen"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0035383.g005", "stats"=>{"downloads"=>1, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Kinetics_of_CWD_amplification_using_PMCAb_/324454", "title"=>"Kinetics of CWD amplification using PMCAb.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-13 01:14:14"}
  • {"files"=>["https://ndownloader.figshare.com/files/653718"], "description"=>"<p>CWD-positive brain homogenate (BH; 10% w/v) was serially diluted in normal BH (NBH). A 10 µL aliquot of the seed dilution was transferred into 90 µL NBH with 0.05% saponin in five-fold increments and subjected to PMCA (96 cycles) in the presence or absence of two 2.32 mm Teflon® beads (PMCAb). The no-seed control was 100 µL NBH treated as the other samples. MWM, molecular weight marker.</p>", "links"=>[], "tags"=>["beads", "pmca"], "article_id"=>324209, "categories"=>["Physics", "Virology", "Infectious Diseases", "Biophysics"], "users"=>["Chad J. Johnson", "Judd M. Aiken", "Debbie McKenzie", "Michael D. Samuel", "Joel A. Pedersen"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0035383.g001", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Teflon_beads_in_PMCA_reaction_increase_sensitivity_and_yield_of_PrP_CWD_/324209", "title"=>"Teflon® beads in PMCA reaction increase sensitivity and yield of PrP<sup>CWD</sup>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-13 01:10:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/653908"], "description"=>"<p>Eleven 10 µL CWD-negative white-tailed deer, homozygous for the wild-type <i>Prnp</i>, gene brain homogenate (BH; 10% w/v) samples (lanes 1–11) or 10 µL 3.2×10<sup>−12</sup> dilution of wt/wt CWD+ white-tailed deer BH (lane 13) was added to 90 µL NBH with 0.05% saponin and subjected to six successive rounds of 96-cycle PMCAb. Samples from the sixth round were digested with proteinase K (PK; 50 µg·mL<sup>−1</sup> final concentration) for 1 h at 37°C except one no-PK control.</p>", "links"=>[], "tags"=>["pmcab"], "article_id"=>324401, "categories"=>["Physics", "Virology", "Infectious Diseases", "Biophysics"], "users"=>["Chad J. Johnson", "Judd M. Aiken", "Debbie McKenzie", "Michael D. Samuel", "Joel A. Pedersen"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0035383.g004", "stats"=>{"downloads"=>1, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Serial_PMCAb_has_high_specificity_/324401", "title"=>"Serial PMCAb has high specificity.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-13 01:13:21"}
  • {"files"=>["https://ndownloader.figshare.com/files/336292", "https://ndownloader.figshare.com/files/336336"], "description"=>"<div><p>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<sup>−13</sup> 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<sup>+/−</sup> mice) allowed detection of CWD agent from the 10<sup>−6</sup> dilution of 10% brain homogenate. PMCAb is, thus, more sensitive than bioassay in transgenic mice by a factor exceeding 10<sup>5</sup>. Additionally, we are able to amplify CWD agent from brain tissue and lymph nodes of CWD-positive white-tailed deer having <em>Prnp</em> alleles associated with reduced disease susceptibility.</p> </div>", "links"=>[], "tags"=>["amplification", "wasting", "misfolding", "cyclic", "beads"], "article_id"=>126448, "categories"=>["Physics", "Cancer", "Biophysics"], "users"=>["Chad J. Johnson", "Judd M. Aiken", "Debbie McKenzie", "Michael D. Samuel", "Joel A. Pedersen"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0035383.s001", "https://dx.doi.org/10.1371/journal.pone.0035383.s002"], "stats"=>{"downloads"=>3, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Highly_Efficient_Amplification_of_Chronic_Wasting_Disease_Agent_by_Protein_Misfolding_Cyclic_Amplification_with_Beads_PMCAb_/126448", "title"=>"Highly Efficient Amplification of Chronic Wasting Disease Agent by Protein Misfolding Cyclic Amplification with Beads (PMCAb)", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-04-13 01:47:28"}
  • {"files"=>["https://ndownloader.figshare.com/files/653847"], "description"=>"<p>CWD-positive BH (10% w/v) was diluted using serial five-fold dilutions in NBH and subjected to serial PMCAb in the presence of 0.05% saponin for four 96-cycle rounds. Results from the (A) third and (B) fourth rounds are shown. No-seed controls contained 100 µL NBH. MWM, molecular weight marker.</p>", "links"=>[], "tags"=>["pmcab", "allows", "detection", "femtogram", "levels", "infected"], "article_id"=>324328, "categories"=>["Physics", "Virology", "Infectious Diseases", "Biophysics"], "users"=>["Chad J. Johnson", "Judd M. Aiken", "Debbie McKenzie", "Michael D. Samuel", "Joel A. Pedersen"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0035383.g003", "stats"=>{"downloads"=>1, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Serial_PMCAb_allows_detection_of_PrP_CWD_from_femtogram_levels_of_infected_brain_/324328", "title"=>"Serial PMCAb allows detection of PrP<sup>CWD</sup> from femtogram levels of infected brain.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-13 01:12:08"}
  • {"files"=>["https://ndownloader.figshare.com/files/654028"], "description"=>"<p>Brain homogenates (BH; 10% w/v) from CWD-positive deer with allelic variations of <i>Prnp</i> were tested for their ability to seed PMCAb. CWD-positive 10% BH from (A) wt/wt or wt/95H deer and (B) wt/96S or 95H/96S white-tailed deer were serially diluted in normal BH (NBH) from Tg(CerPrP)1536<sup>+/−</sup> mice, and 10 µL of the 25-, 625- and 1.6×10<sup>4</sup>-fold dilutions from each were used to seed 90 µL fresh NBH and subjected to one 96-cycle PMCAb round. (C) For testing the homozygous 96S allelic variant, retropharyngeal lymph nodes (LN) or BH from two separate hunter-harvested CWD-positive deer were used to seed NBH for PMCAb. As controls, homogenates of lymph nodes from a hunter-harvested CWD-positive wt/wt deer or orally inoculated wt/wt CWD clinically affected deer was used to seed a 96-cycle round of PMCAb. (D) Immunoblotting three five-fold dilutions of 10% BH from the end-stage wt/wt deer used for these studies after proteinase K digestion demonstrates that the PrP<sup>CWD</sup> concentrations used in the PMCAb reactions (<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035383#pone-0035383-g006\" target=\"_blank\">Fig. 6A</a>) were not detectable without amplification.</p>", "links"=>[], "tags"=>["amplifies", "white-tailed", "allelic", "variations", "non-cns"], "article_id"=>324511, "categories"=>["Physics", "Virology", "Infectious Diseases", "Biophysics"], "users"=>["Chad J. Johnson", "Judd M. Aiken", "Debbie McKenzie", "Michael D. Samuel", "Joel A. Pedersen"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0035383.g006", "stats"=>{"downloads"=>1, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_PMCAb_amplifies_PrP_CWD_from_white_tailed_deer_with_allelic_variations_and_from_non_CNS_tissues_/324511", "title"=>"PMCAb amplifies PrP<sup>CWD</sup> from white-tailed deer with allelic variations and from non-CNS tissues.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-13 01:15:11"}

PMC Usage Stats | Further Information

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Relative Metric

{"start_date"=>"2012-01-01T00:00:00Z", "end_date"=>"2012-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences", "average_usage"=>[322, 550, 671, 773, 864, 955, 1048, 1135, 1223, 1308, 1387, 1465, 1534, 1602, 1673, 1744, 1813, 1885, 1955, 2026, 2093, 2160, 2228, 2290, 2349]}, {"subject_area"=>"/Biology and life sciences/Organisms", "average_usage"=>[331, 557, 677, 777, 868, 960, 1050, 1136, 1223, 1307, 1390, 1466, 1536, 1603, 1673, 1741, 1814, 1889, 1954, 2028, 2096, 2164, 2233, 2305, 2362]}, {"subject_area"=>"/Biology and life sciences/Veterinary science", "average_usage"=>[368, 635, 753, 861, 975, 1103, 1205, 1302, 1411, 1512, 1630, 1714, 1767, 1840, 1918, 1992, 2095, 2196, 2254, 2308, 2385, 2438, 2520, 2604, 2682]}, {"subject_area"=>"/Medicine and health sciences/Anatomy and physiology", "average_usage"=>[308, 539, 659, 759, 852, 941, 1028, 1113, 1197, 1275, 1353, 1423]}, {"subject_area"=>"/Medicine and health sciences/Infectious diseases", "average_usage"=>[328, 580, 713, 821, 914, 1012, 1107, 1202, 1293, 1381, 1477, 1551, 1633, 1694, 1769, 1847, 1917, 1986, 2056, 2121, 2194, 2267, 2329, 2410, 2461]}]}
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