An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis
Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate...
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| Veröffentlicht in: | PloS one 2011-11, Vol.6 (11), p.e27369-e27369 |
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| creator | Travis, Emma R Gaze, William H Pontiroli, Alessandra Sweeney, Francis P Porter, David Mason, Sam Keeling, Matthew J C Jones, Rebecca M Sawyer, Jason Aranaz, Alicia Rizaldos, Elena Castellanos Cork, Jennifer Delahay, Richard J Wilson, Gavin J Hewinson, R Glyn Courtenay, Orin Wellington, Elizabeth M H |
| description | Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 10(5) cells g(-1) and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife. |
| doi_str_mv | 10.1371/journal.pone.0027369 |
| format | Article |
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In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 10(5) cells g(-1) and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0027369</identifier><identifier>PMID: 22110634</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Agriculture ; Analysis ; Animals ; Assaying ; Bacillus Calmette-Guerin vaccine ; Bacteria ; Badgers ; BCG ; Beef cattle ; Biology ; Bovidae ; Cattle ; Data processing ; Data recovery ; Deoxyribonucleic acid ; Design optimization ; Dilution ; DNA ; E coli ; Epidemiology ; Escherichia coli ; Excretion ; Experimental design ; False Negative Reactions ; Feces ; Feces - microbiology ; Health aspects ; Infection ; Infections ; Laboratories ; Latrines ; Life sciences ; Mammals ; Medicine ; Meles meles ; Mustelidae - microbiology ; Mycobacterium ; Mycobacterium bovis ; Mycobacterium bovis - genetics ; Mycobacterium bovis - isolation & purification ; Operators ; Panels ; Polymerase chain reaction ; Population ; Quantitative analysis ; Real time ; Real-Time Polymerase Chain Reaction - methods ; Real-Time Polymerase Chain Reaction - standards ; Reliability analysis ; Reproducibility ; Science Policy ; Studies ; Sustainable development ; Tuberculosis ; Veterinary Science ; Wildlife ; Wildlife management ; Zoonoses</subject><ispartof>PloS one, 2011-11, Vol.6 (11), p.e27369-e27369</ispartof><rights>COPYRIGHT 2011 Public Library of Science</rights><rights>2011 Travis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>Travis et al. 2011</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c691t-dd1ecef851f8d10e36d39b5b3b471e7277a508cf4025e09e91bedf23277505f53</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3215712/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3215712/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2102,2928,23866,27924,27925,53791,53793,79600,79601</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22110634$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Kaltenboeck, Bernhard</contributor><creatorcontrib>Travis, Emma R</creatorcontrib><creatorcontrib>Gaze, William H</creatorcontrib><creatorcontrib>Pontiroli, Alessandra</creatorcontrib><creatorcontrib>Sweeney, Francis P</creatorcontrib><creatorcontrib>Porter, David</creatorcontrib><creatorcontrib>Mason, Sam</creatorcontrib><creatorcontrib>Keeling, Matthew J C</creatorcontrib><creatorcontrib>Jones, Rebecca M</creatorcontrib><creatorcontrib>Sawyer, Jason</creatorcontrib><creatorcontrib>Aranaz, Alicia</creatorcontrib><creatorcontrib>Rizaldos, Elena Castellanos</creatorcontrib><creatorcontrib>Cork, Jennifer</creatorcontrib><creatorcontrib>Delahay, Richard J</creatorcontrib><creatorcontrib>Wilson, Gavin J</creatorcontrib><creatorcontrib>Hewinson, R Glyn</creatorcontrib><creatorcontrib>Courtenay, Orin</creatorcontrib><creatorcontrib>Wellington, Elizabeth M H</creatorcontrib><title>An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 10(5) cells g(-1) and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.</description><subject>Agriculture</subject><subject>Analysis</subject><subject>Animals</subject><subject>Assaying</subject><subject>Bacillus Calmette-Guerin vaccine</subject><subject>Bacteria</subject><subject>Badgers</subject><subject>BCG</subject><subject>Beef cattle</subject><subject>Biology</subject><subject>Bovidae</subject><subject>Cattle</subject><subject>Data processing</subject><subject>Data recovery</subject><subject>Deoxyribonucleic acid</subject><subject>Design optimization</subject><subject>Dilution</subject><subject>DNA</subject><subject>E coli</subject><subject>Epidemiology</subject><subject>Escherichia coli</subject><subject>Excretion</subject><subject>Experimental design</subject><subject>False Negative Reactions</subject><subject>Feces</subject><subject>Feces - microbiology</subject><subject>Health aspects</subject><subject>Infection</subject><subject>Infections</subject><subject>Laboratories</subject><subject>Latrines</subject><subject>Life sciences</subject><subject>Mammals</subject><subject>Medicine</subject><subject>Meles meles</subject><subject>Mustelidae - microbiology</subject><subject>Mycobacterium</subject><subject>Mycobacterium bovis</subject><subject>Mycobacterium bovis - genetics</subject><subject>Mycobacterium bovis - isolation & purification</subject><subject>Operators</subject><subject>Panels</subject><subject>Polymerase chain reaction</subject><subject>Population</subject><subject>Quantitative analysis</subject><subject>Real time</subject><subject>Real-Time Polymerase Chain Reaction - methods</subject><subject>Real-Time Polymerase Chain Reaction - standards</subject><subject>Reliability analysis</subject><subject>Reproducibility</subject><subject>Science Policy</subject><subject>Studies</subject><subject>Sustainable 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Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Travis, Emma R</au><au>Gaze, William H</au><au>Pontiroli, Alessandra</au><au>Sweeney, Francis P</au><au>Porter, David</au><au>Mason, Sam</au><au>Keeling, Matthew J C</au><au>Jones, Rebecca M</au><au>Sawyer, Jason</au><au>Aranaz, Alicia</au><au>Rizaldos, Elena Castellanos</au><au>Cork, Jennifer</au><au>Delahay, Richard J</au><au>Wilson, Gavin J</au><au>Hewinson, R Glyn</au><au>Courtenay, Orin</au><au>Wellington, Elizabeth M H</au><au>Kaltenboeck, Bernhard</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2011-11-14</date><risdate>2011</risdate><volume>6</volume><issue>11</issue><spage>e27369</spage><epage>e27369</epage><pages>e27369-e27369</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 10(5) cells g(-1) and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>22110634</pmid><doi>10.1371/journal.pone.0027369</doi><tpages>e27369</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2011-11, Vol.6 (11), p.e27369-e27369 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1312154215 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Public Library of Science (PLoS) Journals Open Access; EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Agriculture Analysis Animals Assaying Bacillus Calmette-Guerin vaccine Bacteria Badgers BCG Beef cattle Biology Bovidae Cattle Data processing Data recovery Deoxyribonucleic acid Design optimization Dilution DNA E coli Epidemiology Escherichia coli Excretion Experimental design False Negative Reactions Feces Feces - microbiology Health aspects Infection Infections Laboratories Latrines Life sciences Mammals Medicine Meles meles Mustelidae - microbiology Mycobacterium Mycobacterium bovis Mycobacterium bovis - genetics Mycobacterium bovis - isolation & purification Operators Panels Polymerase chain reaction Population Quantitative analysis Real time Real-Time Polymerase Chain Reaction - methods Real-Time Polymerase Chain Reaction - standards Reliability analysis Reproducibility Science Policy Studies Sustainable development Tuberculosis Veterinary Science Wildlife Wildlife management Zoonoses |
| title | An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis |
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