Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic
Yersinia pestis has been identified as the causative agent of the Black Death pandemic in the 14(th) century. However, retrospective diagnostics in human skeletons after more than 600 years are critical. We describe a strategy following a modern diagnostic algorithm and working under strict ancient...
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| description | Yersinia pestis has been identified as the causative agent of the Black Death pandemic in the 14(th) century. However, retrospective diagnostics in human skeletons after more than 600 years are critical. We describe a strategy following a modern diagnostic algorithm and working under strict ancient DNA regime for the identification of medieval human plague victims. An initial screening and DNA quantification assay detected the Y. pestis specific pla gene of the high copy number plasmid pPCP1. Results were confirmed by conventional PCR and sequence analysis targeting both Y. pestis specific virulence plasmids pPCP1 and pMT1. All assays were meticulously validated according to human clinical diagnostics requirements (ISO 15189) regarding efficiency, sensitivity, specificity, and limit of detection (LOD). Assay specificity was 100% tested on 41 clinically relevant bacteria and 29 Y. pseudotuberculosis strains as well as for DNA of 22 Y. pestis strains and 30 previously confirmed clinical human plague samples. The optimized LOD was down to 4 gene copies. 29 individuals from three different multiple inhumations were initially assessed as possible victims of the Black Death pandemic. 7 samples (24%) were positive in the pPCP1 specific screening assay. Confirmation through second target pMT1 specific PCR was successful for 4 of the positive individuals (14%). A maximum of 700 and 560 copies per µl aDNA were quantified in two of the samples. Those were positive in all assays including all repetitions, and are candidates for future continuative investigations such as whole genome sequencing. We discuss that all precautions taken here for the work with aDNA are sufficient to prevent external sample contamination and fulfill the criteria of authenticity. With regard to retrospective diagnostics of a human pathogen and the uniqueness of ancient material we strongly recommend using a careful strategy and validated assays as presented in our study. |
| doi_str_mv | 10.1371/journal.pone.0075742 |
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However, retrospective diagnostics in human skeletons after more than 600 years are critical. We describe a strategy following a modern diagnostic algorithm and working under strict ancient DNA regime for the identification of medieval human plague victims. An initial screening and DNA quantification assay detected the Y. pestis specific pla gene of the high copy number plasmid pPCP1. Results were confirmed by conventional PCR and sequence analysis targeting both Y. pestis specific virulence plasmids pPCP1 and pMT1. All assays were meticulously validated according to human clinical diagnostics requirements (ISO 15189) regarding efficiency, sensitivity, specificity, and limit of detection (LOD). Assay specificity was 100% tested on 41 clinically relevant bacteria and 29 Y. pseudotuberculosis strains as well as for DNA of 22 Y. pestis strains and 30 previously confirmed clinical human plague samples. The optimized LOD was down to 4 gene copies. 29 individuals from three different multiple inhumations were initially assessed as possible victims of the Black Death pandemic. 7 samples (24%) were positive in the pPCP1 specific screening assay. Confirmation through second target pMT1 specific PCR was successful for 4 of the positive individuals (14%). A maximum of 700 and 560 copies per µl aDNA were quantified in two of the samples. Those were positive in all assays including all repetitions, and are candidates for future continuative investigations such as whole genome sequencing. We discuss that all precautions taken here for the work with aDNA are sufficient to prevent external sample contamination and fulfill the criteria of authenticity. With regard to retrospective diagnostics of a human pathogen and the uniqueness of ancient material we strongly recommend using a careful strategy and validated assays as presented in our study.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0075742</identifier><identifier>PMID: 24069445</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Accident prevention ; Archaeology - methods ; Assaying ; Bacteria ; Bacterial Proteins - genetics ; Biodiversity ; Biology ; Bone and Bones - microbiology ; Bubonic plague ; Contamination ; Copy number ; Death ; Deoxyribonucleic acid ; Diagnostic equipment (Medical) ; Diagnostic systems ; DNA ; DNA sequencing ; DNA, Bacterial - genetics ; Epidemics ; Gene sequencing ; Genetic testing ; Genomes ; Genomics ; Germany ; History ; Human remains ; Humans ; Mortality ; Pandemics ; Pathogens ; Pla gene ; Plague ; Plague - diagnosis ; Plague - epidemiology ; Plasmids ; Plasmids - genetics ; Plasminogen Activators - genetics ; Polymerase chain reaction ; Polymerase Chain Reaction - methods ; Pseudotuberculosis ; Reproducibility of Results ; Screening ; Sensitivity and Specificity ; Strains (organisms) ; Strategy ; Switzerland ; Uniqueness ; Veterinary medicine ; Virulence ; Yersinia pestis ; Yersinia pestis - genetics</subject><ispartof>PloS one, 2013-09, Vol.8 (9), p.e75742-e75742</ispartof><rights>COPYRIGHT 2013 Public Library of Science</rights><rights>2013 Seifert 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>2013 Seifert et al 2013 Seifert et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-c27fccd8a09b358e716c209fe87ceaca52a9800c7f9cd31cabe317be133555583</citedby><cites>FETCH-LOGICAL-c692t-c27fccd8a09b358e716c209fe87ceaca52a9800c7f9cd31cabe317be133555583</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3775804/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3775804/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79342,79343</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24069445$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Seifert, Lisa</creatorcontrib><creatorcontrib>Harbeck, Michaela</creatorcontrib><creatorcontrib>Thomas, Astrid</creatorcontrib><creatorcontrib>Hoke, Nadja</creatorcontrib><creatorcontrib>Zöller, Lothar</creatorcontrib><creatorcontrib>Wiechmann, Ingrid</creatorcontrib><creatorcontrib>Grupe, Gisela</creatorcontrib><creatorcontrib>Scholz, Holger C</creatorcontrib><creatorcontrib>Riehm, Julia M</creatorcontrib><title>Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Yersinia pestis has been identified as the causative agent of the Black Death pandemic in the 14(th) century. However, retrospective diagnostics in human skeletons after more than 600 years are critical. We describe a strategy following a modern diagnostic algorithm and working under strict ancient DNA regime for the identification of medieval human plague victims. An initial screening and DNA quantification assay detected the Y. pestis specific pla gene of the high copy number plasmid pPCP1. Results were confirmed by conventional PCR and sequence analysis targeting both Y. pestis specific virulence plasmids pPCP1 and pMT1. All assays were meticulously validated according to human clinical diagnostics requirements (ISO 15189) regarding efficiency, sensitivity, specificity, and limit of detection (LOD). Assay specificity was 100% tested on 41 clinically relevant bacteria and 29 Y. pseudotuberculosis strains as well as for DNA of 22 Y. pestis strains and 30 previously confirmed clinical human plague samples. 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With regard to retrospective diagnostics of a human pathogen and the uniqueness of ancient material we strongly recommend using a careful strategy and validated assays as presented in our study.</description><subject>Accident prevention</subject><subject>Archaeology - methods</subject><subject>Assaying</subject><subject>Bacteria</subject><subject>Bacterial Proteins - genetics</subject><subject>Biodiversity</subject><subject>Biology</subject><subject>Bone and Bones - microbiology</subject><subject>Bubonic plague</subject><subject>Contamination</subject><subject>Copy number</subject><subject>Death</subject><subject>Deoxyribonucleic acid</subject><subject>Diagnostic equipment (Medical)</subject><subject>Diagnostic systems</subject><subject>DNA</subject><subject>DNA sequencing</subject><subject>DNA, Bacterial - genetics</subject><subject>Epidemics</subject><subject>Gene sequencing</subject><subject>Genetic testing</subject><subject>Genomes</subject><subject>Genomics</subject><subject>Germany</subject><subject>History</subject><subject>Human remains</subject><subject>Humans</subject><subject>Mortality</subject><subject>Pandemics</subject><subject>Pathogens</subject><subject>Pla gene</subject><subject>Plague</subject><subject>Plague - diagnosis</subject><subject>Plague - epidemiology</subject><subject>Plasmids</subject><subject>Plasmids - genetics</subject><subject>Plasminogen Activators - genetics</subject><subject>Polymerase chain reaction</subject><subject>Polymerase Chain Reaction - methods</subject><subject>Pseudotuberculosis</subject><subject>Reproducibility of Results</subject><subject>Screening</subject><subject>Sensitivity and Specificity</subject><subject>Strains (organisms)</subject><subject>Strategy</subject><subject>Switzerland</subject><subject>Uniqueness</subject><subject>Veterinary medicine</subject><subject>Virulence</subject><subject>Yersinia pestis</subject><subject>Yersinia pestis - genetics</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNk12L1DAUhoso7rr6D0QDgujFjEnTNumNsCx-DCwsuCp4FdLT05nsdpLapIv7701nustU9sLkIiF53vckJzlJ8pLRJeOCfbhyQ291u-ycxSWlIhdZ-ig5ZiVPF0VK-eOD-VHyzPsrSnMui-JpcpRmtCizLD9OzGXodcD1LWlcTzxab4K5QaJtTXyHYBoDpMaAEIyzxDXkF_beWKNJhz4YT4wl_hpbDM76cT9skFSthuso02FDumiFWwPPkyeNbj2-mMaT5MfnT9_Pvi7OL76szk7PF1CUaVhAKhqAWmpaVjyXKFgBKS0blAJQg85TXUpKQTQl1JyBrpAzUSHjPI9N8pPk9d63a51XU5a8YhmPjZVFFonVnqidvlJdb7a6v1VOG7VbcP1a6T4YaFGB0JUWosqlZBnktQQmOM2gKVKB-2gfp2hDtcUa0MZ8tjPT-Y41G7V2N4oLkUs6HubdZNC730NMqdoaD9i22qIbduceQVnyiL75B334dhO11vECxjYuxoXRVJ1mQqYRo2Wklg9Qse_eKn6pxsT1meD9TBCZgH_CWg_eq9Xlt_9nL37O2bcH7AZ1GzbetcP43fwczPYg9M77Hpv7JDOqxoq4y4YaK0JNFRFlrw4f6F50VwL8L_CyBr8</recordid><startdate>20130917</startdate><enddate>20130917</enddate><creator>Seifert, Lisa</creator><creator>Harbeck, Michaela</creator><creator>Thomas, Astrid</creator><creator>Hoke, Nadja</creator><creator>Zöller, Lothar</creator><creator>Wiechmann, Ingrid</creator><creator>Grupe, Gisela</creator><creator>Scholz, Holger C</creator><creator>Riehm, Julia M</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20130917</creationdate><title>Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic</title><author>Seifert, Lisa ; Harbeck, Michaela ; Thomas, Astrid ; Hoke, Nadja ; Zöller, Lothar ; Wiechmann, Ingrid ; Grupe, Gisela ; Scholz, Holger C ; Riehm, Julia M</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-c27fccd8a09b358e716c209fe87ceaca52a9800c7f9cd31cabe317be133555583</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Accident prevention</topic><topic>Archaeology - 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Academic</collection><collection>PubMed Central (Full 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>Seifert, Lisa</au><au>Harbeck, Michaela</au><au>Thomas, Astrid</au><au>Hoke, Nadja</au><au>Zöller, Lothar</au><au>Wiechmann, Ingrid</au><au>Grupe, Gisela</au><au>Scholz, Holger C</au><au>Riehm, Julia M</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2013-09-17</date><risdate>2013</risdate><volume>8</volume><issue>9</issue><spage>e75742</spage><epage>e75742</epage><pages>e75742-e75742</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Yersinia pestis has been identified as the causative agent of the Black Death pandemic in the 14(th) century. However, retrospective diagnostics in human skeletons after more than 600 years are critical. We describe a strategy following a modern diagnostic algorithm and working under strict ancient DNA regime for the identification of medieval human plague victims. An initial screening and DNA quantification assay detected the Y. pestis specific pla gene of the high copy number plasmid pPCP1. Results were confirmed by conventional PCR and sequence analysis targeting both Y. pestis specific virulence plasmids pPCP1 and pMT1. All assays were meticulously validated according to human clinical diagnostics requirements (ISO 15189) regarding efficiency, sensitivity, specificity, and limit of detection (LOD). Assay specificity was 100% tested on 41 clinically relevant bacteria and 29 Y. pseudotuberculosis strains as well as for DNA of 22 Y. pestis strains and 30 previously confirmed clinical human plague samples. The optimized LOD was down to 4 gene copies. 29 individuals from three different multiple inhumations were initially assessed as possible victims of the Black Death pandemic. 7 samples (24%) were positive in the pPCP1 specific screening assay. Confirmation through second target pMT1 specific PCR was successful for 4 of the positive individuals (14%). A maximum of 700 and 560 copies per µl aDNA were quantified in two of the samples. Those were positive in all assays including all repetitions, and are candidates for future continuative investigations such as whole genome sequencing. We discuss that all precautions taken here for the work with aDNA are sufficient to prevent external sample contamination and fulfill the criteria of authenticity. With regard to retrospective diagnostics of a human pathogen and the uniqueness of ancient material we strongly recommend using a careful strategy and validated assays as presented in our study.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>24069445</pmid><doi>10.1371/journal.pone.0075742</doi><tpages>e75742</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2013-09, Vol.8 (9), p.e75742-e75742 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1433331964 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry; Public Library of Science (PLoS) |
| subjects | Accident prevention Archaeology - methods Assaying Bacteria Bacterial Proteins - genetics Biodiversity Biology Bone and Bones - microbiology Bubonic plague Contamination Copy number Death Deoxyribonucleic acid Diagnostic equipment (Medical) Diagnostic systems DNA DNA sequencing DNA, Bacterial - genetics Epidemics Gene sequencing Genetic testing Genomes Genomics Germany History Human remains Humans Mortality Pandemics Pathogens Pla gene Plague Plague - diagnosis Plague - epidemiology Plasmids Plasmids - genetics Plasminogen Activators - genetics Polymerase chain reaction Polymerase Chain Reaction - methods Pseudotuberculosis Reproducibility of Results Screening Sensitivity and Specificity Strains (organisms) Strategy Switzerland Uniqueness Veterinary medicine Virulence Yersinia pestis Yersinia pestis - genetics |
| title | Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-09T22%3A55%3A34IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Strategy%20for%20sensitive%20and%20specific%20detection%20of%20Yersinia%20pestis%20in%20skeletons%20of%20the%20black%20death%20pandemic&rft.jtitle=PloS%20one&rft.au=Seifert,%20Lisa&rft.date=2013-09-17&rft.volume=8&rft.issue=9&rft.spage=e75742&rft.epage=e75742&rft.pages=e75742-e75742&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0075742&rft_dat=%3Cgale_plos_%3EA478233109%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1433331964&rft_id=info:pmid/24069445&rft_galeid=A478233109&rft_doaj_id=oai_doaj_org_article_c7aba77b58814c5d8c17304cf627e558&rfr_iscdi=true |