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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Autores principales:	Lisa Seifert, Michaela Harbeck, Astrid Thomas, Nadja Hoke, Lothar Zöller, Ingrid Wiechmann, Gisela Grupe, Holger C Scholz, Julia M Riehm
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/c7aba77b58814c5d8c17304cf627e558
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spelling	oai:doaj.org-article:c7aba77b58814c5d8c17304cf627e5582021-11-18T08:54:50ZStrategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic.1932-620310.1371/journal.pone.0075742https://doaj.org/article/c7aba77b58814c5d8c17304cf627e5582013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24069445/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Yersinia 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.Lisa SeifertMichaela HarbeckAstrid ThomasNadja HokeLothar ZöllerIngrid WiechmannGisela GrupeHolger C ScholzJulia M RiehmPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 9, p e75742 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Lisa Seifert Michaela Harbeck Astrid Thomas Nadja Hoke Lothar Zöller Ingrid Wiechmann Gisela Grupe Holger C Scholz Julia M Riehm Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic.
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.
format	article
author	Lisa Seifert Michaela Harbeck Astrid Thomas Nadja Hoke Lothar Zöller Ingrid Wiechmann Gisela Grupe Holger C Scholz Julia M Riehm
author_facet	Lisa Seifert Michaela Harbeck Astrid Thomas Nadja Hoke Lothar Zöller Ingrid Wiechmann Gisela Grupe Holger C Scholz Julia M Riehm
author_sort	Lisa Seifert
title	Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic.
title_short	Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic.
title_full	Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic.
title_fullStr	Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic.
title_full_unstemmed	Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic.
title_sort	strategy for sensitive and specific detection of yersinia pestis in skeletons of the black death pandemic.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/c7aba77b58814c5d8c17304cf627e558
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Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic.

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