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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...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3775804/ https://www.ncbi.nlm.nih.gov/pubmed/24069445 http://dx.doi.org/10.1371/journal.pone.0075742 |
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author | Seifert, Lisa Harbeck, Michaela Thomas, Astrid Hoke, Nadja Zöller, Lothar Wiechmann, Ingrid Grupe, Gisela Scholz, Holger C. Riehm, Julia M. |
author_facet | Seifert, Lisa Harbeck, Michaela Thomas, Astrid Hoke, Nadja Zöller, Lothar Wiechmann, Ingrid Grupe, Gisela Scholz, Holger C. Riehm, Julia M. |
author_sort | Seifert, Lisa |
collection | PubMed |
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 | Online Article Text |
id | pubmed-3775804 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-37758042013-09-25 Strategy for Sensitive and Specific Detection of Yersinia pestis in Skeletons of the Black Death Pandemic Seifert, Lisa Harbeck, Michaela Thomas, Astrid Hoke, Nadja Zöller, Lothar Wiechmann, Ingrid Grupe, Gisela Scholz, Holger C. Riehm, Julia M. PLoS One Research Article 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. Public Library of Science 2013-09-17 /pmc/articles/PMC3775804/ /pubmed/24069445 http://dx.doi.org/10.1371/journal.pone.0075742 Text en © 2013 Seifert et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Seifert, Lisa Harbeck, Michaela Thomas, Astrid Hoke, Nadja Zöller, Lothar Wiechmann, Ingrid Grupe, Gisela Scholz, Holger C. Riehm, Julia M. Strategy for Sensitive and Specific Detection of Yersinia pestis in Skeletons of the Black Death Pandemic |
title | 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_short | 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 |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3775804/ https://www.ncbi.nlm.nih.gov/pubmed/24069445 http://dx.doi.org/10.1371/journal.pone.0075742 |
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