Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic.
Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic.
复制标题
黑死亡大流行骨骼中对耶尔森氏念珠菌的敏感和特异性检测的策略。
DOI：
10.1371/journal.pone.0075742
复制
发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Riehm JM
中科院分区：
文献类型：
--
作者：
Seifert L;Harbeck M;Thomas A;Hoke N;Zöller L;Wiechmann I;Grupe G;Scholz HC;Riehm JM
鼠疫耶尔森氏菌已被确定为14世纪黑死病大流行的病原体。然而，对600多年后的人类骨骼进行回顾性诊断是至关重要的。我们描述了一种策略，遵循现代诊断算法，并在严格的古代DNA制度下工作，以识别中世纪人类鼠疫受害者。初步筛选和DNA定量分析检测到高拷贝数质粒pPCP1的鼠疫菌特异性pla基因。针对鼠疫菌特异性毒力质粒pPCP1和pMT1进行常规PCR和序列分析证实了结果。根据人类临床诊断要求（ISO 15189）对效率、灵敏度、特异性和检出限（LOD）进行了严格验证。对41种临床相关细菌和29株假结核杆菌菌株以及22株鼠疫杆菌菌株和30份先前确认的临床人类鼠疫样本的DNA进行了100%的检测特异性。优化后的LOD降至4个基因拷贝。最初评估了来自三种不同的多次埋葬的29人可能是黑死病大流行的受害者。7例（24%）pPCP1特异性筛选阳性。通过第二目标pMT1特异性PCR确认4例阳性个体（14%）成功。在两种样品中，每µl aDNA最多定量为700和560拷贝。这些基因在包括所有重复在内的所有分析中均呈阳性，是未来连续研究（如全基因组测序）的候选基因。我们讨论了这里为aDNA工作所采取的所有预防措施足以防止外部样品污染并满足真实性标准。关于人类病原体的回顾性诊断和古代材料的独特性，我们强烈建议使用我们研究中提出的谨慎策略和经过验证的分析方法。
Yersinia pestis has been identified as the causative agent of the Black Death pandemic in the 14th 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.
登录
查看更多内容
影响因子：
64.8
作者：
Bos, Kirsten I.;Schuenemann, Verena J.;Golding, G. Brian;Burbano, Hernan A.;Waglechner, Nicholas;Coombes, Brian K.;McPhee, Joseph B.;DeWitte, Sharon N.;Meyer, Matthias;Schmedes, Sarah;Wood, James;Earn, David J. D.;Herring, D. Ann;Bauer, Peter;Poinar, Hendrik N.;Krause, Johannes
通讯作者：
Krause, Johannes
DOI：
10.1073/pnas.95.21.12637
发表时间：
1998-10-13
影响因子：
11.1
作者：
Drancourt, M;Aboudharam, G;Raoult, D
通讯作者：
Raoult, D
影响因子：
2.2
作者：
Campos, Paula F.;Craig, Oliver E.;Gilbert, M. Thomas P.
通讯作者：
Gilbert, M. Thomas P.
DOI：
10.1016/j.palaeo.2011.03.021
发表时间：
2011-09-15
影响因子：
3
作者：
Hoke, Nadja;Burger, Joachim;Harbeck, Michaela
通讯作者：
Harbeck, Michaela
影响因子：
6.7
作者：
Haensch S;Bianucci R;Signoli M;Rajerison M;Schultz M;Kacki S;Vermunt M;Weston DA;Hurst D;Achtman M;Carniel E;Bramanti B
通讯作者：
Bramanti B