Harkins Kelly M, Buikstra Jane E, Campbell Tessa, Bos Kirsten I, Johnson Eric D, Krause Johannes, Stone Anne C
School of Human Evolution and Social Change, Arizona State University, Tempe, AZ, USA.
Department of Archaeology, University of Cape Town, Cape Town, South Africa.
Philos Trans R Soc Lond B Biol Sci. 2015 Jan 19;370(1660):20130622. doi: 10.1098/rstb.2013.0622.
The field of ancient DNA (aDNA) has rapidly accelerated in recent years as a result of new methods in next-generation sequencing, library preparation and targeted enrichment. Such research is restricted, however, by the highly variable DNA preservation within different tissues, especially when isolating ancient pathogens from human remains. Identifying positive candidate samples via quantitative PCR (qPCR) for downstream procedures can reduce reagent costs, increase capture efficiency and maximize the number of sequencing reads of the target. This study uses four qPCR assays designed to target regions within the Mycobacterium tuberculosis complex (MTBC) to examine 133 human skeletal samples from a wide geographical and temporal range, identified by the presence of skeletal lesions typical of chronic disseminated tuberculosis. Given the inherent challenges working with ancient mycobacteria, strict criteria must be used and primer/probe design continually re-evaluated as new data from bacteria become available. Seven samples tested positive for multiple MTBC loci, supporting them as strong candidates for downstream analyses. Using strict and conservative criteria, qPCR remains a fast and effective screening tool when compared with screening by more expensive sequencing and enrichment technologies.
近年来，由于新一代测序、文库制备和靶向富集等新方法的出现，古代DNA（aDNA）领域发展迅速。然而，此类研究受到不同组织中DNA保存情况高度可变的限制，尤其是在从人类遗骸中分离古代病原体时。通过定量PCR（qPCR）鉴定用于下游程序的阳性候选样本，可以降低试剂成本、提高捕获效率并最大化目标测序读数的数量。本研究使用了四种针对结核分枝杆菌复合群（MTBC）内区域设计的qPCR检测方法，来检测133份来自广泛地理和时间范围的人类骨骼样本，这些样本通过慢性播散性结核病典型的骨骼病变得以识别。鉴于处理古代分枝杆菌存在固有的挑战，必须使用严格的标准，并且随着细菌新数据的出现，引物/探针设计要不断重新评估。七个样本对多个MTBC基因座检测呈阳性，这表明它们是下游分析的有力候选样本。与更昂贵的测序和富集技术相比，使用严格和保守的标准时，qPCR仍然是一种快速有效的筛选工具。
