Forensic Science Program, Department of Biochemistry & Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA.
Genes (Basel). 2024 Aug 27;15(9):1129. doi: 10.3390/genes15091129.
To date, studies on microbial forensics have focused mainly on sequence analysis and generally do not include information on the quantification of and comparison between the human and bacterial DNA present in forensic samples. Knowing the amount of each type of DNA can be important for determining when and how best to employ bacterial DNA analysis, especially when there is insufficient human DNA for successful short tandem repeat (STR) typing. The goal of this work was to develop a quantitative PCR (qPCR) assay that simultaneously quantifies human and bacterial DNA that would be simple and cost-effective for laboratories to implement. Through a reproducibility study and several small-scale experiments, the reliability of a custom qPCR assay was established. A reproducibility study illustrated that the multiplex assay produced data comparable to that of previously established bacterial DNA and human DNA qPCR assays. The small-scale experiments showed that common surfaces such as keyboards (6.76 pg/μL), elevator buttons (11.9 pg/μL), cleaning supplies (7.17 pg/μL), and dispensers (16.4 pg/μL) failed to produce human DNA quantities sufficient for quality STR analysis (≥250 pg). However, all tested surfaces produced bacterial DNA quantities suitable for reaching 1 ng of amplified bacterial targets necessary for sequence analysis. In fact, bacterial DNA concentrations down to 10 ng/uL produce enough amplified product for sequencing. The newly developed qPCR multiplex tool will allow scientists to make better decisions regarding whether human or bacterial DNA analysis methods can be pursued during forensic or other investigations.
迄今为止，微生物取证的研究主要集中在序列分析上，通常不包括法医样本中存在的人类和细菌 DNA 的定量和比较信息。了解每种类型 DNA 的数量对于确定何时以及如何最好地进行细菌 DNA 分析非常重要，尤其是在法医样本中人类 DNA 含量不足以致无法成功进行短串联重复序列（STR）分型时。这项工作的目的是开发一种可同时定量人类和细菌 DNA 的定量聚合酶链反应（qPCR）检测方法，该方法简单且对实验室具有成本效益。通过可重复性研究和几项小规模实验，建立了定制 qPCR 检测方法的可靠性。可重复性研究表明，多重检测方法产生的数据与先前建立的细菌 DNA 和人类 DNA qPCR 检测方法的数据相当。小规模实验表明，常见的表面（如键盘（6.76 pg/μL）、电梯按钮（11.9 pg/μL）、清洁用品（7.17 pg/μL）和分配器（16.4 pg/μL））未能产生足够数量的人类 DNA 进行质量 STR 分析（≥250 pg）。然而，所有测试的表面都产生了足够用于扩增细菌靶标达到 1ng 的细菌 DNA 数量，这是进行序列分析所必需的。实际上，细菌 DNA 浓度低至 10ng/uL 即可产生足够用于测序的扩增产物。新开发的 qPCR 多重工具将使科学家能够更好地决定在法医或其他调查中是否可以采用人类或细菌 DNA 分析方法。
