使用ONT MinION测序器识别细菌的九种提取方法的比较
Kari A Graham1, Javier Gomez2, Todd P Primm2
1Department of Forensic Science, College of Criminal Justice, Sam Houston State University, 1003 Bowers Blvd., Huntsville, TX, 77340-2525, USA.
International journal of legal medicine
|September 29, 2023
概括
没有单一的DNA提取方法可以在微生物法医中使用牛津纳米孔技术 (ONT) 的MinION测序快速识别细菌. DNeasy PowerSoil Kit和Chelex-100方法显示出对基于现场的分析具有前景.
科学领域:
- 微生物法医学 微生物法医学
- 分子生物学分子生物学
- 生物信息学是一种生物信息学.
背景情况:
- 2001年炭病袭击突出了对未知的生物证据进行快速微生物法医分析的需要.
- 传统的细菌鉴定方法,如培养,耗时.
- 目前的分子技术,如16S rRNA基因测序,通常是实验室限制和漫长的.
研究的目的:
- 确定一种"通用"的细菌DNA提取方法,用于快速的现场微生物法医分析.
- 评估牛津纳米孔技术 (ONT) 的适用性,用于法医应用.
- 评估基于云的EPI2ME软件,用于在法医背景下进行数据分析.
主要方法:
- 测试了九种不同的细菌DNA提取方法.
- 使用 16S 条码套件和 16S FASTQ 工作流的 ONT MinION 测序.
- 在各种提取方法和生物体中评估物种级别的识别准确性.
主要成果:
- 没有任何一种"通用"的DNA提取方法被发现对所有使用ONT MINION测序的细菌群体都是最佳的.
- 德尼西电源土壤套件和诺达等人. 采用Chelex-100方法获得了类似的结果,可以用作快速提取技术.
- 精确的物种级别识别仅在63%的案例中实现,大多数对齐度约为89%的精度,这表明法医使用的局限性.
结论:
- 目前的DNA提取方法对于使用ONT MinION测序快速法医微生物识别并不普遍.
- 在现场环境中,DNeasy PowerSoil Kit和Chelex-100方法提供了快速DNA提取的潜力.
- 需要进一步优化提取和测序工作流程,以提高法医微生物识别的准确性.
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