Department of Anesthesia Kyoto University Hospital Japan.
Department of Human Stress Response Science Institute of Biomedical Science Kansai Medical University Hirakata Japan.
FEBS Open Bio. 2019 Jan 29;9(3):548-557. doi: 10.1002/2211-5463.12590. eCollection 2019 Mar.
Rapid identification of bacterial pathogens is crucial for appropriate and adequate antibiotic treatment, which significantly improves patient outcomes. 16S ribosomal RNA (rRNA) gene amplicon sequencing has proven to be a powerful strategy for diagnosing bacterial infections. We have recently established a sequencing method and bioinformatics pipeline for 16S rRNA gene analysis utilizing the Oxford Nanopore Technologies MinION™ sequencer. In combination with our taxonomy annotation analysis pipeline, the system enabled the molecular detection of bacterial DNA in a reasonable time frame for diagnostic purposes. However, purification of bacterial DNA from specimens remains a rate-limiting step in the workflow. To further accelerate the process of sample preparation, we adopted a direct PCR strategy that amplifies 16S rRNA genes from bacterial cell suspensions without DNA purification. Our results indicate that differences in cell wall morphology significantly affect direct PCR efficiency and sequencing data. Notably, mechanical cell disruption preceding direct PCR was indispensable for obtaining an accurate representation of the specimen bacterial composition. Furthermore, 16S rRNA gene analysis of mock polymicrobial samples indicated that primer sequence optimization is required to avoid preferential detection of particular taxa and to cover a broad range of bacterial species. This study establishes a relatively simple workflow for rapid bacterial identification via MinION™ sequencing, which reduces the turnaround time from sample to result, and provides a reliable method that may be applicable to clinical settings.
快速鉴定细菌病原体对于适当和充分的抗生素治疗至关重要，这可以显著改善患者的预后。16S 核糖体 RNA（rRNA）基因扩增测序已被证明是诊断细菌感染的有效策略。我们最近建立了一种利用 Oxford Nanopore Technologies MinION™测序仪进行 16S rRNA 基因分析的测序方法和生物信息学管道。结合我们的分类注释分析管道，该系统能够在合理的时间框架内从诊断目的的标本中分子检测到细菌 DNA。然而，从标本中纯化细菌 DNA 仍然是工作流程中的一个限速步骤。为了进一步加快样品制备过程，我们采用了一种直接 PCR 策略，该策略无需 DNA 纯化即可从细菌细胞悬浮液中扩增 16S rRNA 基因。我们的结果表明，细胞壁形态的差异显著影响直接 PCR 的效率和测序数据。值得注意的是，在直接 PCR 之前进行机械细胞破碎对于获得标本细菌组成的准确表示是必不可少的。此外，对模拟多微生物样本的 16S rRNA 基因分析表明，需要优化引物序列以避免对特定分类群的优先检测，并涵盖广泛的细菌物种。本研究通过 MinION™测序建立了一种相对简单的快速细菌鉴定工作流程，从样本到结果的周转时间缩短，提供了一种可靠的方法，可能适用于临床环境。
