Rapid bacterial identification by direct PCR amplification of 16S rRNA genes using the MinION nanopore sequencer
Rapid bacterial identification by direct PCR amplification of 16S rRNA genes using the MinION nanopore sequencer
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DOI：
10.1002/2211-5463.12590
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发表时间：
2019-03-01
期刊：
影响因子：
2.6
通讯作者：
Hirota, Kiichi
中科院分区：
文献类型：
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作者：
Kai, Shinichi;Matsuo, Yoshiyuki;Hirota, Kiichi
细菌病原体的快速鉴定对于适当和充分的抗生素治疗至关重要，这将显著改善患者的预后。16S核糖体RNA(RRNA)基因扩增序列已被证明是诊断细菌感染的有效策略。我们最近利用牛津纳米孔技术的Minion测序仪建立了用于16S rRNA基因分析的测序方法和生物信息学流水线。与我们的分类注释分析流水线相结合，该系统能够在合理的时间框架内对细菌DNA进行分子检测，以用于诊断目的。然而，从样本中提纯细菌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的制备过程，我们采用了直接聚合酶链式反应的方法，从细菌细胞悬液中扩增出16S rRNA基因，而不需要进行DNA纯化。我们的结果表明，细胞壁形态的不同显著影响直接聚合酶链式反应效率和测序数据。值得注意的是，直接聚合酶链式反应之前的机械细胞破坏对于获得样本细菌组成的准确表示是必不可少的。此外，对模拟多菌样品的16S rRNA基因分析表明，需要对引物序列进行优化，以避免对特定类群的优先检测，并覆盖广泛的细菌种类。本研究建立了一种相对简单的Minion测序快速细菌鉴定流程，缩短了从样品到结果的周转时间，并提供了一种可能适用于临床的可靠方法。
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.