Rapid identification of pathogens from positive blood culture bottles with the MinION nanopore sequencer
Rapid identification of pathogens from positive blood culture bottles with the MinION nanopore sequencer
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DOI：
10.1099/jmm.0.000855
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发表时间：
2018-11-01
影响因子：
3
通讯作者：
Maeda, Takuya
中科院分区：
文献类型：
--
作者：
Ashikawa, Sae;Tarumoto, Norihito;Maeda, Takuya
目的。血液感染是发病率和死亡率的主要原因，导致住院时间延长和医疗费用增加。在这项研究中，我们旨在评估Minion纳米孔测序仪在鉴定阳性血培养瓶中最主要的病原体方面的作用。从阳性血瓶中分离到9株临床分离株和11株包括5种念珠菌在内的模式菌株，用条形码引物进行聚合酶链式反应，扩增出16S和ITS1-5.8S-ITS2 rRNA基因。将条形码扩增物混合，用Minion测序仪进行多重测序。此外，对条形码的PCR扩增产物进行了Sanger测序，以验证MinION的性能。细菌和假丝酵母菌。根据来自NCBI基因数据库的参考序列的最高同源性，经Minion测序鉴定，与基质辅助激光解吸电离飞行时间质谱仪的结果一致，但密切相关的物种链球菌和大肠杆菌除外。在测序后约10分钟内获得的“通过”读数足以识别病原体。与一维(2)化学和R9.5流式细胞的序列一致性平均值在99%左右，因此，频繁的序列错误不会影响基于扩增子序列的物种鉴定。我们已经建立了一种快速、便携和经济的技术来鉴定阳性血培养瓶中的病原体，通过一种新的Minion纳米孔测序器扩增序列测序方案，它取代了传统的Sanger测序。
Purpose. Bloodstream infections are major causes of morbidity and mortality that lead to prolonged hospital stays and higher medical costs. In this study, we aimed to evaluate the MinION nanopore sequencer for the identification of the most dominant pathogens in positive blood culture bottles.Methodology. 16S and ITS1-5.8S-ITS2 rRNA genes were amplified by PCR reactions with barcoded primers using nine clinical isolates obtained from positive blood bottles and 11 type strains, including five types of Candida species. Barcoded amplicons were mixed, and multiplex sequencing with the MinION sequencer was performed. In addition, barcoded PCR amplicons were sequenced by Sanger sequencing to validate the performance of the MinION.Results. The bacterial and Candida spp. identified by MinION sequencing, based on the highest homology of reference sequences from the NCBI gene databases, agreed with the matrix-assisted laser desorption ionization time of flight mass spectrometry results, excepting the closely related species Streptococcusand Escherichia coli. The 'pass' reads obtained within about 10 min of sequencing were sufficient to identify the pathogens. The average values of sequence identities with 1D(2) chemistry and the R9.5 flow cell were around 99 %; thus, frequent sequence errors did not affect species identification based on amplicon sequencing.Conclusion. We have established a rapid, portable and economical technique for the identification of pathogens in positive blood culture bottles through a novel MinION nanopore sequencer amplicon sequencing scheme, which replaces traditional Sanger sequencing.