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首页> 美国卫生研究院文献>Frontiers in Cellular and Infection Microbiology >Development and Validation of a Reference Data Set for Assigning Staphylococcus Species Based on Next-Generation Sequencing of the 16S-23S rRNA Region
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Development and Validation of a Reference Data Set for Assigning Staphylococcus Species Based on Next-Generation Sequencing of the 16S-23S rRNA Region

机译：基于16S-23S rRNA区域下一代测序的葡萄球菌种类参考数据集的开发和验证

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Many members of the Staphylococcus genus are clinically relevant opportunistic pathogens that warrant accurate and rapid identification for targeted therapy. The aim of this study was to develop a careful assignment scheme for staphylococcal species based on next-generation sequencing (NGS) of the 16S-23S rRNA region. All reference staphylococcal strains were identified at the species level using Sanger sequencing of the 16S rRNA, sodA, tuf, and rpoB genes and NGS of the 16S-23S rRNA region. To broaden the database, an additional 100 staphylococcal strains, including 29 species, were identified by routine diagnostic methods, 16S rRNA Sanger sequencing and NGS of the 16S-23S rRNA region. The results enabled development of reference sequences encompassing the 16S-23S rRNA region for 50 species (including one newly proposed species) and 6 subspecies of the Staphylococcus genus. This study showed sodA and rpoB targets were the most discriminative but NGS of the 16S-23S rRNA region was more discriminative than tuf gene sequencing and much more discriminative than 16S rRNA gene sequencing. Almost all Staphylococcus species could be distinguished when the max score was 99.0% or higher and the sequence similarity between the best and second best species was equal to or >0.2% (min. 9 nucleotides). This study allowed development of reference sequences for 21 staphylococcal species and enrichment for 29 species for which sequences were publicly available. We confirmed the usefulness of NGS of the 16S-23S rRNA region by identifying the whole species content in 45 clinical samples and comparing the results to those obtained using routine diagnostic methods. Based on the developed reference database, all staphylococcal species can be reliably detected based on the 16S-23S rRNA sequences in samples composed of both single species and more complex polymicrobial communities. This study will be useful for introduction of a novel diagnostic tool, which undoubtedly is an improvement for reliable species identification in polymicrobial samples. The introduction of this new method is hindered by a lack of reference sequences for the 16S-23S rRNA region for many bacterial species. The results will allow identification of all Staphylococcus species, which are clinically relevant pathogens.
机译：金黄色葡萄球菌属的许多成员是临床相关的机会病原体，需要准确，快速地鉴定靶向治疗。这项研究的目的是基于16S-23S rRNA区域的下一代测序（NGS），开发针对葡萄球菌物种的精心分配方案。使用16S rRNA，sodA，tuf和rpoB基因的Sanger测序和16S-23S rRNA区域的NGS在物种水平上鉴定了所有参考葡萄球菌菌株。为了拓宽数据库，通过常规诊断方法，16S rRNA Sanger测序和16S-23S rRNA区域的NGS鉴定了另外100种葡萄球菌菌株，包括29种。结果使得能够开发涵盖50个物种（包括一个新近提出的物种）和葡萄球菌属6个亚种的16S-23S rRNA区域的参考序列。这项研究表明，sodA和rpoB靶标具有最高的判别力，但16S-23S rRNA区域的NGS比tuf基因测序的判别力更大，而与16S rRNA基因测序的判别力更大。当最大得分为99.0％或更高，并且最佳和次佳物种之间的序列相似性等于或大于0.2％（最少9个核苷酸）时，几乎所有葡萄球菌都可以被区分。这项研究允许开发21种葡萄球菌物种的参考序列，并丰富其序列可公开获得的29种物种。我们通过鉴定45种临床样品中的整个物种含量并将结果与​​使用常规诊断方法获得的结果进行比较，从而确认了16S-23S rRNA区域NGS的有用性。基于开发的参考数据库，可以基于由单一物种和更复杂的微生物群落组成的样品中的16S-23S rRNA序列可靠地检测所有葡萄球菌物种。这项研究将有助于引入一种新颖的诊断工具，这无疑是对微生物样品中可靠物种鉴定的一种改进。由于缺少许多细菌物种的16S-23S rRNA区参考序列，因此阻碍了这种新方法的引入。结果将鉴定所有与临床相关的病原体的葡萄球菌。

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