16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota.
16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota.
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DOI：
10.1371/journal.pone.0052241
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Hilty M
中科院分区：
文献类型：
--
作者：
Brugger SD;Frei L;Frey PM;Aebi S;Mühlemann K;Hilty M
利用限制性内切酶Tsp509I和Hpy166II，建立了一种新的基于非培养的16S rRNA末端限制性片段长度多态性(T-RFLP)方法，用于鼻咽微生物区系的鉴定，并使用最近公布的454焦磷酸测序数据进行了验证。在153例婴幼儿急性中耳炎(AOM)的临床鼻咽标本中，检出5个Tsp509I和6个Hpy166II末端片段，发生率为10%。克隆和测序鉴定了所有TF的流行率>；6%，从而充分描述了最重要的细菌分类群的细菌群落变化。结合的7价肺炎球菌多糖疫苗(PCV-7)和先前的抗生素暴露对细菌组成的影响符合加性主效应和乘性相互作用模型(AMMI)，与16S rRNA454焦磷酸测序数据一致。此外，所提出的T-RFLP方法能够将肺炎链球菌与链球菌的其他成员区分开来，因此可以识别最重要的人类呼吸道病原体之一。这通常不是通过当前的高通量测序协议实现的。总之，所提出的16S rRNA基因T-RFLP方法是一种高度健壮、易于操作的方法，是计算要求高的下一代测序分析的一种廉价的替代方法。如果需要鉴定大量鼻咽标本，建议首先进行16S rRNA T-RFLP，只有在T-RFLP鼻咽模式不同或显示未知的TF时，才使用下一代测序。
A novel non-culture based 16S rRNA Terminal Restriction Fragment Length Polymorphism (T-RFLP) method using the restriction enzymes Tsp509I and Hpy166II was developed for the characterization of the nasopharyngeal microbiota and validated using recently published 454 pyrosequencing data. 16S rRNA gene T-RFLP for 153 clinical nasopharyngeal samples from infants with acute otitis media (AOM) revealed 5 Tsp509I and 6 Hpy166II terminal fragments (TFs) with a prevalence of >10%. Cloning and sequencing identified all TFs with a prevalence >6% allowing a sufficient description of bacterial community changes for the most important bacterial taxa. The conjugated 7-valent pneumococcal polysaccharide vaccine (PCV-7) and prior antibiotic exposure had significant effects on the bacterial composition in an additive main effects and multiplicative interaction model (AMMI) in concordance with the 16S rRNA 454 pyrosequencing data. In addition, the presented T-RFLP method is able to discriminate S. pneumoniae from other members of the Mitis group of streptococci, which therefore allows the identification of one of the most important human respiratory tract pathogens. This is usually not achieved by current high throughput sequencing protocols. In conclusion, the presented 16S rRNA gene T-RFLP method is a highly robust, easy to handle and a cheap alternative to the computationally demanding next-generation sequencing analysis. In case a lot of nasopharyngeal samples have to be characterized, it is suggested to first perform 16S rRNA T-RFLP and only use next generation sequencing if the T-RFLP nasopharyngeal patterns differ or show unknown TFs.
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影响因子：
3.7
作者：
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影响因子：
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作者：
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