Profiling system of oral microbiota utilizing polymerase chain reaction-restriction fragment length polymorphism analysis
Profiling system of oral microbiota utilizing polymerase chain reaction-restriction fragment length polymorphism analysis
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DOI：
10.1016/j.job.2021.05.003
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发表时间：
2021-09-08
影响因子：
2.4
通讯作者：
Sato, Takuichi
中科院分区：
文献类型：
--
作者：
Sano, Hiroto;Wakui, Anna;Sato, Takuichi
目的：口腔微生物群的分析传统上使用常规方法进行。这些方法相对耗时且劳动密集。使用高速下一代测序对口腔微生物群进行宏基因组分析是非常有前途的技术。然而，它是昂贵的。本研究旨在开发一种简单，经济有效的分析方法，为口腔微生物群使用16 S rRNA基因聚合酶链反应-限制性片段长度多态性（PCR-RFLP）分析的PCR扩增的16 S核糖体RNA基因。方法：口腔分离的59种细菌从人类唾液中，包括链球菌，放线菌，韦荣氏球菌，厌氧培养CDC厌氧5%羊血琼脂平板。从单菌落中提取基因组DNA，并使用27 F和1492 R通用引物对16 S rRNA基因进行PCR扩增。PCR产物经纯化后用Hpall限制性内切酶单酶切鉴定。从GenBank数据库中获得16 SrRNA基因序列，并与琼脂糖凝胶电泳获得的RFLP图谱进行比较。基于GenBank 16 S rRNA基因序列计算这些物种的预期片段大小。对GenBank序列进行分析，共获得59种模式。用Hpall产生的RFLP模式区分了许多口腔细菌物种。产生了能够鉴定口腔细菌的RFLP模式。结论：基于16 S rRNA基因的PCR-RFLP分析方法可作为小型实验室口腔菌群分析的一种替代方法。(C)2021日本口腔生物学协会。Elsevier B. V.出版，保留所有权利。
Objectives: Profiling of oral microbiota has traditionally been performed using conventional methods. These methods are relatively time-consuming and labor-intensive. Metagenomic analysis of oral microbiota using high-speed next-generation sequencing is a highly promising technology. However, it is expensive. This study sought to develop a simple and cost-effective profiling method for oral microbiota using 16S rRNA gene polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of PCR-amplified 16S ribosomal RNA genes.Methods: Oral isolates of 59 bacterial species from human saliva, including Streptococcus, Actinomyces, and Veillonella, were cultured anaerobically on CDC Anaerobe 5% sheep blood agar plates. Genomic DNA was extracted from single colonies and 16S rRNA genes were PCR-amplified using the 27F and 1492R universal primers. The PCR products were purified and characterized by single digestion with Hpall restriction endonuclease. 16S rRNA gene sequences were obtained from the GenBank database, and the expected restriction profiles were compared with the RFLP patterns obtained from agarose gel electrophoresis.Results: Sixty-five RFLP patterns were obtained from 27 genera and 59 species. The expected fragment sizes of these species were calculated based on GenBank 16S rRNA gene sequences. Fifty-nine patterns were obtained from the analysis of GenBank sequences. The RFLP patterns produced with Hpall distinguished many oral bacterial species. RFLP patterns enabling identification of oral bacteria were generated. The 16S rRNA gene PCR-RFLP analysis did not require expensive equipment and reagents and was cost-effective.Conclusion: PCR-RFLP analysis based on 16S rRNA genes could be an alternative method for oral microbiota analysis in smaller laboratories. (C) 2021 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.