Profiling system of oral microbiota utilizing polymerase chain reaction-restriction fragment length polymorphism analysis

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.

Automated summary

This study developed a simple and cost-effective method for profiling oral microbiota using PCR-RFLP analysis of PCR-amplified 16S rRNA genes. The analysis successfully identified 65 RFLP patterns representing 27 genera and 59 species from human saliva. The PCR-RFLP method is a cost-effective alternative for oral microbiota analysis in smaller laboratories.