Combination of Genus-specific PCR and RFLP Analysis for Identification of Oral Streptococci

Objective: Oral streptococci are frequently isolated from the oral cavity such as dental plaque. These bacteria have been identified by conventional methods, e.g., biochemical and serological tests, which are sometimes inconsistent. To reliably identify oral streptococci, we firstly constructed genus Streptococcus-specific PCR primers of 16S ribosomal RNA genes, and then generated restriction fragment length polymorphism (RFLP) profiles of reference and clinical strains of oral streptococci including mutans, mitis and anginosus groups. Methods: 16S rRNA gene sequences of oral streptococci were obtained from the GenBank database, and the alignment and design of genus-specific primers were performed by the Clustal W and the DNASIS program. We extracted genomic DNA from bacterial strains, and amplified 16S rRNA gene by PCR using genus Streptococcus-specific primers: Str-F and Str-R, and then digested the PCR products with the restriction endonucleases, HaeIII, HpaII, MseI, or RsaI. Results: We were able to obtain PCR products (790 bp) only from streptococcal strains. The combination of the results of RFLP analysis using HaeIII, HpaII, MseI and RsaI allowed the respective reference strains of S. mutans, S. sobrinus, S. cricetus, S. mitis, S.oralis, S. sanguinis, S. gordonii, S. anginosus, S. constellatus and S. intermedius to be discriminated. Clinical isolates of mutans and anginosus groups were assigned based on this PCR-RFLP analysis. Furthermore, the restriction profiles predicted from the 16S rRNA gene sequences in the GenBank database agreed with the actual RFLP patterns produced in the present study. Conclusion: Therefore, the combination of PCR amplification using genus Streptococcus-specific primers and RFLP analysis of the 16S rRNA gene is a reliable and useful identification method for oral streptococci. Supported in part by the KAKENHI (14771000 to TS; 14571944 to JM; 14370687 to NT; and 13470446) from the MEXT, and a Health Science Research Grant (H12-RHTA-005) from the MHLW.