Primer Design for the Identification of Ten Oral Actinomyces Species Using Multiplex PCR

Background: Actinomyces is one of the predominant genera in the oral cavity. Oral Actinomyces species play a central role in the initial stages of biofilm formation on teeth; however, limited information is currently available on the distribution of individual species in different sites or clinical conditions. Moreover, a suitable method has yet to be developed to identify oral Actinomyces species because of the phenotypic and genetic similarities between these microorganisms. Objective: Actinomyces naeslundii, A. odontolyticus, A. oris, A. georgiae, A. gerencseriae, A. graevenitzii, A. dentalis, A. johnsonii, A. israelii, and A. meyeri among the genus Actinomyces are regarded as normal human oral Actinomyces species. The purpose of the present study was to design primers to identify oral Actinomyces species using multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 16S rDNA genes of the representative oral Actinomyces species. The 16S rDNA genes of the representative oral Actinomyces species were obtained from the DNA Data Bank of Japan, and a multiple sequence alignment analysis was performed with the CLUSTAL W program. Homology among the primers selected for oral Actinomyces species and their respective 16S rRNA sequences was confirmed by a BLAST search. Results: These primers were able to distinguish each oral Actinomyces species and did not display cross-reactivity with representative oral bacteria or other Actinomyces species. Moreover, we developed a multiplex PCR method with the ability to identify and differentiate oral Actinomyces species (i.e., A. naeslundii, A. johnsonii, A. oris, A. odontolyticus, A. israelii, A. georgiae, A. dentalis, A. graevenitzii, A. gerencseriae, and A. meyeri) using only two PCR tubes per sample. Conclusion: The present results indicate that our multiplex PCR method with these primers is useful for identifying the representative oral Actinomyces species. This method is easy because the use of Mighty Amp DNA Polymerase Ver.2 (Takara) means that DNA extraction may be avoided, and species identification using this method only takes approximately 2 hours. Thus, the method described herein will allow the prevalence of oral Actinomyces species and their involvement in oral infections to be fully clarified in future studies.