Development of quantitative real-time PCR primers for detecting 42 oral bacterial species
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Original Paper
Development of quantitative real‑time PCR primers for detecting 42 oral bacterial species Soon‑Nang Park · Yun Kyong Lim · Joong‑Ki Kook
Received: 1 March 2013 / Revised: 9 April 2013 / Accepted: 23 April 2013 © Springer-Verlag Berlin Heidelberg 2013
Abstract In this study, we introduced species-specific quantitative real-time PCR (qPCR) primers designed based on a DNA-dependent RNA polymerase beta-subunit gene (rpoB) for detecting 42 oral bacterial species. The specificity of the qPCR primers was confirmed by conventional PCR with the genomic DNAs of 73–79 strains regarding 73–75 bacterial species including the type strain for the target species. The standard curves revealed the lower detection limits of 42 bacterial species-specific qPCR primers ranged from 4 to 40 fg below a cycle threshold (CT) value of 35, except Atopobium rimae, Fusobacterium nucleatum, Neisseria meningitidis, and Porphyromonas asaccharolytica which were 400 fg. These results suggest that 42 bacterial species-specific qPCR primers are suitable for applications in epidemiological studies related to oral infectious diseases such as periodontal diseases, endodontic infection, and dental caries. Keywords Quantitative real-time PCR primers · Oral bacteria · rpoB
Communicated by Erko Stackebrandt. Electronic supplementary material The online version of this article (doi:10.1007/s00203-013-0896-4) contains supplementary material, which is available to authorized users. S.-N. Park · Y. K. Lim · J.-K. Kook (*) Korean Collection for Oral Microbiology and Department of Oral Biochemistry, School of Dentistry, Chosun University, 375 Seo‑suk Dong, Dong‑Gu, Gwangju 501‑759, Republic of Korea e-mail: [email protected]
Introduction Approximately, 700 bacterial species or phylotypes might exist in human subgingival plaque (Aas et al. 2005). The golden standard method for identifying bacterial species is DNA–DNA hybridization and comparison of the 16S ribosomal RNA gene (16S rDNA) sequence (Krieg 2001). However, these methods are not appropriate to use in epidemiological or clinical studies for detecting or identifying bacteria at the species level, because they are time-consuming, labor-intensive, and are not economic. The conventional polymerase chain reaction (PCR) method has been widely used to overcome these problems (Ashimoto et al. 1996; Conrads et al. 1999). However, the conventional PCR can only be detected qualitatively. Considering theses problems, quantitative real-time PCR (qPCR) assay is strongly recommended for molecular epidemiological studies related to oral infectious diseases. The 16S rDNA gene has been generally used as a target gene to design a conventional PCR or qPCR primers to detect bacterial species (Ashimoto et al. 1996; Conrads et al. 1999; Kato et al. 2005; Nonnenmacher et al. 2005; Saygun et al. 2008; Bizhang et al. 2011). The 16S rDNA nucleotide sequences are well conserved among bacterial species (Krieg 2001). However, the homogeneity between some bacterial species, such as Streptococcus mitis and Strep
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