Quantitative detection of periodontopathogens by real-time PCR Claudia Nonnenmacher a , Alexander Dalpke a , Reinier Mutters a , Klaus Heeg b, * a Institute of Medical Microbiology and Hygiene, Philipps University, Pilgrimstein 2, Marburg D-35037, Germany b Department of Hygiene and Medical Microbiology, University of Heidelberg, Im Neuenheimerfeld 324, 69120 Heidelberg, Germany Received 22 January 2004; received in revised form 3 June 2004; accepted 17 June 2004 Available online Abstract Specific bacteria are believed to play an important role in chronic periodontitis, yet the significance of their relative numbers in initiation and progress of the disease is still unclear. We report here the development of a sensitive, quantitative PCR technique for enumerating Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Dialister pneumosintes (Dp) and Micromonas micros (Mm) as well as total eubacteria in subgingival plaque samples from subjects with periodontitis. Quantification was performed with specific 16S rRNA target sequences with double fluorescence labeled probes and serial dilutions of plasmid standard by real-time PCR. This method showed a broad quantification range from 10 2 to 10 8 and accurate sensitivity and specificity. Fifty subgingival plaque samples from periodontitis patients and 33 from periodontally healthy subjects were subsequently examined. Higher levels of total bacteria numbers, Aa, Pg, Dp and Mm were found in samples from periodontitis subjects in comparison to samples from periodontally healthy subjects. Quantitative real-time PCR thus provides a reliable and valuable method for quantification of periodontopathogens in subgingival plaque samples. D 2004 Elsevier B.V. All rights reserved. Keywords: Periodontopathogens; Subgingival plaque; Real-time PCR 1. Introduction Periodontitis differs from many other types of infections since it is not caused by a single bacterium but by a group of bacteria. Although more than 500 different types of bacteria have been isolated from the oral cavity (Paster et al., 2001), only a small fraction of these bacteria has the potential to cause destruction of periodontal tissues (Socransky et al., 1999). Actino- bacillus actinomycetemcomitans and Porphyromonas gingivalis are considered the major pathogenic species in destructive periodontal disease (Slots and Ting, 1999). However, other subgingival species have also been related to periodontal disease as described for Prevotella intermedia, Prevotella nigrescens (former- ly P. intermedia), Bacteroides forsythus, Fusobacte- rium nucleatum, Campylobacter rectus, Eikenella corrodens, Treponema denticola, Micromonas micros (formerly Peptostreptococcus micros) and some other species. Detection of these putative pathogens in peri- odontal health and disease strongly depends on the 0167-7012/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2004.06.006 * Corresponding author. Tel.: +49-6221-568310; fax: +49- 6221-565857. E-mail address: klaus.heeg@med.uni-heidelberg.de (K. Heeg). www.elsevier.com/locate/jmicmeth Journal of Microbiological Methods 59 (2004) 117 – 125