Quantitative Microbiological Study of Subgingival Plaque by Real-Time PCR Shows Correlation between Levels of Tannerella forsythensis and Fusobacterium spp

Abstract

A TaqMan-based real-time PCR assay was established to quantify the periodontopathic bacteria Tannerella forsythensis and Fusobacterium spp. With this assay, the prevalence and proportion of these bacteria in clinical specimens were evaluated. Our preliminary results suggest a positive colocalization of T. forsythensis and Fusobacterium spp. in periodontal pockets.Periodontitis is thought to arise from the complex microflora consisting of putative periodontopathic bacteria, such as Porphyromonas gingivalis, Treponema denticola, Tannerella forsythensis (formerly Bacteroides forsythus), and Fusobacterium spp.(1, 5). These bacterial species are isolated together from the affected periodontal sites (3, 4), and it has been suggested that bacterial cooperation might promote the infectious process of periodontitis. Consequently, it is important to know the composition of microorganisms in periodontal pockets for diagnosis and rational treatment. A TaqMan-based real-time PCR assay recently developed for the quantitative detection of periodontopathic species has actually contributed to the diagnosis and monitoring of periodontal disease (2,8). One advantage of using a real-time PCR assay is the detection of both absolute and relative amounts of bacteria. This assay is also advantageous for detecting species such as T. forsythensis that are extremely difficult to grow in broth. In this study, we developed a real-time PCR assay to quantify T. forsythensis and Fusobacterium spp. and evaluated the prevalence and proportion of these bacteria by using clinical specimens to describe their colocalization in periodontal pockets.The oligonucleotide primers and TaqMan probes used, designed from the 16S rRNA genes with the Primer Express 1.5 software (Applied Biosystems, Foster City, Calif.), are listed in Table 1. The specificities of the primers and probes were confirmed by conventional PCR and by dot blot analysis with chromosomal DNAs extracted from T. forsythensis ATCC 43037; F. nucleatum ATCC 10953, ATCC 25586, ATCC 49256, and ATCC 51191; Fusobacterium russii ATCC 25533; Fusobacterium periodonticum ATCC 33693; and 18 other oral bacteria (data not shown).Amplification and detection by real-time PCR were performed with the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). For each real-time PCR, 20-l reaction mixtures containing 1 l of lysed cells, 1ϫ qPCR Master Mix (Eurogentec S.A., Seraing, Belgium), each sense and antisense primer at 200 nM, and the TaqMan probe at 250 nM were placed in each well of a 96-well plate. The reaction conditions were set at 50°C for 2 min and 95°C for 10 min, followed by 60 cycles of 95°C for 15 s and 58°C for 1 min. The critical threshold cycle (Ct) is defined as the cycle at which the fluorescence becomes detectable above the background and is inversely proportional to the logarithm of the initial number of template molecules. Standard curves for each organism were plotted for each primer-probe set with the Ct values obtained by amplifying successive 10-fold dilutions of a known conc...

Loop-Mediated Isothermal Amplification Method for Rapid Detection of the Periodontopathic BacteriaPorphyromonas gingivalis,Tannerella forsythia, andTreponema denticola

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Assessment of oral malodor and tonsillar microbiota after gargling with benzethonium chloride

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