Quantitative real-time PCR for detecting oral bacteria

Objective: The aim of the study was to establish and validate a quantitative real-time PCR assay (RQ-PCR) for typing a broad spectrum of caries and health associated oral bacteria. Methods: 140 PCR primers for relevant caries and health associated species were designed by aligning primer sequences against data from the Ribosomal Database Project and against human genome sequence. Samples of plaque (n=5 patients without caries) and carious dentin (n=5 patients with caries) were collected and bacterial DNA was isolated using QIAamp® DNA Mini Kit (Qiagen). RQ-PCR was performed in duplicates (reaction volume 12 µl - 1x SYBR Green PCR Master Mix, 0.33 µM specific primer, 3 ng extracted template DNA) on an ABI PRISM 7900 Sequence Detection System in 384-well PCR plates (95°C for 15 min, 40 cycles of 95°C for 15 s, 60°C for 1 min). Data analysis was conducted with Sequence Detection Software version 2.1. Specificity of PCR products was tested by melting-curve analysis with Sequence Detection Software version 2.1 and agarose gel analysis. Primers that produced unique PCR products of the correct size were used and cloned using CloneJET™ PCR Cloning Kit. pJET1.2/blunt vectors containing the RQ-PCR product were sequenced by Eurofins MWG Operon (Germany). Results: Out of 140 primers 68 produced unique PCR products. Those were cloned and sequenced. 53 primers were sequence verified; amongst them are species of the genera Abiotrophia, Neisseria, Fusobacterium, Actinomyces, Prevotella, Streptococcus, Lactobacillus, Bifidobacterium, Propionobacterium, Veillonella, Campylobacter, Capnocytophaga, Eubacterium, Rothia, Treponema, Corynebacterium and Selenomonas. Conclusion: The RQ-PCR assay was established and validated on human clinical samples. It can be used as a tool for typing health and caries associated bacterial species in further clinical investigations. (The study was supported by the German Association of Conservative Dentistry (DGZ))