Detection of Sulfate-Reducing Bacteria (SRB) using RT-PCR

Introduction and aims: Sulfate-reducing bacteria (SRB) are strictly anaerobic microbes. In periodontitis patients the frequency of SRB presence was significantly higher in deep pockets in comparison with healthy sites. The presence of periodontal SRB is related to active tissue degradation, and in this way can indicate lesions that may demand further therapy. In this study, the occurrence of the SRB strain Desulfomicrobium orale was determined using quantitative real-time PCR on samples collected from 15 patients undergoing periodontal treatment. Materials and method: Using the method of Slots samples were taken introducing paper tips into gingival pockets before SPT as well as 3 and 6 month afterwards. The DNA extraction was performed using the high pure PCR Template Preparation Kit (Roche). For the production of a DNA-standard for quantification, a 350 bp rDNA fragment was amplified by PCR using D. orale rDNA specific primer. The resulting fragment was cloned into pCR2.1 cloning vector using the TOPO TA Cloning Kit (Invitrogen). The quantitative real time PCR was performed using QuantiTect Probe PCR Kit (Qiagen). Results: Out of 15 patients, all samples investigated (baseline, 3 month and 6 month) contained D. orale although in different amounts. Conclusion: Our clinical pilot studies indicate the SRB as “late colonizers” in the subgingival biofilm formation. Quantitative real-time PCR using dual labelled probes is a suitable method to reliably investigate the presence of SRB in bacterial probes isolated from gingival pockets.