Detection and quantification of periodontal pathogens in the subgingival microbiota of smokers and non-smokers

• Grant number: 13/14278-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: December 01, 2013
• End date: November 30, 2014
• Field of knowledge: Health Sciences - Dentistry - Periodontology
• Principal Investigator: Cláudio Mendes Pannuti
• Grantee: Gabriela Elsbeth Bitiati Schwab
• Host Institution: Faculdade de Odontologia (FO). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

The aim of this cross-sectional study will be to assess the presence and to quantify subgingival periodontal pathogens in smokers and nonsmokers. Thirty heavy smokers and 30 nonsmokers with severe chronic periodontal disease will be recruited. Subjects will be matched regarding sex, age + 5 years) and periodontal pocket depth (+1mm). Smoking status will be confirmed by means of subjects´ expired carbon monoxide levels. After screening and inclusion of the subjects, they will receive a complete periodontal examination and will fulfill a structured questionnaire comprising demographic and smoking status data. A pool of subgingival plaque samples will be collected from the deepest periodontal pockets. Tubes containing the samples will be kept at -20°C until the extraction of bacterial DNA. Identification and quantification of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, and Tanerella forsythia will be performed with real-time PCR technique, using the Taqman® system. The mini kit QIAamp® DNA Will be used for DNA extraction. Identification and quantification of bacteria will be performed by the Taqman® system with detection with the ABI PRISM Fast Sequence Detection System. Primers and probes designs were based on species-specific regions of 16S rRNA, by means of Primer ExpressTM software, and their specificity was confirmed with BLAST (Basic Local Alignment Search Tool) software, from National Center for Biotechnology Information Sever (http://ncbi.nlm.nih.gov/blast/). Serial dilutions from the plasmid will serve as positive controls for amplification reactions, and sterile water as the negative control. All clinical samples will be processed by real-time PCR technique, along with the standard curve of each bacteria in triplicate. Smokers and non smokers Will be compared regarding the presence and levels of subgingival periodontal pathogens. (AU)