In-house PCR: DETECTION TECNIQUE OF SUBGINGIVAL BIOFILM PERIODONPATHIC MARKERS

OBJECTIVE: To determine the detection capacity of the in-house end point PCR in the detection of periodontopathic markers in subgingival sampling of patients with periodontitis studied by pool and site. METHODS: The sample included adult volunteers with clinical/instrumental confirmed diagnostic of chronic periodontal disease (CP=38 Age: 51.8+13.69) and aggressive periodontal disease (AP=7; Age: 26.50+7.66) that do not present systemic diseases, antibiotic and/or dental treatment 6 months before the sample was taken. Calibrated personal chose the sites and the taking of samples. The samples were obtained using 4 sterile paper cones Nº35 till reaching bag depth and transported in RTF fluid media. DNA extraction was made with a commercial kit and PCR with specific primers (preserved regions of 16 S ARNr) for periodontopathic markers detection Group Red (BGR:Porphyromonas gingivalis(Pg),Treponema denticola(Td); Tannerella forsythia(Tf)); of Group Orange (BGN: Fusobacterium nucleatum(Fn), Prevotella intermedia(Pi)) y Aggregatibacter actinomycetemcomitans (Aa). The amplicons were visualized through electrophoresis in 2% agarose/buffer TAE gel and were colored (Gold view). Positive/negative controls and detection of bacterial genomic DNA were used. RESULTS: The amplicons distribution in CP samples was: Total /pool/ site(%): Aa: 0.61/21.21/50; Fn: 75.51/75.76/75; Pi: 12.24/18, 18/0; Pg :55.10/36.36/93.75; Td: 30.61/27.27/56.25; Tf: 62.27/45.45/93.75. In AP samples: Aa: 42.31/45. 83/0; Fn: 7.69/0/25; Pi: 19.23/20.83/0; Pg: 69.23/75/50; Td: 65.38/75/50;Tf:73.08/83.33/50. CONCLUSIONS: The detection of BGR – BGN markers by in house PCR technique allowed to determine the statistically significant difference in the detection frequency of Pg and Tf (Chi2 p=0.0005 y 0.0033 respectively) when studying individual sites in patients with CP. In work conditions the PCR usage in population study by pool, did not incremented the detection rate of the studied markers. The implementation of the technique of in house PCR at a site level would give important information regarding the ecological composition of each study site.