Detection Of P.i And P.n. In Chrnic Pulpitis And Chronic Apical Periodontitis Using Real Time Quantitative PCR

Background and Objective:The microbial populations involved in pulpitis and apical periodontitis are known to be highly complex and variable, dominated by anaerobic gram-negative bacteria. Prevotella intermedia (Pi.) and Prevolella nigrescens(P.n.). belonging to Prevotella, are black-pigmented anaerobic gram-negative rods and can be detected frequently in the infected root canals. Attempts to culture anaerobic bacteria from infected root canals could result in a significant underestimation of the numbers of bacteria present and most molecular techniques could not be used to quantify bacterial numbers. Real time quantitative PCR using SYBR GreenⅠpattern allows for rapid detection and quantification of bacterial DNA and therefore bacterial number. It has become a popular tool due to its rapidity, sensitivity and specificity. Pulpitis are mainly caused by caries and traumas, while apical periodontitis are usually secondary to the inflammations of the pulp. Microbial colonization in the infected root canals is a dynamic process and can be affected by the time of the infections.The aim of this study was to investigate the distribution and quantitative levels of P.i.and Pn in patients with chronic pulpitis and chronic apical periodontitis using real time quantitative PCR of SYBR GreenⅠpattern and compare the distribution conditions of thses two pathogens in chronic pulpitis and chronic apical periodontitis.Methods:The root canal levels of P. intermedia, P. nigrescens, and total bacteria were investigated by a quantitative real time PCR assay based on 16SrDNA. A total of 40 chronic pulpitis (n=20) and chronic apical periodontitis (n=20) cases were analyzed. Root canal samples were collected; genomic DNA was extracted and detected by SYBR Green I real-time PCR targeted the 16SrDNAof P.i., P.n., and total bacteria, separately. Statistical analysis was achieved by SPSS.16.0.Result:In samples with chronic pulpitis, the number of P.i., P.n. and the 16SrDNA of the total bacteria ranged from 1.3198x102 to 4.8067x102,7.9269×102to 9.9902×103,1.3432xl04to 3.4112xl06, respectively; positive rates were 55%,40%, and 100%. The proportions of P.i. and P.n. in total bacterial load ranged from 0.000103%-3.37% and 0.259%-4.25%, separately. While in samples with chronic apical periodontitis the number of Pi., P.n. and the 16SrDNA of the total bacteria ranged from 2.4051xl02 to 1.2841xl03,1.6241×102 to 1.8136x103,3.5732x104 to 1.6997x106, respectively; positive rates were 45%,30%,and 100%. The proportions of P.i. and P.n. in total bacterial load ranged from 0.014%-1.50%and 0.0401%-0.122%, separately.The difference of the proportion of P.n. in total bacterial load was significant in statistics. The proportion of P.n. in total bacterial count in chronic pulpitis was higher than that in chronic apical periodontitis.Conclusion:In quantitative level, P.i. and P.n. were not absolute pathogens of chronic pulpitis and chronic apical periodontitis. The results indicated that the colonization condition of P.n. varied with the time of the infection in the root canasl.