Distribution Of Actinobacillus Actinomycetemcomitans And Cdt Gene In Subgingival Plaque

【Objective】Real-time quantitative PCR technique for enumerating Actinobacillus actinomycetemcomitans (Aa ) and cytolethal distending toxin gene(cdt) in subgingival plaque samples from subjects with aggressive periodontitis(AgP) and chornic periodontitis(CP).【Methods】Primers for specific PCR amplification were designed based on the published sequence of the 16s rDNA and cdtB gene of Aa.The following procedures were taken,including Aa anaerble culture,PCR,PCR products purified.The purified DNA product was then linked with pMD19-T Vector to construct recombined plasmids and transformed into E.coli competent cells Top10.Target plasmids in white colonies selected by arnpicillin screening were identified by colony PCR and DNA sequencing.The concentration was identified by OD value and the copy numbers were calculated by value of OD in A260.The purified plasmid DNA was diluted into serial gradient concentrations as standard to plot a standard curve for Real-time PCR to predict the load of Aa and cdt gene contaminant in subgingival plaque samples. 105 subgingival plaque samples from 10 aggressive periodontitis(AgP) patients and 79 subgingival plaque samples from 10 chornic periodontitis(CP) patients and 15 from 5 subjects of periodontally healthy.【Results】The correlation coefficient of the standard curve reached 0.987 and 0.980 respected for 16s rDNA and cdtB gene.This method showed a broad quantification range from 10 to 107 copies and accurate sensitivity and specificity. Higher levels of Aa were found in samples from periodontitis subjects in comparison to samples from periodontally healthy subjects.【Conclusion】The primer pairs and probe were specific to the Aa 16s rDNA and cdtB gene did not crossreact with other bacteria.Successfully prepared the target fragment of 16s rDNA and cdtB gene of Aa,constructed the recombined plasmid contained the target fragment which is stable and keeps its sequential integrity and specificity ,specificity. Reference standards for Real-time PCR were constructed successfully and Real-time PCR can be applied to quickly detect bacterial burden of Aa and cdt gene in clinic subgingival plaque samples. Aa and its cdt gene was detected in higher numbers and frequency in aggressive periodontitis than in chronic periodontitis.The cdt gene are frequently found in the genome of Aa.