Quantitative real-time PCR based on single copy gene sequence for detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis.

Abstract

To establish a method for quantification of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis from subgingival plaque by real-time polymerase chain reaction (PCR) technique. Bacterial cells from both species were obtained from type culture and counted microscopically. Cellular suspension in sterile distilled water was used for DNA extraction by boiling for 20 min, with a mineral oil cover. Primers for PCR were selected from sequences of LktC gene (A. actinomycetemcomitans) and Arg-gingipain (P. gingivalis) to yield amplicons below 100 bp. SYBR Green I based real-time PCR was adjusted to quantify separately both species. A good sensitivity and specificity were obtained for both species, although the yield was better for A. actinomycetemcomitans. A good repeatability of cycle threshold (CT) was encountered, so coefficient of variation was below 6% at every initial copy number. A new method of quantification of A. actinomycetemcomitans and P. gingivalis based on SYBR Green real-time PCR is presented. Its good sensibility and repeatability will allow its application to analysis of subgingival plaque samples.