Development of a 5' fluorogenic nuclease-based real-time PCR assay for quantitative detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis

Abstract

A 5' nuclease TaqMan PCR was developed for the quantitative detection of the periodontopathic bacteria Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. The relative numbers of bacteria were measured by the comparative threshold cycle method. This simplified method is a way of obtaining the relative quantities of these organisms from specimens and of monitoring the effect of therapy.

FIG. 1.

Amplification of genomic DNA from lysed cells. Serial dilutions of genomic DNA were from A. actinomycetemcomitans (A) or P. gingivalis (B). The relative fluorescence (ΔRn) was monitored as the increase in the intensity of the reporter dye relative to the intensity of the passive internal reference dye. The threshold fluorescence, or the level at which the threshold cycle was determined, is shown. The standard curves were generated from the amplification plots in the insets (correlation coefficients, 0.997 for A. actinomycetemcomitans and 0.983 for P. gingivalis). Ct is the cycle number at which the threshold fluorescence is reached.