Development of a 5' nuclease-based real-time PCR assay for quantitative detection of cariogenic dental pathogens Streptococcus mutans and Streptococcus sobrinus

Abstract

A 5' nuclease TaqMan PCR assay was developed for the quantitative detection of the major cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus. The absolute and relative numbers of bacteria were measured by this method. This assay will be useful for quantifying these organisms in oral specimens and for analyzing biofilm formation.

FIG. 1.

Amplification plots of chromosomal DNA from lysed cells. Serial dilutions of chromosomal DNA were from S. mutans (A) or S. sobrinus (B). The log-transformed relative fluorescence [ΔRn(log)] was monitored as the increase in reporter dye intensity relative to the passive internal reference dye. The threshold fluorescence, or level at which the threshold cycle was determined, is shown. The standard curves were generated from the amplification plots in the small panels (correlation coefficient = 0.996 for S. mutans and 1.00 for S. sobrinus). Ct is the cycle number at which the threshold fluorescence was reached.

FIG. 2.

Standard curves generated by known numbers of S. mutans (A) and S. sobrinus (B); linearity is shown for these organisms. The correlation coefficients are 0.982 for S. mutans and 0.958 for S. sobrinus. Ct is the cycle number at which the threshold fluorescence was reached.