Real-time quantitative polymerase chain reaction for enumeration of Streptococcus mutans from oral samples

Abstract

This study compared SYBR Green real-time quantitative PCR (qPCR) with standard plate counting for the enumeration of Streptococcus mutans in oral samples. Oral samples (n = 710) were collected from high-caries-risk children for quantification of S. mutans by qPCR using primer pairs. The S. mutans copy number was calculated with reference to a qPCR quantification cycle (Cq) standard curve and compared with the absorbance value at 600 nm of a standard suspension of S. mutans UA159. The S. mutans copy number results were evaluated in relation to standard plate count (SPC) results obtained from each sample following culture on Petri plates containing S. mutans selective media and reported as colony-forming units (CFUs). The mean S. mutans copy number calculated from qPCR was higher than the SPC CFUs (1.3 × 10(6) and 1.5 × 10(5) CFUs, respectively). The qPCR values were usually higher in individual samples and qPCR detected the presence of S. mutans 84% (231/276) of the time that the SPC did not, compared with 33% (4/12) of the time when qPCR failed to detect S. mutans and the SPC did. The qPCR technique was found to be more sensitive for detection of S. mutans from oral samples, a method that is not dependent on the viability of the sample taken and therefore is proposed as a more reliable and efficient means of quantification of S. mutans.

Fig. 1. UA159 SPC from known cell densities

Comparison of log SPC (CFU/ml) with log cells/ml determined from Ab₆₀₀ of S. mutans UA159 grown in Todd Hewitt broth. S. mutans broth culture from late log phase culture was adjusted to Ab₆₀₀ = 1.0, representing 10⁹ cells/ml based on electronic enumeration. Viable counts of S. mutans were obtained (CFU/ml) using average of 1-3 agar plates of 10-fold dilutions (10⁸ – 10³). Line is linear regression line through the average points generated.

Fig. 2. qPCR of standardized S. mutans DNA

Quantification cycle (Cq) values are plotted against DNA copy number (CN/ml). Two-fold dilutions of DNA extracted from electronically enumerated S. mutans cells (10⁹ cells/ml) were used as the CN standard. Cq values were determined from the diluted CN (n = 5). Insert graph illustrates representative amplification chart for a series of dilutions from the qPCR results (arrow indicates the crossover threshold used for determination of Cq).

Fig. 3. Comparison of Log SPC (CFU/ml) vs Log qPCR (CN/ml) for All Oral Samples

Plot of Log (1 + average) of duplicate CFU/ml and CN/ml. Values for each assay are presented as generated from SPC and qPCR assays. Line represents identity line where SPC and qPCR plots would correspond (i.e., intercept 0,0 with slope of 1). 8* is number of data points in which both SPC and qPCR equal zero. Other numbers along the X and Y axis are the number of data points between log values that differ between SPC and qPCR, respectively, where one indicated zero CFU or CN of S. mutans and the other assay indicated >0 S. mutans.

Fig. 4. Comparison of Log SPC (CFU/ml) vs Log qPCR (CN/ml) for Each Sample Type

Plot of Log of 1+ average of duplicate CFU/ml and CN/ml. Values for each sample type {whole saliva (WS), cotton swab (CS), plaque (PL), tongue (TS), for panels a-d, respectively} are presented as generated from SPC and qPCR assays. Line represents identity line where SPC and qPCR plots would correspond (i.e., intercept 0,0 with slope of 1). Number with ‘*’ is number of data points in which both SPC and qPCR equal zero. Other numbers along the X and Y axis are the number of data points between log values that differ between SPC and qPCR, respectively, where one indicated zero CFU or CN of S. mutans and the other assay indicated >0 S. mutans.