Multiple Primer Set Performance Suitability for Multiplex Q-PCR Assay Design

Real time quantitative PCR (Q-PCR) has increased the speed and productivity of biological investigation. This technology promises to erase traditional quantitative burdens of time, cost, reproducibility, labor intensity and sample viability. The quantitative power of Q-PCR assays can be multiplied when a multiplex assay is designed. Purpose:This study proposes to use Q-PCR to investigate the individual and group performance suitability of three target-specific primer sets for use in subsequent multiplex Q-PCR assay designs. Methods: Two microliters of ten-fold serially diluted Streptococcus mutans and Streptococcus sobrinus DNA were used as template in 25µl Q-PCR mixtures that were thermalcycled and subjected to melting curve analysis using a Bio-Rad IQ5 gradient thermalcycler. A cycle threshold (Ct) value versus copy number standard curve was constructed for S. mutans and S. sobrinus. Likewise, a complex equivalent mixture of genomic DNA isolated from prototype strains of S.mutans, S.sobrinus, Streptococcus sanguis, Streptococcus oralis, Streptococcus mitis, Streptococcus salivarius and Streptococcus parasanguis was used to construct a total streptococci standard curve. Ct values of two mixtures, A and B, consisting of known concentrations of purified genomic DNA, were determined using two microliters of each sample in a separate Q-PCR. Stimulated saliva samples were serially diluted to 10^-4, subjected to standard plate counting on MS and Gold's agar and processed for use as template (2µl) in a separate Q-PCR. Results: Each primer set exhibited unique specificity and sensitivity over the dynamic range of its standard curve and under varied known and unknown template complexity. Quantitative relationships were found between the Q-PCR assays and the traditional standard plate counts. Conclusion: All primer sets were suitable for use in subsequent multiplex Q-PCR assay designs. Acceptable quantitative agreement between Q-PCR based methods and traditional standard plate counts may be obtained using these primers in individual SYBR green assays.

Supported by from grant #DE016684 from the NIDCR