How quantitative is quantitative PCR with respect to cell counts?

Quantitative diagnostic PCR systems based upon rDNA targeted primer and pro be combinations were developed for the detection of Escherichia coli, Pseud omonas fluorescens, Pseudomonas alcaligenes, Pseudomonas alcaligenes, enter ococci, Staphylococcus aureus, and Staphylococcus epidermidis. Primers and probes were designed in silico using the ARE software package (TU Munich) i n combination with Primer Design software of PE Applied Biosystems. Purifie d genomic DNA or bacterial cells of target and reference organisms were use d for the evaluation of the PCR assays applying the TaqMan technique on an API PRISM TM 7700 Sequence Detection System (PE Applied Biosystems). Sensit ive, reliable and reproducible quantification of target rDNA could be achie ved applying primer - probe combinations that mediate in vitro amplificatio n of DNA fragments smaller than 100 base pairs. Large amounts of non target DNA (1 mg per sample) remarkably affected the quantification potential of the approach resulting in an underestimation of the amounts of target DNA. One of the principal goals was to use quantitative PCR to study the correla tion of gene and cell numbers depending on the growth behavior of target or ganisms and to explore the potential to estimate cell numbers from target D NA quantification. A clear correlation of rDNA quantification and bacterial growth was observed, however cell numbers cannot directly be estimated fro m quantitative PCR data, given that the cellular genome content varies with the growth phase of the organisms. In the case of Escherichia coli the cel l numbers which could be assigned to a certain number of rDNA targets varie d reasonably depending upon the growth phase of batch cultures.