How quantitative is quantitative PCR with respect to cell counts?
Quantitative diagnostic PCR systems based upon rDNA targeted primer and probe combinations were developed for the detection of Escherichia coli, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, enterococci, Staphylococcus aureus, and Staphylococcus epidermidis. Primers and probes were designed in silico using the ARB software package (TU Munich) in combination with Primer Design software of PE Applied Biosystems. Purified genomic DNA or bacterial cells of target and reference organisms were used for the evaluation of the PCR assays applying the TaqMan technique on an ABI PRISM TM 7700 Sequence Detection System (PE Applied Biosystems). Sensitive, reliable and reproducible quantification of target rDNA could be achieved applying primer-probe combinations that mediate in vitro amplification 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 correlation of gene and cell numbers depending on the growth behavior of target organisms and to explore the potential to estimate cell numbers from target DNA quantification. A clear correlation of rDNA quantification and bacterial growth was observed, however, cell numbers cannot directly be estimated from quantitative PCR data, given that the cellular genome content varies with the growth phase of the organisms. In the case of Escherichia coli the cell numbers which could be assigned to a certain number of rDNA targets varied reasonably depending upon the growth phase of batch cultures.
Lehrstuhl für Mikrobiologie, Technische Universität München, Freising, Germany. ludwig@mikro.biologie.tu-muenchen.de
W
W
Ludwig
K H
KH
Schleifer
D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis; D018628:Gene Dosage; D020459:Genes, rRNA / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019914:Taq Polymerase
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012338:RNA, Ribosomal, 23S; D019914:Taq Polymerase