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Related Experiment Videos

How quantitative is quantitative PCR with respect to cell counts?

W Ludwig1, K H Schleifer

  • 1Lehrstuhl für Mikrobiologie, Technische Universität München, Freising, Germany. ludwig@mikro.biologie.tu-muenchen.de

Systematic and Applied Microbiology
|March 16, 2001
PubMed
Summary

Developed quantitative PCR assays accurately detect specific bacteria like E. coli and S. aureus. Bacterial cell numbers cannot be directly estimated from DNA quantification due to growth phase variations.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biotechnology

Background:

  • Accurate bacterial detection is crucial for diagnostics and research.
  • Quantitative PCR (qPCR) offers a sensitive method for microbial quantification.
  • Standardization of qPCR assays for diverse bacterial targets remains a challenge.

Purpose of the Study:

  • To develop and validate quantitative diagnostic PCR systems for key bacterial species.
  • To assess the correlation between ribosomal DNA (rDNA) quantification and bacterial cell numbers.
  • To investigate the influence of growth phase on qPCR-based cell enumeration.

Main Methods:

  • Design of rDNA-targeted primers and probes using in silico tools (ARB, Primer Design).
  • Development of TaqMan-based qPCR assays on ABI PRISM 7700.

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  • Evaluation using purified genomic DNA and bacterial cells of target and reference organisms.
  • Assessment of assay sensitivity, reliability, and reproducibility with DNA fragments <100 bp.

    • Main Results:

    • Sensitive, reliable, and reproducible quantification of target rDNA achieved.
    • High concentrations of non-target DNA (1 mg/sample) led to underestimation of target DNA.
    • A clear correlation observed between rDNA quantification and bacterial growth.
    • Bacterial cell numbers could not be directly estimated from qPCR data due to variable genome content across growth phases.

    Conclusions:

    • Quantitative diagnostic PCR systems provide accurate rDNA quantification for specific bacteria.
    • Non-target DNA interference impacts quantification accuracy.
    • While rDNA quantification correlates with growth, direct cell number estimation is limited by organism physiology.