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Related Experiment Videos

Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection by PCR

N G Walter1, P Schwille, M Eigen

  • 1Department of Biochemical Kinetics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

Proceedings of the National Academy of Sciences of the United States of America
|November 12, 1996
PubMed
Summary
This summary is machine-generated.

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A new method, amplified probe extension detected by fluorescence correlation spectroscopy (APEX-FCS), offers sensitive and automatable detection of pathogen DNA. This technique accurately quantifies target molecules, minimizing false positives in molecular diagnostics.

Area of Science:

  • Molecular Biology
  • Biotechnology
  • Analytical Chemistry

Background:

  • Accurate detection of pathogen genomic sequences is crucial for diagnostics.
  • Existing methods may lack sensitivity, automation, or are labor-intensive.
  • Nonradioactive detection methods are preferred for safety and ease of use.

Purpose of the Study:

  • To establish a sensitive, labor-saving, and automatable nonradioactive procedure for detecting specific in vitro amplification of pathogen genomic sequences.
  • To demonstrate the utility of amplified probe extension detected by fluorescence correlation spectroscopy (APEX-FCS) for pathogen detection.
  • To achieve sensitive quantification of target molecules with minimal risk of false positives.

Main Methods:

  • Developed the APEX-FCS assay utilizing a rhodamine-labeled probe (third primer) at nanomolar concentrations during PCR amplification.

Related Experiment Videos

  • Employed the Stoffel fragment of Thermus aquaticus DNA polymerase for PCR.
  • Monitored changes in probe diffusion properties using fluorescence correlation spectroscopy (FCS) to reflect amplification kinetics.

    • Main Results:

    • Achieved sensitive detection and quantification of specific amplification of Mycobacterium tuberculosis genomic DNA.
    • Demonstrated quantification down to 10 target molecules in a background of 2.5 micrograms of unspecific DNA.
    • The low probe concentration effectively avoided false-positives due to unspecific hybridization.
    • Observed a measurable change in probe mobility correlating with amplification progression.

    Conclusions:

    • APEX-FCS is a sensitive, labor-saving, and automatable method for pathogen genomic sequence detection.
    • The assay allows for real-time monitoring of amplification kinetics via changes in probe diffusion.
    • APEX-FCS shows promise for integrated amplification, hybridization, and detection in a closed system, enhancing diagnostic workflows.