FLUORESCENCE CORRELATION-ANALYSIS OF PROBE DIFFUSION SIMPLIFIES QUANTITATIVE PATHOGEN DETECTION BY PCR

A sensitive, labor-saving, and easily automatable nonradioactive proce dure named APEX-FCS (amplified probe extension detected by fluorescenc e correlation spectroscopy) has been established to detect specific in vitro amplification of pathogen genomic sequences. As an example, Myc obacterium tuberculosis genomic DNA was subjected to PCR amplification with the Stoffel fragment of Thermus aquaticus DNA polymerase in the presence of nanomolar concentrations of a rhodamine-labeled probe (thi rd primer), binding to the target in between the micromolar amplificat ion primers, The probe becomes extended only when specific amplificati on occurs, Its low concentration avoids false-positives due to unspeci fic hybridization under PCR conditions. With increasing portion of ext ended probe molecules, the probe's average translational diffusion pro perties gradually change over the course of the reaction, reflecting a mplification kinetics, Following PCR, this change from a stage of high to a stage of low mobility can directly be monitored during a 30-s me asurement using a fluorescence correlation spectroscopy device, Quanti tation down to 10 target molecules in a background of 2.5 mu g unspeci fic DNA without post-PCR probe manipulations could be achieved with di fferent primer/probe combinations, The assay holds the promise to conc urrently perform amplification, probe hybridization, and specific dete ction without opening the reaction chamber, if sealable foils are used .