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

摘要

A sensitive, labor-saving, and easily auto- matable nonradioactive procedure named APEX-FCS (ampli- fied probe extension detected by fluorescence correlation spectroscopy) has been established to detect specific in vitro amplification of pathogen genomic sequences. As an example, Mycobacterium 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 (third primer), binding to the target in between the micromolar amplification primers.