Exonuclease-released Fluorescence Detection of Human Parvovirus B19 DNA

Background: The 5;-->3;-exonuclease activity of Thermus aquaticus (Taq) DNA polymerase permits polymerase chain reaction (PCR) product detection immediately after amplification using a fluorogenic probe. This approach eliminates the requirement for gel electrophoresis or enzyme immunoassays (EIA). The ligonucleotide probe is labeled with a reporter dye at its 5' terminus and a quencher dye at its 3' terminus and is present during DNA amplification. The exonuclease cleaves the reporter molecule from the probe-template hybrid, releasing it from the influence of the quencher molecule. The result is an increase in reporter fluorescence that can be read directly in a fluorescence spectormeter. In contrast to time-consuming gel electrophoresis and Southern blot hybridization or an EIA, this method can produce results from an entire 96-well microtiter plate in 15 minutes. Methods and Results: A B19-specific fluorogenic probe was synthesized containing a 5'-FAM label and a 3'-TAMRA label. Thirty clinical samples were analyzed for Human parvovirus B19 DNA by PCR amplification using both the fluorogenic and EIA method. Conclusions: Results generated with the fluorogenic probe correlated perfectly with those of the EIA, and the method would be particularly useful for high-volume work loads where gel or EIA-based approaches would be cumbersome.