Different real-time PCR formats compared for the quantitative detection ofhuman cytomegalovirus DNA

Background: The aim of this study was to compare the ABI PRISM 7700 Sequenc e Detection System and the LightCycler to develop a quantitative real-time PCR assay for the detection of human cytomegalovirus (HCMV) DNA suitable fo r routine hospital application. Methods: We used one exonuclease probe and five different hybridization pro be sets as sequence-specific fluorescence detection formats. For the exonuc lease assay and two hybridization probe sets, reproducibility and the detec tion limit were determined. To keep the total assay time to a minimum, we g radually shortened individual reaction steps on both instruments. Results: The exonuclease assay can be interchangeably performed on the 7700 and the LightCycler. No change of reaction conditions is required, except for the addition of bovine serum albumin to the LightCycler reaction. The s hortest possible total assay time is 80 min for the ABI PRISM 7700 Sequence Detection System and 20 min for the LightCycler. When the LightCycler is u sed, the exonuclease probe can be replaced by a set of hybridization probes . All assays presented here detected HCMV DNA in a linear range from 10(1) to 10(7) HCMV genome equivalents/assay (r >0.995) with low intraassay (<5%) and interassay (<10%) variation. Conclusions: The ABI PRISM 7700 Sequence Detection System as well as the Li ghtCycler are useful instruments for rapid and precise online PCR detection . Moreover, the two principles of fluorescence signal production allow HCMV quantification with the same accuracy. (C) 1999 American Association for C linical Chemistry.