Enhanced analytical sensitivity of a quantitative PCR for CMV using a modified nucleic-acid extraction procedure

Accurate and rapid diagnosis of CMV disease in immunocompromised individual s remains a challenge. Quantitative polymerase chain reaction (QPCR) method s for detection of CMV in peripheral blood mononuclear cells (PBMC) have im proved the positive and negative predictive value of PCR for diagnosis of C MV disease. However, detection of CMV in plasma has demonstrated a lower ne gative predictive value for plasma as compared with PBMC. To enhance the se nsitivity of the QPCR assay for plasma specimens, plasma samples were centr ifuged before nucleic-acid extraction and the extracted DNA resolubilized i n reduced volume. Optimization of the nucleic-acid extraction focused on de creasing or eliminating the presence of inhibitors in the pelleted plasma. Quantitation was achieved by co-amplifying an internal quantitative standar d (IS) with the same primer sequences as CMV, PCR products were detected by hybridization in a 96-well microtiter plate coated with a CMV or IS specif ic probe. The precision of the QPCR assay for samples prepared from untreat ed and from pelleted plasma was then assessed. The coefficient of variation for both types of samples was almost identical and the magnitude of the co efficient of variations was reduced by a factor of ten if the data were log transformed. Linearity of the QPCR assay extended over a 3.3-log range for both types of samples but the range of linearity for pelleted plasma was 2 0 to 40,000 viral copies/ml (vc/ml) in contrast to 300 to 400,000 vc/ml for plasma. Thus, centrifugation of plasma before nucleic-acid extraction and resuspension of extracted CMV DNA in reduced volume enhanced the analytical sensitivity approximately tenfold over the dynamic range of the assay. (C) 2000 Wiley-Liss, Inc.