Development of a real-time PCR procedure including an internal control forthe measurement of HCMV viral load

Human cytomegalovirus (HCMV) infections are frequent in immune-compromised patients. The recent develop ment of real-time PCR procedures that allow th e rapid quantification of genome load will be helpful for accurate monitori ng of these infections. Two extraction procedures were evaluated using 30 b lood samples that were processed pure and diluted (1/10). Repeatability and reproducibility of the quantitative PCR procedure using an internal contro l for amplification were analysed, and its sensitivity compared to a qualit ative PCR procedure using 50 HCMV culture positive blood samples. The real- time PCR and qualitative PCR procedures were positive in 46 and 48 of the s amples tested, respectively. Discrepancies were observed for samples with a low viral load. The sensitivity of the real-time PCR procedure was evaluat ed at 500 HCMV DNA copies per mi of sera. The use of an internal control co ncomitantly processed during the HCMV quantification did not alter the sens itivity of the procedure, and was relevant for the detection of putative PC R inhibitors that may interfere with the amplification process. This proced ure was used to measure genome load in two bone marrow transplant patients with HCMV disease, confirming that this new PCR procedure should be used wi dely for diagnosing and monitoring HCMV infections in transplant patients. (C) 2001 Elsevier Science B.V. All rights reserved.