Development of a real-time PCR procedure including an internal control for the measurement of HCMV viral load.
Human cytomegalovirus (HCMV) infections are frequent in immuno-compromised patients. The recent development of real-time PCR procedures that allow the rapid quantification of genome load will be helpful for accurate monitoring of these infections. Two extraction procedures were evaluated using 30 blood samples that were processed pure and diluted (1/10). Repeatability and reproducibility of the quantitative PCR procedure using an internal control for amplification were analysed, and its sensitivity compared to a qualitative 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 samples tested, respectively. Discrepancies were observed for samples with a low viral load. The sensitivity of the real-time PCR procedure was evaluated at 500 HCMV DNA copies per ml of sera. The use of an internal control concomitantly processed during the HCMV quantification did not alter the sensitivity of the procedure, and was relevant for the detection of putative PCR inhibitors that may interfere with the amplification process. This procedure was used to measure genome load in two bone marrow transplant patients with HCMV disease, confirming that this new PCR procedure should be used widely for diagnosing and monitoring HCMV infections in transplant patients.
Najioullah
F
F
Laboratoire de Virologie, EA 3085, Domaine Rockefeller, 69373 Lyon cedex 08, France. najiou@rockefeller.univ-lyon1.fr
Thouvenot
D
D
Lina
B
B
D016026:Bone Marrow Transplantation; D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000821:virology*; D004279:DNA, Viral / Q000032:analysis; D006801:Humans; D016031:Liver Transplantation; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load
D004279:DNA, Viral