title: A real-time TaqMan PCR for routine quantitation of cytomegalovirus DNA in crude leukocyte lysates from stem cell transplant patients.
abstract: A real-time TaqMan PCR based on the cytomegalovirus (CMV) polymerase (pol) gene was developed for quantitation of CMV DNA in crude peripheral blood leukocyte (PBL) lysate from stem cell transplantation (SCT) patients. The dynamic range of the assay was between 10 and 4x10(6) copies. Both intra- and inter-assay variability were well within +/-0.25 log10 S.D. Thus, a pooled PBL sample that was used as positive control in 57 consecutive TaqMan PCR runs over 7 months showed a stable CMV quantity (4.12+/-0.13, log10 mean+/-S.D.). The sensitivity of the pol TaqMan PCR was validated by parallel analysis of 177 PBL samples with a nested PCR. The use of crude PBL lysate as PCR input did not cause PCR inhibition. We demonstrated further the clinical utility of the newly developed TaqMan PCR by monitoring changes in CMV levels in eight patients receiving antiviral therapy. This TaqMan PCR was highly sensitive, reproducible, and stable and has served a useful tool for monitoring CMV DNA levels in large number of clinical samples in a routine diagnostic setting for over 1 year.
authors.affiliation: Department of Clinical Virology, Karolinska Institutet, Huddinge University Hospital, F68, S-141 86 Huddinge, Stockholm, Sweden. zhyu@labd01.hs.sll.se <zhyu@labd01.hs.sll.se>
authors.forename: Zhibing
authors.initials: Z
authors.lastname: Yun
authors.forename: Ilona
authors.initials: I
authors.lastname: Lewensohn-Fuchs
authors.forename: Per
authors.initials: P
authors.lastname: Ljungman
authors.forename: Lotta
authors.initials: L
authors.lastname: Ringholm
authors.forename: Jerker
authors.initials: J
authors.lastname: Jonsson
authors.forename: Jan
authors.initials: J
authors.lastname: Albert
mesh_terms: D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000175:diagnosis / Q000821:virology; D004279:DNA, Viral / Q000097:blood*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D033581:Stem Cell Transplantation / Q000009:adverse effects*; D019914:Taq Polymerase / Q000378:metabolism*
chemical_list: D004279:DNA, Viral; D019914:Taq Polymerase