Comparison of a duplex quantitative real-time PCR assay and the COBAS Amplicor CMV Monitor test for detection of cytomegalovirus.
A duplex quantitative real-time PCR (qPCR) assay was designed to detect both the polymerase gene (pol) and the glycoprotein gene (gB) of cytomegalovirus (CMV). The detection limit of the qPCR was determined to be 1 to 3 copies/reaction and the linear measure interval was 10(3) to 10(8) copies/ml. The qPCR system was compared to the COBAS Amplicor CMV Monitor test (COBAS) by an analysis of 138 plasma samples. Both systems detected CMV in 71 cases and had negative results for 33 samples. In addition, 34 samples were positive by qPCR and negative by the COBAS assay, but in no case was the COBAS result positive and the qPCR result negative. Thus, qPCR detected 48% more positive cases than the COBAS method. For samples with > or = 10(5) copies/ml by qPCR, a saturation effect was seen in the COBAS assay and quantification required dilution. Copy numbers for pol and gB by qPCR generally agreed. However, the reproducibility of qPCR assays and the need for an international standard are discussed. Discrepant copy numbers for pol and gB by qPCR were found for samples from two patients, and sequence analysis revealed that the corresponding CMV strains were mismatched at four nucleotide positions compared with the gB fragment primer sequences. In conclusion, a duplex qPCR assay in a real-time format facilitates quantitative measurements and minimizes the risk of false-negative results.
Herrmann
Björn
B
Section of Virology, Department of Medical Sciences, Uppsala University, Sweden. bjorn.herrmann@medsci.uu.se
Larsson
Viviana Cavaglia
VC
Rubin
Carl-Johan
CJ
Sund
Fredrik
F
Eriksson
Britt-Marie
BM
Arvidson
Johan
J
Yun
Zhibing
Z
Bondeson
Kåre
K
Blomberg
Jonas
J
D001483:Base Sequence; D003587:Cytomegalovirus / Q000235:genetics* / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000175:diagnosis* / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D018628:Gene Dosage; D005814:Genes, Viral; D015751:Genes, pol; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D014759:Viral Envelope Proteins / Q000235:genetics; D014773:Virology / Q000379:methods* / Q000706:statistics & numerical data
D017931:DNA Primers; D004279:DNA, Viral; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus