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Journal of Clinical Microbiology, May 2004, p. 1909-1914, Vol. 42, No. 5
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.5.1909-1914.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Comparison of a Duplex Quantitative Real-Time PCR Assay and the COBAS Amplicor CMV Monitor Test for Detection of Cytomegalovirus

Björn Herrmann,1* Viviana Cavaglia Larsson,1 Carl-Johan Rubin,1 Fredrik Sund,2 Britt-Marie Eriksson,2 Johan Arvidson,3 Zhibing Yun,4 Kåre Bondeson,1 and Jonas Blomberg1

Sections of Virology,1 Infectious Diseases, Department of Medical Sciences,2 Section of Pediatrics, Department of Women's and Children's Health, Uppsala University, Uppsala,3 Department of Clinical Virology, Huddinge University Hospital, Stockholm, Sweden4

Received 4 July 2003/ Returned for modification 7 September 2003/ Accepted 16 February 2004

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 103 to 108 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 >=105 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.

* Corresponding author. Mailing address: Department of Clinical Microbiology, Academical Hospital, SE-751 85 Uppsala, Sweden. Phone: (46) 18-611 39 52. Fax: (46) 18-51 56 15. E-mail: bjorn.herrmann{at}medsci.uu.se.

Journal of Clinical Microbiology, May 2004, p. 1909-1914, Vol. 42, No. 5
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.5.1909-1914.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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