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Journal of Clinical Microbiology, May 1999, p. 1489-1497, Vol. 37, No. 5
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

A Highly Sensitive Assay for Detection and Quantitation of Human Cytomegalovirus DNA in Serum and Plasma by PCR and Electrochemiluminescence

René Boom,* Cees Sol, Jan Weel, Yvette Gerrits, Monique de Boer, and Pauline Wertheim-van Dillen

Laboratory of Medical Microbiology, Department of Virology, Section of Clinical Virology, Academic Medical Center, University of Amsterdam, 1100 DD Amsterdam, The Netherlands

Received 28 April 1998/Returned for modification 12 June 1998/Accepted 5 February 1999

We describe a diagnostic PCR assay (D-PCR) and a quantitative PCR assay (Q-PCR) for the detection of human cytomegalovirus (CMV) in plasma and serum. In the D-PCR, DNA was purified from plasma or serum together with internal control (IC) DNA, which monitored both DNA extraction efficiency and PCR efficiency. DNA was subjected to PCR with a single primer pair, and the amount of PCR products was determined by electrochemiluminescence (ECL) in the QPCR System 5000 (Perkin-Elmer) after hybridization with Tris (2,2'-bipyridine) ruthenium (II) chelate-labeled probes. The lower limit of sensitivity of the D-PCR was reached at about 25 CMV particles/ml. Even with extremely low DNA inputs (four molecules of IC DNA/200 µl of plasma), very high yields (near 100%) were reached. DNA extracted from specimens that were CMV positive by the D-PCR was subsequently used in the Q-PCR, which was similar to the D-PCR. The viral load was calculated directly from the ratio of CMV and IC signals obtained by ECL. The Q-PCR assay is quantitative in the range of 100 to 150,000 copies of CMV/ml, independent of the anticoagulant. Interassay variation, intra-assay variation, and interspecimen variation were about 25%, suggesting that the Q-PCR will reliably detect fourfold differences in viral load. Comparison of paired serum and plasma specimens from CMV-infected individuals showed that serum CMV loads were frequently more than 10-fold lower than plasma CMV loads.

* Corresponding author. Mailing address: Laboratory of Medical Microbiology, Department of Virology, Section of Clinical Virology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1100 DD Amsterdam, The Netherlands. Phone: (31-20)-5665472. Fax: (31-20)-6974005.

Journal of Clinical Microbiology, May 1999, p. 1489-1497, Vol. 37, No. 5
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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