A highly sensitive assay for detection and quantitation of human cytomegalovirus DNA in serum and plasma by PCR and electrochemiluminescence.
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 microl 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.
Boom
R
R
Laboratory of Medical Microbiology, Department of Virology, Section of Clinical Virology, Academic Medical Center, University of Amsterdam, 1100 DD Amsterdam, The Netherlands.
Sol
C
C
Weel
J
J
Gerrits
Y
Y
de Boer
M
M
Wertheim-van Dillen
P
P
D000925:Anticoagulants / Q000494:pharmacology; D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification*; D004279:DNA, Viral / Q000097:blood*; D006801:Humans; D008163:Luminescent Measurements; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D000925:Anticoagulants; D004279:DNA, Viral