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,vith internal control (IC) DNA, which monitored both DNA extraction efficiency and PCR ef ficiency. DNA was subjected to PCR with a single primer pair, and the amoun t of PCR products was determined by electrochemiluminescence (ECL) in the Q PCR System 5000 (Perkin-Elmer) after hybridization with Tris (2,2'-bipyridi ne) ruthenium (II) chelate-labeled probes. The lower limit of sensitivity o f the D-PCR was reached at about 25 CMV particles/ml. Even with extremely l ow; DNA inputs (four molecules of IC DNA/200 mu l of plasma), very high yie lds (near 100%) were reached. DNA extracted from specimens that were CMV po sitive by the D-PCR was subsequently used in the Q-PCR, which was similar t o the D-PCR. The viral load was calculated directly from the ratio of CMV a nd 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. Inte rassay variation, intra-assay variation, and interspecimen variation were a bout 25%, suggesting that the Q-PCR will reliably detect fourfold differenc es 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.