A highly sensitive assay for detection and quantitation of human cytomegalovirus DNA in serum and plasma by PCR and electrochemiluminescence

Abstract

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.

FIG. 1

Recovery of IC DNA from plasma at the single-molecule level. (A) Reference plasma was supplemented with IC DNA to a concentration of 4 molecules per 200 μl of plasma. DNA was extracted from 20 200-μl aliquots, and one-quarter of the extracted DNA was used in the PCR (bars 1 to 20). DNA was also extracted from reference plasma only, which served as a negative control (bars c). The amount of amplimers was determined and expressed in LU. The cutoff level is indicated with a broken line. (B) In the same experiment IC DNA was used directly in 10 separate PCRs at an input expected to contain a mean of 1 molecule of IC DNA per PCR (bars 1 to 10). The amount of amplimers was determined by ECL and expressed in LU. The cutoff level is indicated with a broken line.

FIG. 2

Lower limit of detection of CMV DNA in plasma. DNA was purified from 200-μl reference plasma samples containing 20, 10, 5, or 0 CMV particles; 35 molecules of IC DNA were coextracted. Extractions were done four times for the specimens containing 20 and 0 particles and six times for specimens containing 10 and 5 particles. One-fifth of the extracted DNA was subjected to PCR, and the amount of CMV amplimers (filled triangles) and IC amplimers (open squares) was determined by ECL and expressed in LU. The cutoff level is indicated with a broken line.

FIG. 3

Diagnostic PCR. Sequential serum specimens from a renal transplant patient were subjected to D-PCR. Two negative controls (nl, human DNA + 35 molecules IC DNA; n2, human DNA only) and one positive control (pc, reference plasma seeded with CMV to 400 particles per milliliter) were included. Filled bars, CMV probe; hatched bars, IC probe. The amount of amplimers was expressed in LU. The cutoff level is indicated with a broken line.

FIG. 4

Quantitative PCR. Reference plasma was supplemented with CMV particles to a concentration of 150,000 CMV copies/ml, serial threefold dilutions were made in the same plasma, and Q-PCR was done for this dilution series. (A) ECL signals (LU) obtained with the CMV DNA probe (open squares) and the IC DNA probe (filled squares). (B) Values of the CMV DNA/IC DNA ratios after background correction (R). (C) CMV loads (number of copies of CMV per milliliter of plasma) were calculated by amplifying R by a constant factor (1,050). Negative controls (reference plasma) were included; the calculated loads for these controls varied between 0 and −2 copies/ml. The correlation coefficient (r) and slope were obtained by least-squares linear regression analysis.

FIG. 5

Quantitation of CMV load by Q-PCR. The sequential serum specimens from the renal transplant patient described in Fig. 3 were subjected to Q-PCR. Points for specimens found to be negative were arbitrarily plotted as 2 copies of CMV/ml for graphical representation.

FIG. 6

Serum and plasma specimens are equivalent substrates for Q-PCR. Serum, plasma containing heparin, plasma containing EDTA, and plasma containing citrate were tested. The plasma was obtained from each of four healthy volunteers (subjects a, b, c, and d; subject a, CMV seronegative; subjects b to d, CMV seropositive) and was seeded with CMV to 15,000 particles/ml (a1, b1, c1, and d1), 1,000 particles/ml (a2, b2, c2, and d2), and 185 particles/ml (a3, b3, c3, and d3), and the CMV loads were determined by Q-PCR. Open bars, plasma containing heparin; hatched bars, plasma containing citrate; stippled bars, plasma containing EDTA; filled bars, serum.

FIG. 7

CMV loads observed in patient serum may be significantly lower than those observed in plasma. (A) Plasma containing citrate (C), plasma containing EDTA (E), and serum (S) obtained in parallel from the same patient (subject A in Table 2) were subjected to Q-PCR (in duplicate). (B) Plasma containing citrate (C), plasma containing EDTA (E), and serum (S) obtained from a CMV-negative healthy volunteer and the serum from the patient (S+) were seeded to 750 CMV particles/ml, and the CMV loads were determined by Q-PCR. nc, negative controls (reference plasma).