Evaluation of PCR Primers for Cytomegalovirus Detection

We read with great interest the article by Mendez et al. (2) published in a recent issue evaluating different PCR primers for cytomegalovirus (CMV) detection. As CMV PCR becomes a frequently used assay in the management of immunocompromised patients, such as transplant recipients and human immunodeficiency virus-infected individuals, an increasing number of laboratories are establishing in-house CMV PCR assays. In designing a PCR assay, the choices of specimen, sample preparation method, primers, probes, and amplification and detection conditions play a critical role in the sensitivity and specificity of the assay. Mendez et al. (2) compared primer pairs for HindIII-X fragment, EcoRI fragment D, immediate-early antigen 1 gene (IEA1), and major immediate-early gene (MIE) and found sensitivites of 94, 87, 32, and 20%, respectively. Prompted by their paper, we evaluated the HindIII-X primer pair versus EcoRI fragment D (4) and MIE 2783-3114 (1), both of which are routinely used in our laboratory. For this comparison, we used 10 plasma samples containing a low level of CMV DNA (<1,000 copies/ml). DNA was extracted from each plasma sample by using four protocols: protocols A and B involved DNA extraction from 5 and 10 μl of plasma, respectively, with Chelex resin (Bio-Rad); protocol C started with 300 μl of plasma, which was centrifuged at 24,000 × g for 1 h after which DNA was extracted from the pellet with Chelex resin; and in protocol D, plasma samples were heated at 100°C for 50 to 55 s. The amounts of template DNA used in each PCR tube in protocols A, B, C, and D corresponded to 1, 2, 60, and 2 μl of plasma, respectively. The amplification was performed as previously described (3), by using 1 U of Pyrococcus furiosus (Pfu) DNA polymerase (Stratagene) in appropriate buffer, 50 μΜ each deoxynucleoside triphosphate (dNTP), and 0.5 μM each primer in 50-μl reaction volumes. PCR products were separated according to their molecular weight by agarose gel electrophoresis, stained with ethidium bromide, and visualized under UV light. Positive and negative controls were included in each run. In contrast to Mendez et al., we found a sensitivity of 78% for the EcoRI primers, which was superior to the MIE and HindIII primers’ sensitivities of 18 and 5%, respectively. All the specimens in which CMV was detected by HindIII primers also had CMV detected by EcoRI and MIE primers. The differences between primer pair sensitivities were stable across sample preparation protocols. Protocol D, which used 2 μl of heat-inactivated plasma sample/reaction tube, yielded the highest number of positive results across primer pairs, whereas protocol A, Chelex extraction of DNA from 10 μl of plasma, had the lowest yield. There are several differences between our assay and that described by Mendez et al. which might account for the discrepant results, mainly the utilization of different specimens, plasma versus peripheral-blood leukocytes (PBL), and different DNA polymerases, buffers, dNTP and primer concentrations, and cycling conditions. In addition, for this comparison we did not perform a Southern blot, although the assay used for selecting the 10 specimens relied on Southern blot confirmation. These discrepancies emphasize the importance of performing extensive evaluations of in-house PCR methods and adapting assay conditions to the primers used and the DNA template area that is interrogated.