Development and optimization of a sensitive TaqMan® real‐time PCR with synthetic homologous extrinsic control for quantitation of Human cytomegalovirus viral load

Human cytomegalovirus ( Human herpesvirus 5 , HCMV) causes frequent asymptomatic infections in the general population. However, in immunosuppressed patients or congenitally infected infants, HCMV is related to high morbidity and mortality. In such cases, a rapid viral detection is crucial for monitoring the clinical outcome and the antiviral treatment. In this study, we optimized a sensitive biplex TaqMan® real‐time PCR for the simultaneous detection and differentiation of a partial HCMV UL97 sequence and homologous extrinsic control (HEC) in the same tube. HEC was represented by a plasmid containing a modified HCMV sequence retaining the original primer binding sites, while the probe sequence was substituted by a phylogenetically divergent one (chloroplast CF 0 subunit plant gene). It was estimated that the optimal HEC concentration, which did not influence the HCMV amplification is 1,000 copies/reaction. The optimized TaqMan® PCR demonstrated high analytical sensitivity (6.97 copies/reaction, CI = 95%) and specificity (100%). Moreover, the reaction showed adequate precision (repeatability, CV = 0.03; reproducibility, CV = 0.0027) and robustness (no carry‐over or cross‐contamination). The diagnostic sensitivity (100%) and specificity (97.8%) were adequate for the clinical application of the molecular platform. The optimized TaqMan® real‐time PCR is suitable for HCMV detection and quantitation in predisposed patients and monitoring of the applied antiviral therapy. J. Med. Virol. 88:1604–1612, 2016 . © 2016 Wiley Periodicals, Inc.