Anchor-based fluorescent amplicon generation assays (FLAG) for real-time measurement of human cytomegalovirus, Epstein-Barr virus, and varicella-zoster virus viral loads.

BACKGROUND Monitoring the human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), or varicella-zoster virus (VZV) viral load is an important factor in the management of immunosuppressed patients, such as recipients of solid-organ or bone marrow transplants. The advent of real-time PCR technologies has prompted the widespread development of quantitative PCR assays for the detection of viral loads and other diagnostic purposes. METHODS The fluorescent amplicon generation (FLAG) technology uses the PspGI restriction enzyme to monitor PCR product generation. We modified the FLAG technology by introducing an accessory oligonucleotide "anchor" that stabilizes the binding of the forward primer to the target sequence (a-FLAG). We developed assays for HCMV, EBV, and VZV that incorporated an internal amplification-control reaction to validate negative results and extensively analyzed the performance of the HCMV a-FLAG assay. RESULTS The 3 assays performed similarly with respect to reaction efficiency and linear range. Compared with a commercially available kit, the HCMV a-FLAG assay results showed good correlation with calculated concentrations (r = 0.9617), excellent diagnostic sensitivity and specificity (99% and 95%, respectively), and similar values for the linear range (1-10(7) copies/microL), analytical sensitivity (0.420 copies/microL), and intra- and interassay imprecision. CONCLUSIONS The a-FLAG assay is an alternative real-time PCR technology suitable for detecting and quantifying target-DNA sequences. For clinical applications such as the measurement of viral load, a-FLAG assays provide multiplex capability, internal amplification control, and high diagnostic sensitivity and specificity.