Development of a highly sensitive quantitative competitive PCR assay for the detection of murine cytomegalovirus DNA

Abstract

Viral persistence and molecular latency are characteristic of infection by the human cytomegalovirus (HCMV). Using the murine cytomegalovirus (MCMV) as a model for human infection, a quantitative-competitive polymerase chain reaction (QC-PCR) assay was developed to detect and quantify MCMV-DNA in the salivary glands of infected mice. The QC-PCR detected high numbers of MCMV DNA copies in the absence of infectious virus. By comparing the DNA content and the results obtained from a standard semiquantitative plaque assay, it is concluded that 1 plaque-forming unit (pfu) is the equivalent of approximately 1500 viral genomes. By day 42-post infection (pi) 4×10 3 copies of DNA/1 mg tissue were sufficient to reactivate infectious virions after cyclophosphamide immunosupression. By day 90 pi, however, when the DNA load was decreased to <1.2×10 2, reactivation was not observed. These results indicate that viral reactivation will occur when the number of infectious DNA copies is equivalent about 2–3 pfu. This quantitative test may therefore help to detect CMV and the risk of reactivation in immunosupressed patients.