Development of multiplexed real-time quantitative polymerase chain reaction assay for detecting human adenoviruses

Adenoviruses (AdVs) have been associated with a wide variety of human disease and are increasingly recognized as viral pathogens that can cause significant morbidity and mortality in immunocompromised patients. Early detection of AdV DNA in plasma and sterile fluids has been shown to be useful for identifying patients at risk for invasive AdV disease. Because of the large number of existing Adv types, few real-time quantitative AdV polymerase chain reaction (PCR) assays published effectively cover all AdV types. We designed a series of AdV PCR primers and probes and empirically multiplexed them into 2 separate real-time PCR assays to quantitatively detect all 49 serotypes of human AdV (types 1-49) available from American Type Culture Collection. We then subsequently multiplexed all the primers and probes into 1 reaction. The sensitivity of these assays was determined to be less than 10 copies per reaction (500 copies/mL plasma). In a retrospective evaluation, we detected all 84 clinical AdV isolates isolated in cell culture from patients undergoing hematopoietic stem cell transplantation between 1981 and 1987. Prospective analysis of 46 consecutive clinical samples submitted for AdV testing showed greater sensitivity and equal specificity of the AdV PCR than viral culture. This real-time PCR assay allows rapid, sensitive, and specific quantification of all currently defined AdVs into either 2 or 1 multiplex assay for clinical samples.