Molecular detection of the entire spectrum of human adenoviruses by a two-reaction RQ-PCR assay

Abstract

Adenoviruses (AdV) can cause life-threatening infections in immunosuppressed patients, particularly in the allogeneic stem cell transplantation (SCT) setting. Reliable diagnostic tests, covering the entire spectrum of the currently known 51 human AdV serotypes, are therefore of paramount importance. We have recently published a five-reaction real-time (RQ-) PCR assay detecting the AdV species A-F, and have demonstrated its ability to predict disseminated AdV disease. However, for routine diagnostic purposes it was desirable to develop a more economic approach to reducing cost and labor. We have sequenced presumably conserved regions of the adenoviral genome, and developed a two-reaction RQ-PCR assay. Due to the genetic similarities between the species B, D and E we could establish an assay facilitating detection and quantification of these three species. Similarly, a second assay covering the species A, C and F, has been established. The detection systems were tested using reference strains from each serotype and clinical samples derived from peripheral blood and stool of pediatric patients after SCT. The specificity and sensitivity of the two-reaction assay is comparable to the published five-reaction RQ-PCR assay, and permits quantification of AdV-DNA down to 1fg per reaction. From the perspective of routine clinical diagnosis, the assay provides an improvement by facilitating early detection and monitoring of adenoviral infections at a substantially reduced cost.