A hexon-based fluorogenic polymerase chain reaction (PCR) assay utilizing the 5'-nuclease activity of DNA Taqpolymerase was developed as a rapid and type-specific diagnostic system for adenovirus type 4 (Ad4) detection and quantification. The assay consists of a pair of flanking primers and an internal fluorescence labeled probe that allows real time amplification to quantify the Ad4 virus. One out of 12 flanking primer pairs evaluated (combinations of three forward primers and four reverse primers) was found to be optimal for Ad4 virus detection that yielded background-free operation, i.e., no fluorescent signal generated by non-template controls. The assay was employed to detect Ad4 reference virus strain RI-67, Wyeth Ad4 vaccine strain and 71 different clinical Ad4 isolates from US military recruits used in this study with consistent sensitivity (lower detection limit) of 2-4 pfu per PCR reaction. The assay showed linear Ad4 detection with a dynamic range of greater than five logs (from 2-4 pfu/assay to greater than 10(5) pfu/assay). This Ad4-specific assay did not crossreact with representative members of Ad subgroups A, B, C, D and F at viral concentrations greater than 10(8) pfu/ml. It was also demonstrated that Ad4 viruses could be efficiently detected from throat swabs (71/72 specimens or 98.6% detection sensitivity) of infected patients by the Ad4-specific PCR. In general, there was a good correlation between PCR determined viral titers in throat swabs and time required to detect viral cytopathic effects (CPE) in cell culture. Evaluation of the simple Ad4 specific assay developed in this study could be used to provide a rapid clinically relevant diagnosis of Ad4 infections in patients with acute respiratory disease (ARD).