Human adenoviruses (HAdVs) are ubiquitous double-stranded DNA viruses that cause a wide array of diseases in humans including pharyngitis, pneumonia, gastroenteritis, hemorrhagic cystitis, and keratoconjunctivitis. They also cause life-threatening opportunistic infections in immunocompromised individuals and are responsible for outbreaks in certain populations. Diagnosis is traditionally by cell culture or antigen detection methods. However, some HAdVs can take up to 4 weeks to isolate, and diarrheagenic types 40 and 41 will not grow in routine cell culture. Therefore, the goal of this study was to design a rapid, real-time PCR assay to detect all known 57 HAdV types from multiple different specimen sources. Primers and fluorescence resonance energy transfer hybridization probes were designed to target a 185-bp region of the penton base gene of HAdV. The analytical sensitivity was determined to be 10 copies/l for HAdV types showing exact primer/probe homology in specimen matrix. Using whole-virus strains, the analytical sensitivity for representative HAdV types ranged from 10 ؊1 to 10 3 50% tissue culture infective dose (TCID 50 )/ml. The assay demonstrated 100% sensitivity and 99% specificity. This real-time PCR assay provides a rapid method for the detection of all 57 known HAdV types from respiratory specimens, blood, stool, urine, and ocular swabs. Human adenoviruses (HAdVs) are ubiquitous doublestranded DNA viruses that cause a wide array of diseases in humans, including pharyngitis, pneumonia, gastroenteritis, hemorrhagic cystitis, and keratoconjunctivitis (23). Adenoviruses may also cause life-threatening opportunistic infections in immunocompromised individuals and are responsible for outbreaks in certain populations such as day care attendees and military recruits (23).Adenoviruses are classified in the genus Mastadenovirus, which contains 7 known HAdV species (HAdV-A to HAdV-G). Historically, HAdVs were further classified by hemagglutination and serum neutralization reactions into 51 serotypes (29). New adenovirus types have since been identified by several authors based on genomic data, including several emerging and recombinant viruses, for a total of 57 unique HAdV types (1, 14, 16-18, 27, 29, 32, 33). Most recently, a primate adenovirus from New World monkeys was detected which crossed the species barrier to infect humans (2).Diagnosis is traditionally by cell culture or antigen detection methods. Most HAdVs grow readily in cell culture and can be detected in a variety of specimen types including nasopharyngeal, throat and conjunctival swabs, urine, cerebrospinal fluid (CSF), and stool. However, some types can take up to 4 weeks to isolate, and diarrheagenic types 40 and 41 will not grow in routine cell culture. Direct antigen detection from clinical specimens may be used for diagnosis of acute infections but provides markedly low sensitivity compared to culture (30). Less commonly, serology is used to detect a rise in adenovirus-specific antibodies following infection (23).Real-time PCR offers a ...