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To further understand the biology of adeno-associated virus (AAV) and identify the presence of AAV in laboratory samples, we have developed a sensitive PCR-based assay using degenerate primers based on the sequence of seven diverse AAV isolates. Using these primers, we can detect free virus in viral stocks, cleared cell lysate, as well as in latently infected cells. The method can detect as little as 10 viral copies/µL of sample and can be adapted for high-throughput screening technology. With this method, we have also detected a new AAV isolate from a stock of bovine adenovirus.