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Patient care providers face an enormous challenge in diagnosing vial respiratory diseases because of similar clinical manifestations, as well as insensitivity and/or slow conventional laboratory detection methods. Nucleic acid–targeted molecular assays are playing critical roles in rapid detection, screening, and identification of respiratory viral pathogens due to their high sensitivity and specificity, short test turnaround time, as well as automatic and high-throughput processing. User-developed and commercial molecular methods have gradually been developed and become available for detection and identification of common viral pathogens causing respiratory tract infections. Incorporated with cutting edge techniques, these methods can be used to detect one or more pathogens in a single reaction tube qualitatively and quantitatively. Although the molecular assays have been increasingly used in the clinical setting, laboratorians and clinicians should well know the limitations of these molecular assays to wisely choose the right tests and correctly interpret test results.