Simultaneous Molecular Detection and Confirmation of Influenza AH5, with Internal Control

Influenza viruses continue to be a major cause of respiratory tract infection, resulting in substantial morbidity and mortality throughout the world. Accurate and rapid differential diagnosis of influenza virus infections, particularly associated with zoonotic infections, is important for public health actions, patient management, and treatment. Real-time PCR is widely considered the gold standard for molecular detection of influenza viruses owing to its high assay specificity, extreme detection sensitivity, and wide linear dynamic range. This protocol describes the use of a real-time RT-PCR assay for identification of influenza A and B viruses, detection of H5 subtype viruses, and an internal control, in a multiplexed, single-tube format. The inclusion of an internal bacteriophage control allows the efficiency of the extraction and amplification process to be monitored, so that false-negative results may be avoided. The primers and probe sets in this multiplex assay have been validated with a panel of influenza A viruses of different subtypes (including swine influenza viruses), and influenza B viruses, and specificity further confirmed with non-related respiratory viruses.