Development and evaluation of a real‐time nucleic acid sequence based amplification assay for rapid detection of influenza A

The development and introduction of effective treatment for influenza A in the form of neuraminidase inhibitors have made the rapid diagnosis of infection important especially in high‐risk populations. The aim of this study was to develop a real‐time nucleic acid sequenced based amplification (NASBA) using a molecular beacon that could detect a wide range of influenza A subtypes and strains in a single reaction by targeting a conserved region of the influenza genome, and to evaluate its sensitivity and specificity against traditional laboratory techniques on a range of clinical samples usefulness during the 2003/2004 influenza season. The results demonstrated the assay to be highly sensitive and specific, detecting <0.1 TCID 50 of virus stock. Three hundred eighty‐nine clinical samples were tested in total from two patient groups. Overall, the real‐time NASBA assay detected 64% (66/103) more influenza positive samples than cell culture and direct immunofluorescence (IF) and, therefore, was shown to be more sensitive in detecting influenza A in a wide range of respiratory samples than traditional methods. In conclusion, the real‐time influenza A assay demonstrated clinical usefulness in both hospital and community populations. J. Med. Virol. 74:619–628, 2004. © 2004 Wiley‐Liss, Inc.