Qualitative detection of avian influenza A (H5N1) viruses: A comparative evaluation of four real-time nucleic acid amplification methods

The aim of this study was to determine the performance of real-time amplification based methods NASBA, TaqMan, RT-FRET, and RT-PCR LUX (TM) formats - for the detection of influenza A (H5N1) virus RNA. In an analysis of 54 samples obtained from a range of animal species in Thailand during the period 2003 2006, results showed that the NASBA (H5 = 98.2%, N1 = 96.3%), TaqMan (H5 = 98.2%, N1 = 96.3%) and FRET (H5 = 98.2%, N1 = 96.3%) had significantly higher rates of positive detection than LUX (H5 = 94.4%, N1 = 50.0%; P < 0.001) for influenza A, H5 and N1 isolates. There were no false-positive results from any methods used in the negative-control group of samples. The limits of analytical detection were at least 10 copies/reaction in real-time NASBA and LUX assays, while FRET and TaqMan assay appeared to be less sensitive at >= 100 copies/reaction. The assays were relatively specific without cross-reactivity to a number of other influenza strains or viral pathogens. In conclusion, our study demonstrated that real-time NASBA, TaqMan and FRET assays can be used to detect influenza A (H5N1) from a wide range of hosts, and be specific for H5N1 samples obtained during different outbreaks (2003-2006). All assays provided the benefit of rapid influenza H5N1 identification for early diagnosis, in the range of hours, and they are well suited to high throughput analyses. (C) 2008 Elsevier Ltd. All rights reserved.