Detection of H5N1 avian influenza A virus by one-step RT-PCR. A. Amplification of serially diluted in vitro-transcribed single-stranded RNA (lanes 2 to 11) measured by RiboGreen RNA quantitation reagent and H5N1 RNA extracted from allantoic fluid of infected egg (lane 19). The non-template control is (sterile water) illustrated as "NTC". The viral load is indicated by the number of copies per reaction, (lane 2) 1 × 109 copies per reaction, (lane 3) 1 × 108 copies per reaction, (lane 4) 1 × 107 copies per reaction, (lane 5) 1 × 106 copies per reaction, (lane 6) 1 × 105 copies per reaction, (lane 7) 1 × 104 copies per reaction, (lane 8) 1 × 103 copies per reaction, (lane 9) 1 × 102 copies per reaction, (lane 10) 10 copies per reaction, and (lane11) 1 copy per reaction. Viral RNA extracted from human specimens that were previously confirmed respiratory syncytial virus (RSV) B (lane 12), dengue virus 1 (lane 13), dengue virus 2 (lane 14), dengue virus 3 (lane 15), dengue virus 4 (lane 16) and severe acute respiratory syndrome (SARS) (lane 17) were also tested as known negatives. RNA extracted from healthy individual (lane 18) was also performed. B. Specific detection of H5N1 avian influenza A virus. Reference strains of different subtypes of avian influenza A viruses (lanes 20 to 24), including non-influenza viruses such as Newcastle disease virus (NDV, lane 25) are indicated. The H5N1, H3N8, H9N2 and NDV isolates were isolated from field samples by the Veterinary Research Institute, Malaysia, the H5N3 and H7N5 isolates were provided by the Department of Veterinary Pathology of Tottori University, Japan. Negative signals from non-H5N1 isolates and the non-template control (water) are shown.