RESUMO
BACKGROUND: the influenza viruses cause morbidity and mortality annually among children and elderly. Surveillance and rapid diagnosis is imperative in the reference laboratory, as clinical symptoms are insufficient for proper diagnosis. OBJECTIVES: this study involved the design of a rapid detection method for influenza A and B viruses using real time RT-PCR from clinical specimens. Methods were specifically designed for use on the Light Cycler. The sensitivity and specificity were also to be determined. STUDY DESIGN: the identification and discrimination of influenza A and B viruses employs two dual probe systems based on fluorescence resonance energy transfer (FRET) technology. Following submission by physicians participating in the Florida sentinel influenza network, 58 specimens were chosen for testing using both tissue culture and Light Cycler methods. RESULTS: of the 35 identified positive for influenza virus via tissue culture isolation, the Light Cycler results matched identification and typing with 100% agreement. However, the Light Cycler recognized 16 additional specimens that were positive for the presence of the virus. RT-PCR and nucleotide sequencing confirmed the presence of influenza A virus in these specimens. Using tenfold serial dilutions, the sensitivity of the Light Cycler method was determined to be 0.01 TCID50. The lower limit of RNA detection was determined as 1.6 x 10(-7) microg for influenza A virus, and 1.2 x 10(-7) microg for influenza B virus. Specificity of the Light Cycler method was determined by testing specimens containing adenovirus, parainfluenza virus and echovirus, all of which yielded negative results with no discernible background. CONCLUSIONS: overall, this newly developed method of simultaneous detection and typing of influenza types A and B using the Light Cycler proves to be more sensitive than tissue culture isolation, with corresponding specificity. This technique may be valuable for surveillance and rapid identification of influenza for early diagnosis.
