RESUMO
Fluorescence resonance energy transfer probes were selected for three sets of reverse transcription -polymerase chain reaction (RT-PCR), each in duplex format to detect: (1). Influenza virus type A or B; (2). Neuraminidase subtypes N1 or N2; and (3). Hemagglutinin subtypes H1 or H3 using LightCycler Instrument. A pair of probes targeted a type or subtype specific RT-PCR amplified gene segment. The presence of a target in a set of amplification reaction was detected by increased fluorescence over background emitted from the appropriate reporter fluorophore on probe-target hybrid formation and by melting temperature (T(m)) analysis of probe-target hybrid. The T(m) of a probe-target in a duplex amplification was distinctly different from the other, and thus T(m) value allowed specific detection of a target. Amplified product in each set of amplification was also examined by conventional agarose gel electrophoresis. The sensitivities of detection by fluorescence signal analysis were equal or ten times better than that detected by agarose gel electrophoresis.
Assuntos
DNA Viral/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Vírus da Influenza B/classificação , Vírus da Influenza B/genética , Sequência de Bases , Primers do DNA/genética , Sondas de DNA/genética , Eletroforese em Gel de Ágar , Humanos , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/isolamento & purificação , Desnaturação de Ácido Nucleico , RNA Viral/química , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) for the simultaneous detection of influenza virus type and subtypes was evaluated for its use in clinical specimens. One part of each specimen was tested using an established standard culture and/or monoclonal antibody-based immunofluorescence assay method, and the other part was tested by the multiplex RT-PCR method for the presence or absence of the influenza virus, and its type and subtype. Sixty-seven specimens were examined. The results revealed a strong agreement between the data obtained by the established method and those obtained by the multiplex RT-PCR.
Assuntos
Influenza Humana/diagnóstico , Orthomyxoviridae/genética , Orthomyxoviridae/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Antígenos Virais/genética , Estudos de Avaliação como Assunto , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/genética , Vírus da Influenza B/isolamento & purificação , Gammainfluenzavirus/genética , Gammainfluenzavirus/isolamento & purificaçãoRESUMO
Influenza virus type and subtype specific primers were selected for use in reverse transcription polymerase chain reaction (RT-PCR). The selected primer sets were used in a single step RT-PCR of influenza virus RNA in multiplex format for the detection of virus type and subtypes. Three one step reaction conditions are optimized: (1) multiplex typing only, (2) multiplex subtyping of influenza A, and (3) multiplex typing and subtyping simultaneously. RNA from strains of influenza virus type A of subtypes H1N1, H3N2 and H5N1 and influenza virus type B was used for amplification and detection. The amplified DNA fragments were size analyzed for the presence of specific type and subtypes target DNA by agarose gel electrophoresis. The sensitivity of detection was about 0.01 TCID(50) for both influenza type A and B when amplification was for typing alone. In multiplex subtyping or multiplex typing and subtyping simultaneously, the sensitivities for types and subtypes specific bands were 0.01-0.1 TCID (50) in the tested volume.
