RESUMO
Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) is a unique gene amplification method that can be completed within 35 min at 62.5 degrees C. In the present study, RT-LAMP was used to develop a rapid and sensitive laboratory diagnostic system for the H5N1 highly pathogenic avian influenza (HPAI). The sensitivity of the system was 0.1-0.01 plaque-forming units per reaction for HPAI-H5N1 viruses belonging to the genetically and antigenically distinct clade 1, represented by A/Vietnam/JP1203/2004, and clade 2, represented by A/Indonesia/JP283/2006. This RT-LAMP sensitivity is 10-fold higher than the sensitivity of standard one-step RT-PCR. By using viral RNAs extracted from avian influenza viruses of H1-H15 hemagglutinin (HA) subtypes and human pathogenic respiratory viruses, it was confirmed that the RT-LAMP system amplifies specifically RNA of the H5 subtype virus. The system detected H5-HA genes in throat swabs collected from humans as well as from wild birds. These results suggest that the present RT-LAMP system is a useful diagnostic tool for surveillance of recent outbreaks of the HPAI-H5N1 virus.
Assuntos
Virus da Influenza A Subtipo H5N1/isolamento & purificação , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/diagnóstico , Influenza Humana/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Adolescente , Adulto , Idoso , Animais , Antígenos Virais/genética , Sequência de Bases , Criança , Pré-Escolar , Corvos , Primers do DNA/genética , Genes Virais , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Virus da Influenza A Subtipo H5N1/genética , Vírus da Influenza A/genética , Influenza Aviária/virologia , Influenza Humana/virologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
We developed a rapid and sensitive diagnosis system for H5N1 highly pathogenic avian influenza (HPAI) virus infection using an unique gene amplification method, reverse transcriptase loop-mediated isothermal amplification (RT-LAMP). The sensitivity of the system was found to be 100-fold higher than that of ordinary one-step RT-PCR. Moreover, by using viral RNAs extracted from influenza viruses of all 15 HA subtypes, the RT-LAMP system was confirmed to amplify only the RNA of H5 subtype virus. In the surveillance of H5N1 virus infection of wild birds, we detected two positive cases from dead crows found near the affected area with H5N1-HPAI by using RT-LAMP system, although one of two positive cases was missed by RT-PCR. These results suggested that our newly developed RT-LAMP system specific for H5 virus would be a beneficial diagnostic tool for surveillance of recent outbreaks caused by H5N1-HPAI viruses.
