RESUMO
Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase PCR (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 10(1.5), 10(2.3), and 10(3.1) 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the NA genes were genetically subtyped, while only 45% of HA genes and 74% of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples.
Assuntos
Hemaglutininas Virais/genética , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Influenza Aviária/virologia , Neuraminidase/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Proteínas Virais/genética , Animais , Benzotiazóis , Aves , Primers do DNA/genética , Diaminas , Genótipo , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/diagnóstico , Compostos Orgânicos/metabolismo , Quinolinas , Sensibilidade e Especificidade , Coloração e Rotulagem/métodosRESUMO
Real-time reverse transcription-PCR (RT-PCR) was developed for broad detection of diverse H5 and H7 genes in Eurasian and American lineages of avian influenza viruses by using primer and probe sets containing mixed bases. Optimal use of the mixed bases enabled us to minimize sequence mismatches and to broaden the gene detection spectrum without decreasing sensitivity.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/isolamento & purificação , Influenza Aviária/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Virologia/métodos , Animais , Aves , Primers do DNA/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Influenza Aviária/virologia , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/genética , RNA Viral/genética , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
Reverse transcriptase PCR designed to amplify the N1 to N9 neuraminidase (NA) genes of avian influenza viruses detected 118 of the 119 NA genes tested (99.2%) in a subtype-specific manner. This technique successfully subtyped all 167 recent avian influenza viruses isolated from birds. Subtype specificity was confirmed by sequence analyses of all 285 PCR products.