RESUMO
Microarrays are suitable for multiplexed detection and typing of pathogens. Avian influenza virus (AIV) is currently classified into 16 H (hemagglutinin) and 9 N (neuraminidase) subtypes, whereas Newcastle disease virus (NDV) strains differ in virulence and are broadly classified into high and low pathogenicity types. In this study, three assays for detection and typing of poultry viruses were developed on an automated microarray platform: a multiplex assay for simultaneous detection of AIV and detection and pathotyping of NDV, and two separate assays for differentiating all AIV H and N subtypes. The AIV-NDV multiplex assay detected all strains in a 63 virus panel, and accurately typed all high pathogenicity NDV strains tested. A limit of detection of 10(1)-10(3) TCID(50)/mL and 200-400 EID(50)/mL was obtained for NDV and AIV, respectively. The AIV typing assays accurately typed all 41 AIV strains and a limit of detection of 4-200 EID(50)/mL was obtained. Assay validation showed that the microarray assays were generally comparable to real-time RT-PCR. However, the AIV typing microarray assays detected more positive clinical samples than the AIV matrix real-time RT-PCR, and also provided information regarding the subtype. The AIV-NDV multiplex and AIV H typing microarray assays detected mixed infections and could be useful for detection and typing of AIV and NDV.
Assuntos
Vírus da Influenza A/isolamento & purificação , Influenza Aviária/diagnóstico , Doença de Newcastle/diagnóstico , Vírus da Doença de Newcastle/isolamento & purificação , Análise de Sequência com Séries de Oligonucleotídeos/veterinária , Animais , Galinhas/virologia , Genes Virais , Glicoproteínas de Hemaglutininação de Vírus da Influenza/análise , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Influenza Aviária/virologia , Neuraminidase/análise , Neuraminidase/genética , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/classificação , Vírus da Doença de Newcastle/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Sensibilidade e Especificidade , Perus/virologia , Proteínas Virais de Fusão/análise , Proteínas Virais de Fusão/genéticaRESUMO
A vesicular disease multiplex reverse transcription (RT)-PCR with an accompanying microarray assay was developed for simultaneous detection and typing of foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV), and for the detection of swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV). The multiplex RT-PCR successfully detected viral RNA from a collection of 49 strains of vesicular viruses, including multiple strains from all seven serotypes of FMDV and both serotypes of VSV. The multiplex RT-PCR was also able to produce amplified products from the RNA genome of all four viruses simultaneously in mixed samples. An indirect (post-PCR labelling) amplicon labelling method and a direct (concurrent labelling with PCR) amplicon labelling method were compared for the purpose of microarray detection and typing. Accurate detection and typing was achieved with all strains tested in the microarray assay which utilized 163 virus- and serotype-specific probes. It was observed that microarray increased detection for some samples compared to using multiplex RT-PCR alone. This was most likely due to signal amplification resulting from fluorescent labelling. The limit of detection of the microarray assay was as low as 4.6TCID(50)/mL for FMDV. No amplification products or microarray reactivity was observed with non-target livestock pathogens tested or with samples collected from healthy cattle, sheep and pigs. All FMDV and VSV serotypes were detected as early as 2 days post-inoculation from oral swabs obtained from cattle infected experimentally.
