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
We report a novel method that allows simultaneous in situ amplification and then genotyping of single nucleotide polymorphism (SNP) for multiple samples on a single electronic microarray. The locus coding for one of the common inherited thrombosis risk factors, Factor V Leiden (FVL), was chosen as a model system for SNP analysis. This method combines strand displacement amplification (SDA) with electrophoretic movement and concentration of DNA on electronic microarrays to provide a single platform for DNA amplification and analysis. The method includes: electronic anchoring of allele-specific SDA amplifiable primers (APs) and a nonamplifiable primer (NAP) to different electrodes, electronic hybridization of genomic DNA from different samples to those primers, in situ amplification of target DNA, and genotyping of FVL. Compared to previous anchored SDA methods, the addition of a NAP improves detection signals by at least 20-fold. The sensitivity of this method is dependent on the amplification time. Using this method, nine different genomic DNA samples with known FVL genotypes were amplified and correctly genotyped on a single electronic microarray without any contamination between samples. The present method could streamline development of nucleic acid-based assays in applications of molecular diagnostic, point-of-care testing, and forensic detection, which often require the capability to analyze multiple samples efficiently.
