RESUMO
A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for visual detection of European (EU) and North American (NA) porcine reproductive and respiratory syndrome viruses (PRRSVs) were established and evaluated with reference PRRSV strains and clinical samples. The assay was performed in two reaction tubes containing each set of primers specific for EU or NA-PRRSV at 58°C for 40min, and the results could be visually detected by the naked eye, using hydroxynaphthol blue dye. The detection limit of the assay was 1 or 0.1 TCID50/0.1mL for EU or NA PRRSV, respectively, which was comparable to that of the previously described real-time RT-PCR (qRT-PCR). The detection rate of the assay on 130 field samples was 72.3%, relatively higher than that of qRT-PCR (70.8%), and there was high overall percentage agreement between the two assays. The high specificity, sensitivity, and reliability of the RT-LAMP assay described in this study renders it useful for the rapid and differential diagnosis of EU and NA PRRSVs, even in under-equipped laboratories.
Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Síndrome Respiratória e Reprodutiva Suína/diagnóstico , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Transcrição Reversa , Animais , Corantes , Primers do DNA , Europa (Continente) , Limite de Detecção , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/ultraestrutura , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Suínos , Temperatura , Estados UnidosRESUMO
Here, we describe a uracil-DNA glycosylase (UNG)-treated reverse transcription loop-mediated isothermal amplification (uRT-LAMP) for the visual detection of all subtypes of avian influenza A virus (AIV). The uRT-LAMP assay can prevent unwanted amplification by carryover contamination of the previously amplified DNA, although the detection limit of the uRT-LAMP assay is 10-fold lower than that of the RT-LAMP without a UNG treatment. To the best of our knowledge, this is the first successful application of deoxyuridine triphosphate/UNG strategy in RT-LAMP for AIV detection, and the assay can be applied for the rapid, and reliable diagnosis of AIVs, even in contaminated samples.
