RESUMEN
A robust, ultrasensitive, and accurate quantitative assay was developed for avian reovirus (ARV) with the Light Cycler SYBR Green-based real-time reverse transcription-PCR (real-time LC RT-PCR). The assay exhibited high specificity as all negative controls and other avian pathogens, such as Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), avian influenza virus (AIV), and mycoplasma synovia (MS), failed to show any positive detection. A minimum of 39 copies/microl of ARV genomic RNA could be detected by the assay. By dilution analysis, the real-time LC RT-PCR developed in this study was 3-log more sensitive than the conventional RT-PCR for the detection of ARV. The vaccine and field isolates of ARV were detected by the real-time LC RT-PCR. As a result of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, the real-time LC RT-PCR will be useful as a routine assay for the clinical diagnosis of ARV infection.
Asunto(s)
Orthoreovirus Aviar/aislamiento & purificación , ARN Viral/análisis , Infecciones por Reoviridae/diagnóstico , Infecciones por Reoviridae/veterinaria , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Animales , Benzotiazoles , Diaminas , Electroforesis en Gel de Poliacrilamida , Fluorescencia , Colorantes Fluorescentes , Dosificación de Gen , Técnicas de Dilución del Indicador , Desnaturalización de Ácido Nucleico , Compuestos Orgánicos , Orthoreovirus Aviar/clasificación , Quinolinas , ARN Viral/genética , Infecciones por Reoviridae/virología , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/instrumentación , Sensibilidad y Especificidad , Serotipificación , TemperaturaRESUMEN
Avian reovirus (ARV) is a non-enveloped virus with a segmented double-stranded RNA genome surrounded by a double icosahedral capsid shell. ARVs are associated with viral arthritis, immunosuppression, and enteric diseases in poultry. The sigma C protein was involved in induction of apoptosis and neutralization antibody. In the present study, sigma C-His protein was expressed in Sf9 insect cells and purified by immobilized metal affinity chromatography. Eight monoclonal antibodies (mAbs) against sigma C-His and three mAbs against His were screened from hybridoma cells produced by fusion of splenocytes from immunized mice with NS1 myeloma cells. Among the eight mAbs against sigma C protein, all belonged to the IgG isotype except three for IgM. It was discovered that all anti-His mAbs were mixtures of IgG and IgM isotypes. mAbs reacted with sigma C-His protein in a conformation-independent manner based on dot blot and western blotting assays. The competitive binding assay indicated that all mAbs recognized the same epitope on sigma C protein that was conserved in different isolates. Compared with the commercial anti-ARV S1133 polyclonal antibody, mAb (D15) had universal reactivity to all serotypes or genotypes of ARVs tested. This monoclonal antibody may therefore be useful for the development of an antigen-capture enzyme-linked immunosorbent assay for rapid detection of field isolates.