Development of a reliable assay protocol for identification of diseases (RAPID)-bioactive amplification with probing for detection of avian reovirus

Avian reovirus (ARV) causes several disease syndromes in poultry including arthritis, malabsorption syndrome and chronic respiratory disease that result in major economic losses. Early detection is very important for the control of the ARV-induced infections. This study was therefore aimed at developing a reliable assay protocol for identification of diseases (RAPID)-bioactive amplification with probing (BAP) assay for detection of ARV. This assay combines nested polymerase chain reaction (PCR) and magnetic bead-based DNA probing systems greatly increasing its sensitivity and specificity. Alignment of ARV S2 gene from different ARV genotypes and serotypes was done to find the highly conserved regions for primer and probe design. Two reverse transcription (RT)-PCR primer pairs, six nested PCR primer pairs, and one magnetic probe were tested to find the most specific ones for ARV detection. The optimal conditions for RT-PCR, nested PCR, and hybridization of magnetic probe were established. The optimal annealing temperatures for RT-PCR and nested PCR were 62.1 and 54.8 °C, respectively. The optimal hybridization temperature was 51.2 °C using hybridization buffer (5× SSC and 0.5% SDS). The sensitivity of the kit was 5 copies/μl of ARV genomic RNA. The kit was very specific as all negative controls failed to show any positive reactions. The kit shows good reproducibility with intra- and inter-assay coefficient of variation (CV) of 1.3 and 1.7%, respectively. In addition, diferent serotypes and genotypes of ARV were tested by RAPID-BAP assay to estimate the practicability of the kit in clinical samples. All of ARV serotypes and genotypes tested could be detected by this kit proving that the kit is suitable for clinical application.