Development and Validation of a SYBR Green I Real-time RT-PCR Assay for Detecting Duck Reovirus

Abstract

SYBR Green I based real-time RT-PCR assay was developed for the detection and quantification of duck reovirus (DRV) using ABI PRISM 7500 sequence detection system. The assay was carried out using a set of primer designed to amplify highly conserved sequences of S2 gene of DRV. A 10-fold dilution series assay using a plasmid containing the cDNA of DRV S2 gene demonstrated the high sensitivity of the assay with a lowest detection limit of ≤1.48 copies/μL Standard deviation and coefficient of variation were low for both intra-assay and inter-assay variability. The assay performance was evaluated on 80 samples obtained from artificially infected Cherry Valley ducklings and 10 field specimens compared with the conventional RT-PCR assay. It was shown that 10 artificially infected samples tested negative in gel-based assay were positive for the real-time RT-PCR. DRV could be detected in all eight different tissues collected from the ducklings infected artificially. In contrast, the higher detection rate was obtained in the bursa of fabricius (90%), lung (90%), spleen (80%), and thymus (70%) than that in the liver (30%) as well as in the pancreas (10%). This method was rapid, specific, and sensitive for the detection of DRV and will be useful in veterinary diagnostic applications.