Detection and Differentiation of Avian Reoviruses Using SYBR-Green I-Based Two-Step Real-Time Reverse Transcription PCR with Melting Curve Analysis

A two-step SYBR-Green I based real-time PCR with melting curve analysis was developed to detect and differentiate the avian reovirus (ARV) sigma C gene in field and vaccine ARVs. Three primer sets were used to amplify the sigma C gene from its 5', center, and 3' regions and analyze the melting point temperatures of nine ARVs. By combining the melting curves of the three ARV sigma C gene regions, melting curve analysis could accurately distinguish the ARVs of different subtypes, and the results were consistent with phylogenetic analysis. The ARV sigma C gene polymorphisms from different strains were also used to explain the differences in melting point temperatures. Compared with traditional subtyping methods, the current melting curve analysis provided an acculate test for separating ARVs, thereby making it a useful method for the improved selection of ARV vaccines.