A real-time reverse-transcription isothermal recombinase polymerase amplification assay for the rapid detection of genotype III grass carp (Ctenopharyngodon idella) reovirus.

Grass carp (Ctenopharyngodon idella) hemorrhagic disease, which is characterized by external and internal hemorrhage, is a serious infectious disease affecting grass carp production. Strains of the causative agent, grass carp reovirus (GCRV), are divided into genotypes I, II and III, which are represented by the isolates GCRV-873, GCRV-HZ08 and GCRV-104, respectively. In this study, a real-time reverse-transcription recombinase polymerase amplification (real-time RT-RPA) assay was developed to detect the genotype III grass carp reovirus GCRV-104. The assay was based on the detection of the vp55 gene which encodes the outer fiber protein of the virus. A portable ESE-Quant Tube scanner, with a dimension of 17.4 × 18.8 cm, weighing about 1 kg, and equipped with temperature settings to amplify the DNA isothermally and spectral devices to detect the amplified products using fluorescence, was used to complete the assay. Under the optimal conditions, the assay took approximately 10 min to complete at 37 °C and showed no cross-reactions with other aquatic viruses. Consequently, this rapid real-time RT-RPA assay is a useful method for the simple, rapid and reliable detection of genotype III GCRV strains in resource-limited diagnostic laboratories.