Development of a Reverse Transcription Loop-mediated Isothermal Amplification(RT-LAMP) Method for Grass Carp Reovirus

A RT-LAMP virus detection method was established for rapid,sensitive detection and early clinical diagnosis of grass carp reovirus(GCRV). Based on the VP6 gene sequences of GCRV strain HZ08 from GenBank, a set of four specific primers was designed, and the total RNA of GCRV attenuated vaccine strain was extracted as template for RT-LAMP reaction. The amplification products were confirmed by PvuⅡ restriction enzyme digestion. Then the LAMP reaction system, including Mg2+, dNTPs, betaine, and the concentration ratio of inner to outer primers was optimized. The results of the sensitivity test using recombinant plasmid contained VP6 gene as template showed that the minimum detection limit is 10 copies·L-1, a tenth the limit of RT-PCR. No cross-reactivity was observed among white spot syndrome virus(WSSV), Aeromonas hydrophila and viral nervous necrosis virus(VNNV) in RT-LAMP. Sixteen suspected grass carp of hemorrhagic disease were detected by the established assay, and consistent with the RT-PCR result, six samples got positive amplification.