关键词: 草鱼出血病, 呼肠孤病毒, RT-PCR, 检测方法

Abstract: To understand the prevalence of Grass carp reovirus (GCRV)， and for the purpose of early clinical diagnosis， the viral RT-PCR detective method was established. Based on the VP6 gene sequences published in GenBank， a pair of specific primers was designed， and the total RNA of GCRV attenuated vaccine strain GCHV892 was extracted as template to perform RT-PCR reaction， the obtained target band was 712 bp. Then the PCR reaction system and procedure were optimized， the sensitivity and specificity of the method were also considered. The results showed that the minimum detection limit of the sensitivity test was 102 copies·μL-1， in which the recombinant plasmid contained VP6 gene， was adopted as reaction template. The method has good specificity because no crossreactivity was observed between white spot syndrome virus (WSSV)， Aeromonas hydrophila and viral nervous necrosis virus(VNNV). Seven grass carp suspected with hemorrhagic disease were detected by the established assay， and three samples got positive amplification， then one of the positive specimens was cloned and sequenced， the results indicated that the sequence possesses maximum 99% similarity with known sequences in GenBank.

Key words: hemorrhage disease of grass carp, reovirus, RT-PCR, detection method