摘要:
为更加快速、灵敏地检测草鱼呼肠孤病毒(Grass Carp Reovirus, GCRV),建立环介导等温扩增(RT-LAMP)检测方法。根据GenBank中GCRV HZ08等毒株VP6蛋白基因序列设计引物,以GCRV弱毒疫苗株(GCHV-892)总RNA为模板建立RT-LAMP反应,对扩增产物用限制性内切酶PvuⅡ进行酶切鉴定,所得酶切产物大小符合预期值。随后对LAMP反应体系包括Mg2+、dNTPs、甜菜碱(betaine)和内外引物浓度比进行了优化,并以含VP6蛋白基因的重组质粒(pEASY-VP6)为模板考量了该方法的敏感性,结果显示最低检测限为10 copies·μL-1,比RT-PCR检测方法敏感10倍。特异性试验表明与白斑综合征病毒(WSSV)、嗜水气单胞菌(Aeromonas hydrophila)、病毒性神经坏死病毒(VNNV)无交叉反应。应用RT-LAMP方法检测16份疑似出血病草鱼,6份病料获得了阳性扩增,与RT-PCR检测结果一致。
Abstract:
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.
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