To realize the rapid detection of duck reovirus, four primers were designed according to the highly conserved se-quence of avian reovirusσC protein gene published in GenBank. A sensitive and convenient reverse transcription loop-mediated iso-thermal amplification(RT-LAMP) method was established by optimizing reaction time, temperature and the concentration of each component in the reaction system. Then, the genome of duck common virus was used as template to detect specificity. Finally, a visu-alized LAMP detection method was established by adding SYBR Green I fluorescent dyes. The result showed that 25μl reaction sys-tem, Mg2+ 5×10-5 mmol, dNTP 1.25×10-5 mmol, Bst DNA polymerase 8 U, F3/B35×10-6μmol, FIP/BIP 3×10-5μmol, 62℃, 45 min was the optimum reaction condition, the influence of betaine on the reaction system was not obvious under the optimal condition. The positive rate of the method was consistent with that of the conventional RT-PCR assay, but the sensitivity was higher than the PCR method, and the minimum detection limit was 10 pg. The results could be determined directly by the naked eye ob-servation. This method laid the foundation for development of rapid detection kit of the RT-LAMP method for the visualization of muscovy duck reovirus.%为实现番鸭呼肠孤病毒的快速检测,本研究根据GenBank中公布的番鸭呼肠孤病毒σC蛋白基因的高度保守序列设计了4条引物,通过优化反应体系中各组分的浓度、反应时间和温度,建立了一种灵敏、便捷的RT-LAMP扩增方法.然后以鸭常见病毒基因组为模板,开展特异性检测,最后通过添加SYBR Green I荧光染料完成可视化LAMP检测方法的建立.结果显示:25μl LAMP反应体系,镁离子5×10-5 mmol,dNTP 1.25×10-5 mmol,Bst DNA聚合酶8 U,F3/B35×10-6μmol,FIP/BIP 3×10-5μmol,62℃,45 min为最优反应条件;在最佳条件下甜菜碱对反应体系影响不明显;对临床样品的阳性检出率与常规RT-PCR检测方法一致,但灵敏度高于PCR方法,最低检测限为10 pg,检测结果可直接通过肉眼观察来判断.该方法为番鸭呼肠孤病毒的可视化RT-LAMP快速检测试剂盒研制奠定了基础.
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