牛病毒性腹泻病毒可视化一步RT-LAMP检测方法的建立

Establishment of a visual one-step RT-LAMP for detection of bovine viral diarrhea virus

为建立可视化一步RT-LAMP检测牛病毒性腹泻病毒(BVDV)的方法,根据GenBank中的BVDV的5′UTR序列,设计2组RT-LAMP引物,经过反应温度、反应时间、Mg^(2+)和甜菜碱的终浓度的优化,特异性及灵敏性试验,对3种常用显色指示剂钙黄绿素-MnCl_(2)、羟基萘酚蓝和中性红的显色效果进行评估,并使用商品化ELISA试剂盒与本方法进行对照试验。结果显示,经优化本方法在66℃30 min即可完成反应,采用中性红作为显色指示剂,检测BVDV可产生特异阳性反应;对BVDV RNA检测下限最低达0.021 pg/μL;对13份临床样品检测结果与商品化ELISA检测试剂盒相符率100%。结果表明,所建立的BVDV可视化一步RT-LAMP检测方法的特异性、灵敏性较好,可为BVDV临床快速检测提供帮助。

To establish a visual one-step RT-LAMP detection method for bovine viral diarrhea virus(BVDV),two sets of RT-LAMP primers were designed according to the 5′UTR sequence of BVDV from Gen Bank.The temperature,time,final concentration of Mg^(2+)and betaine were optimized.Moreover the specificity,sensitivity and color-developing effects of three common color indicators (calcein-MnCl_(2),HNB and neutral red) were evaluated.The clinical samples were detected by commercial ELISA kit and this method,respectively.The results showed that the optimal conditions were that the reaction was completed at 66℃for30 min,and neutral red as color indicator could cause only specific positive reaction to BVDV.The minimal detection limit was 0.021 pg/μL BVDV RNA.The coincidence rate of 13 clinical samples between commercial ELISA kit and this method was 100%.The results demonstrated that a visual one-step RT-LAMP detection method with high specificity and sensitivity was established,which can provide help for rapid clinical detection of BVDV.