鸭坦布苏病毒可视化RT-LAMP快速检测方法的建立与初步应用

Establishment and Application of Visual RT-LAMP for Rapid Detection of Duck Tembusu Virus

为实现鸭坦布苏病毒(duck tembusu virus,DTMUV)的快速检测,本研究根据GenBank中公布的DTMUV E蛋白基因的高度保守序列设计了1套引物,通过优化反应体系各组分的浓度、反应时间和温度,建立了一种灵敏、便捷的RT-LAMP扩增方法。结果显示,在最佳条件下甜菜碱对反应体系影响不明显;该方法灵敏度是常规RT-PCR的100倍;只能特异性扩增坦布苏病毒,对于其他常见的禽源病毒无特异性扩增;检测结果可直接通过肉眼观察来判断;对临床74份疑似病料,可检出65份。本试验建立的方法灵敏性高、特异性强,可用于鸭坦布苏病毒病的临床诊断和流行病学调查。

To realize the rapid detection of duck tembusu virus(DTMUV),a set of specific primers was designed according to the highly conserved sequence of DTMUV E protein gene published in GenBank.A sensitive and convenient RT LAMP amplification method was established by optimizing the concentrations of each component,reaction time and temperature in the reaction system.The results showed that the influence of betaine on the reaction system was not obvious under the optimal conditions.This method was100times more sensitive than the conventional RT PCR.The RT LAMP method specifically amplified tembusu virus but not other common avian viruses.The results could be differentiated by naked eyes.74clinic suspicious samples were tested and65of them were positive.These results suggested that the RT LAMP assay could be used as a method for the diagnosis and detection of clinical cases,and molecular epidemiological investigation of duck tembusu virus disease.