实时荧光技术在鸭坦布苏病毒RT-LAMP检测方法中的应用

Application of real-time fluorescent technique in the detection of duck Tembusu virus by reverse transcription loop-mediated isothermal amplification

由鸭坦布苏病毒(DTMUV)引起的蛋鸭产蛋急剧下降给我国的养鸭业造成了巨大的经济损失。针对DTMUV E基因保守区域设计一套LAMP引物,利用实时荧光技术建立了DTMUV的实时RT-LAMP病原检测方法。该方法检测RNA的最低检测极限可达到7.8 copies,其灵敏度是普通RT-PCR的100倍,且只能特异性扩增DTMUV的RNA,对其他常见鸭源病毒核酸均无特异性扩增。该方法简单快速、特异性强和灵敏度高,判定结果只需要观察扩增曲线,适用于DTMUV早期感染的诊断、分子流行病学调查和疫情监测。

Duck Tembusu virus（DTMUV） has caused huge losses to the poultry industry in China. A loop-mediated isothermal amplification（LAMP） assay based on real-time fluorescent technique was developed for the rapid detection of DTMUV using a set of specific primers according to the conserved region of DTMUV E gene. The sensitivity of real-time RT-LAMP was 100 times more sensitive than the conventional RT-PCR, with a detection limit of 7.8 copies. The specificity of real-time RT-LAMP was confirmed using RNAs and DNAs extracted from related viruses which caused duck infections. This method is specific and sensitive, and can quickly detect pathogen by observing the amplification curve. As a practical molecular diagnostic method, it can be used for rapid detection of DTMUV early infection and epidemiological investigation.