狂犬病病毒核酸RT-LAMP检测方法的建立

Reverse transcription-loop-mediated isothermal amplification method for rapid detection of rabies virus

目的建立RT-LAMP方法,实现对狂犬病病毒核酸的快速、灵敏、简便的检测。方法针对狂犬病病毒的核衣壳蛋白基因(N基因)和大转录酶蛋白基因(L基因)中的高度保守区段分别设计了一套引物,建立了检测狂犬病病毒的快速一步式反转录-环介导恒温扩增(RT-LAMP)方法。样品提取总RNA后,加入BstDNA聚合酶、各种反应成分,于63℃恒温水浴箱中放置60min,80℃5min终止反应,加入荧光染料SYBR GREEN I,根据颜色变化肉眼判定结果。结果两套引物对狂犬病病毒RNA进行RT-LAMP检测,均有良好的特异性,灵敏度均比RT-巢式PCR高10倍以上。结论该方法简单、快速,不需电泳,利用染色剂肉眼判定结果,不需要大型仪器,能在70min内完成对狂犬病病毒RNA的检测,适用于可疑动物的快速检测,尤其适合基层使用。

Detection of pathogen plays an important role in the control to rabies.In this article a rapid one-step reverse transcription-loop-mediated isothermal amplification（RT-LAMP） technique was developed with two sets of primers aiming to the N gene and the L gene of rabies virus respectively.Both two sets of primers showed fairly good specificity and more than 10 times sensitivity than RT-nest PCR.The whole protocol finished in less than 70mins with only a water-bath.Results were decided by naked-eyes without electrophoresis.This technique is fit for rapid diagnosis to suspectable RNA specimen especially in laboratory lack of special equipments.