坦布苏病毒是新发现的一种可引起家禽产蛋和采食量下降及死亡的黄病毒属病毒。根据GenBank中登录的鹅源坦布苏病毒NS5基因序列,利用Primer Explorer V 4软件在序列保守区域设计1套特异识别NS5基因序列中6个不同区段的环介导等温扩增( loop-mediated isothermal amplification , LAMP)引物,并以此套引物建立一种基于LAMP技术的鹅源坦布苏病毒检测方法。结果表明,该方法灵敏度极高,是普通PCR方法的100倍,只能特异性扩增坦布苏病毒的RNA,对于其他常见禽RNA病毒均无特异性扩增。在反应体系中加入SYBR Green Ⅰ染料后,通过肉眼观察有无绿色荧光可直接判定结果。该方法特异性强,灵敏度高,无需特殊仪器,简便,快速,适用于鹅源坦布苏病毒的临床快速检测。
In this study , a RT-LAMP ( reverse transcription loop-mediated isothermal amplification ) assay was de-veloped to rapidly detect and diagnose goose tembusu virus (GTMUV).Using Primer Explorer V4 software, a series of specific primers were designed based on the nucleotide sequences of GTMUV NS5 gene available in GenBank . These primers targeted 6 distinct conserved sequences according to the conserved region of NS5 gene.The results showed the RT-LAMP method specifically amplified GTMUV but not other common avian disease RNA viruses , and the optimum reaction temperature and time were verified to be 60℃and 60 min.Compared with other methods , this method was 100 times more sensitive than the conventional PCR .Meanwhile , it did not require complicated thermal cycling steps and denaturation , with the addition of SYBR GreenⅠdye in the reaction mixture , and the results could be differentiated by naked eyes .Thus, the results proved that RT-LAMP was a reliable, accurate, simple and practi-cal method for the detection of avian Tembusu virus in the field .