H9N2亚型禽流感病毒二重荧光RT-LAMP检测方法的建立  

Development of multiple fluorescence-based reverse transcript loop-mediated isothermal amplification assay for detection of H9N2 avian influenza virus

作　　者：李云燕 谢芝勋 李孟 罗思思 谢丽基 张民秀 黄娇玲 谢志勤 邓显文 范晴 曾婷婷 王盛 LI Yun-yan;XIE Zhi-xun;LI Meng;LUO Si-si;XIE Li-ji;ZHANG Min-xiu;HUANG Jiao-ling;XIE Zhi-qin;DENG Xian-wen;FAN Qing;ZENG Ting-ting;WANG Sheng(College of Animal Science and Techonlogy,Guangxi University,Nanning 530005,China;Guangxi Key Laboratory of Veterinary Biotechnology,Guangxi Veterinary Research Institute,Nanning 530001,China)

机构地区：[1]广西大学动物科学技术学院,广西南宁530005 [2]广西壮族自治区兽医研究所广西兽医生物技术重点实验室,广西南宁530001

出　　处：《中国兽医学报》2020年第10期1970-1975,共6页Chinese Journal of Veterinary Science

基　　金：广西科技重大专项资助项目(桂科AA17204057);广西科技基地和人才专项资助项目(桂科AD17195083);“广西八桂学者”专项资助项目(2019-79);国家“万人计划”领军人才专项资助项目(2016-37-88)。

摘　　要：根据H9N2亚型禽流感病毒(AIV)的HA和NA基因的保守序列,设计2套特异性LAMP引物和2条探针引物,同时在2条探针的5′端分别标记不同的荧光基团,在3′端都标记猝灭基团,然后使用多色荧光成像仪观察反应后是否有荧光产生来判断反应管里是否有阳性扩增,通过荧光颜色的不同来分析阳性扩增产物的类型。特异性试验结果显示该方法能特异性扩增H9亚型和N2亚型AIV,对其他15个HA亚型和其余8个NA亚型AIV以及禽类常见病原体均无扩增;干扰性试验结果显示H9和N2两种亚型AIV可以在同一反应管中独立完成扩增,二者互不干扰;敏感性试验显示其最低检测限度为100拷贝/μL。试验结果表明本试验建立的二重荧光RT-LAMP检测方法具备反应快速、灵敏、特异性强同时可以用肉眼直接观察辨别扩增产物的优点,对H9N2亚型AIV临床快速检测具有较好的应用前景。Two sets of specific lamp primers and two pairs of probe primers were designed for the conserved regions of the HA and NA genes from H9 N2 avian influenza virus(AIV),the probes were labeled with different fluorescent groups respectively at the 5′ end,and both of the probes contained a quenching group,at the 3′ end.The fluorescent products were detected by a multicolor flurescence imager to determine the positive amplification in the response tube,the type of the positive amplification products was analyzed by fluorescence color.The specificity experiments results showed the reverse transcript loopmediated isothermal amplification(RT-LAMP) assay was specific,H9 subtype and N2 subtype AIV were specifically amplified and did not detect other AIVs or avian respiratory pathogens;interference experiments results showed that H9 subtype and N2 subtype AIV can be amplified in the same reaction tubu without any interfererce;the sensitivity experiments results showed the amplification was achieved at 62℃ constant temperature for 80 minutes and the minimum detection limit was 100 copies/μL of RNA transcript containing LPAIV H9 N2 target genes.All the results demonstrate that this method is rapid,specific,sensitive and the amplification products can be analyed by naked eyes.Rapid clinical detection of H9 N2 subtype AIV by RT-LAMP would be a promising tool for surveillance.

关 键 词：H9N2亚型禽流感病毒 二重荧光RT-LAMP HA基因 NA基因 

分 类 号：S852.65[农业科学—基础兽医学] R535[农业科学—兽医学]