二重荧光RT-LAMP鉴别检测禽呼肠孤病毒和鸡滑液囊支原体  

Identification of avian reovirus and Mycoplasma synoviae using duplex fluorescence RT-LAMP

作　　者：曾婷婷[1] 谢芝勋[1] 谢丽基[1] 王盛[1] 谢志勤[1] 黄娇玲 万丽军 任红玉 张艳芳[1] 张民秀[1] 范晴[1] 邓显文[1] ZENG Tingting;XIE Zhixun;XIE Liji;WANG Sheng;XIE Zhiqin;HUANG Jiaoling;WAN Lijun;REN Hongyu;ZHANG Yanfang;ZHANG Minxiu;FAN Qing;DENG Xianwen(Guangxi Veterinary Research Institute,Nanning 530001,China)

机构地区：[1]广西壮族自治区兽医研究所,广西南宁530001

出　　处：《畜牧与兽医》2023年第9期66-75,共10页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(31660715);国家“万人计划”领军人才专项(W02060083);广西八桂学者专项(2019A50)。

摘　　要：本研究旨在建立一套通过添加荧光探针FD,反应后可使用成像仪直接观测结果的鉴别检测禽呼肠孤病毒(ARV)和鸡滑液囊支原体(MS)的二重荧光RT-LAMP检测方法。根据ARV和MS的保守基因片段,设计了2套LAMP引物,并各添加1条FD荧光探针,2条FD探针分别标记CY5和6-FAM荧光基团。结果:ARV阳性对照在成像仪下发出红色荧光,MS阳性对照发出绿色荧光,双重模板经过图像融合后为黄色荧光;特异性结果显示,该二重荧光RT-LAMP检测方法仅能扩增ARV和MS两种病原,对其他常见病原如鸡毒支原体(MG)、禽流感病毒(AIV)、新城疫病毒(NDV)等无扩增反应;敏感性结果显示,该二重荧光RT-LAMP检测方法对ARV和MS的最低检测量分别为1.7×10^(2)copies和1.9×10^(2)copies;使用该方法检测40份临床样品,检出率与本实验室前期建立的二重荧光RT-PCR检测结果相同。综上,该二重荧光RT-LAMP检测方法可特异、敏感地鉴别检测ARV和MS,通过添加FD荧光探针和成像仪观测结果,为不同条件的实验室提供了新的检测手段。In this study,a duplex fluorescence RT-LAMP assay was established which was able to identify and detect avian orthoreovirus(ARV)and Mycoplasma synoviae(MS)by adding a fluorescence probe FD,which could be directly observed by imager after reaction.Two sets of LAMP primers were designed according to the conserved regions of ARV and MS,and an FD fluorescent probe was added.Fluorophores were labeled at the 3′end of FD with CY5 and 6-FAM,respectively.The specificity results showed that the ARV positive control presented red fluorescence under the imager,the MS positive control displayed green fluorescence,and the dual template exhibited yellow fluorescence,after image fusion;and no cross reaction with other pathogens such as MG,AIV,NDV and so on was observed.The sensitivity results showed that the minimum detection volume of this dual fluorescence RT-LAMP assay for ARV and MS was 1.7×10^(2) copies and 1.9×10^(2) copies,respectively.This method was then used to detect clinical samples,and the detection rate was the same as that of the dual fluorescence RT-PCR previously established in our laboratory.In conclusion,this dual fluorescent RT-LAMP method would be useful for specific and sensitive identification of ARV and MS,and it would offer a new route for laboratory detection under different conditions by adding a FD fluorescent probe and imager observation results.

关 键 词：FD荧光探针 二重荧光RT-LAMP 禽呼肠孤病毒 鸡滑液囊支原体 

分 类 号：S852.6[农业科学—基础兽医学]