可视化牛支原体和传染性鼻气管炎二重荧光LAMP诊断方法的建立  

Establishment of a Visual Duplex Fluorescence-based LAMP Method for the Diagnosis of Mycoplasma Bovis and Infectious Rhinotracheitis

作　　者：范晴[1] 谢芝勋[1] 谢志勤[1] 谢丽基[1] 黄娇玲 张艳芳[1] 曾婷婷[1] 王盛[1] 罗思思[1] 邓显文[1] 刘加波[1] Fan Qing;Xie Zhixun;Xie Zhiqin;Xie Liji;Huang Jiaoling;Zhang Yanfan;Zeng Tingting;Wang Sheng;Luo Sisi;Deng Xianwen;Liu Jiabo(Guangxi Key Laboratory of Veterinary Biotechnology,Guangxi Veterinary Research Institute,Nanning,530001)

机构地区：[1]广西兽医研究所,广西兽医生物技术重点实验室,南宁530001

出　　处：《基因组学与应用生物学》2021年第1期448-456,共9页Genomics and Applied Biology

基　　金：广西科技重大专项(桂科AA17204057);广西八桂学者专项(2019A50);国家万人计划领军人才专项(W02-060083)共同资助。

摘　　要：牛支原体(mycoplasma bovis, MB)和牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)是引起牛呼吸综合症的主要病原体。本研究根据MB和IBRV的保守基因序列,设计并合成两套特异性LAMP引物,在每条内引物的5’端标记荧光基团,通过扩增产物的颜色判断检测结果,建立了用于检测MB和IBRV的二重荧光LAMP检测方法。该方法与其他牛病原体无交叉反应,检测敏感度达100拷贝/μL。应用该方法检测125份样品,MB的感染率为44.8%,IBRV的感染率为13.6%,2种病原混合感染率为1.6%;与OIE推荐的荧光定量PCR检测方法相比,此二重LAMP方法敏感性为94.4%~96.6%,特异性为100%。结果表明该方法灵敏度高,特异性好,重复性好,能同时检测大量样本,可用于MB和IBRV的临床检测和流行病学调查。Mycoplasma bovis(MB) and infectious bovine rhinotracheitis virus(IBRV) are common cattle pathogens that can cause bovine respiratory disease complex. Two sets of specific primers were designed according to the conserved gene sequences of MB and IBRV, and the inner primers were synthesized with fluorophore at its 5’ end. A dual fluorescent LAMP method for MB and IBRV detection was established based on the color determination results of the amplification products. The method had no cross-reaction with other bovine pathogens, and the detection sensitivity was up to 100 copies/μL. In a clinical test for 125 samples, the positive rates detecting MB, IBRV and their co-infection were 44.8%, 12.6% and 1.6%, respectively. Furthermore, duplex fluorescence-based LAMP was 94.4% ~96.6% sensitive and 100% specific compared with OIE recommended real-time PCR in detection of clinical samples. The results indicated that the duplex fluorescence-based LAMP is a rapid, sensitive and specific method for identification of M B and IBRV in clinical detection and epidemiological investigation.

关 键 词：牛支原体(MB) 牛传染性鼻气管病毒(IBRV) 二重荧光LAMP 

分 类 号：S858.23[农业科学—临床兽医学]