鸭坦布苏病毒快速实时荧光RT-LAMP方法的建立及初步应用  

Establishment and Preliminary Application of a Rapid Real-time Fluorescent RT-LAMP for Duck Tembusu Virus

作　　者：王巧萍 粟柯衡 元正菊 常志顺 张薇 张以芳 信爱国 Wang Qiaoping;Su Keheng;Yuan Zhengju;Chang Zhishun;Zhang Wei;Zhang Yifang;Xin Aiguo(College of Animal Medicine,Yunnan Agricultural University,Kunming,Yunnan 650201,China;Department of Poultry Husbandry and Disease Research,Yunnan Animal Science and Veterinary Institute,Kunming,Yunnan 650224,China;Yunnan Rare Biotechnology Co.,Ltd.,Kunming,Yunnan 650041,China)

机构地区：[1]云南农业大学动物医学院,云南昆明650201 [2]云南省畜牧兽医科学院养禽与禽病研究所,云南昆明650224 [3]云南瑞栢泰生物科技有限公司,云南昆明650041

出　　处：《中国动物检疫》2022年第5期103-109,共7页China Animal Health Inspection

基　　金：国家现代农业产业技术(水禽)体系资助项目(CARS-42);云南省廖明专家工作站项目(202105AF150077);云南省重大科技专项(202102AE090029)。

摘　　要：为建立检测鸭坦布苏病毒(DTMUV)的荧光逆转录环介导等温扩增(reverse transcription loop-mediated isothermal amplification,RT-LAMP)检测方法,根据DTMUV NS5基因保守序列设计1套特异性引物,对反应时间和温度进行优化,并评价该方法的特异性、敏感性、重复性和临床适用性。结果显示:建立的荧光RT-LAMP最优反应条件为65℃25 min;只特异性扩增DTMUV,而不扩增其他几种禽类病毒;最低检测限为4×10^(2)copies/μL,与qRT-PCR方法敏感性一致,是普通RT-PCR的100倍;批内和批间重复性高;应用该方法检测DTMUV不同剂量人工感染鸭肝脏样品,对接毒剂量为10^(3-7)TCID_(50)和10^(2-7)TCID_(50)组的阳性检出率均为100%,对接毒量10^(1.7)TCID_(50)组的阳性检出率为90%(9/10)。结果表明,本研究建立的荧光RT-LAMP检测方法特异性强、重复性好、敏感性高,可成为快速诊断DTMUV的方法之一。In order to develop a real-time fluorescent reverse transcription loop-mediated isothermal amplification(RTLAMP)assay for detection of duck Tembusu virus(DTMUV),one set of special primers were designed based on the highly conservative sequence of DTMUV NS5 gene,the reaction time and temperature were optimized,followed by evaluation on specificity,sensitivity,repeatability and clinical applicability. The results showed that the optimal conditions for the established method were 65 ℃ for 25 minutes,only DTMUV could be specifically amplified,and no reaction with other avian viruses was observed;its minimum detection limit was 4×10^(2)copies/μL,which was consistent with qRT-PCR and 100 times higher than that of conventional RT-PCR;with high intra and inter-assay repeatability,the method was applied to detect the liver samples from artificially infected ducks at different doses,the positive detection rates of the samples vaccinated with the challenge doses of 10^(3-7)TCID_(50)and 10^(2-7)TCID_(50)were 100%,that of those with 10^(1.7)TCID_(50)was 90%(9/10). In conclusion,the established method was with high specificity,repeatability and sensitivity,and could be used for rapid diagnosis of DTMUV.

关 键 词：鸭坦布苏病毒 实时荧光 环介导等温扩增技术 NS5基因 

分 类 号：S855.3[农业科学—临床兽医学]