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当前位置: 首页 > 文章 > 鸭坦布苏病毒与鸭瘟病毒二重RT-PCR检测方法的建立 南方农业学报 2014,45 (2) 314-317
Position: Home > Articles > Development of a duplex RT-PCR assay for detection of duck tembusu virus and duck plague virus Journal of Southern Agriculture 2014,45 (2) 314-317

鸭坦布苏病毒与鸭瘟病毒二重RT-PCR检测方法的建立

作  者：
张艳芳;谢芝勋;谢丽基;刘加波;庞耀珊;邓显文;谢志勤;范晴;罗思思
单  位：
广西兽医研究所/广西畜禽疫苗新技术重点实验室
关键词：
鸭坦布苏病毒;鸭瘟病毒;二重RT-PCR;特异性;敏感性
摘  要：
【目的】建立可同时检测鸭坦布苏病毒(Duck tembusu virus,DTMUV)与鸭瘟病毒(Duck plague virus,DPV)的二重RT-PCR,为有效防控DTMUV与DPV提供技术支撑。【方法】根据GenBank中DTMUV E基因和DPV UL6基因的保守序列,设计合成两对引物,优化反应体系条件,并通过特异性、敏感性试验评价建立的二重RT-PCR。【结果】优化后的二重RT-PCR反应体系为:2×PCR Mix 12.5μL,其中DTMUV上、下引物各0.5μL,DPV上、下引物各0.5μL,混合模板2.0μL,ddH2O补足至25.0μL;最佳反应程序:95℃预变性5 min;95℃1 min,54.4℃1 min,72℃1 min,进行35个循环;72℃延伸10 min。该方法对DTMUV与DPV的检测敏感性分别达500和600 fg,但对鸭源新城疫病毒、鸭肝炎病毒、番鸭细小病毒、鸭圆环病毒、H9亚型禽流感病毒和减蛋综合症病毒等病原体不敏感。【结论】建立的二重RT-PCR可用于DTMUV与DPV感染的快速鉴别诊断。
译  名：
Development of a duplex RT-PCR assay for detection of duck tembusu virus and duck plague virus
作  者：
ZHANG Yan-fang;XIE Zhi-xun;XIE Li-ji;LIU Jia-bo;PANG Yao-shan;DENG Xian-wen;XIE Zhi-qin;FAN Qing;LUO Si-si;Guangxi Veterinary Research Institute,Guangxi Key Laboratory of Animal Vaccines and Diagnostics;
关键词：
duck tembusu virus;;duck plague virus;;duplex RT-PCR;;specificity;;sensibility
摘  要：
【Objective】A duplex RT-PCR assay for detecting both duck tembusu virus(DTMUV) and duck plague virus was developed to provide technical support for effective control of DTMUV and DPV. 【Method】According to the gene sequences of duck tembusu virus E gene and duck plague virus UL6 gene in GenBank, two sets of specific primers were designed. The duplex PCR assay was developed through optimization of reaction conditions and validation of specificity, sensitivity and repetitiveness of the method. 【Result】The optimized RT-PCR reaction system was 2×PCR Mix 12.5 μL, of which tupstream and downstream primer of DTMUV and DPV was 0.5 μL each. The mixed template was 2.0 μL and added to 25.0 μL with ddH2O. The optimal reaction procedure was 95 ℃ initial denaturation for 5 min, 95 ℃ for 1 min, 54.4 ℃ for 1 min, 72 ℃ for 1 min with 35 cycles and 72 ℃ extension for 10 min. The sensitivity of the assay was 600 fg for duck tembusu virus and 500 fg for duck plague virus, but it was not sensitive to other duck pathogens, such as duck newcastle disease virus, duck hepatitis virus, Muscovy duck parvovirus, duck circovirus, H9 subtype avian influenza virus and egg drop syndrome virus. 【Conclusion】The duplex RT-PCR assay is a quick, sensitive, and specific test for detection of duck tembusu virus and duck plague virus.

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