水泡性口炎病毒血清型特异LUX实时荧光RT-PCR检测方法的建立  

Development of Serotype Specific LUX Real-time RT-PCR Assay for Rapid Detection of Vesicular Stomatitis Virus

作　　者：陈茹[1] 刘中勇[1] 曾碧健[1] 曹永长[2] 陈俊伟[2] 赵吟[1] 林志雄[1] 毕英佐[2] 

机构地区：[1]广东出入境检验检疫局,广州510623 [2]华南农业大学动物科学学院,广州510642

出　　处：《畜牧兽医学报》2008年第4期522-528,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家质检总局2006年科研项目(2006IK019)

摘　　要：采用LUX荧光核酸扩增技术原理,以水泡性口炎病毒(VSV)NJ、IND型NS基因为模板分别设计并合成单标记LUX荧光引物,建立血清型特异的VSV荧光RT-PCR检测方法。试验表明,两种型特异的LUX荧光RT-PCR能分别特异地鉴定VSV血清型,对口蹄疫、猪水泡病等病毒以及对照细胞、健康动物组织RNA样品的检测结果均为阴性。对比检测试验表明,LUX荧光RT-PCR的检测敏感性比常规RT-PCR提高达10倍以上,对VSV细胞增殖病毒液的检测灵敏度可达1 TCID50。对人工感染豚鼠样品以及临床样品的检测试验证实,该LUX荧光RT-PCR可有效检测到人工感染动物组织以及进口牛临床样品中的水泡性口炎病毒,并能鉴定感染病毒血清型。所报道的检测方法,包括样品核酸提取、LUX荧光RT-PCR以及熔解曲线分析,可在3 h内完成。Serotype specific LUX （Light Upon Extension） real-time RT-PCR assays were developed for rapid detection of vesicular stomatitis virus （VSV）. The LUX primers were designed based on the NS gene sequence of the NJ and IND serotype respectively. The real-time PCR assay could differentiate VSV from FMDV and other related RNA viruses. The detection limit of the assay could reach 1 TCID50 as showed by tests on serial ten fold dilution of cell propagated virus samples. Comparison tests indicated that the LUX real-time RT-PCR assay had at least ten-fold increase of sensitivity than the gel-based RT-PCR method. The assay could detect VSV infection and identify the serotype of the virus from import bovine clinical samples and artificial infected guinea-pig tissues. The whole procedure, including RNA extraction, real-time amplification and dissociation analysis, could complete within 3 hours.

关 键 词：水泡性口炎病毒 实时荧光RT—PCR LUX荧光引物 

分 类 号：S858.285.3[农业科学—临床兽医学]