单一引物标记LUX荧光RT-PCR鉴别BTV和EHDV  

Simultaneous Detection and Differentiation of Bluetongue and Epizootic Hemorrhagic Disease Viruses Using Multiplex LUX Real-time PCR

作　　者：张彩虹[1] 花群义[1] 阮周曦[1] 杨俊兴[1] 曾少灵[1] 陶虹[1] 陈兵[1] 曹琛福[1] 林庆燕[1] 周晓黎[2] 

机构地区：[1]深圳出入境检验检疫局,广东深圳518010 [2]云南出入境检验检疫局,云南昆明650228

出　　处：《动物医学进展》2010年第3期6-11,共6页Progress In Veterinary Medicine

基　　金：国家863计划项目(2006AA10Z445)

摘　　要：采用在线LUXTM专业软件,根据BTV-NS3基因序列和EHDV-NS3基因序列,通过特异性单一引物序列3′末端的荧光标记,分别设计出两对BTV和EHDV的LUX荧光PCR引物,并采用BLAST软件对各引物进行匹配性和特异性分析,根据分析结果选择合适的引物合成。经过各反应条件的优化和特异性、敏感性试验,并对BTV、EHDV、VSV、PPRV、BVDV、AKV的BHK-21细胞培养物和临床样品检测,与常规RT-PCR进行对比检测,建立了能同时鉴别检测BTV和EHDV的二重LUXTM荧光PCR方法。该二重LUXTM荧光PCR的BTV和EHDV各自引物只对相应的病毒呈阳性反应,两者没有交叉反应现象,对健康牛、羊和猪基因组DNA、BKH-21细胞对照,以及其他几种相似疾病如VSV、PPRV、BVDV、AKV均呈阴性反应。该法对病毒的细胞培养液鉴别检测敏感性可达1TCID50C以上,比常规RT-PCR敏感性提高10倍以上,从样品核酸纯化到完成二重LUXTM荧光PCR反应和熔解曲线分析,仅需3h,在进出口动物检验检疫中快速鉴别BTV和EHDV具有实际应用价值。A real-time reverse transcriptase（RT）-PCR assay,applying light upon extension（LUX）fluorogenic primers,was developed for rapid detection and identification of bluetongue virus（BTV）and epizootic hemorrhagic disease virus（EHDV）.The LUX primers were designed based on the NS3 gene sequence of BTV and EHDV respectively.The assay described here was designed to allow simultaneous detection of BTV and EHDV in a multiplex format.The detection limit of the assay was approximately 10TCID50 and 1TCID50 for BTV and EHDV respectively.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 real-time PCR assay could differentiate BTV and EHDV from other related disease viruses,such as VSV,PPRV,BVDV,AKV.The whole procedure,including RNA extraction,real-time amplification and dissociation analysis,can be completed within 3 hours.Due to its high specificity,sensitivity,and relative simplicity,the LUX RT-PCR assay provides a novel,rapid,and practical tool for the identification detection and of BTV and EHDV.

关 键 词：LUX荧光RT-PCR 蓝舌病病毒 流行性出血病病毒 

分 类 号：S852.659.4[农业科学—基础兽医学]