脑心肌炎病毒(EMCV)RT-PCR检测方法的建立及初步应用.pdfVIP

脑心肌炎病毒(EMCV)RT-PCR检测方法的建立及初步应用 王吉 付瑞 卫礼 李晓波冯育芳 巩薇 王淑菁 岳秉飞 贺争鸣 中国食品药品检定研究院、国家实验动物微生物、遗传检测中心，北京100050 [摘要]目的：建立脑心肌炎病毒(EMCV)RT-PCR检测方法，应用于长爪沙鼠、小鼠等实验动物EMCV的检测。方 法：根据已发表的EMCVVP1基因序列设计合成引物。建立RT-PCR方法，并对方法的特异性、敏感性、稳定性等进行 验证。用该方法检测62只长爪沙鼠、12只小鼠。结果：建立的EMCVRT-PCR检测方法特异、敏感、稳定。以EMCV RNA逆转录产物为模板，所能检测RNA最小模板浓度为4.1pg/μl，可检测病毒最小滴度为10-7.ml-1。经RT-PCR检测， 62只沙鼠均为阴性；12只小鼠中有1只EMCV核酸阳性，测序结果与GenBank中EMCV标准株核苷酸序列进行比对，其 同源性为85％。结论：建立的脑心肌炎病毒(EMCV)RT-PCR检测方法特异、敏感、稳定，可用于长爪沙鼠、小鼠等实验 动物EMCV的检测。 [关键词]：脑心肌炎病毒；反转录聚合酶链式反应；长爪沙鼠；小鼠 Q95-33 A DevelopmentandPreliminaryApplicationofRT-PCRMethodforDeterminationof Encephalomyocarditisvirus WangJiFuRuiWeiLiLiXiaoboFengYufangGongWeiWangShujingYueBingfeiHeZhengming NationalInstitutesforfoodanddrugControl,NationalCenterforqualityofLaboratoryAnimal,Beijing100050,China Abstract:ObjectiveTodevelopaRT-PCRmethodfordeterminationofEncephalomyocarditisvirus(EMCV) inMongoliangerbilsandLaboratorymice.MethodTheprimersweredesignedandsynthesisedaccordingtothepub lishedEMCVspecificsequencesofVP1gene.RT-PCRmethodisestablished,carriesonthespecificity,sensitivi ty,stabilitytest.Themethodisusedtodetected62Mongoliangerbilsand12mice.ResultsThedevelopedRT-PCR methodisgoodinspecificity,ensitivity,stability;anditsminimumdetectionlimitusingtherecombinantplasmid containingEMCVgeneasatemplatewas4.1pg/μl,andthelowestdetectionvirustiteris10-7.ml-1.The62Mongoli angerbilsafterRT-PCRdetectionwerenegative;The12miceafterRT-PCRdetection,therewereoneEMCV positive,comparedwiththeEMCVinGenebank,Thehomologiesinnucleotidesequenceofonepositivemiceis 85％ ;therewere5micecanbedetectedEMCVinthebodyofthe6micebyartificialinfectionEMCV.Conclusion ThedevelopedRT-PCRmethodisgoodinspecificity,ensitivity,stability,canbeusedindetectingtheEMCVinla boratoryanimal,suchasMongoliangerbilsandmice. Keywords:Encephalomyocarditisvirus;RT-PCR;Mongoliangerbil;Mouse