猪瘟病毒野生株和疫苗株荧光RT-PCR鉴别方法的建立  

Development of Real-time RT-PCR using MGB Fluorescence Probe for Differentiation of Wild-type and Vaccine Strains of Classical Swine Fever Virus

作　　者：余以刚[1] 黄韵[1] 陶文扬[1] 章丽[1] 吴晖[1] 肖性龙[1] 赖富饶[1] 杨锡洪 

机构地区：[1]华南理工大学轻工与食品学院,广东广州510640 [2]广东温氏佳润食品有限公司,广东云浮527400

出　　处：《现代食品科技》2013年第8期1978-1983,共6页Modern Food Science and Technology

基　　金：国家自然科学基金青年科学基金资助项目(31101279);教育部高等学校博士学科点专项科研基金新教师类资助课题(20110172120034);广东省科技计划资助项目(2011B020314004);广东省省部产学研结合项目(2012B091100113);广东省天然产物绿色加工与产品安全重点实验室开放课题(201101)

摘　　要：为了建立猪瘟病毒野生株和疫苗株荧光RT-PCR鉴别方法,设计了1对通用引物和2条特异性MGB探针。此外,为防止假阴性结果出现,制备了假病毒用于全程检测监控。测试了本方法的特异性,灵敏度,重复性等技术指标。结果显示,在选定的猪病相关病毒组内特异性符合率达100%;对野毒株和疫苗株的检测灵敏度分别达到1TCID50/mL和0.1 TCID50/mL;重复性实验表明组内和组间变异系数均在0.7-2.2%之间。临床比对试验结果显示,荧光RT-PCR对猪肉、脾脏和血液病料进行猪瘟野毒检测的阳性率分别为66.7%、60.0%、77.8%,而传统方法的阳性率分别为52.4%、40.0%、50.0%;检出率比传统方法要高。此外,临床试验样本中,PCR抑制物在猪肉、脾脏和血液病料中存在的比例分别为9.5%、10.0%和5.6%,显示了假病毒作为内标对PCR检测监控的重要作用。In order to the develop multiples real-time PCR for detection and differentiation of wild-type and vaccine strains of classical swine fever virus, a pair of common primers and two differently-labeled specific MGB Fluorescence probes were designed against conserved domain of the 5＇-UTR of wild-type viruses and C-strain vaccine. Moreover, in order to prevent the false negative result, pseudovirus was prepared for monitoring. The multiples real-time PCR method showed 100% specificity for the selected panel with the sensitivity being of 1 TCID50/mL for wild-type virus and 0.1 TCID 50/mL for C-strain vaccine. The reproducibility experiments showed that the variation coefficients （%CV） of intra-assay and inter-assay ranged from 0.7% to 2.2%. By using this method, CSFV RNA was detected in specimens of meat, spleen and blood with positive rates of 66.7%, 60.0% and 77.8%, respectively, while the positive rates obtained by using the standard assay was 52.4%, 40.0% and 50.0%, respectively. The result statistics showed the existence of PCR inhibitos in the clinical specimens and specimens with inhibition ratio being of 5.6--10.0%, indicating the importance ofpseudovirus as IAC when PCR was used to detect the RNA of wild-type viruses and C-strain vaccine.

关 键 词：猪瘟病毒 猪瘟弱毒疫苗株 荧光RT-PCR 假病毒 检测 荧光 

分 类 号：S858.28[农业科学—临床兽医学]