现代食品科技2013，Vol.29Issue(8)：1978-1983,6.
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
摘要
Abstract
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 monitoting.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 of pseudovirus 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Key words
Viruses/ C-strain vaccine/ Polymerase chain reaction/ pseudovirus/ detection/ fluorescence引用本文复制引用
余以刚,黄韵,陶文扬,章丽,吴晖,肖性龙,赖富饶,杨锡洪..猪瘟病毒野生株和疫苗株荧光RT-PCR鉴别方法的建立[J].现代食品科技,2013,29(8):1978-1983,6.基金项目
国家自然科学基金青年科学基金资助项目(31101279) （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) （20110172120034）
广东省科技计划资助项目(2011B020314004) （2011B020314004）
广东省省部产学研结合项目(2012B091100113) （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) （201101）
