PRRSV高致病性毒株和经典毒株SYBR GreenⅠ实时荧光PCR鉴别方法的建立  

Establishment of SYBR GreenⅠ real-time PCR assay for differentiating highly pathogenic PRRSV strains from classical PRRSV strains

作　　者：柴政[1] 李曦[1] 王小武 符芳[1] 张永欣[1,3] 肖晶[1,3] 蔡雪辉[1] 周艳君[1] 宋淑萍[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室猪传染病研究室,黑龙江哈尔滨150001 [2]北京资源亚太药品公司,北京102600 [3]东北农业大学,黑龙江哈尔滨150030

出　　处：《中国兽医科学》2009年第2期140-144,共5页Chinese Veterinary Science

基　　金：黑龙江省科技攻关计划重点项目(GB05B501-1)

摘　　要：根据猪生殖与呼吸综合征病毒高致病性毒株、经典毒株Nsp2基因序列的差异,应用Oligo软件设计了2对特异引物,建立了鉴别PRRSV高致病性毒株和经典毒株的SYBR GreenⅠ实时荧光PCR方法。用该方法检测JEV、CSFV、FMDV和PRCV均呈阴性,表明该方法特异性良好;敏感性试验结果表明,该方法的最低检出量为1 TCID50/0.1 mL;批内、批间重复性试验显示,其变异系数均低于0.3%,具有良好的重复性。应用该方法对人工感染动物进行检测,自感染后第3、5、7、10、142、1 d分别采集全血,检测结果均为阳性。证实,本试验所建立的SYBR GreenⅠ实时荧光PCR方法具有良好的特异性、敏感性和重复性,可以快速、准确地鉴别PRRSV高致病性毒株和经典毒株。Two pairs of differential primers were designed according to the differences of Nsp2 gene sequences between highly pathogenic and classical strains of porcine reproductive and respiratory sydrome virus（PRRSV） available in GenBank. Then,a duplex SYBR Green Ⅰ real-time PCR for differential diagnosis was established. The specific analyses using cDNA of Japanese encephalitis virus, classical swine fever virus,foot-and-mouth disease virus, and porcine respiratory coronavirus as the templates were negative while amplifications of highly pathogenic and classical strains of PRRSV were positive. The sensitivity was 1 TCID50/0.1 mL. Inter- and intra-assay variables of CV were less than 0.30/oo. The results of the whole blood samples from pigs infected-experimentally with PRRSV were positive. The experiments revealed that the developed SYBR Green Ⅰ real-time PCR was specific,sensitive and reproducible,and it could be used for rapid and accurate differentiation of highly pathogenic（PRRSV） strains from classical PRRSV strains.

关 键 词：猪生殖与呼吸综合征病毒 SYBR Green Ⅰ实时荧光PCR 高致病性毒株 经典毒株 

分 类 号：S852.659.6[农业科学—基础兽医学] Q503[农业科学—兽医学]