仔猪免疫PRRSV疫苗后病毒血症的荧光定量RT-PCR检测方法的建立  

Establishment of fluorescence quantitative RT-PCR method for detecting viremia in piglets immunized with PRRSV vaccines

作　　者：丁尊俄 周碧君[1] 赵双成 欧伟业 秦文学 李如举 罗引幸 刘丽娟[1] 张旭[1] 文明[1] 程振涛 王开功[1] DING Zun-e;ZHOU Bi-jun;ZHAO Shuang-cheng;OU Wei-ye;QIN Wen-xue;LI Ru-ju;LUO Yin-xing;LIU Li-juan;ZHANG Xu;WEN Ming;CHENG Zhen-tao;WANG Kai-gongl(College of A nimal Science,Guizhou University,Guiyang 550025,China;Shanghai Hile Bio-technology Co.,Ltd.,Shanghai 201403,China;Guizhou Ruite New A agriculture and Animal Husbandrr Science and Technology Limited Company,Guiyang 550001,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]上海海利生物技术股份有限公司,上海201403 [3]贵州瑞特新科农牧科技有限公司,贵州贵阳550001

出　　处：《中国兽医科学》2018年第12期1522-1528,共7页Chinese Veterinary Science

基　　金：贵州省科学技术厅农业攻关项目[黔科合NY(2015)3009-1号];贵州省研究生工作站计划项目(GZZ2017002);贵州省动物疫病防控与兽医公共卫生保障科技创新人才团队项目[黔科合人才团队(2015)4016号]

摘　　要：为建立一种快速检测猪繁殖与呼吸综合征病毒（PRRSV）疫苗免疫后在猪体内病毒血症持续时间的方法。根据GenBank中PRRSV基因序列,设计1对针对N基因扩增的特异性引物,经RT-PCR扩增后连接到pMD19-T载体上,构建含有N基因片段的重组质粒。采用SYBR GreenⅠ染料法进行荧光定量RT-PCR扩增,建立标准曲线并进行特异性、敏感性及重复性试验,比较分析该方法与商品化试剂盒检测结果的阳性符合率。结果显示,所构建方法的扩增效率为1.063,标准品各稀释度质粒拷贝数与Ct值之间相关系数为0.999,引物特异性强,可检测的最低核酸模板浓度为1.0×10~2copies/L,比常规PCR敏感性高10倍,重复性试验的变异系数均小于1%,与商品化试剂盒检测的阳性符合率达100%。两种方法检测结果均表明,在猪体内PRRSV疫苗病毒血症可持续3周。本研究建立的猪繁殖与呼吸综合征病毒荧光定量RT-PCR具有快速、特异、敏感和重复性好等优点,可为PRRSV感染（包括疫苗毒）的调查、监测和防控提供可行性和操作性强的技术支持。To establish a method to rapidly detect the duration of viremia in pigs after immunizationwithporcinereproductiveandrespiratorysyndromevirus（PRRSV）vaccines,accordingto the PRRSV gene sequence in Gen Bank,a pair of specific primers for the amplification of N gene were designed,and then the N gene was amplified by RT-PCR and ligated into pMD19-T vector to construct a recombinant plasmid containing the N gene fragment.SYBR GreenⅠdye method was used for quantitative RT-PCR amplification,standard curve was established,and specificity,sensitivity and repeatability tests were carried out,then the positive rate of this method and the commercial kit were compared and analyzed.In result, the amplification efficiency of the established method was 1.063,the correlation coefficient between the plasmid copy number of each dilution degree of the standard and Ctvalue was 0.999,and the primer specificity was strong.The detectable minimum nucleic acid template concentration was 1.0×10~2 copies/L,which was 10 times higher than that of the conventional PCR,and the coefficient of variation of the repeatability test was less than 1%.The positive coincidence rate of this method with the commercial kit detection reached 100%.The results of both methods showed that the duration of viremia in pigs after vaccination was 3 weeks.The PRRSV fluorescent quantitative RT-PCR established in this study has the advantages of rapidity,specificity,sensitivity and good repeatability,and can be used for the investigation,monitoring and control of PRRSV infection（including vaccine virus） to provide feasible and operational technical support.

关 键 词：猪繁殖与呼吸综合征病毒 疫苗 N基因 荧光定量RT-PCR 病毒血症 

分 类 号：S852.659.6[农业科学—基础兽医学]