绵羊肺炎支原体TaqMan实时荧光定量PCR检测方法的建立与应用  

Development and application of TaqMan real-time fluorescent quantitation PCR for the detection of Mycoplasma ovipneumoniae

作　　者：韩瑞鑫 申捷 申之义[1] HAN Rui-xin;SHEN Jie;SHEN Zhi-yi(College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot 010018,China;Inner Mongolia Animal Disease Control Center,Hohhot 010010,China)

机构地区：[1]内蒙古农业大学兽医学院,内蒙古呼和浩特010018 [2]内蒙古自治区动物疫病预防控制中心,内蒙古呼和浩特010010

出　　处：《中国兽医科学》2018年第8期965-970,共6页Chinese Veterinary Science

基　　金：国家科技支撑计划资助项目(2015BAI07B02-06)

摘　　要：本研究旨在建立一种灵敏性高、特异性强的检测绵羊肺炎支原体的TaqMan实时荧光定量PCR方法。根据GenBank上已发表的绵羊肺炎支原体HSP70基因序列设计引物和探针,构建标准阳性质粒,建立实时荧光定量PCR方法。试验结果显示,该方法标准曲线的线性关系良好,标准曲线的相关系数R2=1.000,斜率S=-3.334,扩增效率E=99.5%;敏感性高,可以检测出的最低样品浓度为1×101copies/L,是普通PCR的100倍;特异性强,能有效区分巴氏杆菌等9种病原;重复性好,组内、组间的Ct值变异系数均小于2%。运用本试验建立的实时荧光定量PCR方法可简便、准确地鉴定绵羊肺炎支原体。The study aimed at construction of a prompt Taq Man real-time fluorescent quantitation PCR method for detection of Mycoplasma ovipneumoniae.In the study,the primers and probe were disignedby the sequence of HSP70 gene which had been included in the Gen Bank.And the standard plasmid was recombinanted.In result,linearity of the standard curve was good：R2=1.000,S=-3.334,E=99.5%.The sensitivity of the Taq Man real-time fluorescent quantitation PCR was 100 times higher than that of the regular PCR,and the specificity was good for identifying the Pasteurella,Streptococcus and Mycoplasma bovis,etc.In addition,the coefficient of variation of Ctvalues was all lower than 2% within groups or between groups.Using the Taq Man real-time fluorescent quantitation PCR,Mycoplasma ovipneumoniae could be identified with less time and more accuracy.

关 键 词：绵羊肺炎支原体 HSP70基因 TaqMan实时荧光定量PCR 

分 类 号：S852.62[农业科学—基础兽医学]