羊传染性脓疱病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立  

Development of a SYBR GreenⅠ real-time PCR assay for detection of orf virus

作　　者：李超[1] 赵魁[2] 赵权[1] 贺文琦[2] 宋德光[2] 李晶[3] 解胜男[2] 陈克研[2] 高丰[2] 

机构地区：[1]吉林农业大学动物科技学院,吉林长春130118 [2]吉林大学畜牧兽医学院,吉林长春130062 [3]吉林正业生物制品有限责任公司,吉林吉林132000

出　　处：《中国兽医科学》2010年第3期270-273,共4页Chinese Veterinary Science

基　　金：吉林省科技发展计划重点资助项目(20080217)

摘　　要：针对羊传染性脓疱病毒(ORFV)B2L基因序列设计合成了1对引物,以含有该引物扩增序列的重组质粒作为阳性标准品,建立了检测ORFV核酸的SYBRGreenⅠreal-timePCR方法。结果显示,该方法线性关系良好,标准曲线的相关系数为0.999;检测灵敏度可达9.4×104copies/μL;与绵羊痘病毒(SPV)不发生交叉反应,具有良好的特异性;组内Ct值相同,组间变异系数小于2%。应用建立的方法检测20份疑似羊传染性脓疱病病料,结果17份为阳性,同时用常规PCR进行检测,结果仅10份为阳性。结果表明,建立的实时荧光定量PCR方法具有特异、敏感、快速、定量、重复性好等优点,可初步用于ORFV的检测。A pair of primers was designed according to the sequence of the B2L gene of orf virus（ORFV） and the recombinant plasmid containing the target gene was constructed as a standard control.Then,a real-time PCR assay based on SYBR GreenⅠ for detection of ORFV was established.It had a good linear relationship between initial templates and Ct values,and the correlation coefficient of the standard curve was 0.999.The sensitivity analysis showed that the developed SYBR GreenⅠ real-time PCR could detect 9.4×10^4copies/μL.The specificity assay showed that negative control and the other pathogens such as SPV could not be detected by this PCR.The Ct values of intra-assay were the same and the assay had a coefficient less than 2% for inter-assay.17 out of 20 suspicious positive samples detected by the real-time PCR were positive,but 10 out of the 20 suspicious positive samples detected by the normal PCR were positive.The results showed that the developed SYBR Green Ⅰ real-time PCR assay had the advantages of specificity,sensitivity,specificity,rapidity,quantitativity and reproducibility,and was able to be applied to the clinical diagnosis of ORFV.

关 键 词：羊传染性脓疱病毒 SYBR GreenⅠ实时荧光定量聚合酶链反应 标准曲线 

分 类 号：S852.659.1[农业科学—基础兽医学]