绵羊肺炎支原体荧光定量PCR检测方法的建立与应用  

Development and application of quantitative real-time PCR for the detection of Mycoplasma ovipneumoniae

作　　者：黄坚[1] 尹正军 岳华[1] 汤承[1] 

机构地区：[1]西南民族大学生命科学与技术学院,四川成都610041

出　　处：《中国兽医科学》2016年第10期1270-1276,共7页Chinese Veterinary Science

基　　金："十二五"国家科技支撑计划项目(2012BAD13B06)

摘　　要：为建立快速检测绵羊肺炎支原体的荧光定量PCR方法,根据EF-Tu基因序列设计合成引物,构建含有EF-Tu基因的重组质粒,以其为标准品绘制标准曲线,检验该方法的特异性、敏感性和重复性,并用于临床样本的检测。结果显示,本研究建立的方法能特异性扩增绵羊肺炎支原体,与其他病原无交叉反应,最低检测限为5 copies/L,在5×10^4-5×10^9稀释度范围内具有良好的线性关系（r2=0.996）,检测的批内和批间变异系数均小于5%。对临床肺组织样本中绵羊肺炎支原体的检出率（105/134,78%）与基于P113基因的q RT-PCR方法的相符,而对鼻腔棉拭子样本的检出率（27/50,54%）略高于后者（25/50,50%）。对人工感染绵羊肺炎支原体山羊肺的病变部位、病健交界部位和无明显病变部位的检出率分别为1/6、6/6和4/6,确定肺的病健交界部位为最佳采样部位。对采自四川、新疆、青海的439份表观健康羊肺绵羊肺炎支原体的平均检出率分别为48%（61/126）、63%（103/163）和75%（113/150）,提示受检羊群仍存在严重的绵羊肺炎支原体带菌感染情况。该方法的建立对绵羊肺炎支原体感染的监测与快速诊断有重要意义。To establish a rapid detection assay for Mycoplasma ovipneumoniae,the SYBR Green real-time PCR was developed with specific primers targeting an elongation factor Tu（EF-Tu） gene.The results showed that the assay was specific for amplification of M. ovipneumoniae with a detection limit of 5 copies/ L, but no cross-reaction with other pathogens.The method has a wide linear range from 5 ×104to 5 ×109of M. ovipneumoniae DNA（r2=0.996）. In intra and inter assays,the coefficient of variation was less than 5%. Compared to a previously published q RT-PCR protocol based on the P 113 gene,the assay had equal or slightly improved performance in terms of sensitivity and specificity when detecting 134 lung tissues and 50 nasal swab samples from sheep. In addition,the goats were infected by reference Y98 strain of M. ovipneumoniae in experimental infection tests. The pathological-healthy border was the best detection site（6/6） than both pathological（1/6） and relative healthy site（4/6）. Furthermore,the positive detection rates in healthy sheep and goats were 48%（61/126）,63%（103/163） and 75%（113/150） from Sichuan,Xinjiang,and Qinghai provinces,respectively.The results showed that the detected flocks of goats and sheep were still seriously infected by M. ovipneumoniae. Therefore,these data indicated that the real-time PCR method targeting EF-Tu was more sensitive and reliable method for M. ovipneumoniae infection monitoring and rapid diagnosis.

关 键 词：绵羊肺炎支原体 EF-Tu基因 荧光定量PCR 检测部位 临床应用 

分 类 号：S852.62[农业科学—基础兽医学]