牛传染性鼻气管炎病毒gB和gE基因双重荧光定量PCR检测方法的建立

发布时间：2018-04-20 05:42

本文选题：牛传染性鼻气管炎 + gE、gB基因　；参考：《中国预防兽医学报》2017年07期

【摘要】：为建立牛传染性鼻气管炎病毒(IBRV)的双重荧光定量PCR检测方法,本研究根据Gen Bank中登录的IBRV g E和g B基因序列,设计2对特异性引物及Taq Man探针。结果显示,以含有gB、gE基因的重组质粒为模板绘制标准曲线,其相关系数(R~2)均为0.999,有良好的线性关系。该方法仅对IBRV检测为阳性,而对牛支原体、牛副流感病毒3型、牛病毒性腹泻病毒、巴氏杆菌、牛、羊布鲁氏菌检测结果均为阴性,表明其特异性强。该方法的敏感度约为2拷贝/μL,重复性试验表明该方法的组内和组间变异系数均小于2%。根据被OIE采纳的作为国际贸易规定的检测该病毒的荧光定量PCR方法与本实验建立的方法分别对30份牛肺样品进行检测,符合率可达81.8%。研究表明所建立的荧光定量PCR检测方法具有快速、敏感、准确等优点,可以用于IBRV的检测。
[Abstract]:In order to establish a double fluorescent quantitative PCR method for detection of bovine infectious rhinotracheitis virus (IBRV), two pairs of specific primers and Taq Man probes were designed according to the sequence of IBRV g E and g B genes registered in Gen Bank. The results showed that the standard curve of the recombinant plasmids containing gBngE gene was taken as template, the correlation coefficient was 0.999, and there was a good linear relationship. This method was only positive for IBRV, but negative for Mycoplasma Bovis, Bovine parainfluenza virus type 3, Bovine viral diarrhea virus, Pasteurella, Bovine and Sheep Brucella. The sensitivity of the method was about 2 copies / 渭 L, and the repeatability test showed that the coefficient of variation within and between groups of the method was less than 2 copies / 渭 L. According to the fluorescence quantitative PCR method adopted by OIE as an international trade regulation and the method established in this experiment, 30 bovine lung samples were detected respectively. The coincidence rate was 81.8%. The results show that the established fluorescence quantitative PCR method has the advantages of fast, sensitive and accurate, and can be used for the detection of IBRV.
【作者单位】：内蒙古农业大学兽医学院;农业部动物疾病临床诊疗技术重点实验室;
【基金】：国家科技支撑计划项目(2015BA107B02)子课题(2015BA107B02-07)
【分类号】：S852.653

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