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

Establishment of duplex real-time PCR assay for detection of infectious bovine rhinotracheitis virus gB and gE genes

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。

To establish a sensitive detection method for infectious bovine rhinotracheitis virus（IBRV）, a duplex real-time PCR was developed with 2 pair of primers and TaqMan probes targeting the conserved regions of IBRV gE and gB genes. The result showed that the established assay possessed a good linear relationship and the correlation coefficient of standard curve was 0.999. The assay was highly specific for amplification of IBRV, and no amplification from bovine Mycoplasma, bovine parainfluenza virus type 3, bovine viral diarrhea virus, Pasteurella and Burcella. The sensitivity of the real-time PCR detection method approximated 2 copies/μL, and the reproducibility was high with a coefficient of variation was less than 2%. A real-time PCR method recommended by OIE had been established and adopted as a prescribed test for intemational trade. The coincidence rate of the two real-time PCR methods were 81.8% after the detection on 30 samples of bovine lungs. This research established a real-time PCR detection method that was the advantages of rapid, sensitive, accurate detection and could be used for IBRV detection.