1株禽传染性支气管炎病毒的分离鉴定及其实时荧光定量PCR检测方法的建立

Isolation and identification of a strain of avian infectious bronchitis virus and establishment of real-time fluorescent quantitative PCR for the strain

自禽传染性支气管炎病毒流行较严重地区的患鸡组织病料中分离得到1株疑似毒株,利用鸡胚培养、气管环及动物回归试验、电镜观察、RT-PCR等多种方法及对其S1基因序列进行比较分析,确定该分离毒株为禽传染性支气管炎病毒QX型。同时针对该新分离的传染性支气管炎病毒建立了实时荧光定量PCR检测方法。结果显示:针对该分离毒株的S1基因区设计1对引物和TaqMan探针,制备质粒标准品,建立标准曲线,其线性关系为y=-3.385 7x+42.235,R^2=0.999,扩增效率为97.5%,该法能区分常见禽类流行病毒,检测灵敏度可达42.1拷贝数/μL,比普通PCR检测限度高,重复性良好,因而该实时荧光定量PCR检测方法可靠。本试验同时进行了拷贝数与半数鸡胚感染量(EID50)对应关系研究,证明了在控制病毒代次、冻融次数等条件下,建立线性关系为y=0.249 2x+1.341×10^6(以拷贝数为x轴,EID50为y轴),R^2=0.956 4,可实现拷贝数替代EID50。

One strain of the infectious bronchitis virus was isolated from the infected chickens in an area with serious infection.The strain was identified as the QX type of infectious bronchitis virus by chicken embryo culture, tracheal ring and animal regression tests, electron microscopy observation, RT-PCR detection, and analysis of its S1 gene sequence.Meanwhile, a real-time quantitative PCR assay was established for the newly isolated infectious bronchitis virus to improve the efficiency in detection of the virus.The results showed that a pair of primer and TaqMan probe was designed according to the S1 region of the newly isolated virus.The plasmid standards were prepared and the standard curve was established.The linear relationship was y=-3.385 7x+42.235, R^2=0.999, and the amplification efficiency was 97.5%, which might be used to distinguish common avian epidemic viruses.The sensitivity of the real-time quantitative PCR assay was 42.1 copies/μL.This was higher than that of the common PCR detection limit, implying a good repeatability of the assay.The real-time fluorescent quantitative PCR detection method was proved to be reliable.The correspondence between the copy number and EID50 was also studied, which showed that the establishment of a linear relationship of y=0.249 2x+1.341×10^6(the copy number was x and EID50 y), R^2=0.956 4, under the conditions of controlling virus generation and of freezing and thawing times might achieve detection by the copy number instead of EID50.