鸡传染性支气管炎病毒Real-time PCR方法的建立及其对感染鸡体内病毒的检测

Development of a Real-time PCR assay for detection of infectious bronchitis virus

本研究针对传染性支气管炎病毒(IBV)tl/CH/LDT3/03毒株的N基因(GenBank登录号为AY702975)设计并合成了一对引物,构建重组质粒作为阳性标准品,建立了检测IBV核酸的SYBR GreenⅠ荧光定量PCR方法。该方法可检测到初始模板中6.45×10 copies/μL的病毒核酸。与常规PCR相比,敏感性高100倍。该检测方法特异性强,与其它禽源病毒如新城疫病毒(NDV)、传染性喉气管炎病毒(ILTV)、传染性法氏囊病毒(IBDV)、禽流感病毒(AIV)、马立克氏病毒(MDV)均不发生交叉反应;重复性试验的变异系数小于2.6%。结果表明,本试验建立的荧光定量PCR检测方法灵敏度高、特异性强、重复性好,可用于传染性支气管炎的临床诊断。同时应用本方法对人工感染IBV的SPF鸡的主要脏器盲肠扁桃体、肾脏、肺和气管进行了病毒RNA定量检测,结果表明,肾脏的病毒含量最高,盲肠扁桃体中病毒持续的时间最长,从而揭示了IBV在SPF鸡体内复制的动态变化,证实了感染鸡的临床表现与病毒滴度的时间相关性。

In this study, a SYBR Green I Real-time PCR assay for detecting infectious bronchitis virus （IBV） was established and tested in experimentally infected SPF chickens. A pair of primers was designed according to the sequences of the N gene of tl/CH/LDT3/03 strain and a plasmid containing the target gene was constructed as a standard control. The assay had a detection limit of 6.45 × 10^1 copies/μL viral RNA, which was 100 times more sensitive than the conventional PCR. It was highly specific and no cross-reactions were found with other avian pathogens including NDV, ILTV, IBDV, MDV, AIV. This assay was used to detect the IBV distribution in the tissue samples such as cecal tonsils, kidney, lung and trachea of the chickens infected IBV during the 40-day period post-inoculation （PI）. The results showed that the kidney had the highest virus load and the cecal tonsil had the longest time of virus presence. The clinical symptoms were shown related to viral RNA loads. The high sensitivity and specificity of the assay, coupled with a relatively rapid and simple procedure indicated that the Real-time PCR could be used as a routine assay for the clinical diagnosis of IBV infection.