鸡传染性支气管炎病毒real-time PCR检测方法的建立

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

针对鸡传染性支气管炎病毒(IBV)N基因片段设计并合成了1对引物,将构建的重组质粒作为阳性标准品,成功建立了检测IBV核酸的SYBR GreenⅠreal-time PCR检测方法。结果显示,该方法的线性关系好,标准曲线的相关系数达0.998 3;最低可以检测到1×101 copies/μL的核酸模板;与常见禽源病毒新城疫病毒、传染性喉气管炎病毒、传染性法氏囊炎病毒、马立克氏病病毒均不发生交叉反应,重复性试验的变异系数小于3%。应用建立的方法对人工感染IBV的20只试验鸡的40份气管和肾样品中的病毒核酸进行检测,结果显示,攻毒后第5和第7天肾中病毒含量高于气管,证实了临床表现与病毒载量之间的关系。结果表明,建立的real-time PCR检测方法灵敏度高、特异性强、重复性好,可以用于IBV的病原检测及定量分析。

A pair of primers was designed according to the sequence of the N gene fragment of infectious bronchitis virus（IBV） and a recombinant plasmid containing the target gene was constructed as a standard control.A real-time PCR assay based on SYBR GreenⅠ was developed for detection of IBV.The developed assay had a good linear relationship,and the correlation coefficient of the standard curve was 0.998 3.The assay had a detection limit of 10 copies/μL of initial templates and had no cross-reaction with Newcastle disease virus,infectious laryngotracheitis virus,infectious bursal disease virus and Mark＇s disease virus,and had a coefficient of variations less than 3% for both intra-and interassay.40 samples of trachea and kidney from 20 experimentally infected chickens were examined by the developed real-time PCR assay,and the results showed that the kidney had higher virus loads than the trachea on day 5 and 7 post-infection,indicating that the clinical feature were related to viral RNA loads.Therefore,this real-time PCR assay is sensitive,specific and repeatable and could be used as an effective assay for detection of nucleic acids and quantification of viral loads of IBV.