摘要
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.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2011年第2期193-198,共6页
Chinese Veterinary Science
基金
国家自然科学基金项目(30700599)
广西自然科学基金项目(桂科自0991044)
广西科技攻关项目(0993009-2)
广西大学拔尖创新团队建设计划项目