摘要
【目的】利用SYBR GreenⅠ荧光定量技术建立一种相对定量检测坦布苏病毒的方法。【方法】针对坦布苏病毒NS5、E基因分别设计了1对特异性引物,同时设计1对扩增内参基因β-actin引物,将PCR扩增的片段分别连接到pMD18-T载体上构建重组质粒,经筛选、鉴定纯化后,倍比稀释作为质控样品,用于实时荧光定量PCR中NS5、E基因及内参基因β-actin标准曲线的构建,并进行反应的灵敏性、特异性和重复性试验。【结果】结果显示标准曲线线性关系R2值均在0.99以上,检测极限约为1.0E+01拷贝数质粒DNA;特异性结果表明只能检测到坦布苏病毒的扩增曲线;批内和批间重复性试验的变异系数均小于0.5%;用已建立的方法对临床样品进行3次重复检测,病毒RNA的检出率为100%。【结论】本研究初步建立了基于坦布苏病毒NS5、E基因的SYBR GreenⅠ荧光定量RT-PCR的方法,为养鸭场诊断和监测坦布苏病毒提供了一种新的特异、灵敏的检测方法。
【Objective】The objective of the study is to establish a method for detecting Tembusu virus by SYBR GreenⅠ relative fluorescence quantitative RT-PCR. 【Method】 Special primers based on Tembusu virus NS5 and E gene were designed and a pair of primers of house-keeping gene β-actin was chosen. Then these amplified fragments were cloned into pMD18-T. Using the plasmids NS5-pMD18-T, E-pMD18-T and β-actin-pMD18-T as standard products, a real-time quantitative reverse transcription-polymerase chain reaction （ RT-PCR） was performed to construct the standard curves of NS5, E and β-actin gene and detect the sensitivity, specificity and repeatability. 【Result】 The results showed a precise linear relationship with a correlation coefficient of R2〉0.99. The detection limits was 10 copies of DNA plasmid reaction. The amplification curve showing a single peak could only been detected for Tembusu virus. The variation coefficient was less than 0.5% by within and between the group of repeatability tests. The clinical samples were detected 3 times by this method, and all results were positive.【Conclusion】 The developed real-time PCR assay was highly specific, sensitive, and reproducible and could be an available tool for diagnosis and monitoring of Tembusu virus in duck farms.
出处
《中国农业科学》
CAS
CSCD
北大核心
2012年第21期4492-4500,共9页
Scientia Agricultura Sinica
基金
国家水禽产业技术体系项目(CARS-43-34)
山东省农业重大应用技术创新项目
