禽脑脊髓炎病毒SYBR Green Ⅰ荧光定量RT-PCR检测方法的建立与应用

Establishment and Application of SYBR Green Ⅰ Fluorescent Quantitative RT-PCR for Detecting Avian Encephalomyelitis Virus

利用禽脑脊髓炎病毒(Avian encephalomyelitis virus,AEV)YBF02株VPI基因序列设计一对特异性引物,通过对反应体系条件的确定和阳性质粒标准曲线的建立,建立了用于快速检测AEV含量的SYBR Green I荧光定量RT-PCR。结果显示:该方法能特异性地检测AEV YBF02株,且与其他禽类病毒无交叉反应;敏感性试验表明,该方法最低能检出10拷贝的AEV RNA模板,为普通PCR的100倍;使用该方法和鸡胚半数感染量(EID50)法对样品进行检测,二者具有良好的线性关系,且重复性试验的变异系数均小于4%。研究表明,建立的SYBR Green I荧光定量RT-PCR敏感性高、特异性强、重复性好,为AEV含量的快速检测提供了一种新的技术手段。

A pair of specific primers were designed according to VP1 gene sequence of avian encephalomyelitis virus(AEV)YBF02 strain,then a SYBR Green I fluorescent quantitative RT-PCR method for detection of AEV was established through the determination of reaction system conditions and establishment of positive plasmid standard curve.The results showed that this method could detect AEV YBP'02 strain specifically and had no cross-reaction with other avian viruses.The sensitivity test showed that this method could detect at least 10 copies of AEV RNA template,which was 100 times more than ordinary PCR.This method and EID50 method were used to test the samples,which showed that those two methods showed a good linear relationship,and the coefficient of variation value of the repeatability test was less than 4%.The established SYBR Green I fluorescent quantitative RT-PCR method in this study had high sensitivity,good specificity and good repeatability,providing a new technical method for rapid detection of AEV content.