摘要
根据禽白血病病毒各亚群pol基因序列相对保守的特点,在其保守区内设计了1对引物,进行RT-PCR反应,扩增产物为214 bp,将该产物连接T载体作为Real-time PCR反应的标准品,利用SYBR Green I染料进行Real-time PCR反应,建立了标准曲线,并进行反应灵敏性、特异性和重复性试验。试验结果表明:标准曲线线性关系R值均为0.999以上,检测极限约为8.21E+01拷贝数质粒DNA,比RT-PCR灵敏100倍以上;特异性分析表明只有禽白血病病毒能检测到特异性的熔解度峰值;批内和批间重复性试验的变异系数均小于5%;用已建立的方法对人工感染SPF鸡的不同组织进行3次重复检测,病毒RNA的检出率为100%。以上结果表明:该方法稳定可靠,为禽白血病的早期诊断建立了特异、灵敏和快捷的方法。
A SYBR Green I-based real-time PCR was developed for detection of ALV by using a pair of primers that was designed to target conservative region of pol gene.To establish the standard curve,the product of conventional PCR linking to pMD18-T vector served as standard.The results showed a precise linear relationship with a correlation coefficient of R=0.999;The detection limits was 82 copies of DNA plasmid/reaction,The melting curve showed a single peak could only been detected for avian leukosis virus.The different tissues,blood of 5 chickens challenged with SD were detected repeatly 3 times by this method,All results were positive.The method was shown to be of high linearity,specificity,sensitivity,reproducibility,cheap.The assay method provide a powerful tools for detection of avian leukosis.
出处
《内蒙古农业大学学报（自然科学版）》
CAS
北大核心
2009年第4期15-19,共5页
Journal of Inner Mongolia Agricultural University(Natural Science Edition)
基金
国家重点实验室基金(NKLVBP200829)
