多重实时荧光PCR检测禽网状内皮组织增生症病毒方法的建立以及初步应用

摘要

本文建立了一种快速、灵敏、特异性强的禽网状内皮组织增生症病毒（Reticuloendotheliosis virus，REV）检测方法，并对REV在鸡胚成纤维细胞（chick embryo fibroblast，CEF）上的增殖动态规律进行研究。分别针对gag、env以及LTR基因的保守序列设计3对引物，利用重组质粒作为模板，绘制3种不同基因的标准曲线。检测结果显示，本研究建立了多重实时荧光PCR检测方法，所制备的标准品模板在108~101copies范围内与Ct值之间呈现良好的线性关系，且最低检出量为101copies。REV感染CEF后gag、env、LTR基因的拷贝数均先减少后增多。通过实时荧光PCR技术与间接免疫荧光方法检测，发现REV在CEF上复制的初始阶段呈现病毒适应过程，此后病毒大量复制，表现出明显的增长趋势。该研究不仅为REV的诊断技术提供了一种新的方法，同时也为深入研究REV的感染机制和致病机理奠定了基础。%The objective of the present study was to develop a rapid, sensitive and specific method for detection of Reticuloendotheliosis virus (REV) in host cells and evaluation of replication kinetics for REV in chicken embryo fibroblasts (CEFs). Real-time PCR was developed using SYBR Green I and three pairs of primers specific to the conserved regions of gag, env and LTR genes and used to measure copies of viral genes at different days post infection. The results showed real-time PCR assay detected REV in the range of 108 to 101 copies with the detection limit at 101 copies. The copy numbers of these viral genes were detected in low level at initial infection stage followed by abundant increase. As compared with indirect immunofluorescence assay (IFA), real-time PCR demonstrated the presence of REV in CEFs at early infection immediately following by massive virus replication. In conclusion, development of real-time PCR provided a new method for REV detection and further research on mechanisms of infection and pathogenesis.