摘要
采用LUXTM荧光PCR技术原理,以牛流行热病毒(BEFV)糖蛋白抗原G基因保守序列为模板设计特异荧光PCR扩增引物,建立BEFV LUXTM实时荧光RT-PCR快速检测方法。试验结果显示,所建立的荧光RT-PCR可特异检测牛流行热病毒RNA,而对蓝舌病病毒、牛病毒性腹泻-粘膜病病毒、水泡性口炎病毒、口蹄疫病毒、牛白血病病毒以及与BEFV同种属的狂犬病病毒核酸检测呈阴性反应。敏感性试验表明LUXTM荧光RT-PCR对BEFV RNA的检测敏感性比常规RT-PCR方法提高达100倍以上。该LUXTM荧光RT-PCR的批内检测变异系数0.64%-1.17%,批间检测变异系数均小于2%,表明检测体系稳定、重复性好。通过人工添加灭活病毒液的方法制备30份模拟感染牛血清样品,采用上述所建立的BEFV LUXTM荧光RT-PCR方法检测均呈阳性。
A LUX^TM flourescent RT-PCR/PCR assay was established for detecting bovine ephemeral fever virus.The LUXTM primers were designed based on G gene sequence.BLAST analysis showed that the primer＇s sequences were BEFV specific and highly conserved.The specificity of the assay was confirmed with BEFV RNA,but negative result was obtained by testing other related bovine virus（BLV,BVD,VSV,FMD） and genomic RNA from healthy cattle.The result of LUX^TM RT-PCR assay show 100-fold increase in sensitivity when compared to a conventional PCR method.All of the coefficient of variation of intra-assay and inter-assay were less than 1% in the quantitative tests.It takes less than 3 h to complete the whole procedure,including RNA extraction,real-time amplification and dissociation analysis.
出处
《生物技术通报》
CAS
CSCD
北大核心
2010年第8期174-179,共6页
Biotechnology Bulletin
基金
国家质检总局科研项目(2006IK-019)
