狂犬病病毒荧光定量RT-PCR检测方法的建立与初步应用  

Establishment and application of real-time RT-PCR in detection of rabies virus

作　　者：郑学星[1,2] 杨松涛[2] 侯小强[1,2] 王化磊[1,2] 孙培录[1,2] 刘林立[1,2] 夏咸柱[2] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062 [2]军事医学科学院军事兽医研究所,吉林长春130062

出　　处：《中国病原生物学杂志》2008年第9期644-647,650,共5页Journal of Pathogen Biology

基　　金：国家863计划项目(No．2006AA022456);国家科技部支撑计划项目(No．2006BAD06A09);国家科技攻关计划课题(No．2004BA519A48)。

摘　　要：目的建立狂犬病病毒快速检测法。方法根据狂犬病病毒核蛋白基因的保守序列分别设计两对引物及其相应的TaqMan探针。构建pMD-N重组质粒,建立荧光定量RT-PCR绝对定量标准品,并对反应体系进行优化,建立绝对定量的标准曲线。利用10倍稀释法检验方法的灵敏度并与普通RT-PCR法进行比较;作重复性和特异性检验后进行临床标本的检测。结果构建了绝对定量的参照质粒,标准曲线相关系数为0.998。狂犬病病毒荧光定量RT-PCR检测反应的灵敏度为10个TCID_(50);5种非狂犬病病原体检测均为阴性。结论建立的狂犬病病毒的荧光定量RT- PCR检测方法快速、特异性强、灵敏度高、稳定性好,可以应用于临床样品的检测。Objective To establish a real-time PCR assay for rapid detection of rabies virus. Methods Based on the conservative sequence of rabies virus nucleoprotein gene, a pair of primers and a TaqMan probe were designed, and the recombinant plasmid for N gene was constructed as standard reference used for absolute quantification assay. The reaction conditions of real-time RT-PCR were optimized, and the sensitivity of assay was tested with ten fold serial dilution of rabies virus and compared with conventional RT PCR. The quantitative curve was established using reference standard plasraids. The clinical samples were detected after specificity assay. Results It＇s demonstrated that the plasmid for real-time RT-PCR was favorable. The regression coefficient of the standard curve reaches 0. 998. The detection sensitivity of realtime RT-PCR was 10 TCID50 and the specificity was determined by testing five other specimens, all of which yielded negative results. Conclusion The established real-time RT-PCR method, which was rapid, highly sensitive and reproducible, could be potentially used as routine assay to test clinical samples.

关 键 词：狂犬病病毒 荧光定量RT—PCR TaqMan荧光探针 

分 类 号：R373.9[医药卫生—病原生物学]