猪流感病毒核酸检测装甲RNA质控样品的研制  

Preparation of armored RNA as reference controls for nucleic acid test of swine influenza virus

作　　者：乔彩霞[1,2] 高志强[2] 蒲静[2] 汪琳[2] 刘环[2] 张利峰[2] 张鹤晓[2] 刘金华[1] 

机构地区：[1]中国农业大学动物医学院,北京海淀100193 [2]北京出入境检验检疫局,北京朝阳100026

出　　处：《中国预防兽医学报》2014年第5期371-375,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家质检总局科技计划项目(2012IK002)

摘　　要：为研制一种可以应用于猪流感病毒(SIV)核酸检测的质控品,本研究选取MS2噬菌体装甲RNA(Armored RNA)作为RNA病毒核酸检测质控样品,将MS2噬菌体成熟酶蛋白基因、衣壳蛋白基因、包装位点及SIV M基因克隆于表达载体pET-32a中,经原核表达后,利用Cellufine sulfate层析柱纯化,RT-PCR和实时荧光RT-PCR鉴定表明制备的装甲RNA(AR-SIVM)病毒样颗粒中包装有SIV M基因。稳定性研究显示,AR-SIVM可以耐受RNase的降解,并且在-20℃和4℃至少可以保存3个月。试验结果表明,AR-SIVM作为SIV核酸检测质控样品,能够实现对检测全过程(核酸提取、反转录和PCR)的质量控制。Armored RNA of bacteriophage MS2, used as reference controls for nucleic acid test of RNA virus, was shown to be stable, noninfectious, mimics naturally occurring virus particles and RNase-resistant. Application of armored RNA for nucleic acid tests of swine influenza virus （SIV） has potential application value in the laboratory tests. In the present study, the cDNA fragment for maturation protein （A-protein）, coat protein and pac sites of MS2, and the matrix gene （M gene） of SIV was cloned into vector pET-32a and expressed using prokaryotic expression system. The armored RNA （AR-SIVM）, in which SIV M gene encapsulated by bacteriophage coat proteins was purified by cellufine sulfate mini-column and identified by RT-PCR and real-time RT-PCR. The stability test indicated that AR-SIVM particles were resistant to RNase degradation and produced reproducible results in the real-time RT-PCR assay for 90 days when stored at -20 ℃ or 4 ℃. In conclusion, AR-SIVM acting as the reference control for nucleic acid tests of SIV could be used to monitor the complete detection process including RNA extraction, reverse transcription and PCR amplification.

关 键 词：猪流感病毒 核酸检测 装甲RNA 质控样品 

分 类 号：S852.65[农业科学—基础兽医学]