树鼩呼肠孤病毒RT-nPCR检测方法的建立及初步应用  

Establishment and application of a RT-nPCR assay for detection of orthoreovirus in tree shrews

作　　者：李晓飞[1] 殷安国[1] 张媛[1] 罗军[1] 孙晓梅[1] 代解杰[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所树鼩种质资源中心,云南省重大传染病疫苗研发重点实验室,昆明650118

出　　处：《中国比较医学杂志》2014年第6期63-68,共6页Chinese Journal of Comparative Medicine

基　　金：国家科技支撑计划(2011BAI15B01-21;2012BAI39B01;2014BAI01B01);云南省科技创新平台建设(2013DA002)

摘　　要：目的建立树鼩呼肠孤病毒(TRV)RT-nPCR检测方法,为树鼩的质量控制提供检测方法。方法从三批野外来源的具有感染临床症状的树鼩粪便中分离得到三株病毒,经电镜观察和聚丙烯酰胺凝胶电泳鉴定为哺乳动物呼肠孤病毒(MRV)。根据GenBank中已发表的MRV L1基因的保守区域,设计合成巢式引物,对所分离的三株树鼩呼肠孤病毒(TRV1、TRV2、TRV3)的RNA进行RT-nPCR扩增,优化反应条件,进行特异性、敏感性试验。应用RT-nPCR方法对25只野外来源的相同症状疑似病例样本进行检测。结果针对分离到的三株树鼩呼肠孤病毒的RNA进行RT-nPCR扩增,均得到513 bp的特异性目的片段,培养细胞及甲肝病毒、轮状病毒、单纯疱疹病毒阴性对照均未扩增出条带。敏感性试验结果显示可检测到的最小RNA模板浓度为0.01 pg/μL。25只树鼩粪便样本经RT-nPCR检测,有14只TRV阳性,其中死亡动物组10只,检出率为100%;存活动物组15只,检出率为27%。结论建立的TRV RT-nPCR检测方法特异、敏感、稳定,可用于TRV的常规检测。Objective To establish a reverse transcription nested polymerase chain reaction（RT-nPCR） assay for detection of tree shrews orthoreovirus（TRV）. Methods Three strains of TRV were respectively isolated from fresh feces of three tree shrews that came from the same field at different times. We designed and synthesized two pairs of MRV L1 gene nested primers and established the system of RT-nPCR. The TRV RNA was extracted and reversely transcribed to cDNA as a template for nested-PCR amplification. The developed RT-nPCR was optimized. The specificity and sensitivity were tested. Finally,the RT-nPCR was used to detect TRV in 25 tree shrew samples. Results Taking the genomic RNA of TRV as template,the RT-nPCR was able to amplify a specific fragment band targeting the L1 gene,while there were notarget bands in the normal cell control,（Wa strain rotavirus,hepatitis A virus,and herpes simplex virus）. The RNA of TRV was diluted by 1： 10 to 1： 109. Each dilution sample was analyzed by the RT-nPCR. The minimum detectable concentration of RNA was 0. 01 pg /μL. The results of RT-nPCR detection showed that 4 of the 15 tree shrews were TRVpositive in the survival group,and 10 of 10 tree shrews were TRV-positive in the death group. Conclusions The RTnRCR assay established in this study is accurate,specific and sensitive. Therefore,it can be used for routine detection of TRV in quality assurance testing.

关 键 词：树鼩 哺乳动物呼肠孤病毒 反转录巢式聚合酶链式反应 巢式引物 

分 类 号：R33[医药卫生—人体生理学]