CAJ | 학술논문

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
목적：건립수구호장고병독（ TRV） RT-nPCR검측방법，위수구적질량공제제공검측방법。방법종삼비야외래원적구유감염림상증상적수구분편중분리득도삼주병독，경전경관찰화취병희선알응효전영감정위포유동물호장고병독（MRV）。근거GenBank 중이발표적MRV L1기인적보수구역，설계합성소식인물，대소분리적삼주수구호장고병독（TRV1、TRV2、TRV3）적RNA 진행RT-nPCR 확증，우화반응조건，진행특이성、민감성시험。응용RT-nPCR 방법대25지야외래원적상동증상의사병례양본진행검측。결과침대분리도적삼주수구호장고병독적RNA 진행RT-nPCR 확증，균득도513 bp 적특이성목적편단，배양세포급갑간병독、륜상병독、단순포진병독음성대조균미확증출조대。민감성시험결과현시가검측도적최소RNA 모판농도위0.01 pg／μL。25지수구분편양본경RT-nPCR 검측，유14지TRV 양성，기중사망동물조10지，검출솔위100％；존활동물조15지，검출솔위27％。결론건립적TRV RT-nPCR 검측방법특이、민감、은정，가용우TRV 적상규검
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 L 1 gene, while there were no target 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 TRV-positive in the survival group , and 10 of 10 tree shrews were TRV-positive in the death group . Conclusions The RT-nRCR assay established in this study is accurate , specific and sensitive .Therefore, it can be used for routine detection of TRV in quality assurance testing .