用于MERS-CoV核酸检测的假病毒阳性对照品制备及鉴定

Preparation and characterization of pseudoviral positive control for nucleic acid detection of MERS-CoV

目的制备安全、稳定的含有中东呼吸综合征冠状病毒(MERS-CoV)靶基因片段的假病毒颗粒阳性对照品。方法人工合成含up E、ORF1b以及N基因片段的MERS-CoV核苷酸序列,连入慢病毒表达载体,将重组慢病毒表达载体与包装质粒同时转染HEK293T-17细胞,利用透射电镜观察重组假病毒颗粒形态,用荧光定量RT-PCR方法检测假病毒包装情况,并进行稳定性、均一性以及线性试验。结果测序分析显示,3个基因片段正确克隆至载体pCMVMCS-GFP-PURO,经大肠杆菌表达可形成直径约为100 nm的均一、球形重组假病毒颗粒。不同浓度样品在不同温度条件下存放数日仍保持稳定,不同浓度样品、不同组别的Ct值的变异系数均小于0.1%。结论成功构建了含有upE、ORF1b以及N基因片段的MERS-CoV假病毒颗粒,可作为阳性对照品实现对核酸提取与检测过程全程监控。

Objective To prepare quality control samples for Middle East Respiratory Syndrome Coronavirus(MERSCoV)molecular detection by constructing pseudovirus containing upE,ORF1 b and N gene sequences of MERS-CoV.Methods The nucleic acid sequence containing amplification target of MERS-CoV was synthesized and inserted into a lentiviral vector,the recombinant lentiviral vector and the lentivirus-packaging vector were co-transfected into HEK293 T-17 cells.The pseudoviral particles were characterized by transmission electron microscopy,and Real-time PCR assay was used for the detection of the packaging and production.The determination of stability,homogeneity and linearity was conducted.Result Sequencing analysis confirmed that these upE,ORF1 b and N gene sequence of MERSCoV was cloned into vector pCMV-MCS-GFP-PURO.Transmission electron microscopy showed that homogenous spherical particles with a diameter of about 100 nm.The MERS-CoV genomic RNA within the pseudovirus particles remained stable during different temperature,and the result of homogeneity and linearity showed good.Conclusion The pseudoviral particles containing upE,ORF1 b and N gene produced by using lentiviral packaging technology in this study are constructed successfully,which can be used as positive quality control samples for RNA extraction and molecular detection.