西尼罗病毒装甲RNA质控品的制备

Preparation of armored RNA as reference control for West Nile virus

目的 制备一种西尼罗病毒装甲RNA质控品,用于西尼罗病毒核酸检测的全过程质控。方法 依据行业标准的引物WN233/WN640c、WN1160/WN1209序列,扩增西尼罗病毒衣壳蛋白C部分基因片段,将其克隆至含有MS2噬菌体衣壳蛋白基因的表达质粒pET32a-CP中,经原核表达后获得的病毒样颗粒即为装甲RNA。用DNaseI和RNase A消化装甲RNA,提取RNA,进行RT-PCR和实时荧光定量RT-PCR检测。将装甲RNA在4℃、-20℃和37℃保存15 d,定时提取RNA进行检测。结果 参照行业标准,用RT-PCR和荧光定量PCR均能扩增酶消化后的病毒样颗粒RNA,表明制备的装甲RNA含有插入的目的基因片段C,且耐受核糖核酸酶的降解。装甲RNA在4℃、-20℃和37℃环境中保存5~15 d,用RT-PCR和荧光定量PCR仍能扩增到特异性目的 条带,有扩增曲线,表明制备的病毒样颗粒在4℃、-20℃和37℃可以至少保存15 d。结论 制备的西尼罗病毒装甲RNA病毒样颗粒可以作为西尼罗病毒核酸检测的质控品,实现对检测全过程的质量控制。

Objective To prepare armored RNA as control material for West Nile virus which is used as reference controls in the whole process of detecting West Nile virus nucleic acid. Methods The sequence fragment of West Nile virus capsid gene C was amplified and synthesized according to the primer sequences of WN233/WN640 c,WN1160/WN1209 which provided by the standard and then it was cloned into the expression plasmid pET32 a-CP which contained the gene of MS2 phage capsid protein. The prokaryotic expression products virus-like particles(VLPs) were armored RNA. RT-PCR and Real-time RT-PCR were performed to detect RNA extracted from armored RNA after DNaseI and RNaseA digestion. The armored RNA was kept at 4℃,-20℃ and 37℃,respectively for 15 d,after digestion and detect the RNA extracted on time. Results The RNA extracted from the armored RNA after digestion could be amplified by RT-PCR and Real-time RT-PCR followed the standard method. It indicated that the armored RNA contained the target fragment C could resistant to the degradation of RNase. Aromored RNA stored at4℃,-20℃ and 37℃ for 5-15 d could amplify specific bands and curves by using the primers and methods which indicated that the armored RNA were remain stable stored at these conditions. Conclusion Armored RNA of West Nile virus established in this study can be used as reference control for nucleic acid test of West Nile virus to monitor the whole detection process.