A型禽流感病毒通用型核酸检测标准样品的研制及不确定度分析

Development and uncertainty analysis of the avian influenza virus reference material which used for nucleic acid amplification testing

利用基因克隆和体外转录技术,制备A型禽流感病毒通用核酸检测阳性标准样品。根据禽流感基质蛋白基因M的序列,设计全长开放阅读框的克隆引物,RT-PCR获得相应片段,连接至pGEM-T载体,测序后采用体外转录方法制备RNA纯品,初步定量稀释后,混合分装作为核酸标准样品候选物。采用实时荧光定量RT-PCR方法进行均匀性和稳定性检验,并委托外部实验室采用外标实时定量RT-PCR方法对转录的RNA片段进行定值。通过绘制荧光定量标准扩增曲线和协作标定的方式,计算标准物质含量（拷贝）,并进行不确定度的估算。均一性结果显示瓶间差异小于5%,稳定性试验表明室温20~25℃（相对湿度20%~50%）14d,2~8℃3个月以及-20℃保存1年的含量均无明显变化。核酸标准物质定值为（3.120±0.345）×10^8拷贝数,可用作禽流感病毒通用核酸检测的标准质控品。

Using gene cloning and in vitro transcription technology,avian influenza virus positive reference material were developed for nucleic acid amplification testing.The primers were designed to amplify the complete ORFs from extracted RNA,then were cloned into pGME-T vector and sequenced.The pure RNA were prepared by transcription in vitro and preliminary quantitative diluted and then mixed with equal volume.After aliquot,the homogenity and stability testing were done using real-time RT-PCR.With the standard curves were constructed,the gene copies of F genes were determined and uncertainty estimates were made by commissioned laboratories using real-time RT-PCR by adding external standard substance.The results show vial variation were less than 5%by homogenity tesing.The stability test indicates that the parepared reference materials were stable at room temperature（20-25℃）for 2weeks,2-8℃for 3months and-20℃for 1year.The reference materials are evaluated as（3.120±0.345）×10^8 copies and can be used as positive standard AIV sample for nucleic acid amplification testing.