新型冠状病毒RNA标准物质  

SARS-CoV-2 RNA reference materials

作　　者：许丽[1] 梁文[1] 杨雪 闻艳丽[1] 李兰英[1] 杨镇州 李妍[1] 邓敏 陆青 丁敏[1] 任淑贞[1] 孙洁林 左小磊 王丽华 曹程明[1] 胡钧 刘刚[1] 樊春海 Li Xu;Wen Liang;Xue Yang;Yanli Wen;Lanying Li;Zhenzhou Yang;Yan Li;Min Deng;Qing Lu;Min Ding;Shuzhen Ren;Jielin Sun;Xiaolei Zuo;Lihua Wang;Chengming Cao;Jun Hu;Gang Liu;Chunhai Fan(Biometrology Laboratory,Chemical and Ionizing Radiation Metrology Institute,Shanghai Institute of Measurement and Testing Technology,Shanghai 201203,China;Institute of Molecular Medicine,Institute of Translational Medicine,School of Chemistry and Chemical Engineering,Shanghai Jiao Tong University,Shanghai 200240,China;Shanghai Advanced Research Institute,Chinese Academy of Sciences,Shanghai 201210,China;Shanghai Testing&Inspection Institution for Medical Devices,Shanghai 201318,China)

机构地区：[1]上海市计量测试技术研究院化学与电离辐射计量技术研究所生物计量实验室,上海201203 [2]上海交通大学化学化工学院转化医学研究院分子医学研究院,上海200240 [3]中国科学院上海高等研究院,上海201210 [4]上海市医疗器械检测所,上海201318

出　　处：《科学通报》2020年第22期2363-2370,共8页Chinese Science Bulletin

基　　金：国家重点研发计划(2018YFF0212800,2016YFA0201200);上海交通大学新型冠状病毒防治攻关专项;上海交通大学高峰学科-临床医学项目(20171913);上海市科研计划项目(20441900402);国家自然科学基金(21675167)资助。

摘　　要：研制了一种新型冠状病毒(coronavirus, SARS-CoV-2)体外转录RNA标准物质,用于SARS-CoV-2病毒RNA检测的量值溯源.目标基因范围涵盖已公布的SARS-CoV-2病毒检测的3个主要靶标基因:核壳蛋白N基因全长(基因组坐标:28274-29533, GenBank:MT027064.1)、包膜蛋白E基因全长(基因组坐标:26245-26472, GenBank:MT027064.1)、开放阅读框1ab(ORF1ab)基因片段(基因组坐标:13321-15540, GenBank:MT027064.1).通过采用数字聚合酶链式反应(digital PCR, dPCR)方法进行多实验室联合定值,确定了该标准物质的量值为N、E和ORF1ab基因的拷贝数浓度.研究结果表明,该RNA标准物质量值可靠,均匀性良好,稳定性可满足运输要求.进一步地,通过在临床检验、多家不同原理的病毒检测试剂盒生产企业、方法开发实验室等试用,验证了RNA标准物质的普适性.这一RNA标准物质的研制为新型冠状病毒检测试剂盒的质量控制提供了计量学依据,为防控流行性疾病提供了便利.Coronavirus disease(COVID-19) is an acute infectious disease caused by severe acute respiratory syndrome coronavirus 2(SARS-Co V-2). Reverse transcription real-time fluorescent quantitative polymerase chain reaction(RT-q PCR) was the firstly authorized method for the detection of SARS-Co V-2 RNA. As this method is sensitive, specific, it has been widely recognized as the golden standard for the diagnosis of COVID-19. Unfortunately, several false-negative cases have been reported after the outbreak of COVID-19, probably due to the quality of the kits or the improper operation of RT-q PCR.Nucleic acid reference materials(RM) are the key element for the metrology traceability and quality control of SARS-Co V-2 RNA detection, but the development of RNA RM remains a challenge in the biology metrology field. Two main problems are the low stability of the RNA sample and the lack of proven absolute quantification methods.To establish the measurement traceability for SARS-Co V-2 RNA detection, a novel RNA reference material(RM) was developed. The RM is a mixed solution of 3 in vitro transcribed RNA molecules which cover different key target sequences of SARS-Co V-2 gene: The full-length of nucleoprotein(N) gene(28274-29533, Gen Bank: MT027064.1), the full-length of envelope protein(E) gene(26245-26472, Gen Bank: MT027064.1), and partial sequence of open reading frame 1 ab(ORF1 ab)(13321-15540, Gen Bank: MT027064.1). The purity of the transcribed RNA molecules was verified by a biological analyzer. The results showed that the molecular length of all the RNA molecules were consistent with our design. The clear peaks of our RNA RMs strongly demonstrated good purity.For absolute quantification of RNA RMs, we studied digital PCR(d PCR) for RNA samples. Digital PCR evenly partitioned the sample and PCR reaction solution to a very large number of units, on a microporous chip or in the liquid droplets, etc. After a PCR amplification reaction, the fluorescence signal was detected for each unit individually, with a binary readout o

关 键 词：新型冠状病毒 体外转录RNA 标准物质 数字PCR 实时荧光反转录PCR 

分 类 号：R373.1[医药卫生—病原生物学]