More info
  • ReceivedApr 2, 2020
  • AcceptedApr 26, 2020
  • PublishedMay 6, 2020


Coronavirus disease (COVID-19) is an acute infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Reverse transcription real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was the firstly authorized method for the detection of SARS-CoV-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-qPCR. Nucleic acid reference materials (RM) are the key element for the metrology traceability and quality control of SARS-CoV-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-CoV-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-CoV-2 gene: The full-length of nucleoprotein (N) gene (28274-29533, GenBank: MT027064.1), the full-length of envelope protein (E) gene (26245-26472, GenBank: MT027064.1), and partial sequence of open reading frame 1ab (ORF1ab) (13321-15540, GenBank: 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 (dPCR) 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 of “0” or “1” for negative and positive results respectively. Through the statistics of positive results based on the Poisson distribution, the copy number of RNA sample was accurately determined without standard curves needed. Digital PCR has significantly higher reliability and accuracy. Mainly based on the PCR primers and probes for SARS-CoV-2 detection suggested by the Chinese CDC and WHO, we optimized the key factors of dPCR towards improved amplification efficiency. Through digital PCR measurements by 4 laboratories, the certified values of concentration (copies/μL) were assigned for the N gene, E gene, and ORF1ab gene in the mixed RM.

Single-stranded RNA is unstable and easy to be degraded by RNase in the environment, thus the optimization of RNA protectants is very important for the stability of RNA RMs. During the study of the stability, we found that a proper protector (1 mmol/L DTT and 0.5 U/L Rnase Inhibitor) can effectively increase the valid storage life of our RNA RM. Based on the latest data, the concentration of our RNA RMs was stable for at least 30 d under −80 °C storage and 13 d under −4°C storage.

In order to verify the applicability of our RNA RM in the actual virus detection process, we analyzed our RMs using 9 SARS-CoV-2 nucleic acid detection kits. These virus RNA detection kits were from different manufacturers with various detection principles, that are being applied in laboratories for virus detection. Finally, our RNA RMs showed high generalizability among 9 kits. The development of RNA RM provides the metrological basis for the quality control of SARS-CoV-2 detection kits.

Funded by







[1] Huang C L, Wang Y M, Li X W, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet, 2020, 395: 497− 506. Google Scholar

[2] Zhu N, Zhang D, Wang W, et al. A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med, 2020, 382: 727−733. Google Scholar

[3] Health Emergency Office National Health Commission. Update on COVID-19 at 24:00 March 18 (in Chinese). http://www.nhc.gov.cn/yjb/new_index.shtml [国家卫生健康委员会卫生应急办公室. 截至3月18日24时新型冠状病毒肺炎疫情最新情况, 2020. http://www.nhc.gov.cn/yjb/new_index.shtml]. Google Scholar

[4] World Health Organization. Coronavirus Disease 2019 (COVID-19) Situation Report-58, 2020. https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200318-sitrep-58-covid-19.pdf?sfvrsn=20876712_2. Google Scholar

[5] Holshue M L, DeBolt C, Lindquist S, et al. First case of 2019 novel coronavirus in the United States. N Engl J Med, 2020, 382: 929−936. Google Scholar

[6] Cui J, Li F, Shi Z L. Origin and evolution of pathogenic coronaviruses. Nat Rev Microbiol, 2019, 17: 181-192 CrossRef PubMed Google Scholar

[7] Lu R, Zhao X, Li J, et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: Implications for virus origins and receptor binding. Lancet, 2020, 395: 565−574. Google Scholar

[8] Corman V M, Landt O, Kaiser M, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Eurosurveillance, 2020, 25: 23− 30. Google Scholar

[9] National Health Commission. COVID-19 protocol (trial seventh edition), 2020 (in Chinese). http://www.nhc.gov.cn/yzygj/s7653p/202003/46c9294a7dfe4cef80dc7f5912eb1989.shtml [国家卫生健康委员会. 新型冠状病毒肺炎诊疗方案(试行第七版), 2020. http://www.nhc.gov.cn/yzygj/s7653p/202003/46c9294a7dfe4cef80dc7f5912eb1989.shtml]. Google Scholar

[10] Li Z H, Gao X L, Yang X J, et al. Nucleic acid analysis for the detection of SARS-CoV-2 (in Chinese). Lab Med Clini, 2020, http://kns.cnki.net/kcms/detail/50.1167.R.20200317.1710.002.html [李振昊, 高小玲, 杨小娟, 等. 新型冠状病毒核酸检测分析. 检验医学与临床, 2020, http://kns.cnki.net/kcms/detail/50.1167.R.20200317.1710.002.html]. Google Scholar

[11] Liang W, Xu L, Sui Z, et al. Quantification of plasmid DNA reference materials for Shiga toxin-producing Escherichia coli based on UV, HR-ICP-MS and digital PCR. Chem Cent J, 2016, 10: 55 CrossRef PubMed Google Scholar

[12] Haynes R J, Kline M C, Toman B, et al. Standard reference material 2366 for measurement of human cytomegalovirus DNA. J Mol Diagn, 2013, 15: 177-185 CrossRef PubMed Google Scholar

[13] Corbisier P, Pinheiro L, Mazoua S, et al. DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials. Anal Bioanal Chem, 2015, 407: 1831-1840 CrossRef PubMed Google Scholar

[14] Baume M, Cariou A, Leveau A, et al. Quantification of Legionella DNA certified reference material by digital droplet PCR. J Microbiol Methods, 2019, 157: 50-53 CrossRef PubMed Google Scholar

[15] Zhao Y, Chen F, Li Q, et al. Isothermal amplification of nucleic acids. Chem Rev, 2015, 115: 12491-12545 CrossRef PubMed Google Scholar

[16] Chinese Center for Disease Control and Prevention. Primers and probes for the detection of SARS-CoV-2, 2020 (in Chinese). http://www.chinaivdc.cn/kyjz/202001/t20200121_211337.html [中国疾病预防控制中心. 新型冠状病毒核酸检测引物和探针序列, 2020. http://www.chinaivdc.cn/kyjz/202001/t20200121_211337.html]. Google Scholar

[17] World Health Organization. Diagnostic detection of 2019-nCoV by real-time RT-PCR, 2020. https://www.who.int/docs/default-source/coronaviruse/protocol-v2-1.pdf?sfvrsn=a9ef618c_2. Google Scholar

[18] Pinheiro L B, O’Brien H, Druce J, et al. Interlaboratory reproducibility of droplet digital polymerase chain reaction using a new DNA reference material format. Anal Chem, 2017, 89: 11243-11251 CrossRef PubMed Google Scholar

[19] Corbisier P, Bhat S, Partis L, et al. Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction. Anal Bioanal Chem, 2010, 396: 2143-2150 CrossRef PubMed Google Scholar

[20] Devonshire A S, Whale A S, Gutteridge A, et al. Towards standardisation of cell-free DNA measurement in plasma: Controls for extraction efficiency, fragment size bias and quantification. Anal Bioanal Chem, 2014, 406: 6499-6512 CrossRef PubMed Google Scholar

[21] National Genomics Data Center. 2019 Novel Coronavirus Resource (2019nCoVR), 2020 (in Chinese). https://bigd.big.ac.cn/ncov [国家基因组科学数据中心. 2019新型冠状病毒信息库, 2020. https://bigd.big.ac.cn/ncov]. Google Scholar

[22] ISO Guide 35: 2017, Reference Materials General and Statistical Principles for Certification, 2017. Google Scholar

[23] Zhao W M, Song S H, Chen M L, et al. The 2019 novel coronavirus resource (in Chinese). Hereditas, 2020, 42: 212–221 [赵文明, 宋述慧, 陈梅丽, 等. 2019新型冠状病毒信息库. 遗传, 2020, 42: 212–221]. Google Scholar

  • Figure 1

    (Color online) Analysis of the SARS-CoV-2 RNA RM using a bioanalyzer. (a) Lanes of 3 RNA in our RMs (Lane L: RNA ladder; Lane 1−2: N gene RNA; Lane 3−4: E gene RNA; Lane 5−6: ORF1ab RNA). (b) Normalized peaks of 3 RNA in our RMs

  • Figure 2

    (Color online) Schematic illustration of this work. (a) Traceability chart of SARS-CoV-2 RNA quantification; (b) digital PCR absolute quantification of SARS-CoV-2 RNA RM

  • Figure 3

    (Color online) Homogeneity analysis of SARS-CoV-2 RNA RM: 11 units of 200 RMs were randomly analyzed with 3 replications. Homogeneity analysis result of E gene RNA fragments (a), N gene RNA fragments (b) and ORF1ab RNA fragments (c)

  • Figure 4

    (Color online) Stability of SARS-CoV-2 RNA RM under different temperature. (a) Stability after 13 d under 4°C; (b) stability after 13 d under −20°C; (c) stability after 30 d under −80°C

  • Figure 5

    (Color online) Quantification results of our RNA RM. The analysis results of E (a), N (b) and ORF1ab (c) gene from 4 different laboratories, showing 8 replications, the average and SD of each lab. The dPCR data of E (d), N (e) and ORF1ab (f) gene in lab A

  • Figure 6

    (Color online) Practical verification of our RNA RM. (a) RT-qPCR amplification curve of Kit No.1. (b) All analysis results of 9 kits

  • Table 1   The primer and probe sequences for dPCR certification of RNA reference material

























Copyright 2020  CHINA SCIENCE PUBLISHING & MEDIA LTD.  中国科技出版传媒股份有限公司  版权所有

京ICP备14028887号-23       京公网安备11010102003388号