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

Abstract

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

国家重点研发计划(2018YFF02128002016YFA0201200)

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)


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  • 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

    引物/探针

    序列(5′→3′)

    来源

    N-F

    GGGGAACTTCTCCTGCTAGAAT

    CDC[16]

    N-R

    CAGACATTTTGCTCTCAAGCTG

    N-P

    FAM-TTGCTGCTGCTTGACAGATT-TAMRA

    ORF1ab-F

    CCCTGTGGGTTTTACACTTAA

    CDC[16]

    ORF1ab-R

    ACGATTGTGCATCAGCTGA

    ORF1ab-P

    FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ1

    E-F

    ACAGGTACGTTAATAGTTAATAGCGT

    WHO[17]

    E-R

    ATATTGCAGCAGTACGCACACA

    E-P

    FAM-ACACTAGCCATCCTTACTGCGCTTCG-BHQ1

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