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A culture-free method for rapidly and accurately quantifying active SARS-CoV-2

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Abstract

Determining the quantity of active virus is the most important basis to judge the risk of virus infection, which usually relies on the virus median tissue culture infectious dose (TCID50) assay performed in a biosafety level 3 laboratory within 5–7 days. We have developed a culture-free method for rapid and accurate quantification of active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by targeting subgenomic RNA (sgRNA) based on reverse transcription digital PCR (RT-dPCR). The dynamic range of quantitative assays for sgRNA-N and sgRNA-E by RT-dPCR was investigated, and the result showed that the limits of detection (LoD) and quantification (LoQ) were 2 copies/reaction and 10 copies/reaction, respectively. The delta strain (NMDC60042793) of SARS-CoV-2 was cultured at an average titer of 106.13 TCID50/mL and used to evaluate the developed quantification method. Copy number concentrations of the cultured SARS-CoV-2 sgRNA and genomic RNA (gRNA) gave excellent linearity (R2 = 0.9999) with SARS-CoV-2 titers in the range from 500 to 105 TCID50/mL. Validation of 63 positive clinical samples further proves that the quantification of sgRNA-N by RT-dPCR is more sensitive for active virus quantitative detection. It is notable that we can infer the active virus titer through quantification of SARS-CoV-2 sgRNA based on the linear relationship in a biosafety level 2 laboratory within 3 h. It can be used to timely and effectively identify infectious patients and reduce unnecessary isolation especially when a large number of COVID-19 infected people impose a burden on medical resources.

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References

  1. Boger B, Fachi MM, Vilhena RO, Cobre AF, Tonin FS, Pontarolo R. Systematic review with meta-analysis of the accuracy of diagnostic tests for COVID-19. Am J Infect Control. 2021;49(1):21–9. https://doi.org/10.1016/j.ajic.2020.07.011.

    Article  CAS  PubMed  Google Scholar 

  2. Sule WF, Oluwayelu DO. Real-time RT-PCR for COVID-19 diagnosis: challenges and prospects. Pan Afr Med J, 2020, 35(Suppl 2): 121. 10.11604/pamj.supp.2020.35.24258.

  3. Bustin S, Kirvell S, Huggett JF, Nolan T. RT-qPCR diagnostics: the “Drosten” SARS-CoV-2 Assay Paradigm. Int J Mol Sci. 2021;22(16) https://doi.org/10.3390/ijms22168702.

  4. Wolfel R, Corman VM, Guggemos W, Seilmaier M, Zange S, Muller MA, et al. Virological assessment of hospitalized patients with COVID-2019. Nature. 2020;581(7809):465–9. https://doi.org/10.1038/s41586-020-2196-x.

    Article  CAS  PubMed  Google Scholar 

  5. Dagotto G, Mercado NB, Martinez DR, Hou YJ, Nkolola JP, Carnahan RH, et al. Comparison of subgenomic and total RNA in SARS-CoV-2 challenged rhesus macaques. J Virol. 2021;95(8) https://doi.org/10.1128/JVI.02370-20.

  6. Rayms-Keller A, Powers AM, Higgs S, Olson KE, Kamrud KI, Carlson JO, et al. Replication and expression of a recombinant Sindbis virus in mosquitoes. Insect Mol Biol. 1995;4(4):245–51. https://doi.org/10.1111/j.1365-2583.1995.tb00030.x.

    Article  CAS  PubMed  Google Scholar 

  7. Smither SJ, Lear-Rooney C, Biggins J, Pettitt J, Lever MS, Olinger GG Jr. Comparison of the plaque assay and 50% tissue culture infectious dose assay as methods for measuring filovirus infectivity. J Virol Methods. 2013;193(2):565–71. https://doi.org/10.1016/j.jviromet.2013.05.015.

    Article  CAS  PubMed  Google Scholar 

  8. Santos Bravo M, Berengua C, Marín P, Esteban M, Rodriguez C, Del Cuerpo M, et al. Viral culture confirmed SARS-CoV-2 subgenomic RNA value as a good surrogate marker of infectivity. J Clin Microbiol. 2022;60(1):e0160921. https://doi.org/10.1128/jcm.01609-21.

    Article  CAS  PubMed  Google Scholar 

  9. Sawicki SG, Sawicki DL, Siddell SG. A contemporary view of coronavirus transcription. J Virol. 2007;81(1):20–9. https://doi.org/10.1128/jvi.01358-06.

    Article  CAS  PubMed  Google Scholar 

  10. Sola I, Almazan F, Zuniga S, Enjuanes L. Continuous and discontinuous RNA synthesis in coronaviruses. Annu Rev Virol. 2015;2(1):265–88. https://doi.org/10.1146/annurev-virology-100114-055218.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kim D, Lee JY, Yang JS, Kim JW, Kim VN, Chang H. The architecture of SARS-CoV-2 transcriptome. Cell. 2020;181(4):914–21.e10. https://doi.org/10.1016/j.cell.2020.04.011.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Davidson AD, Williamson MK, Lewis S, Shoemark D, Carroll MW, Heesom KJ, et al. Characterisation of the transcriptome and proteome of SARS-CoV-2 reveals a cell passage induced in-frame deletion of the furin-like cleavage site from the spike glycoprotein. Genome Med. 2020;12(1):68. https://doi.org/10.1186/s13073-020-00763-0.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Finkel Y, Mizrahi O, Nachshon A, Weingarten-Gabbay S, Morgenstern D, Yahalom-Ronen Y, et al. The coding capacity of SARS-CoV-2. Nature. 2021;589(7840):125–30. https://doi.org/10.1038/s41586-020-2739-1.

    Article  CAS  PubMed  Google Scholar 

  14. Taiaroa G, Rawlinson D, Featherstone L, Pitt M, Caly L, Druce J, Purcell D, Harty L, Tran T, Roberts J, Scott N, Catton M, Williamson D, Coin L. Direct RNA sequencing and early evolution of SARS-CoV-2. bioRxiv. 2020. https://doi.org/10.1101/2020.03.05.976167.

  15. Xu J, Kirtek T, Xu Y, Zheng H, Yao H, Ostman E, et al. Digital droplet PCR for SARS-CoV-2 resolves borderline cases. Am J Clin Pathol. 2021;155(6):815–22. https://doi.org/10.1093/ajcp/aqab041.

    Article  CAS  PubMed  Google Scholar 

  16. Nyaruaba R, Li C, Mwaliko C, Mwau M, Odiwuor N, Muturi E, et al. Developing multiplex ddPCR assays for SARS-CoV-2 detection based on probe mix and amplitude based multiplexing. Expert Rev Mol Diagn. 2021;21(1):119–29. https://doi.org/10.1080/14737159.2021.1865807.

    Article  CAS  PubMed  Google Scholar 

  17. Liu X, Feng J, Zhang Q, Guo D, Zhang L, Suo T, et al. Analytical comparisons of SARS-COV-2 detection by qRT-PCR and ddPCR with multiple primer/probe sets. Emerg Microbes Infect. 2020;9(1):1175–9. https://doi.org/10.1080/22221751.2020.1772679.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Nyaruaba R, Mwaliko C, Dobnik D, Neuzil P, Amoth P, Mwau M, et al. Digital PCR Applications in the SARS-CoV-2/COVID-19 era: a roadmap for future outbreaks. Clin Microbiol Rev. 2022;35(3):e0016821. https://doi.org/10.1128/cmr.00168-21.

    Article  CAS  PubMed  Google Scholar 

  19. Dong L, Zhou J, Niu C, Wang Q, Pan Y, Sheng S, et al. Highly accurate and sensitive diagnostic detection of SARS-CoV-2 by digital PCR. Talanta. 2021;224:121726. https://doi.org/10.1016/j.talanta.2020.121726.

    Article  CAS  PubMed  Google Scholar 

  20. Németh B, Fasseeh A, Molnár A, Bitter I, Horváth M, Kóczián K, et al. A systematic review of health economic models and utility estimation methods in schizophrenia. Expert Rev Pharmacoecon Outcomes Res. 2018;18(3):267–75. https://doi.org/10.1080/14737167.2018.1430571.

    Article  PubMed  Google Scholar 

  21. Casagrande M, Fitzek A, Spitzer M, Puschel K, Glatzel M, Krasemann S, et al. Detection of SARS-CoV-2 genomic and subgenomic RNA in retina and optic nerve of patients with COVID-19. Br J Ophthalmol. 2022;106(9):1313–7. https://doi.org/10.1136/bjophthalmol-2020-318618.

    Article  PubMed  Google Scholar 

  22. Niu C, Wang X, Gao Y, Qiao X, Xie J, Zhang Y, et al. Accurate quantification of SARS-CoV-2 RNA by isotope dilution mass spectrometry and providing a correction of reverse transcription efficiency in droplet digital PCR. Anal Bioanal Chem. 2022;414(23):6771–7. https://doi.org/10.1007/s00216-022-04238-6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Huggett JF, Foy CA, Benes V, Emslie K, Garson JA, Haynes R, et al. The digital MIQE guidelines: minimum information for publication of quantitative digital PCR experiments. Clinical chemistry. 2013;59(6):892–902. https://doi.org/10.1373/clinchem.2013.206375.

    Article  CAS  PubMed  Google Scholar 

  24. Weaver S, Dube S, Mir A, Qin J, Sun G, Ramakrishnan R. Taking qPCR to a higher level: analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution. Methods (San Diego, Calif). 2010;50:271–6.

    Article  CAS  PubMed  Google Scholar 

  25. Daniel WT, Kristian L, Marina K, David AA, Patricia EG, Robert LJ, Martin HK, Rudolf ML, Thomas JP, Scassellati GA, Heinz S, Jane T. EP17-A—Protocols for determination of limits of detection and limits of quantitation; Approved Guideline.

  26. Yang M, Cao S, Liu Y, Zhang Z, Zheng R, Li Y, et al. Performance verification of five commercial RT-qPCR diagnostic kits for SARS-CoV-2. Clin Chim Acta. 2022;525:46–53. https://doi.org/10.1016/j.cca.2021.12.004.

    Article  CAS  PubMed  Google Scholar 

  27. Vierbaum L, Wojtalewicz N, Grunert HP, Lindig V, Duehring U, Drosten C, et al. RNA reference materials with defined viral RNA loads of SARS-CoV-2-A useful tool towards a better PCR assay harmonization. PLoS One. 2022;17(1):e0262656. https://doi.org/10.1371/journal.pone.0262656.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Niu C, Wang X, Zhang Y, Lu L, Wang D, Gao Y, et al. Interlaboratory assessment of quantification of SARS-CoV-2 RNA by reverse transcription digital PCR. Anal Bioanal Chem. 2021;413(29):7195–204. https://doi.org/10.1007/s00216-021-03680-2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Tan SYH, Kwek SYM, Low H, Pang YLJ. Absolute quantification of SARS-CoV-2 with Clarity Plus™ digital PCR. Methods (San Diego, Calif). 2022;201:26–33. https://doi.org/10.1016/j.ymeth.2021.07.005.

    Article  CAS  PubMed  Google Scholar 

  30. Hou YJ, Okuda K, Edwards CE, Martinez DR, Asakura T, Dinnon KH 3rd, et al. SARS-CoV-2 reverse genetics reveals a variable infection gradient in the respiratory tract. Cell. 2020;182(2):429–46.e14. https://doi.org/10.1016/j.cell.2020.05.042.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Telwatte S, Martin HA, Marczak R, Fozouni P, Vallejo-Gracia A, Kumar GR, et al. Novel RT-ddPCR assays for measuring the levels of subgenomic and genomic SARS-CoV-2 transcripts. Methods. 2022;201:15–25. https://doi.org/10.1016/j.ymeth.2021.04.011.

    Article  CAS  PubMed  Google Scholar 

  32. Dimcheff DE, Valesano AL, Rumfelt KE, Fitzsimmons WJ, Blair C, Mirabelli C, et al. Severe Acute respiratory syndrome coronavirus 2 total and subgenomic RNA viral load in hospitalized patients. J Infect Dis. 2021;224(8):1287–93. https://doi.org/10.1093/infdis/jiab215.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Moron-Lopez S, Riveira-Munoz E, Urrea V, Gutierrez-Chamorro L, Avila-Nieto C, Noguera-Julian M, et al. Comparison of reverse transcription (RT)-quantitative PCR and RT-droplet digital PCR for detection of genomic and subgenomic SARS-CoV-2 RNA. Microbiol Spectr. 2023;11(2):e0415922. https://doi.org/10.1128/spectrum.04159-22.

    Article  CAS  PubMed  Google Scholar 

  34. Osborn LJ, Chen PY, Flores-Vazquez J, Mestas J, Salas E, Glucoft M, et al. Clinical utility of SARS-CoV-2 subgenomic RT-PCR in a pediatric quaternary care setting. J Clin Virol. 2023;164:105494. https://doi.org/10.1016/j.jcv.2023.105494.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Yang J, Kidd M, Nordquist AR, Smith SD, Hurth C, Modlin IM, et al. A sensitive, portable microfluidic device for SARS-CoV-2 detection from self-collected Saliva. Infect Dis Rep. 2021;13(4):1061–77. https://doi.org/10.3390/idr13040097.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Singanayagam A, Patel M, Charlett A, Lopez Bernal J, Saliba V, Ellis J, et al. Duration of infectiousness and correlation with RT-PCR cycle threshold values in cases of COVID-19, England, January to May 2020. Euro Surveill., 2020, 25(32). https://doi.org/10.2807/1560-7917.ES.2020.25.32.2001483.

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Funding

This work was supported by Basic scientific research project of National institute of metrology (AKYZZ2126, AKYYJ2009/ZYZJ2001) and the Major Science and Technique Programs in Yunnan Province (202102AA310055).

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Author notes

  1. Yi Yang, Xiaoli Feng, and Yang Pan contributed equally to this work.

Authors and Affiliations

  1. Center for Advanced Measurement of Science, National Institute of Metrology, Beijing, 100029, China

    Yi Yang, Xia Wang, Tao Peng, Chunyan Niu, Lianhua Dong, Xinhua Dai & Xiang Fang

  2. Shenzhen Institute for Technology Innovation, National Institute of Metrology, Shenzhen, 518107, China

    Yi Yang, Wang Qu, Qingcui Zou & Minghua Li

  3. Kunming National High-Level Biosafety Research Center for Non-Human Primates, Center for Biosafety Mega-Science, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650107, Yunnan, China

    Xiaoli Feng

  4. Institute for Infectious Disease and Endemic Disease Control, Beijing Center for Disease Control and Prevention, Beijing, 100029, China

    Yang Pan

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  1. Yi Yang
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Contributions

Conceptualization and methodology: Lianhua Dong, Xinhua Dai, Xiang Fang, and Minghua Li. Methodology: Lianhua Dong, Xia Wang, Chunyan Niu, and Xiaoli Feng. Software and formal analysis: Lianhua Dong, Yi Yang, and Tao Peng. Investigation and data curation: Yi Yang, Yang Pan, Xiaoli Feng, Wang Qu, and Qingcui Zou. Resources: Lianhua Dong, Xinhua Dai, and Minghua Li. Writing—original draft preparation: Lianhua Dong, Yi Yang, Xia Wang, and Tao Peng. Writing—review and editing: Lianhua Dong, Yang Pan, and Tao Peng. Supervision: Lianhua Dong, Xinhua Dai, and Xiang Fang. All authors have read and agreed to the final version of the manuscript.

Corresponding authors

Correspondence to Lianhua Dong, Xinhua Dai, Minghua Li or Xiang Fang.

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This study was approved by the Ethics Committee of the Beijing Center for Disease Prevention and Control. The analysis was performed on existing samples collected during standard diagnostic tests, posing no extra burden to patients.

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Yang, Y., Feng, X., Pan, Y. et al. A culture-free method for rapidly and accurately quantifying active SARS-CoV-2. Anal Bioanal Chem 415, 5745–5753 (2023). https://doi.org/10.1007/s00216-023-04855-9

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