Evaluation of Manual, Automated, and Extraction-Free Workflows for SARS-CoV-2 RNA Recovery from Swab Samples

Abstract

Aim: This study aimed to compare the performance of four RNA recovery approaches—two silica column-based kits, an extraction-free vNAT method, and an automated magnetic bead-based extraction system—for the recovery of SARS-CoV-2 RNA from nasopharyngeal, oropharyngeal, and intubated samples. Materials and Methods: Samples collected from 29 patients under biosafety conditions were processed within 12 hours. RNA yield and purity were quantified using A260/280 and A260/230 ratios measured by NanoDrop spectrophotometry. IBM SPSS 21.0 were used for statistical analyses. Normality was assessed with the Shapiro–Wilk test, Wilcoxon signed-rank and Friedman tests were applied. p<0.05 were considered as statistically significant. Results: Silica Column–Based Extraction Kit 1 showed low RNA isolation and low purity indices. Silica Column–Based Extraction Kit 2 produced the highest RNA yields but reflected by high A260/280 and low A260/230 ratios. The automated magnetic bead-based extraction method provided the most consistent purity profile with moderate RNA yield; notably, A260/280 ratios were closest to ideal values, although A260/230 ratios remained below optimal levels. Conclusion: RNA yield and purity differed significantly among the four workflows. The automated magnetic bead-based isolation method was the most suitable for obtaining high-quality RNA for research applications, whereas the extraction-free vNAT method was appropriate only for rapid RT-qPCR–based diagnostic use.

Keywords

• SARS-CoV-2
• RNA isolation
• nasopharyngeal
• oropharyngeal

Supporting Institution

There is no organization supporting our research.

Ethical Statement

Ethical approval was obtained from the Clinical Research Ethics Committee of Istanbul Medeniyet University (Date: 18.11.2020; Approval No: 2020/06/56). Informed consent to participate was obtained from all individual participants included in the study. All procedures involving human participants were conducted in accordance with the ethical standards of the institutional and national research committees and with the principles outlined in the Declaration of Helsinki (as revised in 2013).

Thanks

The author would like to acknowledge Prof. Dr. Güler Öztürk and Prof. Dr. Sadrettin Pençe for their contributions. While their support was appreciated, their involvement did not fulfill the criteria for authorship and is therefore recognized in the acknowledgments.

References

1. [1]. Zhu, N., Zhang, D., Wang, W., Li, X., Yang, B., Song, J., Zhao, X., Huang, B., Shi, W., Lu, R., Niu, P., Zhan, F., Ma, X., Wang, D., Xu, W., Wu, G., Gao, G. F., Tan, W., & China Novel Coronavirus Investigating and Research Team (2020). A Novel Coronavirus from Patients with Pneumonia in China, 2019. The New England journal of medicine, 382(8), 727–733. https://doi.org/10.1056/NEJMoa2001017.
2. [2]. Wu, F., Zhao, S., Yu, B., Chen, Y. M., Wang, W., Song, Z. G., Hu, Y., Tao, Z. W., Tian, J. H., Pei, Y. Y., Yuan, M. L., Zhang, Y. L., Dai, F. H., Liu, Y., Wang, Q. M., Zheng, J. J., Xu, L., Holmes, E. C., & Zhang, Y. Z. (2020). A new coronavirus associated with human respiratory disease in China. Nature, 579(7798), 265–269. https://doi.org/10.1038/s41586-020-2008-3.
3. [3]. Kim, D., Lee, J. Y., Yang, J. S., Kim, J. W., Kim, V. N., & Chang, H. (2020). The Architecture of SARS-CoV-2 Transcriptome. Cell, 181(4), 914–921.e10. https://doi.org/10.1016/j.cell.2020.04.011.
4. [4]. Walls, A. C., Park, Y. J., Tortorici, M. A., Wall, A., McGuire, A. T., & Veesler, D. (2020). Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell, 181(2), 281–292.e6. https://doi.org/10.1016/j.cell.2020.02.058.
5. [5]. Hoffmann, M., Kleine-Weber, H., Schroeder, S., Krüger, N., Herrler, T., Erichsen, S., Schiergens, T. S., Herrler, G., Wu, N. H., Nitsche, A., Müller, M. A., Drosten, C., & Pöhlmann, S. (2020). SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell, 181(2), 271–280.e8. https://doi.org/10.1016/j.cell.2020.02.052
6. [6]. Zou, X., Chen, K., Zou, J., Han, P., Hao, J., & Han, Z. (2020). Single-cell RNA-seq data analysis on the receptor ACE2 expression reveals the potential risk of different human organs vulnerable to 2019-nCoV infection. Frontiers of medicine, 14(2), 185–192. https://doi.org/10.1007/s11684-020-0754-0.
7. [7]. Guan, W. J., Ni, Z. Y., Hu, Y., Liang, W. H., Ou, C. Q., He, J. X., Liu, L., Shan, H., Lei, C. L., Hui, D. S. C., Du, B., Li, L. J., Zeng, G., Yuen, K. Y., Chen, R. C., Tang, C. L., Wang, T., Chen, P. Y., Xiang, J., Li, S. Y., … China Medical Treatment Expert Group for Covid-19 (2020). Clinical Characteristics of Coronavirus Disease 2019 in China. The New England journal of medicine, 382(18), 1708–1720. https://doi.org/10.1056/NEJMoa2002032.
8. [8]. Zhou, F., Yu, T., Du, R., Fan, G., Liu, Y., Liu, Z., Xiang, J., Wang, Y., Song, B., Gu, X., Guan, L., Wei, Y., Li, H., Wu, X., Xu, J., Tu, S., Zhang, Y., Chen, H., & Cao, B. (2020). Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet (London, England), 395 (10229), 1054–1062. https://doi.org/10.1016/S0140-6736(20)30566-3.

9. [9]. Guan, W. J., Liang, W. H., Zhao, Y., Liang, H. R., Chen, Z. S., Li, Y. M., Liu, X. Q., Chen, R. C., Tang, C. L., Wang, T., Ou, C. Q., Li, L., Chen, P. Y., Sang, L., Wang, W., Li, J. F., Li, C. C., Ou, L. M., Cheng, B., Xiong, S., … China Medical Treatment Expert Group for COVID-19 (2020). Comorbidity and its impact on 1590 patients with COVID-19 in China: a nationwide analysis. The European respiratory journal, 55(5), 2000547. https://doi.org/10.1183/13993003.00547-2020.
10. [10]. Polack, F. P., Thomas, S. J., Kitchin, N., Absalon, J., Gurtman, A., Lockhart, S., Perez, J. L., Pérez Marc, G., Moreira, E. D., Zerbini, C., Bailey, R., Swanson, K. A., Roychoudhury, S., Koury, K., Li, P., Kalina, W. V., Cooper, D., Frenck, R. W., Jr, Hammitt, L. L., Türeci, Ö., … C4591001 Clinical Trial Group (2020). Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. The New England journal of medicine, 383(27), 2603–2615. https://doi.org/10.1056/NEJMoa2034577.
11. [11]. Corman, V. M., Landt, O., Kaiser, M., Molenkamp, R., Meijer, A., Chu, D. K., Bleicker, T., Brünink, S., Schneider, J., Schmidt, M. L., Mulders, D. G., Haagmans, B. L., van der Veer, B., van den Brink, S., Wijsman, L., Goderski, G., Romette, J. L., Ellis, J., Zambon, M., Peiris, M., … Drosten, C. (2020). Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin, 25(3), 2000045. https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045.
12. [12]. World Health Organization. (2020). Laboratory testing for coronavirus disease (COVID-19) in suspected human cases: Interim guidance. https://apps.who.int/iris/handle/10665/331501 (accessed at 19 March 2020).
13. [13]. Bruce, E. A., Huang, M. L., Perchetti, G. A., Tighe, S., Laaguiby, P., Hoffman, J. J., Gerrard, D. L., Nalla, A. K., Wei, Y., Greninger, A. L., Diehl, S. A., Shirley, D. J., Leonard, D. G. B., Huston, C. D., Kirkpatrick, B. D., Dragon, J. A., Crothers, J. W., Jerome, K. R., & Botten, J. W. (2020). DIRECT RT-qPCR DETECTION OF SARS-CoV-2 RNA FROM PATIENT NASOPHARYNGEAL SWABS WITHOUT AN RNA EXTRACTION STEP. bioRxiv : the preprint server for biology, 2020.03.20.001008. https://doi.org/10.1101/2020.03.20.001008.
14. [14]. Johnson, F. B. (1990). Transport of viral specimens. Clinical microbiology reviews, 3(2), 120–131. https://doi.org/10.1128/CMR.3.2.120.
15. [15]. McAuley, J., Fraser, C., Paraskeva, E., Trajcevska, E., Sait, M., Wang, N., ve diğerleri. (2021). Optimal preparation of SARS-CoV-2 viral transport medium for culture. Virology journal, 18(1), 53. https://doi.org/10.1186/s12985-021-01525-z.
16. [16]. Zafer, E. İ. (2016). Bölüm 7: Solunum Yolu Enfeksiyonlarında Mikrobiyolojik Tanı. In Tana Aslan, A, ve Kiper, N (Eds.). Çocuk Göğüs Hastalıklarında Tanı Yöntemleri (pp: 95-11). Türkiye Solunum Araştırmaları Derneği. Probiz Ltd. Şti (Content Ed Net Türkiye). e-ISBN: 978-605-66161-2-9. Retrieved April 19, 2022 from https://solunum.org.tr/TusadData/Book/535/3011201611466-01_Giris_Cocuk_Gogus.pdf.
17. [17]. Türkiye Cumhuriyeti, Sağlık Bakanlığı, Halk Sağlığı Genel Müdürlüğü, Mikrobiyoloji Referans Laboratuvarları ve Biyolojik Ürünler Dairesi Başkanlığı, Numune Alma El Kitabı (2022, April 19). Retrieved April 19, 2022 from https://hsgm.saglik.gov.tr/depo/kurumsal/plan-ve-faaliyetler/numune-alma-el-kitabi.pdf.
18. [18]. FDA. Adresss: https://www.fda.gov/media/141823/download, (accessed at 06.10.2025).
19. [19]. Qiagen. https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/rna-purification/total-rna/rneasy-kits?catno=74104 (accessed at 16.11.2025).
20. [20]. Promega. https://worldwide.promega.com/products/lab-automation/automated-dna-rna-extraction-purification-maxwell/maxwell-rsc-48-instrument/?tabset0=0, (accessed at 17.11.2025).
21. [21]. Kirkland, P. D., & Frost, M. J. (2020). The impact of viral transport media on PCR assay results for the detection of nucleic acid from SARS-CoV-2. Pathology, 52(7), 811–814. https://doi.org/10.1016/j.pathol.2020.09.013.
22. [22]. Dimke, H., Larsen, S. L., Skov, M. N., Larsen, H., Hartmeyer, G. N., & Moeller, J. B. (2021). Phenol-chloroform-based RNA purification for detection of SARS-CoV-2 by RT-qPCR: Comparison with automated systems. PloS one, 16(2), e0247524. https://doi.org/10.1371/journal.pone.0247524.
23. [23]. Lázaro-Perona, F., Rodriguez-Antolín, C., Alguacil-Guillén, M., Gutiérrez-Arroyo, A., Mingorance, J., García-Rodriguez, J., & SARS-CoV-2 Working Group (2021). Evaluation of two automated low-cost RNA extraction protocols for SARS-CoV-2 detection. PloS one, 16(2), e0246302. https://doi.org/10.1371/journal.pone.0246302.
24. [24]. Yalley, A. K., Ahiatrogah, S., Moro, I. I., Gmagna, P., Yankson, I. K., Kafintu-Kwashie, A. A., & Nii-Trebi, N. I. (2024). Utility of Extraction-Free SARS-CoV-2 Detection by RT-qPCR for COVID-19 Testing in a Resource-Limited Setting. Diseases (Basel, Switzerland), 12(9), 198. https://doi.org/10.3390/diseases12090198.
25. [25]. Smyrlaki, I., Ekman, M., Lentini, A., Rufino de Sousa, N., Papanicolaou, N., Vondracek, M., Aarum, J., Safari, H., Muradrasoli, S., Rothfuchs, A. G., Albert, J., Högberg, B., & Reinius, B. (2020). Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR. Nature communications, 11(1), 4812. https://doi.org/10.1038/s41467-020-18611-5.