Diagnostic performance of RNA extraction-free dilution and heating method for the detection of severe acute respiratory syndrome coronavirus 2

Abstract

Real-time quantitative polymerase chain reaction remains the gold standard for COVID-19 diagnosis, but RNA extraction is time-consuming, expensive, and associated with increased biosafety requirements. This study evaluated an extraction-free dilution and heating (EFDH) method as a simplified alternative to conventional extraction-based (EB) real-time quantitative polymerase chain reaction for severe acute respiratory syndrome coronavirus 2 detection. A total of 300 archived nasopharyngeal specimens, including 190 positives and 110 negatives, from the National Virology Reference Laboratory at the Ethiopian Public Health Institute, were analyzed. Samples were diluted 1:2 with RNase-free water, heated at 72°C for 15 min, and analyzed using an ABI 7500 Fast instrument. The EFDH method showed a sensitivity of 92%, a specificity of 100%, and an overall accuracy of 85.8%, producing 15 false-negative and 12 invalid results. Agreement with the EB method was high, with 95% concordance and a kappa coefficient of 0.89. Performance was strongest in samples with high viral loads (cycle threshold [Ct] < 20) and declined in those with low viral loads (Ct > 35). A significant correlation was observed between the two methods(R² = 0.99, p=0.001). These findings indicate that the EFDH approach reliably detects moderate-to-high viral loads and may serve as a practical testing option in resource-limited settings, especially during outbreaks when rapid and simplified workflows are needed.

Keywords

SARS-CoV-2 / Extraction free / Heating / Dilution / Diagnostic performance

References

[1] Wu A, Peng Y, Huang B, et al. Genome composition and divergence of the novel coronavirus ( 2019-nCoV) originating in China. Cell Host Microbe. 2020; 27(3):325-328. doi: 10.1016/j.chom.2020.02.001

[2] Huang Y, Yang C, Xu XF, Xu W, Liu SW. Structural and functional properties of SARS-CoV-2 spike protein: Potential antivirus drug development for COVID-19. Acta Pharmacol Sin. 2020; 41(9):1141-1149. doi: 10.1038/s41401-020-0485-4

[3] Harvey WT, Carabelli AM, Jackson B, et al. SARS-CoV-2 variants, spike mutations and immune escape. Nat Rev Microbiol. 2021; 19(7):409-424. doi: 10.1038/s41579-021-00573-0

[4] Corman VM, Landt O, Kaiser M, et al.Detection of 2019 novel coronavirus ( 2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020; 25(3):2000045. doi: 10.2807/1560-7917.es.2020.25.3.2000045

[5] Liu AB, Lee D, Jalihal AP, Hanage WP, Springer M. Quantitatively assessing early detection strategies for mitigating COVID-19 and future pandemics. Nat Commun. 2023; 14(1):8479. doi: 10.1038/s41467-023-44199-7

[6] Zhang C, Zhou C, Xu W, et al. Transmission risk of asymptomatic SARS-CoV-2 infection: A systematic review and meta-analysis. Infect Med (Beijing). 2022; 2(1):11-18. doi: 10.1016/j.imj.2022.12.001

[7] Joynt GM, Wu WK. Understanding COVID-19: What does viral RNA load really mean? Lancet Infect Dis. 2020; 20(6):635-636. doi: 10.1016/S1473-3099(20)30237-1

[8] Sethuraman N, Jeremiah SS, Ryo A. Interpreting diagnostic tests for SARS-CoV-2. JAMA. 2020; 323(22):2249-2251. doi: 10.1001/jama.2020.8259

[9] Loeffelholz MJ, Tang YW. Laboratory diagnosis of emerging human coronavirus infections - the state of the art. Emerg Microbes Infect. 2020; 9(1):747-756. doi: 10.1080/22221751.2020.1745095

[10] Cheng VCC, Lau SKP, Woo PCY, Yuen KY. Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection. Clin Microbiol Rev. 2007; 20(4):660-694. doi: 10.1128/CMR.00023-07

[11] Ferrari D, Motta A, Strollo M, Banfi G, Locatelli M. Routine blood tests as a potential diagnostic tool for COVID-19. Clin Chem Lab Med. 2020; 58(7):1095-1099. doi: 10.1515/cclm-2020-0398

[12] Rodriguez-Morales AJ, Cardona-Ospina JA, Gutiérrez- Ocampo E, et al. Clinical, laboratory and imaging features of COVID-19: A systematic review and meta-analysis. Travel Med Infect Dis. 2020; 34:101623. doi: 10.1016/j.tmaid.2020.101623

[13] Salamanna F, Maglio M, Landini MP, Fini M. Body localization of ACE-2: On the trail of the keyhole of SARS-CoV-2. Front Med. 2020; 7:594495. doi: 10.3389/fmed.2020.594495

[14] Wang MY, Zhao R, Gao LJ, Gao XF, Wang DP, Cao JM. SARS-CoV-2: Structure, biology, and structure-based therapeutics development. Front Cell Infect Microbiol. 2020; 10:587269. doi: 10.3389/fcimb.2020.587269

[15] Marty FM, Chen K, Verrill KA. How to obtain a nasopharyngeal swab specimen. N Engl J Med. 2020; 382(22):e76. doi: 10.1056/NEJMvcm2010260

[16] Böger 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):21-29. doi: 10.1016/j.ajic.2020.07.011

[17] Smyrlaki I, Ekman M, Lentini A, et al. Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR. Nat Commun. 2020; 11(1):4812. doi: 10.1038/s41467-020-18611-5

[18] Bruce EA, Huang ML, Perchetti GA, et al. Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step. PLoS Biol. 2020; 18(10):e3000896. doi: 10.1371/journal.pbio.3000896

[19] Alhamid G, Tombuloglu H, Rabaan AA, Al-Suhaimi E. SARS-CoV-2 detection methods: A comprehensive review. Saudi J Biol Sci. 2022; 29(11):103465. doi: 10.1016/j.sjbs.2022.103465

[20] Bustin SA, Nolan T. RT-qPCR testing of SARS-CoV-2: A primer. Int J Mol Sci. 2020; 21(8):3004. doi: 10.3390/ijms21083004

[21] Aoki MN, Coelho BO, Góes LGB, et al. Colorimetric RT-LAMP SARS-CoV-2 diagnostic sensitivity relies on color interpretation and viral load. Sci Rep. 2021; 11:9026. doi: 10.1038/s41598-021-88506-y

[22] Tombuloglu H, Sabit H, Al-Suhaimi E, Al Jindan R, Alkharsah KR. Development of multiplex real-time RT-PCR assay for the detection of SARS-CoV-2. PLoS One. 2021; 16(4):e0250942. doi: 10.1371/journal.pone.0250942

[23] Lv DF, Ying QM, Weng YS, et al. Dynamic change process of target genes by RT-PCR testing of SARS-Cov-2 during the course of a coronavirus disease 2019 patient. Clin Chim Acta. 2020; 506:172-175. doi: 10.1016/j.cca.2020.03.032

[24] Tahmasebi S, Khosh E, Esmaeilzadeh A. The outlook for diagnostic purposes of the 2019-novel coronavirus disease. J Cell Physiol. 2020; 235(12):9211-9219. doi: 10.1002/jcp.29804

[25] Mouliou DS. Managing viral emerging infectious diseases via current molecular diagnostics in the emergency department: The Tricky cases. Expert Rev Anti Infect Ther. 2022; 20(9):1163-1169. doi: 10.1080/14787210.2022.2089653

[26] Mouliou DS, Gourgoulianis KI. COVID-19 ‘asymptomatic’ patients: An old wives’ tale. Expert Rev Respir Med. 2022; 16(4):399-407. doi: 10.1080/17476348.2022.2030224

[27] Mouliou DS, Gourgoulianis KI. False-positive and false-negative COVID-19 cases: Respiratory prevention and management strategies, vaccination, and further perspectives. Expert Rev Repir Med. 2021; 15(8):993-1002. doi: 10.1080/17476348.2021.1917389

[28] Pizzol JLD, Hora VP, Reis AJ, et al. Laboratory diagnosis for Covid-19: A mini-review. Rev Soc Bras Med Trop. 2020; 53:e20200451. doi: 10.1590/0037-8682-0451-2020

[29] Zhao Y, Cui C, Zhang K, et al. COVID19: A systematic approach to early identification and healthcare worker protection. Front Public Health. 2020; 8:205. doi: 10.3389/fpubh.2020.00205

[30] Fomsgaard AS, Rosenstierne MW. An alternative workflow for molecular detection of SARS-CoV-2 - escape from the NA extraction kit-shortage, Copenhagen, Denmark, March 2020. Eur Surveill. 2020; 25(14):2000398. doi: 10.2807/1560-7917.es.2020.25.14.2000398

[31] Villota SD, Nipaz VE, Carrazco-Montalvo A, et al. Alternative RNA extraction-free techniques for the real-time RT-PCR detection of SARS-CoV-2 in nasopharyngeal swab and sputum samples. J Virol Methods. 2021; 298:114302. doi: 10.1016/j.jviromet.2021.114302

[32] QIAGEN. QIAamp® Viral RNA Mini Handbook; 2020. Available from: https://www.qiagen.com [Last accessed on 2023 Aug 21].

[33] Jayaprakasam M, Aggarwal S, Mane A, et al. RNA-extraction-free diagnostic method to detect SARS-CoV-2: An assessment from two states, India. Epidemiol Infect. 2021; 149:e245. doi: 10.1017/s0950268821002302

[34] Byrnes SA, Gallagher R, Steadman A, et al. Multiplexed and extraction-free amplification for simplified SARS-CoV-2 RT-PCR tests. Anal Chem. 2021; 93(9):4160-4165. doi: 10.1021/acs.analchem.0c03918

[35] Morecchiato F, Coppi M, Baccani I, et al. Evaluation of extraction-free RT-PCR methods for faster and cheaper detection of SARS-CoV-2 using two commercial systems. Int J Infect Dis. 2021; 112:264-268. doi: 10.1016/j.ijid.2021.09.046

[36] Mahmoud SA, Ganesan S, Ibrahim E, et al. Evaluation of RNA extraction-free method for detection of SARS-CoV-2 in salivary samples for mass screening for COVID-19. Biomed Res Int. 2021; 2021:5568350. doi: 10.1155/2021/5568350

[37] Mokhtar KM. Improved COVID-19 Testing by Extraction Free SARS-Cov-2 RT-PCR; 2020. Available from: https://medrxiv.org/lookup/doi/10.1101/2020.08.10.20171512Availablefrom:https://medrxiv.org/lookup/doi/10.1101/2020.08.10.20171512 [Last accessed on 2024 May 02].

[38] Domnich A, De Pace V, Pennati BM, et al. Evaluation of extraction-free RT-qPCR methods for SARS-CoV-2 diagnostics. Arch Virol. 2021; 166(10):2825-2828. doi: 10.1007/s00705-021-05165-0

[39] Bastola MM, Locatis C, Fontelo P. Diagnostic Laboratory Tests for COVID-19 in US: Methodology and Performance. Research Square [Preprint]; 2020. doi: 10.21203/rs.3.rs-43374/v1

[40] Hasan MR, Mirza F, Al-Hail H, et al. Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA. PLoS One. 2020; 15(7):e0236564. doi: 10.1371/journal.pone.0236564