AccScience Publishing / MI / Online First / DOI: 10.36922/MI025410114
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ORIGINAL RESEARCH ARTICLE

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

Getnet Hailu1,2* ,  Molalegne Bitew3* ,  Girmay Medhin1 ,  Adamu Tayachew1,2 ,  Nebiyou Yemanebirhan1,2 ,  Andargachew Gashu1,2 ,  Samuel Ayele4 ,  Admikew Agune2 ,  Assefa Konda2 ,  Mengistu Legesse1
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1 Department of Tropical and Infectious Diseases, Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis Ababa, Ethiopia
2 Department of Infectious Diseases, Ethiopian Public Health Institute, Addis Ababa, Ethiopia
3 Department of Health Biotechnology, Bio and Emerging Technology Institute, Addis Ababa, Ethiopia
4 USAID Eliminate TB Project, Addis Ababa, Ethiopia
MI, 025410114 https://doi.org/10.36922/MI025410114
Received: 9 October 2025 | Revised: 21 November 2025 | Accepted: 25 November 2025 | Published online: 10 December 2025
© 2025 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
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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
Funding
None.
Conflict of interest
The authors declare that they have no competing interests.
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Microbes & Immunity, Electronic ISSN: 3029-2883 Print ISSN: 3041-0886, Published by AccScience Publishing
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