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.

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