Development of a colorimetric RT-LAMP-based microfluidic device for COVID-19 diagnosis

Background: Diagnosing COVID-19, caused by the coronavirus SARS-CoV-2, mainly involves detecting specific viral gene sequences. To effectively control highly contagious diseases such as COVID-19, it is essential to develop quick, accurate, sensitive, and easy-to-use diagnostic methods for medical settings. The current gold standard, reverse transcription quantitative polymerase chain reaction (RT-qPCR), requires specialized equipment and trained personnel and is typically conducted in dedicated laboratories. Loop-mediated isothermal amplification (LAMP) provides a rapid and simple alternative under isothermal conditions. In this study, we developed a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection and integrated it into a microfluidic device. Methods: RT-LAMP primers targeting conserved regions of the SARS-CoV-2 envelope (E) and RNA-dependent RNA polymerase (RdRp) genes were designed. Hydroxy naphthol blue (HNB) was used as a color indicator for visual detection. The microfluidic device, fabricated from polydimethylsiloxane (PDMS), contained five reaction chambers. RNA samples extracted from nasopharyngeal swabs of COVID-19 patients were tested using the RT-LAMP microfluidic device. Diagnostic performance of the device was assessed using RT-qPCR as the reference standard. Results: Among 86 RNA samples, 66 were RT-qPCR positive and 20 were negative. The RT-LAMP microfluidic device achieved 100% specificity (95% CI: 83.2–100%) and an overall sensitivity of 75.8% (95% CI: 63.6–85.5%) for samples with Ct ≤ 35. Sensitivity correlated with viral load, showing 100% for samples with Ct ≤ 25, 88.6% for Ct ≤ 30, and 50% for 30 < Ct ≤ 35. Conclusion: The colorimetric RT-LAMP-based microfluidic device enables simple, rapid, and reliable detection of SARS-CoV-2 RNA with high specificity and clinically relevant sensitivity. The device enables visual detection of results and requires only simple equipment, making it suitable as a practical molecular diagnostic tool for point-of-care and resource-limited settings. In terms of diagnostic performance, our findings, when considered alongside previously published data, indicate that the sensitivity and specificity of the device are comparable to or exceed those of lateral flow tests commonly used for point-of-care COVID-19 diagnostics.