Browsing by Author "Alhamid, Galyah Mohammed Abdullah"

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Development of a Colorimetric SARS-CoV-2 Detection Technology Using Reverse-Transcription Loop-mediated Isothermal Amplification (RT-LAMP) Assay
(2022) Alhamid, Galyah Mohammed Abdullah; اوغلو، حسين كومبول
The ongoing COVID-19 remains to spread worldwide, causing new cases and deaths daily since its declaration as a pandemic over two years ago. Diagnostic tests are the first line of defense against the transmission of this disease, with the reverse-transcription quantitative polymerase chain reaction (RT-qPCR) being the approved gold standard. However, due to the global demand for facilities, reagents, and healthcare workers needed for rapid population-based testing, alternative tests need to be invested. The most common limitation in using RT-qPCR is giving false diagnoses, which puts others at risk of infection. Efficient fighting against this pandemic correlates with easy and large-scale accessibility of detection tools. Therefore, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to diagnose COVID-19 by targeting and amplifying the SARS-CoV-2 E gene. The reaction takes place isothermally in a water bath set at 65 °C, and the positive results are read out with the naked eye via a color change from red to yellow, without requiring expensive and bulky instruments. In addition, a fluorometric RT-LAMP assay was developed to validate the results. Both colorimetric and fluorometric assays performed superbly when testing 150 clinical specimens, yielding 89.5% and 100% sensitivities, respectively. Optimizing the originally designed primer set (E-ID1) successfully detected SARS-CoV-2 in 20 minutes with a limit of detection of 500 copies/reaction, corresponding to 20 copies/µL, with no cross-reactivity observed among other respiratory viruses. No misamplification was evident after prolonged reaction times, which eliminates the most common limitation of potential false-positive diagnoses. Furthermore, we used a color sensor to demonstrate that the RGB codes gradually increase with the color change in positive RT-LAMP reactions, providing a simple alternative monitoring technique. Lyophilization of the colorimetric RT-LAMP reagents showed promising results to develop in-home and easy-to-store testing kit. These findings are essential to support the use of this technique in healthcare systems in fighting the COVID-19 pandemic, especially in resource-limited areas.
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