Molecular diagnostics for clinical respiratory virus on a total integrated centrifugal microsystem using reverse transcription-loop-mediated isothermal amplification

ABSTRACT

Respiratory viruses are highly contagious agents that can cause endemic and epidemic infections in humans. Early detection of these viruses is crucial in preventing economic damage and reducing mortality rates. In this study, we present a total integrated genetic analyzer to perform a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the simultaneous detection of 7 respiratory viruses (Influenza A H1N1 and H3N2, Influenza B, Respiratory syncytial virus A and B, Adenovirus, and COVID-19). The primer sets for the RT-LAMP assay were designed and evaluated in comparison with the RT-PCR assay using clinical samples, confirming high specificity and efficiency. The entire process of viral RNA extraction, reagent mixing, gene amplification, and detection was completed on the device in 1hr 20min. The constructed portable diagnostic instrument is equipped with a rotary motor, two sets of peltier heaters, a fluorescence detector, and a touch screen for inputting experimental parameters and displaying result. The proposed point-of-care (POC) diagnostic platform correctly analyzed a total of 21 clinical samples (3 for each of the 7 viruses). The limit-of-detection (LOD) for Influenza A subtype H3N2 was 101 pfu/mL, which demonstrates the high performance of our proposed centrifugal microsystem for on-site molecular diagnostics in medical centers.