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.
ABSTRACT
Background and Objectives: The coronavirus disease (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to be a pandemic even in 2022. As the initial symptoms of COVID-19 overlap with those of infections from other respiratory viruses, an accurate and rapid diagnosis of COVID-19 is essential for administering appropriate treatment to patients. Currently, the most widely used method for detecting respiratory viruses is based on real-time polymerase chain reaction (PCR) and includes reverse-transcription real-time quantitative PCR (RT-qPCR). However, RT-qPCR assays require sophisticated facilities and are time-consuming. This study aimed to develop a real-time quantitative loop-mediated isothermal amplification (RT-qLAMP) assay and compare its analytical performance with RT-qPCR. Materials and Methods: A total of 315 nasopharyngeal swabs from patients with symptoms of respiratory infections were included in this study. A primary screening of the specimens was performed using RT-qPCR. RNA/DNA from standard strains for respiratory viruses and heat-inactivated preparations of standard strains for SARS-CoV-2 were used to evaluate the accuracy and target specificity of the RT-qLAMP assay. Results: We successfully developed an RT-qLAMP assay for seven respiratory viruses: respiratory syncytial virus (RSV) A, RSV B, adenovirus, influenza (Flu) A (H1N1 and H3N2), Flu B, and SARS-CoV-2. RT-qLAMP was performed in a final reaction volume of 9.6 µL. No cross-reactivity was observed. Compared with the RT-PCR results, the sensitivity and specificity of the RT-qLAMP assay were 95.1% and 100%, respectively. The agreement between the two methods was 97.1%. The median amplification time to RT-qLAMP positivity was 22:34 min (range: 6:80-47:98 min). Conclusions: The RT-qLAMP assay requires a small number of reagents and samples and is performed with an isothermal reaction. This study established a fast, simple, and sensitive test that can be applied to point-of-care testing devices to facilitate the detection of respiratory viruses, including SARS-CoV-2.