Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has caused the outbreak of coronavirus disease 2019 (COVID-19) all over the world. In the absence of appropriate antiviral drugs or vaccines, developing a simple, rapid, and reliable assay for SARS-CoV-2 is necessary for the prevention and control of the COVID-19 transmission. Methods A novel molecular diagnosis technique, named multiplex reverse transcription loop-mediated isothermal amplification, that has been linked to a nanoparticle-based lateral flow biosensor (mRT-LAMP-LFB) was applied to detect SARS-CoV-2 based on the SARS-CoV-2 RdRp and N genes, and the mRT-LAMP products were analyzed using nanoparticle-based lateral flow biosensor. The mRT-LAMP-LFB amplification conditions, including the target RNA concentration, amplification temperature, and time were optimized. The sensitivity and specificity of the mRT-LAMP-LFB method were tested in the current study, and the mRT-LAMP-LFB assay was applied to detect the SARS-CoV-2 virus from clinical samples and artificial sputum samples. Results The SARS-CoV-2 specific primers based on the RdRp and N genes were valid for the establishment of mRT-LAMP-LFB assay to detect the SARS-CoV-2 virus. The multiple-RT-LAMP amplification condition was optimized at 63°C for 30 min. The full process, including reaction preparation, viral RNA extraction, RT-LAMP, and product identification, could be achieved in 80 min. The limit of detection (LoD) of the mRT-LAMP-LFB technology was 20 copies per reaction. The specificity of mRT-LAMP-LFB detection was 100%, and no cross-reactions to other respiratory pathogens were observed. Conclusion The mRT-LAMP-LFB technique developed in the current study is a simple, rapid, and reliable method with great specificity and sensitivity when it comes to identifying SARS-CoV-2 virus for prevention and control of the COVID-19 disease, especially in resource-constrained regions of the world.
【저자키워드】 COVID-19, SARS-CoV-2, limit of detection, lateral flow biosensor, reverse transcription-loop-mediated isothermal amplification, 【초록키워드】 coronavirus disease, severe acute respiratory syndrome coronavirus 2, Coronavirus disease 2019, coronavirus, Vaccines, antiviral drugs, SARS-CoV-2 virus, severe acute respiratory syndrome Coronavirus, antiviral drug, Novel coronavirus, COVID-19 disease, RNA, lateral flow, amplification, clinical samples, RT-LAMP, sensitivity, specificity, Region, Viral, Molecular diagnosis, Sensitivity and specificity, Loop-mediated isothermal amplification, outbreak, nanoparticle, RdRP, Pathogens, temperature, reverse transcription, isothermal amplification, Viral RNA, respiratory, N gene, multiplex, cross-reaction, Prevention and control, SARS-CoV-2 RdRp, Concentration, COVID-19 transmission, acute respiratory syndrome, Reactions, acute respiratory syndrome coronavirus, reaction, respiratory pathogen, primer, N genes, cross-reactions, sputum samples, viral RNA extraction, clinical sample, Result, tested, analyzed, detect, caused, applied, absence, conditions, the SARS-CoV-2, the SARS-CoV-2 virus, 【제목키워드】 detection, biosensor, Rapid, gold, Flow, Visual, Lateral,