One-tube SARS-CoV-2 detection platform based on RT-RPA and CRISPR/Cas12a

Abstract Background COVID-19 has spread rapidly around the world, affecting a large percentage of population. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection SARS-CoV-2 as essential component. Methods We tried to develop one-tube platform based on RT-RPA (Reverse Transcription Recombinase Polymerase Isothermal Amplification) DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology, termed OR-DETECTR, detect SARS-CoV-2. designed primers RdRp N genes following gene sequence. optimized reaction components so that process could carried out in one tube. Specificity was demonstrated by detecting samples from pseudoviruses seven human coronaviruses Influenza A (H1N1). Clinical were used validate all results compared rRT-PCR. RNA standards diluted different gradients demonstrate limit. Additionally, we have developed lateral flow assay OR-DETECTR COVID-19. Results The can completed tube, which takes approximately 50 min. This method specifically (H1N1), low limit 2.5 copies/µl input (RNA standard) 1 copy/µl (pseudovirus). six patients, eight patients fever but no infection, mixed sample 40 negative controls showed is 100% consistent COVID-19, input. Conclusions rapid, accurate, tube closed, easy-to-operate, free instruments.