Simple tests of infectiousness that return results in minutes and directly from samples even with low viral loads could be a potential game-changer in the fight against COVID-19. Here, we describe an improved isothermal nucleic acid amplification assay, termed the RICCA ( R NA I sothermal C o-assisted and C oupled A mplification) reaction, that consists of a simple one-pot format of ‘sample-in and result-out’ with a primary focus on the detection of low copy numbers of RNA virus directly from saliva without the need for laboratory processing. We demonstrate our assay by detecting 16S rRNA directly from E. coli cells with a sensitivity as low as 8 CFU/μL and RNA fragments from a synthetic template of SARS-CoV-2 with a sensitivity as low as 1740 copies/μL. We further demonstrate the applicability of our assay for real-time testing at the point of care by designing a closed format for paper-based lateral flow assay and detecting heat-inactivated SARS-COV-2 virus in human saliva at concentrations ranging from 28,000 to 2.8 copies/μL with a total assay time of 15–30 min.
【저자키워드】 biomedical engineering, nucleic acids, Synthetic biology, 【초록키워드】 COVID-19, SARS-CoV-2, Saliva, point of care, SARS-CoV-2 virus, virus, Laboratory, lateral flow, 16S rRNA, sensitivity, Viral, Viral load, infectiousness, human saliva, RNA virus, nucleic acid amplification, rRNA, Concentration, isothermal, copy numbers, primary focus, applicability, heat, minutes, E. coli cells, simple, heat-inactivated, RNA fragment, E. coli cell, 【제목키워드】 RNA, RNA virus, development, isothermal, Amplification assay, robust,