UnCovid: A versatile, low-cost, and open-source protocol for SARS-CoV-2 RNA detection

Abstract

Here, we describe a detailed step-by-step protocol to detect SARS-CoV-2 RNA using RT-PCR-mediated amplification and Crispr/Cas-based visualization. The optimized assay uses basic molecular biology equipment such as conventional thermocyclers and transilluminators for qualitative detection. Alternatively, a fluorescence plate reader can be used for quantitative measurements. The protocol detects two regions of the SARS-CoV-2 genome in addition to the human RNaseP sample control. Aiming to reach remote regions, this work was developed to use the portable molecular workstation from BentoLab.

A low-cost and open-source protocol to produce key enzymes for molecular detection assays

“…The present work describes adapted protocols for the production of LbCas12a (henceforth Cas12a) ( Chen et al., 2018 ; Zetsche et al., 2015 ), Taq DNA polymerase (Taq) ( Chien et al., 1976 ; Graham et al., 2021 ; Lawyer et al., 1989 ), M-MLV reverse transcriptase (RT) ( Graham et al., 2021 ; Kotewicz et al., 1985 , 1988 ), and TEV protease (Tev)( Kapust et al., 2001 ; Reischl et al., 1997 ). These enzymes were intended to be used for molecular detection of SARS-CoV-2 viral RNA by an RT-PCR coupled to CRISPR/Cas detection ( Alcántara et al., 2021a , 2021b ). However, they can be used in a variety of molecular biology applications, such as nucleic acid amplification, DNA target detection, and in vitro DNA digestion.…”

“…Here, we describe a one-step RT-PCR assay. For protocol details, please refer to ( Alcántara et al., 2021b ). For buffer composition and a recipe for enzyme dilution and RT-PCR reaction see the Data S1 .…”

“…B6002S, NEB) can be used as an alternative for reaction buffer. Local production of the crRNA used here can be found in ( Alcántara et al., 2021b ). Perform the crRNA re-folding by incubation at 65°C for 10 min followed by cooling at 22°C–25°C for 10 min.…”

“…For protein quantification, we used a Nanodrop One spectrophotometer (Thermofisher) and a SynergyTM H1 Hybrid Multi-Mode plate reader (Biotek) for Bradford analysis. The latter was also used for corroborating Cas12a enzymatic activity in the fluorescence detection assay optimized and reported on ( Alcántara et al., 2021a , 2021b ). Details on bacteria strains used are described within the adequate step and in the key resources table .…”

“…For complete details on the use and execution of this protocol, please refer to Alcántara et al. (2021a , 2021b) .…”