Unlocking SARS-CoV-2 detection in low- and middle-income countries

Abstract

Low- and middle-income countries (LMICs) are significantly affected by SARS-CoV-2, partially due to their limited capacity for local production and implementation of molecular testing. Here, we provide detailed methods and validation of a molecular toolkit that can be readily produced and deployed using laboratory equipment available in LMICs. Our results show that lab-scale production of enzymes and nucleic acids can supply over 50,000 tests per production batch. The optimized one-step RT-PCR coupled to CRISPR-Cas12a-mediated detection showed a limit of detection of 10 2 ge/μL in a turnaround time of 2 h. The clinical validation indicated an overall sensitivity of 80%–88%, while for middle and high viral load samples (Cq ≤ 31) the sensitivity was 92%–100%. The specificity was 96%–100% regardless of viral load. Furthermore, we show that the toolkit can be used with the mobile laboratory Bento Lab, potentially enabling LMICs to implement detection services in unattended remote regions.

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 fluorescent detection assay using the target-specific RT-PCR products as input DNA ( Figures 9 C and 9D). For methodology details for SARS-CoV-2 detection, please refer to ( Alcántara et al., 2021a ). For buffer composition and a recipe for the CRISPR/Cas reaction see the Data S1 .…”

“…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 .…”

“…The enzymes produced with this protocol have been successfully used for molecular detection applications. For complete details on the use and execution of this protocol, please refer to Alcántara et al. (2021a , 2021b) .…”

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