RESUMEN
Here, we describe a detailed step-by-step protocol for the expression, purification, quantification, and activity determination of key enzymes for molecular detection of pathogens. Based on previous reports, we optimized the protocol for LbCas12a, Taq DNA polymerase, M-MLV reverse transcriptase, and TEV protease to make it compatible with minimal laboratory equipment, broadly available in low- and middle-income countries. 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).
Asunto(s)
Enzimas , Escherichia coli , Proteínas Recombinantes , Cromatografía de Afinidad , Pruebas de Enzimas , Enzimas/genética , Enzimas/aislamiento & purificación , Enzimas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Tipificación Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Transformación BacterianaRESUMEN
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 102 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.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , Países en Desarrollo , ARN Viral/genética , Sensibilidad y Especificidad , Técnicas de Amplificación de Ácido NucleicoRESUMEN
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. For complete details on the use and execution of this protocol, please refer to Alcántara et al., 2021.