Search

Summary 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)., Graphical abstract, Highlights • Open-source and low-cost production schemes of Taq, M- MLV RT, and Cas12a enzymes • Compatible with basic equipment to produce enzymes for molecular detection • Enzyme preparations validated for SARS-CoV-2 detection, 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.

Diarrheagenic E. coli (DEC) is an important cause of diarrhea in children, immunocompromised patients, and travelers. There are six classic pathotypes: enteropathogenic (EPEC), shiga toxin–producing (STEC), enteroaggregative (EAEC), enterotoxigenic (ETEC), enteroinvasive (EIEC), and diffusely adherent E. coli (DAEC). A new pathotype has been recently reported: adherent invasive E. coli (AIEC). The clinical manifestations include watery diarrhea, persistent diarrhea, and invasive inflammatory diarrhea. STEC can be associated with hemolytic uremic syndrome and AIEC with Crohn's disease. The pathogenesis of DEC is heterogeneous. Molecular-based techniques are used for diagnosis of DEC, but they are restricted to research laboratories. Hygiene, breastfeeding and oral rehydration are the most important tools for prevention and management. Vaccines against DEC pathotypes are under development. The use of antibiotics for DEC diarrhea, especially in children, is restricted because of increasing antibiotic resistance and insufficient efficacy data.