Homebrew reagents for low-cost RT-LAMP

• Published in: Journal of biomolecular techniques Vol. 32; no. 3; pp. 114 - 120
• Main Authors: Matute, Tamara, Nuñez, Isaac, Rivera, Maira, Reyes, Javiera, Blázquez-Sánchez, Paula, Arce, Aníbal, Brown, Alexander J., Gandini, Chiara, Molloy, Jennifer, Ramírez-Sarmiento, César A., Federici, Fernán
• Format: Journal Article
• Language: English
• Published: United States Association of Biomolecular Resources Facilities 01.09.2021
• Subjects: Animals; COVID-19; Humans; Indicators and Reagents; Mice; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; RNA, Viral - genetics; SARS-CoV-2; Sensitivity and Specificity; SARS-CoV-2; diagnostics open source
• ISSN: 1524-0215; 1943-4731; 1943-4731
• DOI: 10.7171/jbt.21-3203-006

Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) has gained popularity for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The high specificity, sensitivity, simple protocols, and potential to deliver results without the use of expensive equipment has made it an attractive alternative to RT-PCR. However, the high cost per reaction, the centralized manufacturing of required reagents, and their distribution under cold chain shipping limit RT-LAMP's applicability in low-income settings. The preparation of assays using homebrew enzymes and buffers has emerged worldwide as a response to these limitations and potential shortages. Here, we describe the production of Moloney murine leukemia virus reverse transcriptase and BstLF DNA polymerase for the local implementation of RT-LAMP reactions at low cost. These reagents compared favorably to commercial kits, and optimum concentrations were defined in order to reduce time to threshold, increase ON/OFF range, and minimize enzyme quantities per reaction. As a validation, we tested the performance of these reagents in the detection of SARS-CoV-2 from RNA extracted from clinical nasopharyngeal samples, obtaining high agreement between RT-LAMP and RT-PCR clinical results. The in-house preparation of these reactions results in an order of magnitude reduction in costs; thus, we provide protocols and DNA to enable the replication of these tests at other locations. These results contribute to the global effort of developing open and low-cost diagnostics that enable technological autonomy and distributed capacities in viral surveillance.