An Open One-Step RT-qPCR for SARS-CoV-2 detection

• Published in: PloS one Vol. 19; no. 1; p. e0297081
• Main Authors: Cerda, Ariel, Rivera, Maira, Armijo, Grace, Ibarra-Henriquez, Catalina, Reyes, Javiera, Blázquez-Sánchez, Paula, Avilés, Javiera, Arce, Aníbal, Seguel, Aldo, Brown, Alexander J., Vásquez, Yesseny, Cortez-San Martín, Marcelo, Cubillos, Francisco A., García, Patricia, Ferres, Marcela, Ramírez-Sarmiento, César A., Federici, Fernán, Gutiérrez, Rodrigo A.
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 25.01.2024; Public Library of Science (PLoS)
• Subjects: Analysis; Biology and life sciences; Collaboration; Confidentiality; COVID-19; Developed countries; Diagnostic systems; Disease transmission; DNA; DNA polymerase; Enzymes; Evaluation; Intellectual property; Laboratories; Medicine and health sciences; Pandemics; Pathogens; Personal protective equipment; Polymerase chain reaction; Reagents; Real time; Research and Analysis Methods; Reverse transcription; Ribonucleic acid; RNA; RNA probes; Severe acute respiratory syndrome coronavirus 2; Supplies; Viral diseases; Chile
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0297081

The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, demonstrating sensitivity comparable to a commercial kit routinely employed in clinical settings for patient diagnosis. Further evaluation on 40 clinical samples (20 positive and 20 negative) confirmed its comparable diagnostic accuracy. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.