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

• Published in: medRxiv : the preprint server for health sciences
• 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 26.05.2023
• Subjects: Recombinant enzymes; One-Step RT-PCR; SARS-CoV-2; Open Source; Diagnostic system
• DOI: 10.1101/2021.11.29.21267000

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, and the kit showed comparable sensitivity to approved commercial kits. The One-Step RT-qPCR was then tested on clinical samples and demonstrated similar performance to commercial kits in terms of positive and negative calls. 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.