An Open One-Step RT-qPCR for SARS-CoV-2 detection
Ariel Cerda, Maira Rivera, Grace Armijo, Catalina Ibarra-Henríquez, Javiera Reyes, Paula Blázquez‐Sánchez, Javiera Avilés, Aníbal Arce, Aldo Seguel, Alexander J. Brown, Yesseny Vásquez, Marcelo Cortez‐San Martín, Francisco A. Cubillos, Patricia García, Marcela Ferrés, César A. Ramírez‐Sarmiento, Fernán Federici, Rodrigo A. Gutiérrez
Abstract
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.