Journal Article

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

Blázquez-Sánchez, Paula, García, Patricia, Avilés, Javiera, Seguel, Aldo, Brown, Alexander J., Ferres, Marcela, Federici, Fernán, Rivera, Maira, Gutiérrez, Rodrigo A., Ibarra-Henriquez, Catalina, Arce, Aníbal, Cortez-San Martín, Marcelo, Cerda, Ariel, Armijo, Grace, Cubillos, Francisco A., Reyes, Javiera, Vásquez, Yesseny, Ramírez-Sarmiento, César A.

2024

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Analysis / Biology and life sciences / Clinical Laboratory Techniques - methods / Collaboration / Confidentiality / COVID-19 / COVID-19 - diagnosis / COVID-19 Testing / Developed countries / Diagnostic systems / Disease transmission / DNA / DNA polymerase / Enzymes / Evaluation / Humans / 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 / RNA, Viral - analysis / RNA, Viral - genetics / SARS-CoV-2 - genetics / Sensitivity and Specificity / Severe acute respiratory syndrome coronavirus 2 / Supplies / Viral diseases