A One-Step open RT-qPCR for SARS-CoV-2 detection

Abstract

AbstractThe Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), etiological agent of the coronavirus disease 2019 (COVID-19), is currently detected by reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) of its viral RNA genome. Within the available alternatives, One-Step procedures are preferred since they are fast and significantly decrease preanalytical errors, minimizing the risk of diagnostic errors.Increasing the testing capacity and tracing contacts are essential steps to control the pandemic. However, high-cost commercial reagents subject to shortage and poor scalability have hindered the use of these technologies and their adoption for a wide population-scale testing, being even more critical in developing countries. In the current context, open-source initiatives have promoted global collaboration to promote accessible solutions for rapid local deployment. As a result, open protocols are being developed for the local production of SARS-CoV-2 diagnostics.This work aimed to produce an open-source system for SARS-CoV-2 diagnostic tests in RNA clinical samples. We provide guidelines for standardizing an open One-Step RT-qPCR master mix using recombinant M-MLV reverse transcriptase together with either Pfu-Sso7d or Taq DNA polymerase. Both were tested on synthetic RNA and clinical samples, observing a good correlation when compared to commercial RT-qPCR kits. Nevertheless, the best results were obtained using M-MLV RT combined with Taq DNA polymerase in a probe-based RT-qPCR assay, allowing successful discrimination between positive and negative samples with accuracies comparable to a CDC-recommended commercial kit.Here, we demonstrate that these open RT-qPCR systems can be successfully used to identify SARS-CoV-2 in clinical samples and potentially be implemented in any molecular diagnostic laboratory.