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

ABSTRACT: The 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.

SUBMITTER: Cerda A

PROVIDER: S-EPMC8669853 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Since December 2019, SARS-CoV-2 has spread extensively throughout the world, with more than 117 million reported cases and 2.6 million deaths (Johns Hopkins coronavirus resource center, https://coronavirus.jhu.edu/map.html). Detecting the virus is the first step in diagnosing the infection, followed by quarantine to prevent transmission. Nasopharyngeal/oropharyngeal swabs (NP/OP) and saliva are two specimen types that are most often analyzed to detect SARS-CoV-2 by molecular tests that detect viral RNA or by antigen/antibody tests that detect viral proteins and/or the host immune response against the virus. Compared to antigen/antibody tests, molecular tests are highly sensitive and specific for detecting the virus. A significant drawback is that specimen collection requirements are specific to each test and cannot be interchanged with another test. Some tests are qualified to be used on NP swabs or saliva, but not both specimen types. Even with NP swabs, a test may be qualified to detect the virus only with swabs collected in viral transport medium (VTM) but not in other media. These restrictive pre-analytic steps are disadvantageous in that a lab would have to develop and validate different tests for SARS-CoV-2 depending on the specimen type and collection media, with added setup cost, infrastructure, and training requirements. To overcome these problems, we developed and validated a cost-effective multiplex reverse-transcription real-time PCR assay that can be used to detect SARS-CoV-2 in different specimen types. The assay is highly sensitive and specific, can be used to detect the virus in saliva as well as NP swabs collected in different media such as VTM, saline, and commercial preservative fluid, and serves as one test for all applications. The protocol also describes an optimal laboratory setup and unidirectional workflow for detecting SARS-CoV-2 by RT-qPCR. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Manual viral nucleic acid extraction from NP/OP swabs collected in different media, and from saliva Alternate Protocol 1: Low-throughput automated extraction on the Qiagen EZ1 Advanced XL machine (1-14 samples) Alternate Protocol 2: High-throughput automated extraction on the Kingfisher Flex machine (1-96 samples) Basic Protocol 2: Multiplex RT-qPCR protocol to detect SARS-CoV-2 Alternate Protocol 3: Multiplex one-step RT-qPCR protocol to detect SARS-CoV-2 with S and E gene probes labeled with the same fluorochrome.

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

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