McQ – An open-source multiplexed SARS-CoV-2 quantification platform

Vonesch, Sibylle C.; Bredikhin, Danila; Dobrev, Nikolay; Villacorta, Laura; Kleinendorst, Rozemarijn; Cacace, Elisabetta; Flock, Julia; Frank, M; Jung, Ferris; Kornienko, Julia; Mitosch, Karin; Osuna-López, Mireia; Zimmermann, Juergen; Goettig, Stephan; Hamprecht, Axel; Kraeusslich, H.-G.; Knop, Michael; Typas, Athanasios; Steinmetz, Lars M.; Beneš, Vladimı́r; Remans, Kim; Krebs, Arnaud R

doi:10.1101/2020.12.02.20242628

Cited by 4 publications

(5 citation statements)

References 55 publications

(65 reference statements)

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“…We hope that continued development and dissemination of open‐source RT‐qPCR methods will help reduce the dependence of clinical testing centers and research labs on black‐box commercial products. Hopeful signs are recent publications describing other testing methods based on homemade enzymes (Bhadra et al., 2020; Mascuch et al., 2020; Vonesch et al., 2020) and growing online resources for open‐source molecular biology (OpenWetWare; Open Enzyme Collection; Pipette Jockey; see Internet Resources).…”

Section: Commentarymentioning

confidence: 99%

“…The advantages of RT‐qPCR for clinical detection of viruses were realized early on (Beuret, 2004; Bustin & Mueller, 2005; Ishiguro et al., 1995; Monpoeho et al., 2002; Ozoemena, Minor, & Afzal, 2004), and the usefulness of this approach was demonstrated, in particular, during the first outbreak of SARS coronavirus (Hui et al., 2004; Jiang et al., 2004; Lau et al., 2003; Ng et al., 2003; Peiris et al., 2003; Poon et al., 2003; Poon et al., 2004). While other methods for rapid viral RNA detection have recently shown promise (Arizti‐Sanz et al., 2020; Bloom et al., 2020; Dao Thi et al., 2020; Vonesch et al., 2020; Zhang et al., 2020), RT‐qPCR has remained the state of the art for clinical diagnostics and has been the primary workhorse for SARS‐CoV‐2 testing.…”

Section: Commentarymentioning

confidence: 99%

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Simple, Inexpensive RNA Isolation and One‐Step RT‐qPCR Methods for SARS‐CoV‐2 Detection and General Use

et al. 2021

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The most common method for RNA detection involves reverse transcription followed by quantitative polymerase chain reaction (RT‐qPCR) analysis. Commercial one‐step master mixes—which include both a reverse transcriptase and a thermostable polymerase and thus allow performing both the RT and qPCR steps consecutively in a sealed well—are key reagents for SARS‐CoV‐2 diagnostic testing; yet, these are typically expensive and have been affected by supply shortages in periods of high demand. As an alternative, we describe here how to express and purify Taq polymerase and M‐MLV reverse transcriptase and assemble a homemade one‐step RT‐qPCR master mix. This mix can be easily assembled from scratch in any laboratory equipped for protein purification. We also describe two simple alternative methods to prepare clinical swab samples for SARS‐CoV‐2 RNA detection by RT‐qPCR: heat‐inactivation for direct addition, and concentration of RNA by isopropanol precipitation. Finally, we describe how to perform RT‐qPCR using the homemade master mix, how to prepare in vitro−transcribed RNA standards, and how to use a fluorescence imager for endpoint detection of RT‐PCR amplification in the absence of a qPCR machine In addition to being useful for diagnostics, these versatile protocols may be adapted for nucleic acid quantification in basic research. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of a one‐step RT‐qPCR master mix using homemade enzymes Basic Protocol 2: Preparation of swab samples for direct RT‐PCR Alternate Protocol 1: Concentration of RNA from swab samples by isopropanol precipitation Basic Protocol 3: One‐step RT‐qPCR of RNA samples using a real‐time thermocycler Support Protocol: Preparation of RNA concentration standards by in vitro transcription Alternate Protocol 2: One‐step RT‐PCR using endpoint fluorescence detection

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Section: Commentarymentioning

confidence: 99%

Section: Commentarymentioning

confidence: 99%

Simple, Inexpensive RNA Isolation and One‐Step RT‐qPCR Methods for SARS‐CoV‐2 Detection and General Use

et al. 2021

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“…In response to these limitations, many groups have described the successful implementation of homemade RT-PCR and RT-LAMP protocols based on homebrewed enzymes. 15,[20][21][22][23][24][25][26][27][28] Here, we describe the in-house production of RT-LAMP reactions for SARS-CoV-2 from homebrewed Moloney murine leukemia virus (M-MLV) reverse transcriptase and BstLF polymerase. We compare their performance to commercial reactions and tested their ability to detect SARS-CoV-2 from RNA extracted from clinical nasopharyngeal samples.…”

Section: Introductionmentioning

confidence: 99%

Homebrew reagents for low-cost RT-LAMP

et al. 2021

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Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) has gained popularity for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The high specificity, sensitivity, simple protocols, and potential to deliver results without the use of expensive equipment has made it an attractive alternative to RT-PCR. However, the high cost per reaction, the centralized manufacturing of required reagents, and their distribution under cold chain shipping limit RT-LAMP's applicability in low-income settings. The preparation of assays using homebrew enzymes and buffers has emerged worldwide as a response to these limitations and potential shortages. Here, we describe the production of Moloney murine leukemia virus reverse transcriptase and BstLF DNA polymerase for the local implementation of RT-LAMP reactions at low cost. These reagents compared favorably to commercial kits, and optimum concentrations were defined in order to reduce time to threshold, increase ON/OFF range, and minimize enzyme quantities per reaction. As a validation, we tested the performance of these reagents in the detection of SARS-CoV-2 from RNA extracted from clinical nasopharyngeal samples, obtaining high agreement between RT-LAMP and RT-PCR clinical results. The in-house preparation of these reactions results in an order of magnitude reduction in costs; thus, we provide protocols and DNA to enable the replication of these tests at other locations. These results contribute to the global effort of developing open and low-cost diagnostics that enable technological autonomy and distributed capacities in viral surveillance.

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“…Other studies provide procedures to prepare reagents and a ‘blueprint’ that can be followed to replicate RT-qPCR kits in academic laboratories to overcome test shortages[ 19 , 20 ]. In addition, a barcoding system has also been proposed to reduce manipulation and scale the number of samples per sequencing run[ 21 ]. Using this open-source multiplexed platform together with homemade enzymes and non-protected buffers considerably reduces the cost per sample.…”

Section: Introductionmentioning

confidence: 99%

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

Cerda

Rivera

Armijo

et al. 2021

Preprint

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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.

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McQ – An open-source multiplexed SARS-CoV-2 quantification platform

Cited by 4 publications

References 55 publications

Simple, Inexpensive RNA Isolation and One‐Step RT‐qPCR Methods for SARS‐CoV‐2 Detection and General Use

Simple, Inexpensive RNA Isolation and One‐Step RT‐qPCR Methods for SARS‐CoV‐2 Detection and General Use

Homebrew reagents for low-cost RT-LAMP

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

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