Evaluation of two automated low-cost RNA extraction protocols for SARS-CoV-2 detection

Lázaro-Perona, Fernando; Rodriguez-Antolín, Carlos; Alguacil-Guillén, Marina; Gutiérrez-Arroyo, Almudena; Mingorance, Jesús; García-Rodríguez, Julio

doi:10.1371/journal.pone.0246302

Cited by 21 publications

(15 citation statements)

References 11 publications

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“…While some reports describe the performance of in-house RNA SARS-CoV-2 extraction protocols, validating their results with the same kit [19]. Others point out its compatibility to detect SARS-CoV-2 in nasopharyngeal samples without prior RNA extraction [20], corroborating its good performance and sensitivity in viral detection. On the other hand, while the BGI kit has shown a sensitivity of ≥ 95% in other studies [6], we obtained only 70.1%, possibly due to the random selection of positive samples without considering high or low viral loads.…”

Section: Discussionmentioning

confidence: 89%

The Comparison of Three Real-Time PCR Kits for Sars-Cov-2 Diagnosis Reveals Discrepancies on the Identification of Positive Covid-19 Cases and Dispersion on the Values Obtained for the Detection of Sars-Cov-2 Variants

Santibáñez

Luraschi

Barrera-Avalos

et al. 2021

Preprint

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The COVID-19 pandemic has generated a huge challenge and threat to public health throughout the world population. Reverse transcription associated with real-time Polymerase Chain Reaction (RT-qPCR) has been the gold-standard molecular tool for diagnosis and detection of the SARS-CoV-2. Currently, it is used as the main strategy for testing, traceability, and control of positive cases For this reason, the on-top high demand for reagents has produced stock-out on several occasions and the only alternative to keep population diagnosis has been the use of different RT-qPCR kits. Therefore, we evaluate the performance of three of the commercial RT-qPCR kits currently in use for SARS-CoV-2 diagnosis in Chile, consisting in: TaqMan 2019-nCoV Assay Kit v1 (Thermo). Real-Time Fluorescent RT-PCR Kit for Detecting SARS-CoV-2 (BGI), and LightCycler Multiplex RNA Virus Master (Roche). Results of quantification cycle (Cq) and relative fluorescence units (RFU) obtained from their RT-qPCR reactions revealed important discrepancies on the total RNA required for the identification of SARS-CoV-2 genes and diagnosis. Marked differences between kits in samples with 30>Cq value< 34 was observed. Samples with positive diagnoses for Covid-19 using the Thermo Fisher kit had different results when the same samples were evaluated with Roche and BGI kits. The displacement on the Cq value for SARS-CoV-2 identification between the three different RT-qPCR kits was also evident when the presence of single nucleotide variants was evaluated in the context of genomic surveillance. Taken together, this study emphasizes the special care adjusting RT-qPCR reaction conditions of the different kits must be taken by all the laboratories before carrying out the detection of SARS-CoV-2 genes from total RNA nasopharyngeal swab (NPS) samples.

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Section: Discussionmentioning

confidence: 89%

The Comparison of Three Real-Time PCR Kits for Sars-Cov-2 Diagnosis Reveals Discrepancies on the Identification of Positive Covid-19 Cases and Dispersion on the Values Obtained for the Detection of Sars-Cov-2 Variants

Santibáñez

Luraschi

Barrera-Avalos

et al. 2021

Preprint

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“…We know that there are other recent molecular approach techniques such as the use of automated systems or the elimination of the extraction stage, which require speci c commercial kits and signi cantly increase the cost of nes 21,22 . Nevertheless, the focus of this study was to expand the diagnostic con rmation alternatives while maintaining the security and quality of the tests.…”

Section: Discussionmentioning

confidence: 99%

Classic RNA extraction is efficient for subsequent detection of SARS-CoV-2 by RT-PCR in nasopharyngeal swab samples

Saldanha

Reis

Azevedo

et al. 2022

Preprint

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Background: Mass testing is a fundamental measure for tracking and controlling the spread of coronavirus disease 2019 (COVID-19) cases. The standard diagnosis for infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) involves the extraction of nucleic acids for the recognition of viral genes present in biological samples collected from the population. This worldwide demand affects the distribution of specific reagents and difficult to expand the number of tests. The aim of the study was to compare the quality of the extracted material using a commercial kit and a phenol-based reagent and the reverse transcription-polymerase chain reaction (RT-PCR) results. We performed these two RNA extraction protocols with 15 samples positive for SARS-CoV-2. Results: Our analysis showed that there was no reduction in the sensitivity of the RT-PCR test or direct impact on the diagnosis of SARS-CoV-2 infection. Conclusions: Both methodologies proved to be useful and show similar results, confirming the possibility of using alternative RNA extraction protocols for the detection of SARS-CoV-2.

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“…In conclusion, this work can help to increase SARS-CoV-2 testing capacity of established laboratories and provide an alternative to proprietary protocols and during acute consumable shortages [15,16]. These open-source solutions might also be a good fit for laboratories that currently do not have the resources for other automated platforms.…”

Section: Plos Onementioning

confidence: 95%

“…OT-2-based circuits are a promising solution to increase SARS-CoV-2 testing capability since it is an affordable open-source platform for liquid handling. The cost of the robots is much lower than other solutions, and the cost of reagents can be significantly reduced by using in-house protocols [15]. However, other associated costs (personnel, labware, etc.)…”

Section: Plos Onementioning

confidence: 99%

“…Therefore, in the design of our circuits where test accuracy and sensitivity were key, RNA extraction was carried out by a KingFisher™ instrument which incorporates an agitator module. However, advances and improvements to the OT-2 platform's capabilities continue, as evidenced by a new in-house extraction protocol which has been recently implemented using OT-2 robots in a hospital [15].…”

Section: Plos Onementioning

confidence: 99%

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Implementation of an open-source robotic platform for SARS-CoV-2 testing by real-time RT-PCR

Villanueva‐Cañas

González‐Roca

Unanue³

et al. 2021

PLoS ONE

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The current global pandemic due to the SARS-CoV-2 has pushed the limits of global health systems across all aspects of clinical care, including laboratory diagnostics. Supply chain disruptions and rapidly-shifting markets have resulted in flash-scarcity of commercial laboratory reagents; this has motivated health care providers to search for alternative workflows to cope with the international increase in demand for SARS-CoV-2 testing. The aim of this study is to present a reproducible workflow for real time RT-PCR SARS-CoV-2 testing using OT-2 open-source liquid-handling robots (Opentrons, NY). We have developed a framework that includes a code template which is helpful for building different stand-alone robotic stations, capable of performing specific protocols. Such stations can be combined together to create a complex multi-stage workflow, from sample setup to real time RT-PCR. Using our open-source code, it is easy to create new stations or workflows from scratch, adapt existing templates to update the experimental protocols, or to fine-tune the code to fit specific needs. Using this framework, we developed the code for two different workflows and evaluated them using external quality assessment (EQA) samples from the European Molecular Genetics Quality Network (EMQN). The affordability of this platform makes automated SARS-CoV-2 PCR testing accessible for most laboratories and hospitals with qualified bioinformatics personnel. This platform also allows for flexibility, as it is not dependent on any specific commercial kit, and thus it can be quickly adapted to protocol changes, reagent, consumable shortages, or any other temporary material constraints.

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Evaluation of two automated low-cost RNA extraction protocols for SARS-CoV-2 detection

Cited by 21 publications

References 11 publications

The Comparison of Three Real-Time PCR Kits for Sars-Cov-2 Diagnosis Reveals Discrepancies on the Identification of Positive Covid-19 Cases and Dispersion on the Values Obtained for the Detection of Sars-Cov-2 Variants

The Comparison of Three Real-Time PCR Kits for Sars-Cov-2 Diagnosis Reveals Discrepancies on the Identification of Positive Covid-19 Cases and Dispersion on the Values Obtained for the Detection of Sars-Cov-2 Variants

Classic RNA extraction is efficient for subsequent detection of SARS-CoV-2 by RT-PCR in nasopharyngeal swab samples

Implementation of an open-source robotic platform for SARS-CoV-2 testing by real-time RT-PCR

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