Dataset Information

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

ABSTRACT:

Background

Two automatable in-house protocols for high-troughput RNA extraction from nasopharyngeal swabs for SARS-CoV-2 detection have been evaluated.

Methods

One hundred forty one SARS-CoV-2 positive samples were collected during a period of 10-days. In-house protocols were based on extraction with magnetic beads and designed to be used with either the Opentrons OT-2 (OT-2in-house) liquid handling robot or the MagMAXTM Express-96 system (MMin-house). Both protocols were tested in parallel with a commercial kit that uses the MagMAXTM system (MMkit). Nucleic acid extraction efficiencies were calculated from a SARS-CoV-2 DNA positive control.

Results

No significant differences were found between both in-house protocols and the commercial kit in their performance to detect positive samples. The MMkit was the most efficient although the MMin-house presented, in average, lower Cts than the other two. In-house protocols allowed to save between 350€ and 400€ for every 96 extracted samples compared to the commercial kit.

Conclusion

The protocols described harness the use of easily available reagents and an open-source liquid handling system and are suitable for SARS-CoV-2 detection in high throughput facilities.

SUBMITTER: Lazaro-Perona F

PROVIDER: S-EPMC7886139 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Publications

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

Lázaro-Perona Fernando F Rodriguez-Antolín Carlos C Alguacil-Guillén Marina M Gutiérrez-Arroyo Almudena A Mingorance Jesús J García-Rodriguez Julio J

PloS one 20210216 2

<h4>Background</h4>Two automatable in-house protocols for high-troughput RNA extraction from nasopharyngeal swabs for SARS-CoV-2 detection have been evaluated.<h4>Methods</h4>One hundred forty one SARS-CoV-2 positive samples were collected during a period of 10-days. In-house protocols were based on extraction with magnetic beads and designed to be used with either the Opentrons OT-2 (OT-2in-house) liquid handling robot or the MagMAXTM Express-96 system (MMin-house). Both protocols were tested i ...[more]

PMID: 33591986

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Project description:The gold standard for the laboratory diagnosis of COVID-19 is the reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) assay, which searches for SARS-CoV-2 target genes in nasopharyngeal/oropharyngeal (NP/OP) samples, and its performance depends on the quantity and quality of the RNA input. This study compared the performance and cost-effectiveness of three different kits/reagents for RNA extraction used in COVID-19 diagnosis in Sao Paulo, Brazil. A total of 300 NP/OP samples belonging to suspected cases of COVID-19 stored in a biorepository were randomly selected, and RNA was extracted using (i) automated extraction (Loccus, Extracta Kit FAST), (ii) manual extraction (BioGene Kit, Bioclin, Quibasa), and (iii) quick extraction methods (Lucigen, Quick DNA Extract Kit). Next, the samples were tested using RT-qPCR for SARS-CoV-2 with the Allplex 2019-nCoV modified assay and the Charité-Berlin protocol. All assays/kits were used according to the manufacturer's instructions. For the Allplex kit, the sensitivity in detecting SARS-CoV-2 with previously extracted RNA by different procedures was 100.0% for Loccus, 100.0% for BioGene and 91.9% for Quick. Using the Charité-Berlin protocol, the sensitivities were 81.4% for Loccus, 81.2% for BioGene and 60.7% for Quick. The least sensitive target gene and the gene most affected by RNA extraction procedures was the RNA-dependent RNA polymerase gene (Charité-Berlin protocol). No false-positive SARS-CoV-2 results were detected using RNA obtained from any of the different protocols. In conclusion, Loccus and BioGene RNA extractions were efficient for RT-qPCR assays, and although the BioGene procedure is less expensive, Loccus is the best choice because it allows the rapid handling of hundreds or thousands of samples, a desirable feature during pandemics. Although less sensitive, the Quick extraction is useful during outbreaks coupled with the Allplex amplification kit for SARS-CoV-2 diagnosis (κ = 0.925).

Dataset Information

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

Background

Methods

Results

Conclusion

Publications

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

Similar Datasets

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