Dataset Information

Homebrew: An economical and sensitive glassmilk-based nucleic-acid extraction method for SARS-CoV-2 diagnostics.

ABSTRACT: Management of COVID-19 and other epidemics requires large-scale diagnostic testing. The gold standard for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains reverse transcription quantitative PCR (qRT-PCR) analysis, which detects viral RNA more sensitively than any other method. However, the resource use and supply-chain requirements of RT-PCR have continued to challenge diagnostic laboratories worldwide. Here, we establish and characterize a low-cost method to detect SARS-CoV-2 in clinical combined nose and throat swabs, allowing for automation in high-throughput settings. This method inactivates virus material with sodium dodecylsulfate (SDS) and uses silicon dioxide as the RNA-binding matrix in combination with sodium chloride (NaCl) and isopropanol. With similar sensitivity for SARS-CoV-2 viral targets but a fraction of time and reagent expenditure compared with commercial kits, our method also enables sample pooling without loss of sensitivity. We suggest that this method will facilitate more economical widespread testing, particularly in resource-limited settings.

SUBMITTER: Page R

PROVIDER: S-EPMC8890991 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Homebrew: An economical and sensitive glassmilk-based nucleic-acid extraction method for SARS-CoV-2 diagnostics.

Page Robert R Scourfield Edward E Ficarelli Mattia M McKellar Stuart W SW Lee Kwok Leung KL Maguire Thomas J A TJA Bouton Clement C Lista Maria Jose MJ Neil Stuart J D SJD Malim Michael H MH Zuckerman Mark M Mischo Hannah E HE Martinez-Nunez Rocio T RT

Cell reports methods 20220303 3

Management of COVID-19 and other epidemics requires large-scale diagnostic testing. The gold standard for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains reverse transcription quantitative PCR (qRT-PCR) analysis, which detects viral RNA more sensitively than any other method. However, the resource use and supply-chain requirements of RT-PCR have continued to challenge diagnostic laboratories worldwide. Here, we establish and characterize a low-cost method to detect ...[more]

PMID: 35262039

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Project description:Nucleic acid extraction (NAE) plays a crucial role for diagnostic testing procedures. For decades, dried blood spots (DBS) have been used for serology, drug monitoring, and molecular studies. However, extracting nucleic acids from DBS remains a significant challenge, especially when attempting to implement these applications to the point-of-care (POC). To address this issue, we have developed a paper-based NAE method using cellulose filter papers (DBSFP) that operates without the need for electricity (at room temperature). Our method allows for NAE in less than 7 min, and it involves grade 3 filter paper pre-treated with 8% (v/v) igepal surfactant, 1 min washing step with 1× PBS, and 5 min incubation at room temperature in 1× TE buffer. The performance of the methodology was assessed with loop-mediated isothermal amplification (LAMP), targeting the human reference gene beta-actin and the kelch 13 gene from P. falciparum. The developed method was evaluated against FTA cards and magnetic bead-based purification, using time-to-positive (min) for comparative analysis. Furthermore, we optimised our approach to take advantage of the dual functionality of the paper-based extraction, allowing for elution (eluted disk) as well as direct placement of the disk in the LAMP reaction (in situ disk). This flexibility extends to eukaryotic cells, bacterial cells, and viral particles. We successfully validated the method for RNA/DNA detection and demonstrated its compatibility with whole blood stored in anticoagulants. Additionally, we studied the compatibility of DBSFP with colorimetric and lateral flow detection, showcasing its potential for POC applications. Across various tested matrices, targets, and experimental conditions, our results were comparable to those obtained using gold standard methods, highlighting the versatility of our methodology. In summary, this manuscript presents a cost-effective solution for NAE from DBS, enabling molecular testing in virtually any POC setting. When combined with LAMP, our approach provides sample-to-result detection in under 35 minutes.

Dataset Information

Homebrew: An economical and sensitive glassmilk-based nucleic-acid extraction method for SARS-CoV-2 diagnostics.

Publications

Homebrew: An economical and sensitive glassmilk-based nucleic-acid extraction method for SARS-CoV-2 diagnostics.

Similar Datasets

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