Phenol-chloroform-based RNA purification for detection of SARS-CoV-2 by RT-qPCR: Comparison with automated systems

Dimke, Henrik; Larsen, Sanne Løkkegaard; Skov, Marianne Nielsine; Larsen, Hanne; Hartmeyer, Gitte Nyvang; Møeller, Jesper Bonnet

doi:10.1371/journal.pone.0247524

Cited by 9 publications

(3 citation statements)

References 11 publications

(13 reference statements)

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“…In recent years, commercially available RNA extraction kits have largely replaced conventional RNA extraction approaches [7]. However, commercial systems and kits are costly, and they are not widely available in many countries [1,9]. Many resource-constrained institutions and laboratories lack enough or no research funding, making the use of commercial kits challenging.…”

Section: Discussionmentioning

confidence: 99%

“…Te use of commercial kits necessitates fewer blood samples and takes less time than existing traditional techniques [7]. However, commercial systems and kits are costly and are not readily available in many countries [1,9]. Many resource-constrained institutions and laboratories lack enough or no research funding, making the use of commercial kits challenging.…”

Section: Introductionmentioning

confidence: 99%

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Comparison of Modified Manual Acid-Phenol Chloroform Method and Commercial RNA Extraction Kits for Resource Limited Laboratories

Sakyi,

Effah,

Naturinda

et al. 2023

International Journal of Clinical Practice

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Background and Aim. RNA extraction is a commonly used technique in molecular biology. In recent years, commercially available RNA extraction kits have largely replaced conventional approaches. However, these commercial kits are expensive and are not readily available in many resource-constrained institutions and laboratories. This study therefore compared the performance of the conventional acid guanidinium thiocyanate-phenol-chloroform (AGPC) extraction method to QIAamp® Viral RNA Mini Kit (QIAGEN, Cat. No. 52906) and OxGEn RNA Kit (OxGEn Molecular Solutions, GE-009) to build an in-house RNA extraction technique from blood and oral swab samples. Method. In a comparative experimental cross-sectional study, RNA was extracted from oral swabs and blood samples from 25 healthy individuals at the Department of Molecular Medicine, KNUST. RNA was extracted by the manual AGPC extraction method and commercial RNA extraction kits. The quantity (ng/μl) and purities (260/280 nm) of the extracted RNA were measured spectrophotometrically using the IMPLEN NanoPhotometer® N60. The presence of RNA in the extracts was confirmed using 2% agarose gel electrophoresis. Statistical analyses were conducted using R language. Results. The yield of RNA extracted from blood and oral swab samples using modified AGPC was significantly higher compared to the commercial methods ( p < 0.0001). However, the purity of RNA extracted by the manual AGPC method from blood was significantly lower than the commercial methods ( p < 0.0001). Moreover, the purity from oral swabs using the manual AGPC method was significantly lower compared to QIAamp ( p < 0.0001) and the OxGEn kits method ( p < 0.001). Conclusion. The modified manual AGPC method has a very high yield of RNA extracts using blood samples, which could serve as an alternate cost-effective method for RNA extraction in resource-limited laboratories; however, its purity may not be suitable for downstream processes. Moreover, the manual AGPC method may not be suitable for extracting RNA from oral swab samples. Future investigation is needed to improve the purity of the manual AGPC RNA extraction method and also confirmation of the obtained results by PCR amplification and RNA purity verification by sequencing.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Comparison of Modified Manual Acid-Phenol Chloroform Method and Commercial RNA Extraction Kits for Resource Limited Laboratories

Sakyi,

Effah,

Naturinda

et al. 2023

International Journal of Clinical Practice

View full text Add to dashboard Cite

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“…These approaches result in the concentration of viral RNA ( Graham et al., 2021 ). The commercial solutions for performing full viral RNA extraction include sophisticated and automated instruments ( Dimke et al., 2021 ; Genoud et al., 2021 ), or more simple, labor-intensive and ready-to-use extraction kits ( Wozniak et al., 2020 ; Ambrosi et al., 2021 ; Dimke et al., 2021 ) based on extractions with organic solvents, as well as solid-phase purification ( Klein et al., 2020 ; Graham et al., 2021 ) ( Table 2 ). The assessment of these instruments and kits already proved in numerous studies that a full extraction process plays a significant role in maximizing the recovery of SARS-CoV-2 RNA, increasing the sensitivity of the diagnostic process ( Israeli et al., 2020 ; Lübke et al., 2020 ).…”

Section: Sample Processingmentioning

confidence: 99%

SARS-CoV-2 Diagnostics Based on Nucleic Acids Amplification: From Fundamental Concepts to Applications and Beyond

Vindeirinho

Pinho

Azevedo

et al. 2022

Front. Cell. Infect. Microbiol.

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COVID-19 pandemic ignited the development of countless molecular methods for the diagnosis of SARS-CoV-2 based either on nucleic acid, or protein analysis, with the first establishing as the most used for routine diagnosis. The methods trusted for day to day analysis of nucleic acids rely on amplification, in order to enable specific SARS-CoV-2 RNA detection. This review aims to compile the state-of-the-art in the field of nucleic acid amplification tests (NAATs) used for SARS-CoV-2 detection, either at the clinic level, or at the Point-Of-Care (POC), thus focusing on isothermal and non-isothermal amplification-based diagnostics, while looking carefully at the concerning virology aspects, steps and instruments a test can involve. Following a theme contextualization in introduction, topics about fundamental knowledge on underlying virology aspects, collection and processing of clinical samples pave the way for a detailed assessment of the amplification and detection technologies. In order to address such themes, nucleic acid amplification methods, the different types of molecular reactions used for DNA detection, as well as the instruments requested for executing such routes of analysis are discussed in the subsequent sections. The benchmark of paradigmatic commercial tests further contributes toward discussion, building on technical aspects addressed in the previous sections and other additional information supplied in that part. The last lines are reserved for looking ahead to the future of NAATs and its importance in tackling this pandemic and other identical upcoming challenges.

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Laboratory-based molecular test alternatives to RT-PCR for the diagnosis of SARS-CoV-2 infection

Arevalo-Rodriguez,

Mateos-Haro,

Dinnes

et al. 2024

Cochrane Database of Systematic Reviews

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Phenol-chloroform-based RNA purification for detection of SARS-CoV-2 by RT-qPCR: Comparison with automated systems

Cited by 9 publications

References 11 publications

Comparison of Modified Manual Acid-Phenol Chloroform Method and Commercial RNA Extraction Kits for Resource Limited Laboratories

Comparison of Modified Manual Acid-Phenol Chloroform Method and Commercial RNA Extraction Kits for Resource Limited Laboratories

SARS-CoV-2 Diagnostics Based on Nucleic Acids Amplification: From Fundamental Concepts to Applications and Beyond

Laboratory-based molecular test alternatives to RT-PCR for the diagnosis of SARS-CoV-2 infection

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