Low-Cost, Real-Time Polymerase Chain Reaction System with Integrated RNA Extraction.

Publication date: May 09, 2023

Rapid, easy-to-use, and low-cost systems for biological sample testing are important for point-of-care diagnostics and various other health applications. The recent pandemic of Coronavirus Disease 2019 (COVID-19) caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) showed an urgent need to rapidly and accurately identify the genetic material of SARS-CoV-2, an enveloped ribonucleic acid (RNA) virus, in upper respiratory specimens from people. In general, sensitive testing methods require genetic material extraction from the specimen. Unfortunately, current commercially available extraction kits are expensive and involve time-consuming and laborious extraction procedures. To overcome the difficulties associated with common extraction methods, we propose a simple enzymatic assay for the nucleic acid extraction step using heat mediation to improve the polymerase chain reaction (PCR) reaction sensitivity. Our protocol was tested on Human Coronavirus 229E (HCoV-229E) as an example, which comes from the large coronaviridae family of viruses that affect birds, amphibians, and mammals, of which SARS-CoV-2 is a member. The proposed assay was performed using a low-cost, custom-made, real-time PCR system that incorporates thermal cycling and fluorescence detection. It had fully customizable reaction settings to allow versatile biological sample testing for various applications, including point-of-care medical diagnosis, food and water quality testing, and emergency health situations. Our results show that heat-mediated RNA extraction is a viable extraction method when compared to commercial extraction kits. Further, our study showed that extraction has a direct impact on purified laboratory samples of HCoV-229E, but no direct impact on infected human cells. This is clinically relevant, as it allows us to circumvent the extraction step on clinical samples when using PCR.

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Concepts Keywords
Amphibians Animals
Basel COVID-19
Coronavirus COVID-19
Expensive COVID-19 Testing
Pcr fluorescence sensing
Nucleic Acids
Nucleic Acids
point-of-care diagnostics
real-time PCR
RNA extraction


Type Source Name
disease VO time
disease MESH Coronavirus Disease 2019
disease VO Severe acute respiratory syndrome coronavirus 2
disease IDO assay
disease VO protocol
disease VO Coronaviridae
disease VO Viruses
disease MESH water quality
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drug DRUGBANK Coenzyme M
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disease VO efficient
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disease VO efficiency
pathway REACTOME Release
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drug DRUGBANK Edetic Acid
drug DRUGBANK Tretamine
disease IDO replication
drug DRUGBANK Cefaclor
disease MESH infection
disease IDO cell
disease VO volume
disease VO Glycoprotein
drug DRUGBANK Uracil
drug DRUGBANK Propylthiouracil
disease VO report
drug DRUGBANK Ranitidine
disease VO device
drug DRUGBANK Albendazole
disease VO effectiveness
drug DRUGBANK Sodium lauryl sulfate
disease VO Bacteria
drug DRUGBANK Ethanol
disease VO organization
drug DRUGBANK Bismuth subgallate
disease IDO quality
disease MESH Salmonellosis
drug DRUGBANK Silicon
disease MESH listeriosis
disease VO Mycobacterium tuberculosis
disease VO Mycobacterium
disease VO population

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