Dataset Information

Ultrafast RNA extraction-free SARS-CoV-2 detection by direct RT-PCR using a rapid thermal cycling approach.

ABSTRACT: The COVID19 pandemic has underlined the need for quick and high-throughput SARS-CoV-2 detection assays. Here we report the development of a direct RT-PCR detection method that can reliably detect SARS-CoV-2 gRNA in nasopharyngeal swab samples in under 27 minutes without needing nucleic acid extraction. Fluorescence readouts were highly linear, robust, and sensitive with a LoD95% of determined at 1.46 copies/μL as determined by RT-PCR on a surrogate sample panel containing clinical samples with varying SARS-CoV-2 viral load. We benchmarked our direct RT-PCR method against a reference qPCR method in 368 nasopharyngeal swab samples, confirming a sensitivity score of 99.4% and a specificity score of 98.5% as compared to the reference method. In summary, we here describe a novel rapid direct RT-PCR method to detect SARS-CoV-2 gRNA in clinical specimens, which can be completed in significantly less time compared to conventional PCR methods making it ideal for large-scale screening applications.

SUBMITTER: Struijk R

PROVIDER: S-EPMC10154057 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Ultrafast RNA extraction-free SARS-CoV-2 detection by direct RT-PCR using a rapid thermal cycling approach.

Struijk Robin R van den Ouden Anton A Louwerse Jeroen J Čurová Katarína K Burggrave Ronald R McNally Brian B de Groot Theun T Mulder Bert B de Vos Gert G

Diagnostic microbiology and infectious disease 20230502 1

The COVID19 pandemic has underlined the need for quick and high-throughput SARS-CoV-2 detection assays. Here we report the development of a direct RT-PCR detection method that can reliably detect SARS-CoV-2 gRNA in nasopharyngeal swab samples in under 27 minutes without needing nucleic acid extraction. Fluorescence readouts were highly linear, robust, and sensitive with a LoD95% of determined at 1.46 copies/μL as determined by RT-PCR on a surrogate sample panel containing clinical samples with v ...[more]

PMID: 37343400

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Project description:Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. However, it generally requires expensive equipment such as RNA isolation instruments and real-time PCR thermal cyclers. As a pandemic, COVID-19 has spread indiscriminately, and many low resource settings and developing countries do not have the means for fast and accurate COVID-19 detection to control the outbreak. Additionally, long assay times, in part caused by slow sample preparation steps, have created a large backlog when testing patient samples suspected of COVID-19. With many PCR-based molecular assays including an extraction step, this can take a significant amount of time and labor, especially if the extraction is performed manually. Using COVID-19 clinical specimens, we have collected evidence that the RT-qPCR assay can feasibly be performed directly on patient sample material in virus transport medium (VTM) without an RNA extraction step, while still producing sensitive test results. If RNA extraction steps can be omitted without significantly affecting clinical sensitivity, the turn-around time of COVID-19 tests, and the backlog we currently experience can be reduced drastically. Furthermore, our data suggest that rapid RT-PCR can be implemented for sensitive and specific molecular diagnosis of COVID-19 in locations where sophisticated laboratory instruments are not available. Our USD 300 set up achieved rapid RT-PCR using thin-walled PCR tubes and a water bath setup using sous vide immersion heaters, a Raspberry Pi computer, and a single servo motor that can process up to 96 samples at a time. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 min using untreated samples, heat-inactivated samples, or extracted RNA templates with our low-cost water bath setup. These findings can help rapid COVID-19 testing to become more accessible and attainable across the globe.

Dataset Information

Ultrafast RNA extraction-free SARS-CoV-2 detection by direct RT-PCR using a rapid thermal cycling approach.

Publications

Ultrafast RNA extraction-free SARS-CoV-2 detection by direct RT-PCR using a rapid thermal cycling approach.

Similar Datasets

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