Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR

Smyrlaki, Ioanna; Ekman, Martin; Lentini, Antonio; Sousa, Nuno Rufino de; Papanicolaou, Natali; Vondracek, Martin; Aarum, Johan; Safari, H; Muradrasoli, Shaman; Rothfuchs, Antonio Gigliotti; Albert, Jan; Högberg, Björn; Reinius, Björn

doi:10.1038/s41467-020-18611-5

Cited by 391 publications

(455 citation statements)

References 14 publications

(15 reference statements)

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“…This dilution seems to provide the optimum balance between input RNA and reducing the concentrations of PCR inhibitors. A similar observation was found by Smyrlaki et al [ 12 ] where they reported that dilution of the clinical samples is essential to dilute the inhibitors that might be present in the samples. Heating the diluted specimen to 95 °C followed by quenching at 4 °C for 5 min was used to deactivate possible PCR inhibitors that might be present in the samples and to denature viral proteins.…”

Section: Discussionsupporting

confidence: 82%

“…While the 5 min denaturation provided a comparable LoD to the standard protocol, the 10-min denaturation resulted in much higher Ct values, possibly due to viral RNA degradation. Heating at 95 °C was reported by Smyrlski et al [ 12 ] to help inactivate the nucleases and inhibitors in the clinical samples and was most importantly found to inactivate SARS-coV-2. Bruce et al [ 11 ] also showed that heating is important for the detection of low viral load samples most probably since it denatured RNases/inhibitors of the enzymes and/or improved the accessibility of viral RNA through direct lysis of cells and virions.…”

Section: Discussionmentioning

confidence: 99%

“…Their results showed that the direct RT-PCR can replace extraction-based SARS-CoV-2 diagnostics. The fourth study [ 12 ] of Ioanna et al found that heat inactivation of the samples prior PCR reactions at 95 °C gave superior results than the incubation at 65 °C and also lead to virus inactivation for better biosafety.…”

Section: Introductionmentioning

confidence: 99%

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A Direct Method for RT-PCR Detection of SARS-CoV-2 in Clinical Samples

et al. 2021

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Introduction: the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of acute respiratory disease (COVID-19). SARS-CoV-2 is a positive-strand RNA virus and its genomic characterization has played a vital role in the design of appropriate diagnostics tests. The current RT-PCR protocol for SARS-CoV-2 detects two regions of the viral genome, requiring RNA extraction and several hours. There is a need for fast, simple, and cost-effective detection strategies. Methods: we optimized a protocol for direct RT-PCR detection of SARS-CoV-2 without the need for nucleic acid extraction. Nasopharyngeal samples were diluted to 1:3 using diethyl pyrocarbonate (DEPC)-treated water. The diluted samples were incubated at 95 °C for 5 min in a thermal cycler, followed by a cooling step at 4 °C for 5 min. Samples then underwent reverse transcription real-time RT-PCR in the E and RdRp genes. Results: our direct detection protocol showed 100% concordance with the standard protocol with an average Ct value difference of 4.38 for the E region and 3.85 for the RdRp region. Conclusion: the direct PCR technique was found to be a reliable and sensitive method that can be used to reduce the time and cost of the assay by removing the need for RNA extraction. It enables the use of the assay in research, diagnostics, and screening for COVID-19 in regions with fewer economic resources, where supplies are more limited allowing for wider use for screening.

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Section: Discussionsupporting

confidence: 82%

Section: Discussionmentioning

confidence: 99%

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A Direct Method for RT-PCR Detection of SARS-CoV-2 in Clinical Samples

et al. 2021

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“…The reported LOD for detecting SARS-CoV-2, evaluated using SARS-CoV-2 positive saliva, was 6,000 virus 90 copies per ml of saliva. In direct RT-PCR tests, addition of non-ionic detergents, such as Tween-20 and Triton X-100, to saliva or swab-derived samples was shown to increase the sensitivity of SARS-CoV-2 RNA detection [7,11].…”

mentioning

confidence: 99%

Rapid processing of SARS-CoV-2 containing specimens for direct RT-PCR

Chomczyński

Chomczynski

Kennedy

et al. 2020

Preprint

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Widespread diagnostic testing is needed to reduce transmission of COVID-19 and manage the pandemic. Effective mass screening requires robust and sensitive tests that reliably detect the SARS-CoV-2 virus, including asymptomatic and pre-symptomatic infections with a low viral count. Currently, the most accurate tests are based on detection of viral RNA by RT-PCR. We developed a method to process COVID-19 specimens that simplifies and increases the sensitivity of viral RNA detection by direct RT-qPCR, performed without RNA purification. In the method, termed Alkaline-Glycol Processing (AG processing), a SARS-CoV-2-containing biological specimen, such as saliva or a swab-collected suspension, is processed at pH 12.2 to 12.8 for 5 min at room temperature. An aliquot of the AG-processed specimen is used for detection of SARS-CoV-2 RNA by direct RT-qPCR. AG processing effectively lyses viruses and reduces the effect of inhibitors of RT-PCR that are present in biological specimens. The sensitivity of detecting viral RNA using AG processing is on par with methods that include a viral RNA purification step. One copy of SARS-CoV-2 virus per reaction, equivalent to 300 copies per ml of saliva, is detectable in the AG-processed saliva. The LOD calculated following U.S. FDA guidelines is 600 viral copies per ml of initial saliva specimen. AG processing works with saliva specimens or swab specimens collected into Universal Transport Medium (UTM), is compatible with heat treatment, and was confirmed to work with a range of CDC-approved RT-qPCR products and kits. Detection of SARS-CoV-2 RNA using AG processing with direct RT-qPCR provides a reliable and scalable diagnostic test for COVID-19 that can be integrated into a range of workflows, including automated settings.

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“…[16][17][18] Furthermore, these drive-through tests are available only for preregistered and eligible patients with access to automobiles. As rapid and affordable tests for COVID-19 are showing promise, 19 the USPS facilities and workforce could be leveraged to broaden access, especially for these underserved groups. With more than 600,000 workers, facilities in every county, and in more than 28,000 of the approximately 33,000 ZIP Code Tabulation Areas (ZCTAs) in the United States, USPS has a nationwide presence (ZCTAs are generalized areal representations of ZIP codes used by the US Census Bureau 20 ).…”

mentioning

confidence: 99%

Expanding Access to COVID-19 Tests through US Postal Service Facilities

et al. 2020

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Widespread, convenient access to COVID-19 testing has been challenging in the United States. We make a case for provisioning COVID-19 tests through the United States Postal Service (USPS) facilities and demonstrate a simple method for selecting locations to improve access. We provide quantitative evidence that even a subset of USPS facilities could provide broad access, particularly in remote and at-risk communities with limited access to health care. Based on daily travel surveys, census data, locations of USPS facilities, and an established care-seeking model, we estimate that more than 94% of the US population would be willing to travel to an existing USPS facility if warranted. For half of the US population, this would require traveling less than 2.5 miles from home; for 90%, the distance would be less than 7 miles. In Georgia, Illinois, and Minnesota, we estimate that testing at USPS facilities would provide access to an additional 4.1, 3.1, and 1.3 million people and reduce the median travel distance by 3.0, 0.8, and 1.2 miles, respectively, compared with existing testing sites per 28 July 2020. We also discuss the option of distributing test-at-home kits via USPS instead of private carriers. Finally, our proposal provides USPS an opportunity to increase revenues and expand its mission, thus improving its future prospects and relevance.

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Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR

Cited by 391 publications

References 14 publications

A Direct Method for RT-PCR Detection of SARS-CoV-2 in Clinical Samples

A Direct Method for RT-PCR Detection of SARS-CoV-2 in Clinical Samples

Rapid processing of SARS-CoV-2 containing specimens for direct RT-PCR

Expanding Access to COVID-19 Tests through US Postal Service Facilities

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