Fast SARS-CoV-2 detection by RT-qPCR in preheated nasopharyngeal swab samples

Alcoba-Flórez, Julia; González-Montelongo, Rafaela; Íñigo-Campos, Antonio; Artola, Diego García-Martínez de; Gil-Campesino, Helena; Ciuffreda, Laura; Valenzuela-Fernandez, Agustin; Flores, Carlos

doi:10.1101/2020.04.08.20058495

Cited by 35 publications

(51 citation statements)

References 10 publications

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“…This approach can be employed as rapid and economical method for diagnosis which does not require any RNA extraction step. Our results are in line with the earlier reports of RNA extraction-free RT-PCR (Alcoba-Florez et al, 2020;Bruce et al, 2020;Smyrlaki et al, 2020), but here we have done a very extensive (n=40) and thorough standardization of this procedure which is now consistent and compelling.…”

Section: Resultssupporting

confidence: 92%

Improved and Simplified Diagnosis of Covid-19 using TE Extraction from Dry Swabs

Kiran

Gokulan

Kuncha

et al. 2020

Preprint

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Rigorous testing is the way forward to fight the Covid-19 pandemic. Here we show that the currently used and most reliable RT-PCR based SARS-CoV-2 procedure can be further simplified to make it faster, safer and economical by bypassing the RNA isolation step. The modified method is not only fast and convenient but also at par with the traditional method in terms of accuracy, and therefore, can be used for mass screening. Our method takes about half the time and is cheaper by about 40% compared to current most widely used method. We also provide a variant of the new method that increases the efficiency of detection by about 20%compared to the currently used method. Taken together, we demonstrate a more effective and reliable method of SARS-CoV-2 detection.

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Section: Resultssupporting

confidence: 92%

Improved and Simplified Diagnosis of Covid-19 using TE Extraction from Dry Swabs

Kiran

Gokulan

Kuncha

et al. 2020

Preprint

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“…The original CDC protocol approved four commercial master mixes for the RT-PCR test from Quantabio, Promega, and ThermoFisher (Centers for Disease Control and Prevention 2020). However, published RT-PCR protocols have also successfully employed 1-step RT-PCR master mixes from a variety of companies including NEB, Applied Biosciences, Qiagen, Roche, Takara, and others and a growing list of approved alternative commercial reagents can be found at the FDA EUA website (Chandler-Brown et al 2020;Kalikiri et al 2020;Zhao et al 2020;Won et al 2020;Merindol et al;Alcoba-Florez et al 2020;Marzinotto et al;Xu et al 2020;Food and Drug Administration). Many commercial master mixes seem to function well in the detection of SARS-CoV-2, although a detailed side-byside comparison of the numerous commercial reagents is lacking.…”

Section: Rt-pcrmentioning

confidence: 99%

Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection

Esbin

Whitney

Chong

et al. 2020

RNA

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474

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The current COVID-19 pandemic presents a serious public health crisis, and a better understanding of the scope and spread of the virus would be aided by more widespread testing. Nucleicacid based tests currently offer the most sensitive and early detection of COVID-19. However, the "gold standard" test pioneered by the United States Center for Disease Control & Prevention, takes several hours to complete and requires extensive human labor, materials such as RNA extraction kits that could become in short supply and relatively scarce qPCR machines. It is clear that a huge effort needs to be made to scale up current COVID-19 testing by orders of magnitude. There is thus a pressing need to evaluate alternative protocols, reagents, and approaches to allow nucleic-acid testing to continue in the face of these potential shortages. There has been a tremendous explosion in the number of papers written within the first weeks of the pandemic evaluating potential advances, comparable reagents, and alternatives to the "gold-standard" CDC RT-PCR test. Here we present a collection of these recent advances in COVID-19 nucleic acid testing, including both peer-reviewed and preprint articles. Due to the rapid developments during this crisis, we have included as many publications as possible, but many of the cited sources have not yet been peer-reviewed, so we urge researchers to further validate results in their own labs. We hope that this review can urgently consolidate and disseminate information to aid researchers in designing and implementing optimized COVID-19 testing protocols to increase the availability, accuracy, and speed of widespread COVID-19 testing.

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“…Our study corroborates the findings from others who have successfully performed COVID-19 RT-qPCR reactions in a benchtop real-time thermal cycler by simply adding a few microliters of the unprocessed sample in transport medium directly into the RT-qPCR assay master mix. [9][10][11][12] The data presented below suggests that it is possible to skip the RNA extraction step and use rapid RT-PCR to deliver fast COVID-19 testing using minimal equipment. While this approach may not be desirable for use in developed countries, it will find applications in many remote regions inside underdeveloped and developing countries where extraction devices and PCR thermal cyclers are not commonly available.…”

Section: Rapid Rt-pcr Detection Of Sars-cov-2 In Untreated Clinical Smentioning

confidence: 99%

A Rapid COVID-19 RT-PCR Detection Assay for Low Resource Settings

Arumugam

Faron

et al. 2020

Preprint

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Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. It has been used by the Centers for Disease Control and Prevention (CDC) and several other companies in their Emergency Use Authorization (EUA) assays. RT-qPCR requires expensive equipment such as RNA isolation instruments and real-time PCR thermal cyclers, which are not available in many low resource settings and developing countries. As a pandemic, COVID-19 has quickly spread to the rest of the world. Many underdeveloped and developing counties do not have the means for fast and accurate COVID-19 detection to control this outbreak. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 minutes using untreated samples, heat-inactivated samples, or extracted RNA templates. Rapid RT-PCR was achieved using thin-walled PCR tubes and a setup including sous vide immersion heaters/circulators. Our data suggest that rapid RT-PCR can be implemented for sensitive and specific molecular diagnosis of COVID-19 in situations where sophisticated laboratory instruments are not available.

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Fast SARS-CoV-2 detection by RT-qPCR in preheated nasopharyngeal swab samples

Cited by 35 publications

References 10 publications

Improved and Simplified Diagnosis of Covid-19 using TE Extraction from Dry Swabs

Improved and Simplified Diagnosis of Covid-19 using TE Extraction from Dry Swabs

Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection

A Rapid COVID-19 RT-PCR Detection Assay for Low Resource Settings

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