Extraction-free COVID-19 (SARS-CoV-2) diagnosis by RT-PCR to increase capacity for national testing programmes during a pandemic

Grant, Paul; Turner, Melanie A; Shin, Gee Yen; Nastouli, Eleni; Levett, Lisa J.

doi:10.1101/2020.04.06.028316

Cited by 74 publications

(90 citation statements)

References 2 publications

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“…Additionally, they found that direct sample addition in VTM without heating to the TaqMan Fast Virus 1-step Master Mix (Thermo Fisher) RT-qPCR reaction allowed detection 3.77 cycles earlier than the same test performed with RNA purified using the EZ1 Qiagen kit. Overall, their test using direct, unheated sample had 98.8% diagnostic accuracy when compared to cartridge-based RNA purification and RT-qPCR using the Panther Fusion system (Grant et al 2020). Intermediate inactivation temperatures seem to perform worse than high heat or no heating at all.…”

Section: Rtmentioning

confidence: 99%

“…Another group reported that directly added samples were detected 3.5 cycles later than RNA isolated using the MagNA Pure kit (Roche), but heating the sample at 95°C for 5 minutes before direct-to-test addition resulted in detection only one cycle later, with 97.4% accuracy compared to tests using purified RNA (Fomsgaard and Rosenstierne 2020). However, Grant et al found the opposite -heating direct-to-test samples in VTM at 95°C for 10 minutes delayed detection of viral RNA compared to directly added samples not heated prior to amplification (Grant et al 2020). Additionally, they found that direct sample addition in VTM without heating to the TaqMan Fast Virus 1-step Master Mix (Thermo Fisher) RT-qPCR reaction allowed detection 3.77 cycles earlier than the same test performed with RNA purified using the EZ1 Qiagen kit.…”

Section: Rtmentioning

confidence: 99%

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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

482

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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Section: Rtmentioning

confidence: 99%

Section: Rtmentioning

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

482

474

View full text Add to dashboard Cite

show abstract

“…The CCC test was validated against the N gene assay developed by the reference laboratory (Health Services Laboratories, HSL) [6]. The N gene assay is a high throughput RT-PCR assay that runs on the Hologic Panther Fusion platform using the Open Access function which allows the use of custom primers and probe targeting the nucleocapsid (N) gene of SARS-…”

Section: N Gene Assaymentioning

confidence: 99%

Scalable and Resilient SARS-CoV-2 testing in an Academic Centre

Aitken¹,

Ambrose²,

Barrell³

et al. 2020

Preprint

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The emergence of the novel coronavirus SARS-CoV-2 has led to a pandemic infecting more than two million people worldwide in less than four months, posing a major threat to healthcare systems. This is compounded by the shortage of available tests causing numerous healthcare workers to unnecessarily self-isolate. We provide a roadmap instructing how a research institute can be repurposed in the midst of this crisis, in collaboration with partner hospitals and an established diagnostic laboratory, harnessing existing expertise in virus handling, robotics, PCR, and data science to derive a rapid, high throughput diagnostic testing pipeline for detecting SARS-CoV-2 in patients with suspected COVID-19. The pipeline is used to detect SARS-CoV-2 from combined nose-throat swabs and endotracheal secretions/ bronchoalveolar lavage fluid. Notably, it relies on a series of in-house buffers for virus inactivation and the extraction of viral RNA, thereby reducing the dependency on commercial suppliers at times of global shortage. We use a commercial RT-PCR assay, from BGI, and results are reported with a bespoke online web application that integrates with the healthcare digital system. This strategy facilitates the remote reporting of thousands of samples a day with a turnaround time of under 24 hours, universally applicable to laboratories worldwide.

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“…Due to the unprecedented high demand in SARS-CoV-2 testing required to identify and isolate infected individuals, many laboratories are now facing shortages in reagents and consumables required for nucleic acid (NA) extraction. This has led us to investigate alternative sample processing methods that would remove the requirements for NA extraction 4,5,6,7 and allow rapid diagnosis.…”

Section: Introductionmentioning

confidence: 99%

Molecular detection of SARS-CoV-2 using a reagent-free approach

Calvez

Taylor

Calvo-Bado

et al. 2020

Preprint

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Shortage of reagents and consumables required for the extraction and molecular detection of SARS-CoV-2 RNA in respiratory samples has led many laboratories to investigate alternative approaches for sample preparation. Fomsgaard et al 2020 5 recently presented results using heat-processing of respiratory samples prior to RT-qPCR as an economical method enabling an extremely fast streamlining of the processes at virtually no cost.Here, we present our results using this method and highlight some major pitfalls that diagnostics laboratories should be aware of before proceeding with this technique. We first investigated various treatments using different temperatures, incubation times and sample volumes based on the above study to optimise the heattreatment conditions. Although the initial data confirmed the published results, further investigations revealed unexpected inhibitory properties of some commonly used virus transport media (VTMs) on some commercially available RT-qPCR mixes, emphasising the critical importance of a thorough validation process to determine the most adapted reagents to be used depending on the sample types to be tested.In conclusion, although the method works, with very consistent Ct values and an excellent sensitivity when compared to a conventional RNA extraction method, it is critical to include an internal control to check each sample for potential inhibition.

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Extraction-free COVID-19 (SARS-CoV-2) diagnosis by RT-PCR to increase capacity for national testing programmes during a pandemic

Cited by 74 publications

References 2 publications

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

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

Scalable and Resilient SARS-CoV-2 testing in an Academic Centre

Molecular detection of SARS-CoV-2 using a reagent-free approach

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