A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing

Kellner, Max J.; Ross, James J.; Schnabl, Jakob; Dekens, Marcus P. S.; Heinen, Robert; Grishkovskaya, Irina; Bauer, Benedikt; Stadlmann, Johannes; Menéndez‐Arias, Luis; Fritsche-Polanz, Robert; Traugott, Marianna; Seitz, Tamara; Zoufaly, Alexander; Foedinger, Manuela; Wenisch, Christoph; Zuber, Johannes; Initiative, Vienna Covid Diagnostics; Pauli, Andrea; Brennecke, Julius

doi:10.1101/2020.06.23.166397

Cited by 68 publications

(118 citation statements)

References 38 publications

Supporting

Mentioning

109

Contrasting

Unclassified

Order By: Relevance

“…Additionally, one thing to be kept in mind when directly comparing sensitivity between the different methods, is the fact that qPCR reaches this standard only in isolated RNA whilst the multiplex RT-LAMP assay attains an optimized detection rate already in primary material. Of note, further addition of a step concentrating RNA using bead-based pulldown to an RT-LAMP-based protocol has also been successful [ 26 ]. Remarkably, here the authors move from swabs, that require trained personnel and personal protection equipment, to a home-based gargle [ 26 ] and a very recent report using qPCR demonstrated, that even saliva could be used as a valid source for viral diagnostics [ 27 ].…”

Section: Discussionmentioning

confidence: 99%

Rapid SARS-CoV-2 testing in primary material based on a novel multiplex RT-LAMP assay

et al. 2020

View full text Add to dashboard Cite

Background Rapid and extensive testing of large parts of the population and specific subgroups is crucial for proper management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and decision-making in times of a pandemic outbreak. However, point-of-care (POC) testing in places such as emergency units, outpatient clinics, airport security points or the entrance of any public building is a major challenge. The need for thermal cycling and nucleic acid isolation hampers the use of standard PCR-based methods for this purpose. Methods To avoid these obstacles, we tested PCR-independent methods for the detection of SARS-CoV-2 RNA from primary material (nasopharyngeal swabs) including reverse transcription loop-mediated isothermal amplification (RT-LAMP) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK). Results Whilst specificity of standard RT-LAMP assays appears to be satisfactory, sensitivity does not reach the current gold-standard quantitative real-time polymerase chain reaction (qPCR) assays yet. We describe a novel multiplexed RT-LAMP approach and validate its sensitivity on primary samples. This approach allows for fast and reliable identification of infected individuals. Primer optimization and multiplexing helps to increase sensitivity significantly. In addition, we directly compare and combine our novel RT-LAMP assays with SHERLOCK. Conclusion In summary, this approach reveals one-step multiplexed RT-LAMP assays as a prime-option for the development of easy and cheap POC test kits.

show abstract

Section: Discussionmentioning

confidence: 99%

Rapid SARS-CoV-2 testing in primary material based on a novel multiplex RT-LAMP assay

et al. 2020

View full text Add to dashboard Cite

show abstract

“…This cost can be potentially reduced by reducing the volume to the 25 µl to achieve ~$2 per reaction, albeit with reduced sensitivity. Additionally, use of open source enzymes 26,33 and pooling the samples could further reduce costs. As the volume of LAMP reaction increases, false positive results seem to increase in frequency which manifested as a few negative samples turning orange in our experiments presented here.…”

Section: Discussionmentioning

confidence: 99%

“…Since it does not require a PCR machine, RT-LAMP is ideal for field testing of viral infections and has been used for Ebola diagnosis and surveillance in Guinea 9,10 , for tracking Zika virus in Brazilian mosquito populations 11 , and for COVID-19 diagnosis during the initial outbreak in Wuhan and Shenyang, China 12,13 . Recent work has solidified RT-LAMP as a simple, inexpensive, and sufficiently sensitive alternative to RT-qPCR for SARS-CoV-2 detection [14][15][16][17][18][19][20][21][22][23][24][25][26] .…”

Section: Introductionmentioning

confidence: 99%

A simple direct RT-LAMP SARS-CoV-2 saliva diagnostic

Flynn

Snitser

Flynn

et al. 2020

Preprint

View full text Add to dashboard Cite

Widespread, frequent testing is essential for curbing the ongoing COVID-19 pandemic. Because its simplicity makes it ideal for widely distributed, high throughput testing, RT-LAMP provides an attractive alternative to RT-qPCR. However, most RT-LAMP protocols require the purification of RNA, a complex and low-throughput bottleneck that has often been subject to reagent supply shortages. Here, we report an optimized RT-LAMP-based SARS-CoV-2 diagnostic protocol for saliva and swab samples. In the protocol we replace RNA purification with a simple sample preparation step using a widely available chelating agent, as well as optimize key protocol parameters. When tested on clinical swab and saliva samples, this assay achieves a limit of detection of 105 viral genomes per ml, with sensitivity close to 90% and specificity close to 100%, and takes 45 minutes from sample collection to result, making it well suited for a COVID-19 surveillance program.

show abstract

“…Thus, much effort has been devoted to developing more streamlined sample prep protocols that match the simplicity of LAMP. Silica-coated magnetic beads [47,48], pH-sensitive magnetic beads [49,50], poly carboxyl-functionalized magnetic beads [51,52], gravity-driven gel filtration [53], and lateral-flow filtration [54] are seemingly more appropriate for field-deployable extraction than spin columns, vacuum manifolds, or robotic systems often employed in laboratory settings. However, these processes still require multiple steps and skilled operators.…”

Section: Sample Preparationmentioning

confidence: 99%

LAMP Diagnostics at the Point-of-Care: Emerging Trends and Perspectives for the Developer Community

Moehling

Choi

Dugan

et al. 2021

Expert Review of Molecular Diagnostics

124

View full text Add to dashboard Cite

Introduction: Over the past decade, loop-mediated isothermal amplification (LAMP) technology has played an important role in molecular diagnostics. Amongst numerous nucleic acid amplification assays, LAMP stands out in terms of sample-to-answer time, sensitivity, specificity, cost, robustness, and accessibility, making it ideal for field-deployable diagnostics in resource-limited regions. Areas covered: In this review, we outline the front-end LAMP design practices for point-of-care (POC) applications, including sample handling and various signal readout methodologies. Next, we explore existing LAMP technologies that have been validated with clinical samples in the field. We summarize recent work that utilizes reverse transcription (RT) LAMP to rapidly detect SARS-CoV-2 as an alternative to standard PCR protocols. Finally, we describe challenges in translating LAMP from the benchtop to the field and opportunities for future LAMP assay development and performance reporting. Expert opinion: Despite the popularity of LAMP in the academic research community and a recent surge in interest in LAMP due to the COVID-19 pandemic, there are numerous areas for improvement in the fundamental understanding of LAMP, which are needed to elevate the field of LAMP assay development and characterization.

show abstract

A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing

Cited by 68 publications

References 38 publications

Rapid SARS-CoV-2 testing in primary material based on a novel multiplex RT-LAMP assay

Rapid SARS-CoV-2 testing in primary material based on a novel multiplex RT-LAMP assay

A simple direct RT-LAMP SARS-CoV-2 saliva diagnostic

LAMP Diagnostics at the Point-of-Care: Emerging Trends and Perspectives for the Developer Community

Contact Info

Product

Resources

About