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A new, 20-minute assay for COVID-19 diagnosis — Melbourne University

Researchers have developed a new test that can diagnose COVID-19 in just 20 minutes. The findings show the rapid molecular test called N1-STOP-LAMP, is 100% accurate in diagnosing samples containing SARS-CoV-2 at high loads.

The test is highly accurate and easy to use, making it a prime candidate for use in settings with limited testing capabilities. The method involves using a small portable machine, which can reliably detect SARS-CoV-2 from just one nasal swab. "In the race to control the COVID-19 pandemic, access to rapid, precision diagnostics is key. We have developed an alternative COVID-19 molecular test that can be readily deployed in settings where access to standard laboratory testing is limited or where ultra-rapid result turnaround times are needed" said University of Melbourne Professor Tim Stinear, laboratory head at the Doherty Institute.

This new test uses only one tube and involves only a single step, making it more efficient and lower cost than many of the current tests for SARS-CoV-2. The N1-STOP-LAMP method was found to be 100% accurate and correctly identified 87% of tests as positive when used to assess 157 confirmed-positive samples. The results were fast, with an average time-to-positive of 14 minutes for 93 of those clinical samples.

"We see this kind of technology having benefit in settings liked aged care facilities, or overseas laboratories with limited resources and equipment," Stinear said. "The test requires a small shoebox-sized machine, as well as reagents, but everything is portable."
"STOP-LAMP is what's referred to as a 'near care' test, it is not intended to replace the current gold standard PCR testing. It's a robust diagnostic test for the specific and rapid detection of COVID-19. But it's important to note however, it trades some detection sensitivity for speed and ease-of-use."

Abstract
Introduction: The SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues.
Aim: To establish and validate a reverse transcription loop-mediated isothermal amplification (RT- LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs.
Methodology: We used a commercial RT-LAMP mastermix from OptiGene in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby 1 µl of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65 °C for 30 min and measure fluorescence in the FAM channel at 1 min intervals.
Results: Assay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87 and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 min (SD±7 min). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 ml−1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay.
Conclusion: With a simplified workflow, The N1 gene Single Tube Optigene LAMP assay (N1-STOP-LAMP) is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19.

Authors
Jean YH Lee, Nickala Best, Julie McAuley, Jessica L Porter, Torsten Seemann, Mark B Schultz, Michelle Sait, Nicole Orlando, Karolina Mercoulia, Susan A Ballard, Julian Druce, Thomas Tran, Mike G Catton, Melinda J Pryor, Huanhuan L Cui, Angela Luttick, Sean McDonald, Arran Greenhalgh, Jason C Kwong, Norelle L Sherry, Maryza Graham, Tuyet Hoang, Marion Herisse, Sacha J Pidot, Deborah A Williamson, Benjamin P Howden, Ian R Monk, Timothy P Stinear

 

[link url="https://microbiologysociety.org/news/press-releases/a-new-20-minute-assay-for-covid-19-diagnosis.html"]Microbiology Society material[/link]

 

[link url="https://www.microbiologyresearch.org/content/journal/jmm/10.1099/jmm.0.001238"]Journal of Medical Microbiology abstract[/link]

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