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

Abstract

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.

Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis

“…After the evaluation of 768 samples the authors observed that samples with Ct > 30 remained negative or after 30 min unspecific amplification started to occur. Schermer et al 2020 established a Ct < 30 cut-off in combination to a primer optimization step by multiplexing RT-LAMP. The study aimed to detect Orf7a and Orf3a genes simultaneously.…”

Ultra-rapid detection of SARS-CoV-2 in public workspace environments

“…Alternatively formation of high molecular weight amplicons allows an intercalating dye to create a fluorescent signal [ 28 , 29 ] or non-fluorescent signal [ 30 , 31 ]. Alternatively, the pH change arising during the amplification is detected by the change in the color of an indicator [ 32 – 34 ].…”

Rapid diagnostics of novel coronavirus infection by loop-mediated isothermal amplification

“…Такой протокол, использующий сразу 2 мишени, может быть особенно востребован с учётом постоянно появляющихся новых мутаций возбудителя, потенциально способных привести к снижению эффективности некоторых тестов (что уже произошло с рядом диагностикумов вследствие «британской мутации») [51]. Наконец, описан ещё один подход с мультиплексированием RT-LAMP и проверена его чувствительность [63]. Мультиплексирование праймеров в реакциях RT-LAMP представляется многообещающим способом дальнейшего повышения чувствительности за счёт детекции нескольких геномных фрагментов.…”