Starting from scratch: Step-by-step development of diagnostic tests for SARS-CoV-2 detection by RT-LAMP

Abstract

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people worldwide. Public health strategies to reduce viral transmission are based on widespread diagnostic testing to detect and isolate contagious patients. Several reverse transcription (RT)-PCR tests, along with other SARS-CoV-2 diagnostic assays, are available to attempt to cover the global demand. Loop-mediated isothermal amplification (LAMP) based methods have been established as rapid, accurate, point of care diagnostic tests for viral infections; hence, they represent an excellent alternative for SARS-CoV-2 detection. The aim of this study was to develop and describe molecular detection systems for SARS-CoV-2 based on RT-LAMP. Recombinant DNA polymerase from Bacillus stearothermophilus and thermostable engineered reverse transcriptase from Moloney Murine Leukemia Virus were expressed using a prokaryotic system and purified by fast protein liquid chromatography. These enzymes were used to set up fluorometric real time and colorimetric end-point RT-LAMP assays. Several reaction conditions were optimized such as reaction temperature, Tris-HCl concentration, and pH of the diagnostic tests. The key enzymes for RT-LAMP were purified and their enzymatic activity was determined. Standardized reaction conditions for both RT-LAMP assays were 65°C and a Tris-HCl-free buffer at pH 8.8. Colorimetric end-point RT-LAMP assay was successfully used for viral detection from clinical saliva samples with 100% sensitivity and 100% specificity compared to the results obtained by RT-qPCR based diagnostic protocols with Ct values until 30. The developed RT-LAMP diagnostic tests based on purified recombinant enzymes allowed a sensitive and specific detection of the nucleocapsid gene of SARS-CoV-2.

A Comparative Analysis of Molecular Biological Methods for the Detection of SARS-CoV-2 and Testing the In Vitro Infectivity of the Virus

“…This has fueled criticism against lobbyism in the new EU legislation for allowing only private companies to perform such activities in the EU [ 56 , 58 ], thus, allegedly, paving the way for the privatization of knowledge and scientific research. Concerns have been voiced that with this legislation, along with the proposed international agreement on pandemic prevention and preparedness [ 57 , 59 ], any efforts for laboratories to develop methods such as this one and others will become futile [ 25 , 26 , 27 , 28 , 58 , 59 , 60 , 61 ], as will their actual application in helping people to adequately control diseases, thus making the EU dependent on corporations and the WHO.…”

“…The samples that produced invalid results in both tests indicate that the sample quality may have been compromised, e.g., during the sampling step, during the NA extraction procedure, or during the detection assays [ 60 , 61 , 62 , 63 , 64 ]. We believe that, because each batch of the LAMP diagnostic tests is validated against a positive clinical sample, it is unlikely that the invalid results are attributable to a failure of the tests, as suggested by Matzkies et al [ 63 , 65 ].…”

Quantitative mRNA expression measurement at home

“…Loop-mediated isothermal amplification (LAMP) protocols 1 have become a prevalent method of developing diagnostic assays 2 – 14 . Diagnostics based on LAMP amplification are a compelling alternative to polymerase chain reaction (PCR) because LAMP can be performed without commercial thermocyclers, significantly reducing cost and time to result 1 , 15 .…”

Laboratory Evaluation of a SARS-CoV-2 RT-LAMP Test

“…The development of loop-mediated isothermal amplification (LAMP) represents an interesting advance in nucleic acid-based diagnostics [ 10 , 11 ] with several advantages. First, the amplification reaction is isothermal (between 60 and 65 °C) and does not require the use of a thermocycler.…”

“…Third, the product can be visualized directly using simple detection methods, from fluorescence to colorimetric to lateral flow assay. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays have now also been developed for the diagnosis of SARS-CoV-2 [ 10 , 11 ]. RT-LAMP combines the advantages of Ag-RDTs as they are easy, rapid, and do not require specialized infrastructures [ 10 ] or real-time RT-PCR (high sensitivity) [ 10 , 11 , 12 , 13 ].…”