Loop-Mediated Isothermal Amplification-Assay Rapidly Diagnoses COVID-19

The loop-mediated isothermal amplification (LAMP) assay is robust, rapid and straightforward, yet retains high sensitivity. These features have seen the LAMP assay and the inclusion of a reverse transcriptase (RT-LAMP) implemented for a broad range of molecular diagnostic applications extending from infectious diseases, virus, bacteria and parasites, to cancer.

Microbiologists at the University of Melbourne (Melbourne, Australia) and their associates collected 157 confirmed-positive nasopharyngeal swabs between the March 23 and April 4 2020. The team developed a rapid molecular test called N1 gene Single Tube Optigene LAMP (N1-STOP-LAMP), a reverse transcriptase loop-mediated isothermal amplification-assay. Copan flocked swabs were collected and directly inoculated on site in either 1 mL or 3 mL of universal transport medium (UTM, Copan Diagnostics, Murrieta, CA, USA).

A 200 µL aliquot of Copan UTM was processed through the QIAsymphony DSP Virus/Pathogen Mini Kit (Qiagen, Hilden, Germany) on the QIAsymphony SP instrument. Each N1-STOP-LAMP reactions were assembled in either 8-tube Genie strips (OptiGene Ltd, Horsham, UK) or 96-MicroAmp-Fast-Optical reaction plate (Applied Biosystems, Foster City, CA, USA). Strip tubes were loaded onto the OptiGene Genie-II or Genie-III and 96-well plates run on an Applied Biosystems’ QuantStudio 7 thermocycler.

The scientists reported that the assay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed the N1-STOP-LAMP assay had sensitivity and specificity of 87% and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 ± 7 minutes. The team 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 inter-laboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay.

The authors concluded that with a simplified workflow, The N1 gene Single Tube Optigene LAMP assay 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. The study was published on August 5, 2020 in the Journal of Medical Microbiology.

Related Links:
University of Melbourne
Copan Diagnostics
Qiagen
OptiGene Ltd
Applied Biosystems