Loop-Mediated Isothermal Amplification-Assay Rapidly Diagnoses COVID-19
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By LabMedica International staff writers Posted on 25 Aug 2020 |
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Image: The Genie III instrument was used for the reverse transcriptase loop-mediated isothermal amplification-assay called N1 gene Single Tube Optigene LAMP (N1-STOP-LAMP) for COVID-19 (Photo courtesy of OptiGene Ltd).
The SARS-CoV-2 (COVID-19) 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.
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
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
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