Diagnostic utility and validation of a newly developed real time loop mediated isothermal amplification method for the detection of SARS CoV-2 infection

Bushran N Iqbal; Shiyamalee Arunasalam; Maduja V M Divarathna; Aaom Jabeer; Pdnn Sirisena; Thamarasi Senaratne; Rohitha Muthugala; Faseeha Noordeen

doi:10.1016/j.jcvp.2022.100081

Diagnostic utility and validation of a newly developed real time loop mediated isothermal amplification method for the detection of SARS CoV-2 infection

J Clin Virol Plus. 2022 Aug;2(3):100081. doi: 10.1016/j.jcvp.2022.100081. Epub 2022 May 5.

Authors

Bushran N Iqbal¹, Shiyamalee Arunasalam¹, Maduja V M Divarathna¹, Aaom Jabeer¹, Pdnn Sirisena², Thamarasi Senaratne³, Rohitha Muthugala⁴, Faseeha Noordeen¹

Affiliations

¹ Diagnostic and Research Virology Laboratory, Department of Microbiology, Faculty of Medicine, University of Peradeniya, Peradeniya 20400, Sri Lanka.
² ImmunifyMe Health Tech Pvt Ltd, New Delhi, India.
³ Department of Multidisciplinary Sciences, Faculty of Allied Health Sciences, General Sir John Kotelawala Defense University, Werahera, Ratmalana 10390, Sri Lanka.
⁴ Diagnostic and Reference Virology Laboratory, National Hospital, Kandy 20000, Sri Lanka.

Abstract

Background: Detecting SARS-CoV-2 using a simple real time molecular assay will be helpful for the mitigation efforts in low / middle income countries during the pandemic. We have developed and validated a rapid and simple real time loop mediated isothermal amplification assay (LAMP) for screening of SARS-CoV-2 infection in known infected and non-infected individuals.

Methods: Six sets of primers were designed targeting the N-gene of the SARS-CoV-2 (Accession ID MN994468). LAMP reactions were performed using Warm Start 2X Master Mix and real-time PCR machine at 65 °C for 60 cycles with 15 s for each cycle. Results were read by visualizing turbidity under ultraviolet light and real time fluorescence detection through FAM channel of the real time PCR machine. We tested a total of 320 including 240 SARS CoV-2 positive (Ct values <40) and 80 SARS CoV-2 negative samples as tested by a real time RT-PCR using the newly developed LAMP assay.

Results: A total of 206 out of 240 SARS CoV-2 positive samples were tested positive by the newly developed LAMP assay with a sensitivity of 86%. All 80 SARS CoV-2 negative samples were tested negative by the newly developed LAMP assay with a specificity of 100%.

Conclusion: The newly developed real time LAMP assay has a sensitivity of 86% and specificity of 100% compared to the real time RT-PCR for the detection of SARS CoV-2. The new assay will be useful to screen large number of samples if adopted to minimize the time and cost.

Keywords: Loop mediated amplification; SARS CoV-2, Severe acute respiratory syndrome coronavirus-2; SARS-CoV-2; Sensitivity; Specificity; rtRT-PCR; rtRT-PCR, Real time reverse transcription polymerase chian reaction.