Development of Duplex and Multiplex Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) Assays for Clinical Diagnosis of SARS-COV-2 in Sri Lanka
M Hewadikaram, K Perera, K Dissanayake, M Ramanayake, S C Isurika, A Panch, A Jayarathne, P Pushpakumara, N Malavige, C Jeewandara, S D N K Bathige, A M Mubarak
International Journal of Infectious Diseases, doi:10.1016/j.ijid.2021.12.095
Purpose: Despite the rollout of several vaccines targeting SARS-CoV-2, attainment of near-universal vaccination is a challenging task, particularly for low-and middle-income nations such as Sri Lanka. Rapid, reliable diagnostics for the detection of the virus is of vital importance for the predominantly export-and tourismbased economy of the country. Herein, we report the development of a RT-LAMP assay as an alternative to the gold-standard RT-qPCR method for diagnostic laboratories in Sri Lanka in a cost-effective and highly reliable manner. Methods & Materials: About 313 nasopharyngeal and oropharyngeal samples from the community were collected and subjected to RNA purification and subjected to simultaneous RT-qPCR and RT-LAMP experiments by using previously published primers in a thermocycler. Duplex (containing N and E gene primers) and multiplex (containing N, E and ORF1ab gene primers) RT-LAMP assay results were compared with standard RT-qPCR results using an agreement attribute statistical test. The effect of guanidine hydrochloride was also analyzed. Results: The limit of detection for the duplex assay was found to be 10 copies μL-1 at a constant temperature of 63 °C, and 5 copies μL-1 for multiplex assays at 66.4 °C. Both types of RT-LAMP assay were specific only for the SARS-COV-2 virus, successfully distinguishing it from multiple other human viruses. Attribute agreement analysis between duplex-and multiplex RT-LAMP vs RT-qPCR yielded 93% and 96.5% scores, respectively. Moreover, both RT-LAMP assays showed 100% agreement with RT-qPCR when Ct was < 25 in positive samples and showed 100% (duplex) or 97.22% (multiplex) at 35 ≥ Ct ≥25. The discrepancy between agreements at higher Ct values was attributed to the higher sensitivity of the multiplex RT-LAMP assay. The addition of guanidine hydrochloride increased the sensitivity and decreased detection time significantly for both the duplex and multiplex assays. Conclusion: Overall, we have demonstrated a potentially rapidly deployable diagnostic test kit not only for widespread community use but particularly for high-risk locations such as ports of entry or manufacturing facilities to mitigate the effects of the SARS-CoV-2 virus in Sri Lanka.
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