Development of Duplex and Multiplex Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) Assays for Clinical Diagnosis of SARS-COV-2 in Sri Lanka. (March 2022)

• Record Type: Journal Article
• Authors: Hewadikaram, M.; Perera, K.; Dissanayake, K.; Ramanayake, M.; Isurika, S.C.; Panch, A.; Jayarathne, A.; Pushpakumara, P.; Malavige, N.; Jeewandara, C.; Bathige, S.D.N.K.; Mubarak, A.M.
• Is Part Of: International journal of infectious diseases. Volume 116(2022)Supplement
• Page Start: S39
• Page End: S40
• Publication Date: 2022-03
• Subjects: Communicable diseases -- Periodicals; Communicable Diseases -- Periodicals; Communicable diseases; Periodicals; Electronic journals; 616.9
• DOI: 10.1016/j.ijid.2021.12.095
• Languages: English
• ISSNs: 1201-9712

Abstract

Abstract : 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 tourism-based 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.