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

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. 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. 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