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

Abstract

PurposeDespite 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 & MaterialsAbout 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.ResultsThe 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.ConclusionOverall, 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.