ABSTRACT
Introduction: The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), identified in December of 2019, is the cause of the coronavirus disease 2019 (COVID-19). Due to the high reproductive rate of the virus, the best way to slow down the spread is to identify and isolate patients at the early stage of infections. The current diagnostic methods are either too expensive, slow or have low accuracy. Variants of SARS-CoV-2 with mutations at the primer binding sites may cause evasion of polymerase chain reaction (PCR) detection using current primers. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has potential as a rapid molecular test that is easy to conduct. Methods: LAMP primers were design based on the highly conserved regions of the SARS-CoV-2 Nucleocapsid (N) gene. RT-LAMP assays were conducted using an optimized Bst 3.0 polymerase protocol on T7 RNA polymerase synthesized RNA template. The LAMP sensitivity assay was tested on 1:10 serial diluted pJET1.2 vector with SARSCoV2 N gene inserts. A specificity test was conducted by running the test on plasmids containing SARS-CoV and MERS-CoV N genes. The results were visualised via gel electrophoresis, SYBR Green staining and Lateral Flow Dipstick (LFD). Results: The optimized protocol is sensitive enough to detect SARS-CoV-2 genetic material within 10 minutes but is most sensitive at 30 minutes. Additionally, it is specific to only the genetic materials of SARS-CoV-2. Furthermore, an LFD with multiple test lines was successful for multiplexed LAMP reactions with different genic regions of the virus. Conclusion: The multiplexed LFD-LAMP is potentially a simple yet specific and sensitive method of rapid molecular diagnostics of COVID-19.