RAPID VISUAL DETECTION OF SARS-COV-2 USING REVERSE TRANSCRIPTION LOOP MEDIATED ISOTHERMAL AMPLIFICATION

Abstract

In December 2019, a pneumonia-like disease outbreak of unknown origin was reported in Wuhan, China. Investigations revealed the causal agent to be the now widely known single stranded RNA virus, severe acute respiratory syndrome 2 (SARS-CoV-2), which has caused millions of infections and fatalities around the world ^1,2. The widespread pandemic resulted in lockdowns and other efforts aimed at controlling the spread of the disease, resulting in adverse effects on almost all sectors of human life. Detection of the viral agent is very important in infection control and management, but the use of real-time RT-PCR which is still the gold standard for detection is affected in resource constrained communities by its high cost, operational complexity and time-consuming protocol, while antibody-based tests are limited in sensitivity ^3,4. This work aims to develop a rapid, specific, and simple RT LAMP assay which can detect SARS-CoV-2. Multiple nucleocapsid (N) gene sequences of SARS-CoV-2 where aligned and LAMP primers designed from conserved regions using the PrimerExplorer 5. Primers were screened for specificity to SARS-CoV-2 N gene using synthetic constructs of related coronaviruses in 60 minutes LAMP reactions. Selected primers were also used in real-time LAMP and RT-LAMP using synthetic (T7 polymerase in vitro transcribed) and clinical samples of SARS-CoV-2 (extracted RNA) previously confirmed by RT-PCR. Amplified products were confirmed by gel electrophoresis and sequencing. The assay was optimized, incorporated with hydroxynaphthol blue dye for visual detection and its limit of detection determined. The optimized assay specifically detects 8 copies/µL of SARS CoV-2 in 45 minutes. Positive samples (sky blue) are visually distinct from negative samples (purple). The colorimetric assay is suitable for use in poor communities, has faster turnaround time and can be performed with a simple water bath or heating block. This outcome can increase community testing and boost surveillance of COVID-19 infections.