Detection of SARS-CoV-2 by RT-LAMP assay in Human COVID-19 patients

ABSTRACT

Background: The novel COVID-19 outbreak has infected human population all around the world. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) diagnosis in a rapid manner remains challenging for health care professionals. Currently, RT-qPCR technique is extensively practiced in SARS-CoV-2 diagnosis and is considered as gold standard. The constraints of RT-qPCR, high cost and need for trained technician, longer detection time, highlighted the need for alternate healthcare diagnostic approaches. They follow the WHO assured standard and offer the health-care sector optimism. One of them is the Loop Mediated isothermal amplification system (LAMP). There is no need for costly equipment like thermal cycler since LAMP assay is performed at a fixed temperature. It can also be implemented as a point of care testing device. RT-LAMP is one of the extensively used isothermal amplification system in pathogen diagnostics.Aims: The current study aims to validate and standardize RT-LAMP assay for rapid diagnosis of SARS-CoV-2 in both lab and field conditions. The reactions can be carried out using a heating vessel including the use of a water bath and end-point detection by colorimetry. A rising middle ground of tiny, more portable technology, that provides most of the capability at less cost and time.Methods and Results: 20 Samples were taken from COVID-19 positive patients. RNA extraction from COVID-19 samples was followed up by one-step reverse transcription and loop-mediated isothermal amplification (LAMP). LAMP primers were designed to amplify the conserved regions of SARS-COV-2 specific genes. The target regions for primer design were selected after genome-wide sequence alignment of SARS-CoV-2 strains isolated in various regions of the world i.e., Europe, Africa, Asia, and North America. RT-LAMP assays were performed at the specific incubation temperature (60°C) for 50 minutes. Assay was optimized as per consumable compatibility, COVID template integrity, primer concentration, template concentration, primer ratio, testing time etc. Sensitivity and specificity of the assay was elucidated. Finally, different end-point analysis i.e., Agarose Gel Electrophoresis and Colorimetry have been used to interpret the results.Conclusion: RT-LAMP assay has shown to be a quick and accurate diagnostic method that can be put to use for SARS-CoV-2 detection in laboratories and Point-of- Care settings. © 2022 IEEE.