Development of Reverse Transcription-loop-mediated Isothermal Amplification (RT-LAMP) for the Detection of the Causative Agent of COVID-19
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Abstract
SARS-CoV-2 virus is the causative agent of COVID-19, which is an emerging infectious disease. The disease has affected a number of infected cases and death worldwide. The current detection standard is real time RT-PCR, a nucleic acid amplification test (NAAT) used to detect the viral RNA in respiratory specimens. However, the method requires the thermocycler machine, which is complicated and costly and causes the limitation of its use in general laboratories. LAMP, an isothermal amplification, is a simple NAAT. It provides the results in a short time, which can be visualized the color change by naked eyes. This study aimed to develop RT-LAMP for SARS-CoV-2 detection by designing primers and evaluating the in silico inclusivity and cross-reactivity analyses based on microbial sequences in GISAID and NCBI databases. The reaction was optimized, and the limit of detection was determined. The results showed that the primer set targeting ORF8 exhibited > 98 % homologous to the sequence of SARS-CoV-2 and < 80 % to those of other microbes. The optimum condition of the reaction was 60 ºC for an hour, and the limit of detection for SARS-CoV-2 was 25 copies/reaction. Overall, the developed RT-LAMP was able to detect SARS-CoV-2. It requires further evaluation using clinical specimens for the determination of its applicability in COVID-19 diagnosis.
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References
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