In Vitro Synthesis Of SARS-Cov-2 Nucleocapsid Gene RNA

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, which was initially discovered in December 2019 in Wuhan, China, is the main cause of the quickly growing COVID-19 pandemic. This virus has affected approximately 608.3 million individuals as of January 2022, however since the severity of the symptoms varies, it is difficult to determine the total infection rate. Rapid and precise diagnostics are required to properly detect and stop the spread of COVID- 19. Currently, quantitative reverse transcription PCR (RT-qPCR) is considered a standard method to detect the presence of viral RNA in the patient sample. However, human error and the shelf life of enzymes used for template amplification may affect how effectively these molecular approaches work. Using a surrogate template as a positive control along with clinical samples is a vital step in the validation process to avoid false negative findings. One of the many positive controls that can be used in nucleic acid-based experiments today is in vitro RNA. The objective of this study is to empower researchers to take an active role in identifying the source of positive control samples, laying the foundation for the design and technical evaluation, and going beyond the strict biosafety regulations. To sum up, this study has prepared the appropriate reagents and enzymes to transcribe RNA in vitro, however, the result can not verify the success in creating the RNA since cross-contamination happened during the handling process.