In-vitro synthesis of RNA fragments specifying envelope protein gene and RdRp gene of SARS-CoV-2 as positive standards for molecular diagnosis tests

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19), has rapidly spread through entire world and become worst pandemic from December until now. The establishment positive standards for molecular diagnostic testing SARS-CoV-2 plays a critical role in development assessment assays associated with shortage specimens viral culture fluids. This study aims to establish novel assay in-vitro RNA fragment synthesis based on amplicons self-priming PCR targeting envelope protein gene RNA-dependent polymerase (RdRp) SARS-CoV-2. cDNA library targeted genes was generated by using long-primers named sMn1 forward/reverse primer (E gene) sMn2 (RdRp assays. synthesised that overlap target sequence World Health Organization (WHO) were cloned into pGEM-T easy vector, then transformed E. coli competent cells conventional methods. recombinant plasmids used as materials transcription. Concentrations transcribed 200-800 ng/μl A260/A280 ratios 2.0-2.2. Gel electrophoresis showed single band each molecule sizes 216 214 bases sMn1- E sMn2- RdRp gene, respectively. Furthermore, we effectively evaluated one-step, real-time RT-PCR according standard WHO protocol. stability over 6-month storage period investigated. In conclusion, our fragments successfully established thus these useful diagnosing