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