Construction of SARS-CoV-2 E gene plasmids for development of an in-house method for quantification of viral RNA in patient samples

Started as a local outbreak in December 2019, SARS-CoV-2 infection has risen into the global pandemic with over 65 million cases of infection and 1.5 million deaths. Although timely set diagnosis allows preventing further transmission of the infection, increasing demand for high-throughput COVID-19 diagnostic testing has resulted in reagent shortage. The aim of the study: To contribute to research for developing COVID-19 testing, we aimed to construct SARS-CoV-2 E gene artificial plasmids and to use plasmid constructs in the development of an in-house RT-PCR assay for quantification of SARS-CoV-2 RNA in patient samples. The RNA isolated from nasal swabs of patients (n=5) with confirmed SARS-CoV-2 infection were received from the national biobank and used as a template for plasmid construction. The cDNA synthesis followed by E gene PCR was used to prepare amplicons for plasmid construction and cloning into E.coli XL-1 Blue cells. The colony PCR and subsequent product sequencing were performed to confirm plasmid inserts. The recombinant plasmids were purified, serially diluted, and used to evaluate the linearity and efficiency of the RT-PCR assay. The analytical sensitivity of the assay was determined by analysis of replicate samples (n=20) containing 3, 5, and 10 gene copies/per well. The 200 bp long E gene amplicons were obtained from 4/5 clinical samples. The colony PCR followed by product sequencing enabled the selection of 15 bacterial colonies containing valid plasmid constructs. The E gene-based RT-PCR assay was tested within the range 10-1000000 gene copies/per well and exhibited acceptable linearity (r2≥0.997) and efficiency (95.8-104.9%). The lower limit of quantification (LLOQ) was 5 gene copies/per reaction. A time- and cost-effective solution was found in response to reference material supply shortage and applied in the development of the in-house SARS-CoV-2 RT-PCR assay.