PRIMERS DESIGN FOR GENE N AMPLIFICATION OF SARS-COV-2 USING THE VIRUS GENOMES OF ARGENTINE PATIENTS

REYES, SARITA ISABEL; SAID ADAMO, MARÍA DEL MILAGRO; CORBALAN, NATALIA; CRISTÓBAL, HÉCTOR ANTONIO

Congreso; LVII SAIB Meeting - XVI SAMIGE Meeting; 2021

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was associated with pandemic disease in 2019 (COVID19). The high diagnostic demand has affected the supply of reagents used in the molecular detection workflow for COVID19. Knowing the nucleotide sequences of genes in SARS-CoV-2, allows us to improve, increase and readjust the diagnostic detection system, i.e. sensitivity and specificity for assays. For this purpose, bioinformatics analysis are crucial for the development of new primer and probe systems that amplify molecular markers in viruses and bacteria. The aim of this work was to design specific primers in order to amplify the complete nucleocapsid gene (gen-N) of SARS CoV-2 to analyze its sequences and detect possible variations in the strains that circulates in Salta-Argentina. Nucleotide sequences of gen-N of Argentina and Wuhan were downloaded from the Global Initiative on Sharing All Influenza Data (GISAID) database. A multiple sequence alignment was carried out with DNA-Man software to identify the consensus regions using default settings.Different primer sets were designed using Primer Express® v.3.0.1 software. The primer sets obtained were tested in silico with a Basic local Alignment Search Tool (primer-BLAST) of public databases. Inactivated clinical samples from COVID-19 positive patients in Salta city were used to obtain RNA viral using commercial kits. Retro-transcription (RT), conventional PCR and electrophoresis were performed using primes design to gen-N amplification. The results obtained with primerBLAST software show an in silico amplification of 1468 bp of gene-N in SARS-CoV-2 strains for the database of NCBI. High specificity was observed with primer designed for the virus target demonstrated by NCBI database analysis. Positive results for RT-qPCR reactions were obtained to amplify N and RNaseP genes from all samples. These results show a correct sample processing, RNA extraction and amplification of SARS-CoV-2 genes by RT-qPCR. Nevertheless, in some positive samples the amplification of the complete gen-N by RT followed by conventional PCR failed. This could be a consequence of the RNA poor integrity or variations in gene-N sequence in the region primers annealing. In this work we show a specific design of primers that amplify the complete gene that encodes the nucleocapsid of SARS-CoV-2, which will be sequenced to carry out a more specific design of the marker of the virus under study in the future.