The development of primers and the installation of PCR for the detection of coronavirus SARS-CoV-2

The relevance of this study stems from the need to improve the accuracy and sensitivity of laboratory diagnostics for COVID-19, particularly in the early stages of infection and with low viral loads. The aim of the study was to validate and experimentally evaluate the performance of a pair of primers targeting the SARS-CoV-2 nucleocapsid gene using a plasmid positive control. The study utilised computer-aided primer design using standard criteria, testing their specificity against nucleotide sequence databases, modelling a plasmid construct with the target fragment, and performing one-step reverse transcription followed by polymerase chain reaction and agarose gel analysis of the products. Primers N9171F and N9368R, 20-22 nucleotides in length, with optimal GC content and consistent melting temperatures, were developed and characterised. Bioinformatics analysis revealed a lack of significant tendency to form stable hairpins and dimers, and confirmed minimal homology with off-target sequences, which increases amplification specificity. Recombinant plasmid DNA pJET1.2, containing a 197-bp insert of the target fragment, was used as a positive control. Visualisation of the construct map confirmed the presence of the diagnostic insert and its ease of use for reaction control. Experimental results demonstrated the production of a specific product of the expected size (197 bp) during SARS-CoV-2 RNA analysis, while the negative amplification control did not produce any, confirming the accuracy of the reaction and reducing the risk of false negative results with a valid control. The practical value of this work lies in the potential for using the proposed primer pair and plasmid control in laboratories for RT-PCR testing in laboratory diagnostics, epidemiological monitoring, and the development of diagnostic test systems for detecting the coronavirus pathogen