TITLE:
PCR-HRM for Genomic Surveillance of SARS-CoV-2: A Variant Detection Tool in Côte d’Ivoire, West Africa
AUTHORS:
Aboubacar Sylla, Solange Kakou-Ngazoa, Tata Gniré Safiatou Coulibaly, Yakoura Karidja Ouattara, Mireille Sylvie Kouamé-Sina, Zeinab Ouattara, David Ngolo Coulibaly, Brice Kouakou Bla, Mireille Dosso
KEYWORDS:
Genomic Surveillance, SARS-CoV-2, PCR-HRM, Variants, Côte d’Ivoire
JOURNAL NAME:
American Journal of Molecular Biology,
Vol.14 No.3,
July
23,
2024
ABSTRACT: The rise of new viruses, like SARS-CoV-2 causing the COVID-19 outbreak, along with the return of antibiotic resistance in harmful bacteria, demands a swift and efficient reaction to safeguard the health and welfare of the global population. It is crucial to have effective measures for prevention, intervention, and monitoring in place to address these evolving and recurring risks, ensuring public health and international security. In countries with limited resources, utilizing recombinant mutation plasmid technology in conjunction with PCR-HRM could help differentiate the existence of novel variants. cDNA synthesis was carried out on 8 nasopharyngeal samples following viral RNA extraction. The P1 segment of the SARS-CoV-2 Spike S protein was amplified via conventional PCR. Subsequently, PCR products were ligated with the pGEM-T Easy vector to generate eight recombinant SARS-CoV-2 plasmids. Clones containing mutations were sequenced using Sanger sequencing and analyzed through PCR-HRM. The P1 segment of the S gene from SARS-CoV-2 was successfully amplified, resulting in 8 recombinant plasmids generated from the 231 bp fragment. PCR-HRM analysis of these recombinant plasmids differentiated three variations within the SARS-CoV-2 plasmid population, each displaying distinct melting temperatures. Sanger sequencing identified mutations A112C, G113T, A114G, G214T, and G216C on the P1 segment, validating the PCR-HRM findings of the variations. These mutations led to the detection of L452R or L452M and F486V protein mutations within the protein sequence of the Omicron variant of SARS-CoV-2. In summary, PCR-HRM is a vital and affordable tool for distinguishing SARS-CoV-2 variants utilizing recombinant plasmids as controls.