Molecular Characterization of SARS-CoV-2 and Evaluation of Extraction-Free RT PCR Detection Methods for the Development of an Alternative and Affordable Diagnostic Tools
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Date
2025
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Addis Ababa Universtity
Abstract
Background: SARS-CoV-2, the etiological agent of COVID-19, first emerged in Wuhan, China, and then disseminated globally at an extraordinary pace. It is classified as a single-stranded, positive-sense RNA virus within the Coronaviridae family and the Betacoronavirus genus. March 2nd, 2025, it has resulted in over 777 million confirmed cases and more than 7 million deaths worldwide. In Africa, over 12 million cases and 256,542 deaths have been reported, with Ethiopia reporting more than 500,000 cases and 7,574 deaths. The primary diagnostic method used for identifying infection is real-time reverse transcription quantitative PCR (RT-qPCR), which is recognized as the gold standard. In Ethiopia the number of SARS-CoV-2 sequences that represented the different pandemic episode and submitted to GISAID is very small compared to other African countries. In Addition, its detection was challenged by limited access to essential laboratory supplies, including RNA extraction kits, alongside the ongoing emergence of new variants. These factors highlight the continuous genomic surveillance of SARS-CoV-2 is essential for tracking of viral mutations, informs public health interventions, and aids in the development of effective diagnostics, vaccines, and therapeutic strategies; and demanding need for the development of an alternative diagnostic methods that are accurate, cost-effective, rapid, and less reliant on imported resources.
Objectives: The study aimed to characterizes circulating SARS-CoV-2 variants during the fifth wave of the pandemic in Ethiopia and identify conserved gene regions through whole genome sequencing, as well as to evaluate the diagnostic performance of an extraction-free RT-qPCR method as a simplified approach for SARS-CoV-2 detection.
Methods: 150 SARS-CoV-2 positive Ct-value of <30) samples were retrieved from the national reference repository of Ethiopian Public Health Institute for whole genome sequencing. These samples were primarily collected from COVID-19 suspected cases for clinical management purpose during the fifth wave of the pandemic (June to August 2022). These samples were retested, and 70 samples that maintained a Ct value ≤30 were selected for whole genome sequencing on the Illumina NextSeq 550 platform. Of the 70 sequenced samples, 63 yielded high-quality genomes that successfully passed all bioinformatics quality control assessments. These genomes
were analyzed for variant classification, single-nucleotide polymorphism (SNP) distances, phylogenetic relationships, and conserved gene regions identification. The analytical workflow incorporated tools such as FastQC, Cutadapt, BWA, Samtools, Pangolin, Nextclade, SNP-dists, MAFFT, and IQ-TREE. Metadata and sequence data were analyzed using STATA v17. In addition, three hundred stored nasopharyngeal specimens (190 SARS-CoV-2 positive and 110 negative) were randomly selected from the national repository. The samples were processed using the standard RNA extraction kits (used as the gold standard) and an extraction-free method that involved dilution and heating (EFDH). In the EFDH protocol, samples were diluted at a 1:2 ratio with RNase-free water and subjected to heating at 72°C for 15 minutes. Then, after master mixing, RT-qPCR was performed using DAAN kits targeting the N and ORF1ab genes by the ABI 7500 platform. The results were stratified by viral load: Ct <20, 20–35, and >35 and diagnostic performance was analyzed by using R-Studio and STATA v17.Simultaneously, 150 SARS-CoV-2 positive samples (Ct ≤30) collected during the fifth wave of the pandemic in Ethiopia for genomic analysis.
Results: Among the 70 samples sequenced, 63 met the quality control criteria, exhibiting a genome completeness of 99.8%. The Omicron variant was the predominant strain, accounting for 97% of the samples, while the Delta variant was present in 3% of the samples. Phylogenetic analysis revealed five principal clades. On average, 69 single-nucleotide variations (SNVs) were identified per genome. Mutations were detected within the S, M, and N genes, as well as in nonstructural proteins, with notable deletions observed in the S gene. Additional mutations were identified in PLpro and 3CLpro, and the transition/transversion (Ti/Tv) ratio was calculated to be 1.97. The sequences from Ethiopian samples exhibited low divergence (9.78×10⁴) in comparison to global sequences (1.54×10³). Two conserved regions within the nucleocapsid (N) gene were identified, encompassing amino acids 45–180 and 247–364. Three linear B-cell epitopes, residues 56–57, 160–180, and 255–275, were located within these regions. These epitopes displayed strong antigenic properties, low variability, and surface accessibility, indicating their potential as targets for future diagnostic applications.The EFDH method demonstrated a sensitivity of 86% and a specificity of 100% when compared to the standard RT-qPCR assay. It accurately identified 163 out of 190 positive samples and all 110 negative samples. The average Ct value recorded for EFDH was 27.7, in contrast to 24.1 for the standard method. A strong correlation between the two methodologies was observed (R² = 0.96, p = 0.001). EFDH exhibited 100% sensitivity for samples with Ct values less than 20 and 98% sensitivity for those with Ct values ranging from 20 to 35. However, sensitivity significantly decreased for samples with Ct values greater than 35. The overall diagnostic accuracy of the EFDH method was determined to be 91%, with a kappa value of 0.82 (p < 0.001), indicating a strong agreement with the standard method for samples with high viral loads.
Conclusions: The genomic analyses revealed that the Omicron variant was the predominant strain during Ethiopia's fifth wave, suggesting ongoing community transmission and the imperative for sustained surveillance efforts. The nucleocapsid protein was validated as a stable and immunogenic diagnostic target, with identified epitope candidates facilitating future diagnostic advancements. The integration of genomic and structural data may enhance the identification of reliable diagnostic markers in the context of viral evolution. The EFDH method exhibited high sensitivity and specificity for the detection of SARS-CoV-2 in samples with elevated viral loads, demonstrating a strong correlation with the established RT-qPCR method. Nevertheless, its diminished sensitivity in cases of low viral load underscores the necessity for confirmatory testing. EFDH presents an alternative and cost-effective that suitable for resource-limited countries.
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SARS-CoV-2 genetic divergence whole genome sequencing EFDH method Ethiopia.