Multiplexed Magnetofluorescent Bioplatform for the Sensitive Detection of SARS-CoV-2 Viral RNA without Nucleic Acid Amplification.

Rapid and sensitive detection of SARS-CoV-2 virus genetic material is of paramount importance to mitigate the COVID-19 pandemic outbreak and lower the death toll. Herein, we report the design of a magnetofluorescent bioplatform for the direct and specific detection of the viral RNA of SARS-CoV-2 in the total RNA extracted from nasopharyngeal swabs of COVID-19-positive patients. A higher fluorescence response was achieved using two capture probes tethered to magnetic beads using a biotin/streptavidin linkage, targeting two specific sites in the ORF1a and S genes. Two horseradish peroxidase (HRP)-conjugated reporter sequences, complementary to the loci of the S and N genes, were used to reveal the presence of the viral RNA through the oxidation of o-phenylenediamine to fluorescent 2,3-diaminophenazine. Under optimal conditions, the bioplatform showed high selectivity and sensitivity and was able to detect as low as 0.01 ng of viral RNA (1 × 103 copies/µL) with a linear dynamic range varying from 0.01 to 3.0 ng (1 × 103 to 9 × 107 copies/µL). The bioplatform was also able to discriminate the SARS-CoV-2 RNA from those of other related viruses such as hepatitis C, West Nile, measles, and non-polio viruses. Furthermore, the developed biosensor was validated in 46 clinical samples (36 COVID-19-positive patients and 10 COVID-19-negative subjects, as assessed with the gold standard RT-qPCR method). Both sensitivity and specificity of the developed method reached 100%. Finally, making such a simple and specific method available in the field, at a primary point of care, can better help the detection of SARS-CoV-2 infection in low-resource settings.

Whole genome sequencing and phylogenetic analysis of six SARS-CoV-2 strains isolated during COVID-19 pandemic in Tunisia, North Africa.

BACKGROUND: In Tunisia a first SARS-CoV-2 confirmed case was reported in March 03, 2020. Since then, an increase of cases number was observed from either imported or local cases. The aim of this preliminary study was to better understand the molecular epidemiology and genetic variability of SARS-CoV-2 viruses circulating in Tunisia and worldwide. METHODS: Whole genome sequencing was performed using NGS approach on six SARS. CoV-2 highly positive samples detected during the early phase of the outbreak. RESULTS: Full genomes sequences of six Tunisian SARS-CoV-2 strains were obtained from imported and locally transmission cases during the COVID-19 outbreak. Reported sequences were non-identical with 0.1% nucleotide divergence rate and clustered into 6 different clades with worldwide sequences. SNPs results favor the distribution of the reported Tunisian sequences into 3 major genotypes. These SNP mutations are critical for diagnosis and vaccine development. CONCLUSIONS: These results indicate multiple introductions of the virus in Tunisia and add new genomic data on SARS-CoV-2 at the international level.