Abstract
Extensive transmission of SARS-CoV-2 during the COVID-19 pandemic allowed the generation of thousands of mutations within its genome. While several of these become rare, others largely increase in prevalence, potentially jeopardizing the sensitivity of PCR-based diagnostics. Taking advantage of SARS-CoV-2 genomic knowledge, we designed a one-step probe-based multiplex RT-qPCR (OmniSARS2) to simultaneously detect short fragments of the SARS-CoV-2 genome in ORF1ab, E gene and S gene. Comparative genomics of the most common SARS-CoV-2 lineages, other human betacoronavirus and alphacoronavirus, was the basis for this design, targeting both highly conserved regions across SARS-CoV-2 lineages and variable or absent in other Coronaviridae viruses. The highest analytical sensitivity of this method for SARS-CoV-2 detection was 94.2 copies/mL at 95% detection probability (~1 copy per total reaction volume) for the S gene assay, matching the most sensitive available methods. In vitro specificity tests, performed using reference strains, showed no cross-reactivity with other human coronavirus or common pathogens. The method was compared with commercially available methods and detected the virus in clinical samples encompassing different SARS-CoV-2 lineages, including B.1, B.1.1, B.1.177 or B.1.1.7 and rarer lineages. OmniSARS2 revealed a sensitive and specific viral detection method that is less likely to be affected by lineage evolution oligonucleotide–sample mismatch, of relevance to ensure the accuracy of COVID-19 molecular diagnostic methods.
Highlights
The coronavirus disease 2019 (COVID-19) is caused by a positive single-strandedRNA virus from the Coronaviridae family, named Severe Acute Respiratory SyndromeCoronavirus 2 (SARS-CoV-2)
Some of these mutations become rare while others reached high frequencies in the population. This raised a diagnostic challenge requiring the redesign of the oligonucleotide sequences used in reverse transcription (RT)-qPCR assays to circumvent the oligonucleotide–sample mismatches caused by the mutations
To maximize the sensitivity and specificity of the method the design of the onestep multiplex RT-qPCR assay was based on the combination of a comparative genomics approach and a widely validated primer design algorithm [19]
Summary
The coronavirus disease 2019 (COVID-19) is caused by a positive single-strandedRNA virus from the Coronaviridae family, named Severe Acute Respiratory SyndromeCoronavirus 2 (SARS-CoV-2). The detection of SARS-CoV-2 nucleic acids by reverse transcription (RT) and quantitative polymerase chain reaction (RT-qPCR) is the current gold standard COVID-19 diagnostic method. This virus transposed the species barrier and Biomedicines 2021, 9, 1314. Some of these mutations become rare while others (with neutral or advantageous consequences for the virus) reached high frequencies in the population This raised a diagnostic challenge requiring the redesign of the oligonucleotide sequences used in RT-qPCR assays to circumvent the oligonucleotide–sample mismatches caused by the mutations. Every mismatch, regardless of the nucleotide or its position in the primer or template, could decrease the thermal stability of the oligonucleotide–template duplex This phenomenon has the potential to affect polymerization efficiency resulting in biased RT-qPCR results or reaction failure
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