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J Clin Microbiol ; 60(4): e0228321, 2022 04 20.
Article in English | MEDLINE | ID: covidwho-1759279


Tools to detect SARS-CoV-2 variants of concern and track the ongoing evolution of the virus are necessary to support public health efforts and the design and evaluation of novel COVID-19 therapeutics and vaccines. Although next-generation sequencing (NGS) has been adopted as the gold standard method for discriminating SARS-CoV-2 lineages, alternative methods may be required when processing samples with low viral loads or low RNA quality. To this aim, an allele-specific probe PCR (ASP-PCR) targeting lineage-specific single nucleotide polymorphisms (SNPs) was developed and used to screen 1,082 samples from two clinical trials in the United Kingdom and Brazil. Probit regression models were developed to compare ASP-PCR performance against 1,771 NGS results for the same cohorts. Individual SNPs were shown to readily identify specific variants of concern. ASP-PCR was shown to discriminate SARS-CoV-2 lineages with a higher likelihood than NGS over a wide range of viral loads. The comparative advantage for ASP-PCR over NGS was most pronounced in samples with cycle threshold (CT) values between 26 and 30 and in samples that showed evidence of degradation. Results for samples screened by ASP-PCR and NGS showed 99% concordant results. ASP-PCR is well suited to augment but not replace NGS. The method can differentiate SARS-CoV-2 lineages with high accuracy and would be best deployed to screen samples with lower viral loads or that may suffer from degradation. Future work should investigate further destabilization from primer-target base mismatch through altered oligonucleotide chemistry or chemical additives.

COVID-19 , SARS-CoV-2 , Alleles , COVID-19/diagnosis , Humans , Polymerase Chain Reaction , SARS-CoV-2/genetics
Science ; 372(6539)2021 04 16.
Article in English | MEDLINE | ID: covidwho-1125076


Extensive global sampling and sequencing of the pandemic virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have enabled researchers to monitor its spread and to identify concerning new variants. Two important determinants of variant spread are how frequently they arise within individuals and how likely they are to be transmitted. To characterize within-host diversity and transmission, we deep-sequenced 1313 clinical samples from the United Kingdom. SARS-CoV-2 infections are characterized by low levels of within-host diversity when viral loads are high and by a narrow bottleneck at transmission. Most variants are either lost or occasionally fixed at the point of transmission, with minimal persistence of shared diversity, patterns that are readily observable on the phylogenetic tree. Our results suggest that transmission-enhancing and/or immune-escape SARS-CoV-2 variants are likely to arise infrequently but could spread rapidly if successfully transmitted.

COVID-19/transmission , COVID-19/virology , Genetic Variation , SARS-CoV-2/genetics , COVID-19/immunology , Coinfection/virology , Coronavirus Infections/virology , Coronavirus OC43, Human , Family Characteristics , Genome, Viral , Humans , Immune Evasion , Mutation , Phylogeny , RNA, Viral/genetics , RNA-Seq , SARS-CoV-2/pathogenicity , SARS-CoV-2/physiology , Selection, Genetic , Spike Glycoprotein, Coronavirus/genetics , United Kingdom , Viral Load