Which genes are targeted in coronavirus disease 2019 (COVID-19) diagnostic testing, and how do SARS-CoV-2 variants affect test accuracy?

Updated: Jun 18, 2021
  • Author: James J Dunn, PhD, D(ABMM), MT(ASCP); more...
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Some of the most frequently tested gene targets for the detection of SARS-CoV-2 include the E, S, and N genes and the open reading frame ORF1a/1b. [14] While some gene targets may also detect SARS-CoV-1, which is no longer known to be circulating in the human population, there is little cross-reactivity with the endemic coronaviruses that are associated with the common cold. [14, 15]  Detection of the E gene appears to have the highest analytic sensitivity, [16]  with detection capabilities for this gene being far below the estimated viral load for SARS-CoV-2–positive patients; [17] NAAT assays can detect as few as 10 copies per reaction. [18] However, different NAAT systems vary in terms of clinical sensitivity, independent of the gene detected. [14] In addition to detecting SARS-CoV-2, many NAATs have been approved as multiplex assays, which can simultaneously detect other respiratory viruses such as influenza A and B, as well as bacteria that cause atypical pneumonia. [9]

With the emergence of SARS-CoV-2 strains with potentially significant genetic variations, certain nucleic acid detection assays could be impacted. There are currently multiple nomenclature systems to describe variants of SARS-CoV-2, but two of the more common ones are the phylogenetic assignment of named global outbreak (PANGO) and World Health Organization (WHO) systems. [19, 20, 21] The latter is based on the Greek alphabet to facilitate communication with non-scientific personnel. Two ways that the CDC and WHO categorize variants are as variants of interest and variants of concern. [19] There are currently five variants of concern in circulation in the United States: Alpha, Beta, Gamma, Delta, and Epsilon (when using the WHO nomenclature). [19]

Some variants of concern, particularly the Alpha variant, may contain a significant number of substitutions and/or deletions in the spike protein (S gene), which can impact the ability of an RNA-based assay to detect the virus if the primer and/or probe sequences are in the affected region(s). The FDA has released a statement that outlines some nucleic acid detection tests that may be impacted when they are used to test a genetic variant. [22, 23] However, while individual gene targets (namely, the S gene) in an assay may be falsely negative due to the presence of substitutions or deletions, the assay’s overall sensitivity may remain unaffected, since most incorporate detection of multiple gene targets. Laboratories are encouraged to reach out to the manufacturers of the assays in use to determine if any of the mutations present in the newly circulating strains are likely to affect test performance. To date, there is little evidence that the nucleotide substitutions and/or deletions in the Beta, Gamma, Delta, or Epsilon variant have an impact on the performance of diagnostic RNA detection assays. [24]


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