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

Updated: Apr 02, 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 recent emergence of SARS-CoV-2 strains with potentially significant genetic variations, certain nucleic acid detection assays could be impacted. The recently described strains include the B.1.1.7. variant, which was originally discovered in the United Kingdom but has since been identified in other countries, including the United States. [19] A variant of SARS-CoV-2 initially found in South Africa, known as 20C/501Y.V2, or B.1.351 lineage, shares some of the B.1.1.7 lineage’s mutations, despite having developed independently of it. Since its emergence, the B.1.351 lineage has been detected outside of South Africa. These different variants may contain substitutions or deletions that 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 testing a genetic variant. [20] However, while individual gene targets 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.

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