S-Gene Target Failure and How PCR Works

The SARS-CoV-2 Omicron variant (B.1.1.529) was first reported to WHO on November 24, 2021 and was classified as a variant of concern two days later [1]. The Omicron variant has more than 60 mutations as compared to the ancestral strain, many of which affect the spike (S) protein and its receptor binding domain (RBD) [2]. These mutations, which contribute to Omicron’s higher degree of contagiousness, can also affect the diagnostic tools designed to detect the presence of the virus in a sample, specifically via S-gene target failure in PCR tests. The possibility of less effective detection is also a concern for any future variants with further mutations.

The most accurate method for detecting SARS-CoV-2 is reverse-transcriptase polymerase chain reaction (RT-PCR), which amplifies viral RNA into many copies of DNA so that it can be detected and identified. A fundamental tool of molecular biology, PCR uses a nucleic acid template (DNA or RNA), DNA primers, free nucleotides, and a polymerase enzyme. PCR involves three steps that are repeated throughout the reaction: denaturation of the template into single strands, annealing of primers to each strand, and synthesis of complementary DNA molecules beginning from the primers [3].

The PCR solution is first heated so that double-stranded DNA denatures, or separates, into single strands. This allows the DNA primers, which are short sequences of DNA, to anneal complementarily to specific regions of the template DNA. Primers are designed to bind upstream, or before, the DNA or RNA sequence of interest, which allows the polymerase enzyme to then bind to the primers and amplify the sequence of interest by attaching free nucleotides to form double-stranded DNA. Each iteration of PCR doubles the amount of DNA, allowing for the amplification of even a small amount of starting material; after 30 cycles of PCR, one molecule of viral RNA can be amplified into over 500 million molecules of double-stranded DNA, all of which contain the same sequence. Diagnostic PCR assays, such as the RT-PCR method used to detect SARS-CoV-2, also involve a probe or dye that allows for the detection of the suspected template [4].

PCR primers are designed to amplify only one particular stretch of genetic material. The primers for a COVID-19 PCR detection test, therefore, will complement certain sequences from the viral genome of SARS-CoV-2. Some assays, such as Thermo Fisher Scientific’s Taqpath Kit, target the S protein, but this method fails to detect it in Omicron infections because the primers can only bind to a particular version of the S protein sequence and Omicron has a mutation (69-70del) in this sequence that interferes with binding [5]. S-gene target failure, however, can actually be advantageous, because most PCR assays, including Taqpath, use multiple SARS-CoV-2 targets, such as its nucleocapsid (N) protein, whose sequence is not significantly mutated in Omicron [6]. The assay will therefore detect the presence of SARS-CoV-2 but will report the absence of the original S protein sequence, signifying the presence of the Omicron variant.

The phenomenon of S-gene target failure reflects the importance of having multiple targets in a PCR diagnostic assay. As SARS-CoV-2 continues to evolve, there may emerge mutations in target proteins that render assays obsolete. One approach to this problem could therefore be to identify other SARS-CoV-2 proteins that can be targeted in diagnostic assays. For example, Wang et al. reported that SARS-CoV-2 orf8 protein can be detected in an RT-PCR assay [7]; as COVID-19 continues to spread, assays such as these could prove helpful in detecting the evolving virus. In the meantime, encouraging data suggest that vaccines continue to be effective in reducing the risk of serious disease, especially after a booster dose.

 

References 

 

  1. Classification of Omicron (B.1.1.529): SARS-CoV-2 Variant of Concern. https://www.who.int/news/item/26-11-2021-classification-of-omicron-(b.1.1.529)-sars-cov-2-variant-of-concern.
  2. He, X., Hong, W., Pan, X., Lu, G. & Wei, X. SARS-CoV-2 Omicron variant: Characteristics and prevention. MedComm 2, 838–845 (2021).
  3. Delidow, B. C., Lynch, J. P., Peluso, J. J. & White, B. A. Polymerase chain reaction : basic protocols. Methods Mol. Biol. Clifton NJ 15, 1–29 (1993).
  4. Introduction to PCR Primer & Probe Chemistries. Bio-Rad Laboratories https://www.bio-rad.com/en-us/applications-technologies/introduction-pcr-primer-probe-chemistries?ID=LUSOJW3Q3.
  5. Metzger, C. M. J. A. et al. PCR performance in the SARS-CoV-2 Omicron variant of concern? Swiss Med. Wkly. (2021) doi:10.4414/smw.2021.w30120.
  6. Thermo Fisher Scientific Confirms Detection of SARS-CoV-2 in Samples Containing the Omicron Variant with its TaqPath COVID-19 Tests. MediaRoom https://thermofisher.mediaroom.com/2021-11-29-Thermo-Fisher-Scientific-Confirms-Detection-of-SARS-CoV-2-in-Samples-Containing-the-Omicron-Variant-with-its-TaqPath-COVID-19-Tests.
  7. Wang, X. et al. Accurate Diagnosis of COVID-19 by a Novel Immunogenic Secreted SARS-CoV-2 orf8 Protein. mBio (2020) doi:10.1128/mBio.02431-20.