Assessing oligonucleotide designs from early lab developed PCR diagnostic tests for SARS-CoV-2 using the PCR_strainer pipeline

J Clin Virol. 2020 Oct;131:104581. doi: 10.1016/j.jcv.2020.104581. Epub 2020 Aug 21.


Introduction: During the first month of the SARS-CoV-2 outbreak, rapid development of PCR-based diagnostic tests became a global priority so that timely diagnosis, isolation, and contact tracing could minimize the advancing pandemic surge. Designing these tests for broad, long-term detection was complicated by limited information about the novel virus' genome sequence and how it might mutate during global spread and adaptation to humans.

Methods: We assessed eight widely adopted lab developed PCR tests for SARS-CoV-2 against 15,001 SARS-CoV-2 genome sequences. Using a custom bioinformatic pipeline called PCR_strainer, we identified all mismatches and sequence variants in genome locations targeted by 15 sets of primer/probe oligonucleotides from these assays.

Results: For 12 out of 15 primer/probe sets, over 98 % of SARS-CoV-2 genomes had no mismatches. Two primer/probe sets contained a single mismatch in the reverse primer that was present in over 99 % of genomes. One primer/probe set targeted a location with extensive polymorphisms with 23 sequence observed variants at the forward primer location. One of these variants, which contains three nucleotide mismatches, arose in February as part of the emergence of a viral clade and was present in 18.8 % of the genomes we analyzed.

Discussion: Most early PCR diagnostic tests for SARS-CoV-2 remain inclusive of circulating viral diversity, but three assays with extensive mismatches highlight assay design challenges for novel pathogens and provide valuable lessons for PCR assay design during future outbreaks. Our bioinformatics pipeline is also presented as a useful general-purpose tool for assessing PCR diagnostics assays against circulating strains.

Keywords: COVID-19; Circulating strains; Laboratory developed test; Molecular diagnostic techniques; Real-time polymerase chain reaction; SARS-CoV-2.

MeSH terms

  • Betacoronavirus / genetics*
  • COVID-19
  • COVID-19 Testing
  • Clinical Laboratory Techniques / methods*
  • Computational Biology
  • Computer Simulation
  • Coronavirus Infections / diagnosis*
  • Coronavirus Infections / virology
  • Genome, Viral
  • Humans
  • Oligonucleotides / genetics*
  • Pandemics
  • Pneumonia, Viral / diagnosis*
  • Pneumonia, Viral / virology
  • RNA, Viral
  • Real-Time Polymerase Chain Reaction / methods*
  • SARS-CoV-2
  • Sensitivity and Specificity


  • Oligonucleotides
  • RNA, Viral