Scientific Reports (Jul 2022)

Direct PCR with the CDC 2019 SARS-CoV-2 assay: optimization for limited-resource settings

  • Christia M. Victoriano,
  • Megan E. Pask,
  • Nicole A. Malofsky,
  • Adam Seegmiller,
  • Steve Simmons,
  • Jonathan E. Schmitz,
  • Frederick R. Haselton,
  • Nicholas M. Adams

DOI
https://doi.org/10.1038/s41598-022-15356-7
Journal volume & issue
Vol. 12, no. 1
pp. 1 – 12

Abstract

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Abstract PCR-based diagnostics generally require nucleic acid extraction from patient specimens prior to amplification. As highlighted early in the COVID-19 pandemic, extraction steps may be difficult to scale during times of massive demand and limited reagent supply. Forgoing an extraction step, we previously reported that the N1 primer/probe-set of the widespread CDC COVID-19 assay maintains high categorical sensitivity (95%) and specificity (100%) with direct inoculation of viral transport media (VTM) into qRT-PCR reactions. In contrast, the N2 set demonstrated a prominent Ct delay and low sensitivity (33%) without extraction. In the current study, we have improved the performance of this modified CDC assay (in particular the N2 set) by incorporating N1/N2/RNase P multiplexing and dissecting the effects of annealing temperature, VTM interference, and inoculum volume. The latter two factors exerted a more prominent effect on the performance of N2 than N1, although these effects were largely overcome through elevated annealing temperature. This unextracted/multiplex protocol was evaluated with 41 SARS-CoV-2 positive and 43 negative clinical samples, demonstrating a categorical sensitivity of 92.7% and specificity of 100% versus the unmodified CDC methodology. Overall, this work offers a generalizable strategy to maximize testing capabilities for COVID-19 or other emerging pathogens when resources are constrained.