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

Autor: Christia M. Victoriano, Megan E. Pask, Nicole A. Malofsky, Adam Seegmiller, Steve Simmons, Jonathan E. Schmitz, Frederick R. Haselton, Nicholas M. Adams
Jazyk: angličtina
Rok vydání: 2022
Předmět:
Zdroj: Scientific Reports, Vol 12, Iss 1, Pp 1-12 (2022)
Druh dokumentu: article
ISSN: 2045-2322
DOI: 10.1038/s41598-022-15356-7
Popis: 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.
Databáze: Directory of Open Access Journals
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