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bioRxiv · 10.1101/2022.10.06.511205

Type-specific EV-D68 real-time RT-PCR assay for the detection of all extant enterovirus D68 strains

Ng, T. F. F.·Nix, W. A.·Rogers, S.·Emery, B.·Chern, S.-W.·Butler, K.·Oberste, M. S.
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Abstract

Enterovirus D68 (EV-D68) has caused recurring respiratory disease outbreaks in the United States since 2014. The dominant circulating EV-D68 strain has evolved from clade B1 to the more recent B2 and B3 clades. As recurrent outbreaks and continued virus evolution are expected for EV-D68, a robust real-time PCR assay that detects known strains as well as potential emerging strains is critical for national surveillance and clinical diagnostics. We describe a type-specific EV-D68 real-time RT-PCR (rRT-PCR) assay termed CDC2022, which targets sequences encoding conserved amino acid regions of all extant EV-D68 strains. We targeted three motifs conserved among all strains in the last 60 years. The assay achieved 100% (270/270) sensitivity and 100% (344/344) specificity when tested with a collection of 613 respiratory specimens, compared to the gold-standard EV semi-nested VP1 PCR and sequencing assay (snPCR/Seq). CDC2022 gave negative results with 289/289 non-target viruses, including 104 EV A-D isolates, 165 rhinovirus (RV) isolates or clinical specimens, and 14 other common respiratory viruses. The assay can detect as few as 0.28 CCID50 per reaction. An in silico "phylo-primer-mismatch" analysis was performed to visualize primer/probe mismatches and to compare CDC2022 with other EV-D68 rRT-PCR assays, including the previous CDC assay (CDC2015) developed in 2014 for clade B1 strains. It showed that CDC2022 has the fewest primer/probe mismatches among all assays analyzed and is suitable for all clades. We additionally tested 11 EV-D68-positive clinical specimens from 2022 that were confirmed by snPCR/Seq, and all were detected. CDC2022 assay could provide a critical tool for molecular surveillance of EV-D68.

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Ng, T. F. F., Nix, W. A., Rogers, S., Emery, B., Chern, S.-W., Butler, K., Oberste, M. S.. 2022-10-06. Type-specific EV-D68 real-time RT-PCR assay for the detection of all extant enterovirus D68 strains. https://doi.org/10.1101/2022.10.06.511205

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