Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR.

Autor: Perchetti GA; Department of Laboratory Medicine, Virology Division, University of Washington, Seattle, WA, United States., Nalla AK; Department of Laboratory Medicine, Virology Division, University of Washington, Seattle, WA, United States., Huang ML; Department of Laboratory Medicine, Virology Division, University of Washington, Seattle, WA, United States., Jerome KR; Department of Laboratory Medicine, Virology Division, University of Washington, Seattle, WA, United States; Vaccine and Infectious Diseases Division, Fred Hutchinson Cancer Research Center, Seattle, WA, United States., Greninger AL; Department of Laboratory Medicine, Virology Division, University of Washington, Seattle, WA, United States; Vaccine and Infectious Diseases Division, Fred Hutchinson Cancer Research Center, Seattle, WA, United States. Electronic address: agrening@uw.edu.
Jazyk: angličtina
Zdroj: Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology [J Clin Virol] 2020 Aug; Vol. 129, pp. 104499. Date of Electronic Publication: 2020 Jun 08.
DOI: 10.1016/j.jcv.2020.104499
Abstrakt: Background: The novel respiratory virus SARS-CoV-2, responsible for over 380,000 COVID-19 related deaths, has caused significant strain on healthcare infrastructure and clinical laboratories globally. The pandemic's initial challenges include broad diagnostic testing, consistent reagent supply lines, and access to laboratory instruments and equipment. In early 2020, primer/probe sets distributed by the CDC utilized the same fluorophore for molecular detection - requiring multiple assays to be run in parallel - consuming valuable and limited resources.
Methods: Nasopharyngeal swabs submitted to UW Virology for SARS-CoV-2 clinical testing were extracted, amplified by our laboratory developed test (LDT) - a CDC-based quantitative reverse transcriptase PCR reaction - and analyzed for agreement between the multiplexed assay. Laboratory- confirmed respiratory infection samples were included to evaluate assay cross-reaction specificity.
Results: Triplexing correctly identified SARS-CoV-2 in 98.4% of confirmed positive or inconclusive patient samples by single-plex LDT (n = 183/186). All 170 SARS-CoV-2 negative samples tested by single-plex LDT were negative by triplexing. Other laboratory-confirmed respiratory infections did not amplify for SARS-CoV-2 in the triplex reaction.
Conclusions: Multiplexing two virus-specific gene targets and an extraction control was found to be comparable to running parallel assays independently, while significantly improving assay throughput.
Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest.
(Copyright © 2020 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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