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Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR.
J Clin Virol. 2020 08; 129:104499.JC

Abstract

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.

Authors+Show Affiliations

Department of Laboratory Medicine, Virology Division, University of Washington, Seattle, WA, United States.Department of Laboratory Medicine, Virology Division, University of Washington, Seattle, WA, United States.Department of Laboratory Medicine, Virology Division, University of Washington, Seattle, WA, United States.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.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.

Pub Type(s)

Evaluation Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

32535397

Citation

Perchetti, Garrett A., et al. "Multiplexing Primer/probe Sets for Detection of SARS-CoV-2 By QRT-PCR." Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology, vol. 129, 2020, p. 104499.
Perchetti GA, Nalla AK, Huang ML, et al. Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR. J Clin Virol. 2020;129:104499.
Perchetti, G. A., Nalla, A. K., Huang, M. L., Jerome, K. R., & Greninger, A. L. (2020). Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR. Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology, 129, 104499. https://doi.org/10.1016/j.jcv.2020.104499
Perchetti GA, et al. Multiplexing Primer/probe Sets for Detection of SARS-CoV-2 By QRT-PCR. J Clin Virol. 2020;129:104499. PubMed PMID: 32535397.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR. AU - Perchetti,Garrett A, AU - Nalla,Arun K, AU - Huang,Meei-Li, AU - Jerome,Keith R, AU - Greninger,Alexander L, Y1 - 2020/06/08/ PY - 2020/06/04/received PY - 2020/06/07/accepted PY - 2020/6/15/pubmed PY - 2020/8/12/medline PY - 2020/6/15/entrez KW - COVID-19 KW - LDT KW - Multiplex KW - RT-PCR KW - SARS-CoV-2 KW - Triplex SP - 104499 EP - 104499 JF - Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology JO - J Clin Virol VL - 129 N2 - 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. SN - 1873-5967 UR - https://www.unboundmedicine.com/medline/citation/32535397/Multiplexing_primer/probe_sets_for_detection_of_SARS_CoV_2_by_qRT_PCR_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1386-6532(20)30241-9 DB - PRIME DP - Unbound Medicine ER -