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Volume 27, Number 7—July 2021
Research

Multiplex Real-Time Reverse Transcription PCR for Influenza A Virus, Influenza B Virus, and Severe Acute Respiratory Syndrome Coronavirus 2

Bo Shu1, Marie K. Kirby1, William G. Davis, Christine Warnes, Jimma Liddell, Ji Liu, Kai-Hui Wu, Norman Hassell, Alvaro J. Benitez, Malania M. Wilson, Matthew W. Keller, Benjamin L. Rambo-Martin, Yamundow Camara, Jörn Winter, Rebecca J. Kondor, Bin Zhou, Stacey Spies, Laura E. Rose, Jonas M. Winchell, Brandi M. Limbago, David E. Wentworth2, and John R. Barnes2Comments to Author 
Author affiliations: Centers for Disease Control and Prevention, Atlanta, Georgia, USA (B. Shu, M.K. Kirby, W.G. Davis, C. Warnes, J. Liddell, J. Liu, K.-H. Wu, N. Hassell, A.J. Benitez, M.M. Wilson, M.W. Keller, B.L. Rambo-Martin, Y. Camara, J. Winter, R.J. Kondor, B. Zhou, S. Spies, L.E. Rose, J.M. Winchell, B.M. Limbago, D.E. Wentworth, J.R. Barnes); Battelle Memorial Institute, Atlanta (W.G. Davis, J. Liddell); Leidos Inc, Atlanta (Y. Camara)

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Table 2

Sensitivity of the Influenza SARS-CoV-2 Multiplex Assay compared with singleplex assays*

Viral titers† Cycle threshold value‡
Multiplex
Singleplex
A/Illinois/20/2018_(H1N1)pdm09
104.1 23.50 23.59 23.17 24.97 24.97 24.66
103.4 26.57 26.50 26.81 27.71 27.49 27.50
102.7 29.90 30.20 30.15 30.50 29.97 29.74
102.0 35.58 35.24 36.17 32.32 32.43 33.63
101.3
42.23
37.28
0

36.01
34.61
34.96
B/Colorado/06/2017_Victoria
104.3 24.47 24.44 24.31 25.80 25.68 25.93
103.6 27.57 27.45 27.71 28.84 28.98 29.30
102.9 31.09 30.17 30.47 32.10 32.10 32.38
102.2 34.38 33.49 34.43 35.19 35.49 35.99
101.5
39.75
0
0

0
0
0
2019-nCoV/USA-WA1/2020
102.4 25.41 25.8 25.42 26.48 26.57 26.46
101.7 28.69 28.87 28.5 30.26 29.77 29.51
101.0 31.31 31.42 31.32 32.74 33.17 32.27
100.3 35.14 36.36 34.58 36.10 35.34 35.81
10–0.4 0 0 0 37.16 0 0

*Multiplex PCR is selective for influenza A virus, influenza B virus, and SARS-CoV-2. Boldface type indicates limits of detection. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. †Viral titers are in relation to 50% tissue culture infectious dose, except for B/Colorado/06/2017, which is in relation to 50% egg infectious dose. ‡The multiplex cycle threshold values were derived from Influenza SARS-CoV-2 Multiplex Assay. The singleplex cycle threshold values were derived from the InfA (for influenza A), InfB (for influenza B), or N1 singleplex assays (for SARS-CoV-2). The N1 singleplex assay is a component of the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel (7). The InfA and InfB singleplex assays are components of the CDC Human Influenza Virus Real-Time RT-PCR Detection and Characterization Panel [510(k) no. K200370] (9). Each assay was performed in triplicate.

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1These first authors contributed equally to this article.

2These senior authors contributed equally to this article.

Page created: May 13, 2021
Page updated: June 16, 2021
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