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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)

Main Article

Table 5

Evaluation of the Influenza SARS-CoV-2 Multiplex Assay specificity*

Virus strain Lineage GISAID accession no. Con† Cycle threshold value
Influenza A
 Influenza B
 SARS-CoV-2
Influenza A
A/Florida/81/2018 A(H1N1) pdm09 EPI1310819 108.1 13.97 14.00 14.02 0 0 0 0 0 0
103.1 27.48 28.16 27.74 0 0 0 0 0 0
A/Kansas/14/2017 A(H3N2) EPI1653963 108.5 13.62 13.66 13.68 0 0 0 0 0 0
105.5 25.07 25.00 25.01 0 0 0 0 0 0
A/Ohio/35/2017 A(H1N2)v EPI1056728 106.9 14.71 14.90 14.84 0 0 0 0 0 0
101.9 30.91 31.25 30.99 0 0 0 0 0 0
A/chicken/Pennsyl-
vania/298101-
4/2004 A(H2N2) EPI229365 109.5 15.60 15.66 15.74 0 0 0 0 0 0
103.5 33.40 33.20 34.71 0 0 0 0 0 0
A/Ohio/13/2017 A(H3N2)v EPI1056648 106.6 20.85 20.96 20.86 0 0 0 0 0 0
101.6 35.48 35.49 33.98 0 0 0 0 0 0
A/canine/Florida/
43/2004 A(H3N2) 108.1 19.61 19.69 19.44 0 0 0 0 0 0
EPI98471 104.1 34.10 35.45 36.87 0 0 0 0 0 0
A/equine/Ohio/01/
2003 A(H3N8) DQ124188§ 108.4 16.50 16.70 16.68 0 0 0 0 0 0
103.4 31.01 31.55 31.07 0 0 0 0 0 0
A/Northern 
pintail/Washington
/40964/2014 A(H5N2) EPI860995 109.4 16.39 16.43 16.49 0 0 0 0 0 0
104.4 36.89 36.45 34.43 0 0 0 0 0 0
A/gyrfalcon/Wash-
ington/41088-
6/2014 A(H5N8) EPI569393 109.75 14.12 14.10 14.13 0 0 0 0 0 0
104.75 29.60 29.38 29.90 0 0 0 0 0 0
A/chicken/Califor-
nia/32213–1/2000		 A(H6N2) EPI1915583 109.2 14.97 15.05 15.08 0 0 0 0 0 0
103.2 34.19 32.52 32.49 0 0 0 0 0 0
A/feline/New 
York/16-040082-
1/2016 A(H7N2) EPI985440 1010.2 15.76 15.94 16.00 0 0 0 0 0 0
105.2 28.16 28.30 28.46 0 0 0 0 0 0
A/Taiwan/1/2017 A(H7N9) EPI917065 109.5 16.69 16.90 16.99 0 0 0 0 0 0
103.5 32.20 33.11 32.53 0 0 0 0 0 0
A/Bangladesh/
0994/2011
 A(H9N2)
 EPI445991 1010.5 18.03 18.14 18.21 0 0 0 0 0 0
104.5
 34.25
 35.32
 36.90
0
 0
 0
0
 0
 0
Influenza B
B/Maryland/15/
2016 B(Victoria) EPI1255266 108.5 0 0 0 13.46 13.49 13.47 0 0 0
102.5 0 0 0 30.82 31.07 31.20 0 0 0
B/Phuket/3073/
2013
 B (Yamagata)
 EPI1799818 108.9 0 0 0 13.66 13.67 13.68 0 0 0
102.9
 0
 0
 0
31.87
 31.77
 32.18
0
 0
 0
SARS-CoV-2
2019-nCoV/USA-
WA1/2020 Beta-coronavirus MT 576563 104.5 0 0 0 0 0 0 18.34 18.55 18.41
100.5 0 0 0 0 0 0 33.11 34.15 34.88

*Multiplex real-time reverse transcription PCR for influenza A virus, influenza B virus, and SARS-CoV-2. Evaluation conducted in triplicate. Con, concentration; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. ‡Concentration in relation to 50% tissue culture infectious dose. §GenBank accession number.

Main Article

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
Page reviewed: June 16, 2021
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