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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 1

Primers and probes used in the Influenza SARS-CoV-2 Multiplex Assay*

Primer or probe
Oligonucleotide sequence, 5′→3′
Target gene or region
Nucleotide position†
Concentration, µM‡
InfA
Forward primer 1 CAA GAC CAA TCY TGT CAC CTC TGAC Matrix protein 143–167 3.33
Forward primer 2 CAA GAC CAA TYC TGT CAC CTY TGAC 143–167 3.33
Reverse primer 1 GCA TTY TGG ACA AAV CGT CTA CG 248–226 5
Reverse primer 2
GCA TTT TGG ATA AAG CGT CTA CG

248–226
1.67
InfB
Forward primer TCC TCA AYT CAC TCT TCG AGC G Nonstructural protein 746–767 6.67
Reverse primer
CGG TGC TCT TGA CCA AAT TGG

848–828
6.67
RP
Forward primer AGA TTT GGA CCT GCG AGC G Human RNase P 50–67 6.67
Reverse primer
GAG CGG CTG TCT CCA CAA GT

50–67
6.67
SARS-CoV-2
Forward primer CTG CAG ATT TGG ATG ATT TCT CC Nucleoprotein–3′ untranslated region 29463–29485 6.67
Reverse primer
CCT TGT GTG GTC TGC ATG AGT TTA G

29554–29530
6.67
InfA probe§
TGC AGT CCT /ZEN/ CGC TCA CTG GGC ACG
Matrix protein
224–201
1.67
InfB probe¶
CCA ATT CGA /ZEN/ GCA GCT GAA ACT GCG GTG
Nonstructural protein
790–817
1.67
RP probe#
TTC TGA CCT /TAO/ GAA GGC TCT GCG CG
Human RNase P
71–93
1.67
SARS-CoV-2 probe** ATT GCA ACA /TAO/ ATC CAT GAG CAG TGC TGA CTC Nucleoprotein–3′ untranslated region 29491–29520 1.67

*Multiplex real-time reverse transcription PCR for influenza A virus, influenza B virus, and SARS-CoV-2. InfA, influenza A; InfB, influenza B; RP, RNase P; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. †Nucleotide positions are indicated as location in the coding domain sequences for the ribonuclease P/MRP subunit p30 (human; GenBank accession no. NM-004613), M gene (GISAID accession no. EPI 1312561) of A/Brisbane/02/2018-H1N1 (InfA), NS gene (GenBank accession no. CY232070) of B/Colorado/06/2017 (InfB), or Wuhan-Hu-1 (SARS-CoV-2; GenBank accession no. NC045512). ‡Multiplex assay is supplied with a tube of primers mixed in 1.5 mL aqueous solution and a tube of probes mixed in 1.5 mL aqueous solution. §Probe labeled with the reporter molecule 6-carboxyfluorescein at the 5′ end, a ZEN quencher between nt 9 and 10, and an Iowa Black FQ quencher at the 3′ end (Integrated DNA Technologies, Inc., https://www.idtdna.com). Probe and primer sequences are identical to InfA sequences used by the CDC Human Influenza Real-Time RT-PCR Diagnostic Panel [510(k) no. K200370] (9). ¶Probe labeled with Yakima Yellow reporter at the 5′ end, a ZEN quencher between nt 9 and 10, and an Iowa Black FQ quencher at the 3′ end (Integrated DNA Technologies, Inc.). Probe and primer sequences identical to InfB sequences used by the CDC Human Influenza Virus Real-Time RT-PCR Detection and Characterization Panel [510(k) no. K200370] (9). #Probe labeled with the CY5 reporter at the 5′ end, a TAO quencher between nt 9 and 10, and an Iowa Black RQ quencher at the 3′ end (Integrated DNA Technologies, Inc.). Probe and primer sequences identical to RP sequences in the CDC Human Influenza Virus Real-Time RT-PCR Detection and Characterization Panel [510(k) no. K200370]. **Probe labeled with the Texas Red-XN reporter at the 5′ end, a TAO quencher between nt 9 and 10, and an Iowa Black RQ quencher at the 3′ end (Integrated DNA Technologies, Inc.)

Main Article

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1These first authors contributed equally to this article.

2These senior authors contributed equally to this article.

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Page updated: June 16, 2021
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