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CDC’s Influenza SARS-CoV-2 Multiplex Assay

Updated Nov. 14, 2022

Summary of Recent Changes

Added information on research use only CDC Flu SC2 Multiplex Assay primers and probes

See previous updates

Overview

The CDC Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay is a real-time reverse-transcription polymerase chain reaction (rRT-PCR) laboratory test that can simultaneously detect and differentiate between influenza A, influenza B, and SARS-CoV-2 in upper or lower respiratory specimens. The test is a highly accurate, nucleic acid-based diagnostic tool to evaluate specimens from patients who are in the acute phase of infection.

Why the CDC Flu SC2 Multiplex Assay is Important

Diagnoses infection with the SARS-CoV-2, influenza A, and/or influenza B viruses in one test
Allows laboratories to conserve important testing materials and process more tests in a given time period
Gives public health officials information they need to control the spread of COVID-19 and flu
Allows for ongoing flu surveillance while also testing for SARS-CoV-2

How Public Health Laboratories Order the CDC Flu SC2 Multiplex Assay

CDC’s International Reagent Resource (IRR) provides CDC Flu SC2 multiplex kits to IRR-registered state and local public health laboratories. IRR does not supply clinicians, hospitals, or healthcare professionals with testing kits directly.

During the COVID-19 pandemic, state public health laboratories can authorize county or city laboratories in their state to perform the SARS-CoV-2 (Flu SC2) Multiplex Assay testing. These laboratories must be certified under the Clinical Laboratory Improvement Amendments (CLIA) to perform high complexity tests, have appropriate laboratory equipment and training, and demonstrate testing proficiency under their state laboratory’s stewardship to maintain their status as an IRR-registered laboratory. CDC has shared the primers and probes sequence specifications so that laboratories and test developers may manufacture their own reagents. Refer to description of kit contents below.

Emergency Use Authorizations

The U.S. Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for use of the Flu SC2 Multiplex Assay on July 2, 2020. The EUA process enables FDA to consider and authorize the use of unapproved but potentially lifesaving medical or diagnostic products during a public health emergency. The U.S. Secretary of Health and Human Services declared SARS-CoV-2 to be a U.S. public health emergency justifying the authorization of emergency use of drugs and biologic products, including in vitro diagnostics, on February 4, 2020.

November 20, 2020

FDA granted an amendment to the EUA for the CDC Flu SC2 Multiplex Assay to expand the list of extraction instruments that may be used to prepare samples for testing. Included in this amendment:

Addition of four automated nucleic acid extraction instrument options
- QIAcube HT
- KingFisher Flex
- MagNA Pure Compact
- NucliSENS® easyMAG®
Recommendation to use the JOE (5’-Dichloro-dimethoxy-fluorescein dye) filter instead of the VIC (2′-chloro-7′phenyl-1,4-dichloro-6-carboxy-fluorescein) filter to view the InfB signal with standard 7500 Fast Dx calibration
Clarification of verification instructions and dilutions

FDA granted an amendment to the EUA for the CDC Flu SC2 Multiplex Assay to expand the list of extraction instruments that may be used to prepare samples for testing. Included in this amendment:

Addition of four automated nucleic acid extraction instrument options
- QIAcube HT
- KingFisher Flex
- MagNA Pure Compact
- NucliSENS® easyMAG®
Recommendation to use the JOE (5’-Dichloro-dimethoxy-fluorescein dye) filter instead of the VIC (2′-chloro-7′phenyl-1,4-dichloro-6-carboxy-fluorescein) filter to view the InfB signal with standard 7500 Fast Dx calibration
Clarification of verification instructions and dilutions

January 8, 2021

FDA granted an amendment to the EUA for the CDC Flu SC2 Multiplex Assay to expand the list of extraction instruments that may be used to prepare samples for testing and to add an additional authorized manufacturer of primer and probe reagents for use with the test. This amendment will enable more laboratories to use the test with automated, high throughput specimen preparation instruments, potentially increasing the speed and throughput of testing and will increase manufacturing capacity of primer and probe reagents by adding a second authorized manufacturer. Included in this amendment:

Addition of two automated nucleic acid extraction instrument options
- Maxwell® RSC 48
- Maxwell® CSC 48
Addition of another authorized manufacturer of primer and probe reagents for use with the CDC Flu SC2 Multiplex Assay

Addition of two automated nucleic acid extraction instrument options
- Maxwell® RSC 48
- Maxwell® CSC 48
Addition of another authorized manufacturer of primer and probe reagents for use with the CDC Flu SC2 Multiplex Assay

July 21, 2021

FDA granted an amendment to the EUA for the CDC Flu SC2 Multiplex Assay to update the in silico analysis of assay primer/probe sequences against available genomic sequences of SARS-CoV-2 viruses/variants circulating in the United States and globally.

Primers and Probes (Research Use Only)

Not for diagnostic testing use: Reagents manufactured from the sequences listed below may not be used for diagnostic testing under FDA’s authorization of the CDC Flu SC2 Multiplex Assay. Only primers and probes labeled for EUA use and distributed by the International Reagent Resource may be used.

The oligonucleotide sequences listed below are intended to be used for the purposes of respiratory virus surveillance and research. The recipient agrees to use them in compliance with all applicable laws and regulations. Every effort has been made to assure the accuracy of the sequences, but CDC cannot provide any warranty regarding their accuracy. The recipient can acknowledge the source of sequences in any oral presentations or written publications concerning the research project by referring to the Genomics and Diagnostics Team, Virology Surveillance and Diagnosis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA (CDCSARS2FluAB@cdc.gov).

Should the recipient use these oligonucleotide sequences in the design of a diagnostic device, the device developed from these sequences and the use of primers and probes synthesized from these sequences is not authorized under CDC’s Flu SC2 Multiplex Assay EUA. The device would be considered a new and separate test from the Flu SC2 Multiplex Assay and must comply with applicable laws and regulations. CDC has extended an open right of reference to CDC’s EUA for the Flu SC2 Multiplex Assay. Any recipient seeking FDA authorization for a device based in whole or in part on the design of the CDC Flu SC2 Multiplex Assay is welcome to reference the CDC EUA (EUA 201781) in their application.

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Primer and Probe Kit Description

Tube 1: Flu SC2 Multiplex Assay: Forward (For) and Reverse (Rev) Primers

Flu SC2 Multiplex Assay: Forward (For) and Reverse (Rev) Primers
Name	Description	Oligonucleotide Sequence (5’ to 3’)	Concentration
InfA-F	InfA For1	CAA GAC CAA TCY TGT CAC CTC TGA C	3.33 µM
InfA-F	InfA For2	CAA GAC CAA TYC TGT CAC CTY TGA C	3.33 µM
InfA-R	InfA Rev1	GCA TTY TGG ACA AAV CGT CTA CG	5.00 µM
InfA-R	InfA Rev2	GCA TTT TGG ATA AAG CGT CTA CG	1.67 µM
InfB-F	InfB For	TCC TCA AYT CAC TCT TCG AGC G	6.67 µM
InfB-R	InfB Rev	CGG TGC TCT TGA CCA AAT TGG	6.67 µM
SC2-F	SC2 For	CTG CAG ATT TGG ATG ATT TCT CC	6.67 µM
SC2-R	SC2 Rev	CCT TGT GTG GTC TGC ATG AGT TTA G	6.67 µM
RP-F	RNase P For	AGA TTT GGA CCT GCG AGC G	6.67 µM
RP-R	RNase P Rev	GAG CGG CTG TCT CCA CAA GT	6.67 µM

Tube 2: Flu SC2 Multiplex Assay: Probes

Flu SC2 Multiplex Assay: Probes
Name	Description	Oligonucleotide Sequence (5’>3’)	Concentration
Option 1: IDT Probes
InfA-P	InfA Probe^[1]	5’-/FAM/TGC AGT CCT /ZEN/ CGC TCA CTG GGC ACG/3IABkFQ/-3’	1.67 µM
InfB-P	InfB Probe^[2]	5’-/YakYel/CCA ATT CGA/ZEN/ GCA GCT GAA ACT GCG GTG/3IABkFQ/-3’	1.67 µM
SC2-P	SC2 Probe^[3]	5’-/TexRd-XN/ATT GCA ACA/TAO/ ATC CAT GAG CAG TGC TGA CTC/3IAbRQSp/-3’	1.67 µM
RP-P	RNase P Probe^[4]	5’-/CY5/TTC TGA CCT /TAO/ GAA GGC TCT GCG CG/3IAbRQSp/-3’	1.67 µM
Option 2: Biosearch Probes
InfA-P	InfA Probe^[5]	5’-/FAM/TGC AGT CCT /Nova/ CGC TCA CTG GGC ACG/BHQ-1/-3’	1.67 µM
InfB-P	InfB Probe^[6]	5’-/CIV-550/CCA ATT CGA/BHQ-1/ GCA GCT GAA ACT GCG GTG/C3/-3’	1.67 µM
SC2-P	SC2 Probe^[7]	5’-/CalFluor610/ATT GCA ACA/Nova/ ATC CAT GAG CAG TGC TGA CTC/BHQ-2/-3’	1.67 µM
RP-P	RNase P Probe^[8]	5’-/Quasar670/TTC TGA CCT /Nova/ GAA GGC TCT GCG CG/BHQ-2/-3’	1.67 µM

Note: Oligonucleotide sequences are continually monitored to ensure performance and may be subject to future changes as the SARS-CoV-2 virus evolves.

Footnotes

¹Probe labeled at the 5′ end with the reporter molecule 6-carboxyfluorescein (FAM), with a ZEN™ quencher between the 9th and 10th nucleotide, and with an Iowa Black FQ quencher (IABkFQ) at the 3′ end (Integrated DNA Technologies, Coralville, IA). InfA probe and primer sequences are identical to InfA sequences in the FDA-cleared CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel (K200370).

²Probe labeled at the 5′ end with the Yakima Yellow (YakYel) reporter, with a ZEN™ quencher between the 9th and 10th nucleotide, and with an Iowa Black FQ quencher (IABkFQ) at the 3′ end (Integrated DNA Technologies, Coralville, IA). Probe and primer sequences are identical to InfB sequences in the FDA-cleared CDC Human Influenza Virus Real-Time RT-PCR Detection and Characterization Panel (K200370).

³Probe labeled at the 5′-end with the Texas Red-XN (TexRd-XN) reporter, with a TAO quencher between the 9th and 10th nucleotide, and with an Iowa Black RQ quencher (IAbRQSp) at the 3′ end (Integrated DNA Technologies, Coralville, IA).

⁴Probe labeled at the 5′-end with the CY5 reporter, with a TAO quencher between the 9th and 10th nucleotide, and with an Iowa Black RQ quencher (IAbRQSp) at the 3′ end (Integrated DNA Technologies, Coralville, IA). Probe and primer sequences are identical to RP sequences in the FDA-cleared CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel (K200370).

⁵Probe labeled at the 5′- end with the reporter molecule 6-carboxyfluorescein (FAM), with a Nova quencher between the 9th and 10th nucleotide, and with a Black Hole Quencher®-1 (BHQ®-1) at the 3′- end (Biosearch Technologies, Novato, CA). InfA probe and primer sequences are identical to InfA sequences in the FDA-cleared CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel (K200370).

⁶Probe labeled at the 5′- end with the CIV-550 reporter, with a Black Hole Quencher®-1 (BHQ®-1) between the 9th and 10th nucleotide, and with a 3’ Spacer C3 (Biosearch Technologies, Novato, CA). Probe and primer sequences are identical to InfB sequences in the FDA-cleared CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel (K200730).

⁷Probe labeled at the 5′-end with the Cal Fluor Red 610 reporter, with a Nova quencher between the 9th and 10th nucleotide, and with a Black Hole Quencher®-2 (BHQ®-2) at the 3′- end (Biosearch Technologies, Novato, CA).

⁸Probe labeled at the 5′-end with the Quasar670 reporter, with a Nova quencher between the 9th and 10th nucleotide, and with a Black Hole Quencher®-2 (BHQ®-2) at the 3′- end (Biosearch Technologies, Novato, CA). Probe and primer sequences are identical to RP sequences in the FDA-cleared CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel (K200730).

Resources

Previous Updates

As of September 30, 2021

Added information on CDC amendment granted by FDA on August 5, 2021

As of February 2, 2021

Added information on CDC amendment granted by FDA on January 8, 2021

As of December 11, 2020

Added information on CDC amendment granted by FDA on November 30, 2020