What to know
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.
Overview
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.
Public health laboratories wanting to perform the SARS-CoV-2 (Flu SC2) Multiplex Assay testing 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.
Primers and Probes (Research Use Only)
Not for diagnostic testing use:
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 are 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.
Primer and Probe Kit Description
| 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 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 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 |
| Name | Description | Oligonucleotide Sequence (5’>3’) | Concentration |
|---|---|---|---|
| Option 1: IDT Probes | |||
| InfA-P | InfA ProbeA | 5’-/FAM/TGC AGT CCT /ZEN/ CGC TCA CTG GGC ACG/3IABkFQ/-3’ | 1.67 µM |
| InfB-P | InfB ProbeB | 5’-/YakYel/CCA ATT CGA/ZEN/ GCA GCT GAA ACT GCG GTG/3IABkFQ/-3’ | 1.67 µM |
| SC2-P | SC2 ProbeC | 5’-/TexRd-XN/ATT GCA ACA/TAO/ ATC CAT GAG CAG TGC TGA CTC/3IAbRQSp/-3’ | 1.67 µM |
| RP-P | RNase P ProbeD | 5’-/CY5/TTC TGA CCT /TAO/ GAA GGC TCT GCG CG/3IAbRQSp/-3’ | 1.67 µM |
| Option 2: Biosearch Probes | |||
| InfA-P | InfA ProbeE | 5’-/FAM/TGC AGT CCT /Nova/ CGC TCA CTG GGC ACG/BHQ-1/-3’ | 1.67 µM |
| InfB-P | InfB ProbeF | 5’-/CIV-550/CCA ATT CGA/BHQ-1/ GCA GCT GAA ACT GCG GTG/C3/-3’ | 1.67 µM |
| SC2-P | SC2 ProbeG | 5’-/CalFluor610/ATT GCA ACA/Nova/ ATC CAT GAG CAG TGC TGA CTC/BHQ-2/-3’ | 1.67 µM |
| RP-P | RNase P ProbeH | 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.
- Probe 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).
- Probe 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).
- Probe 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).
- Probe 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).
- Probe 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).
- Probe 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).
- Probe 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).
- Probe 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).