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

Bo Shu; Marie K. Kirby; 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. Wentworth; John R. Barnes

Disclosures

Emerging Infectious Diseases. 2021;27(7):1821-1830. 

In This Article

Abstract and Introduction

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019, and the outbreak rapidly evolved into the current coronavirus disease pandemic. SARS-CoV-2 is a respiratory virus that causes symptoms similar to those caused by influenza A and B viruses. On July 2, 2020, the US Food and Drug Administration granted emergency use authorization for in vitro diagnostic use of the Influenza SARS-CoV-2 Multiplex Assay. This assay detects influenza A virus at 102.0, influenza B virus at 102.2, and SARS-CoV-2 at 100.3 50% tissue culture or egg infectious dose, or as few as 5 RNA copies/reaction. The simultaneous detection and differentiation of these 3 major pathogens increases overall testing capacity, conserves resources, identifies co-infections, and enables efficient surveillance of influenza viruses and SARS-CoV-2.

Introduction

An outbreak of pneumonia of unknown etiology in Wuhan, China, was reported to the World Health Organization on December 31, 2019.[1] Researchers determined that the illness, later known as coronavirus disease (COVID-19), was caused by a previously unidentified betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).[2] SARS-CoV-2 rapidly spread around the world, and on March 11, 2020, the World Health Organization declared a pandemic.[3] By January 2021, SARS-CoV-2 had infected >96 million persons and caused >2 million deaths worldwide.[4]

The high demand for molecular testing for SARS-CoV-2 has contributed to global shortages of diagnostic resources, including reagents, enzymes used in reverse transcription PCR (RT-PCR), plastic consumables, and staff availability.[5,6] Efficient diagnostic tests can reduce strain on the testing system and decrease turnaround time. To improve testing efficiency, we developed the Centers for Disease Control and Prevention (CDC) Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay, which is selective for influenza A and B viruses and SARS-CoV-2. This quadruplex real-time RT-PCR (rRT-PCR) simultaneously detects and distinguishes RNA of influenza A virus, influenza B virus, and SARS-CoV-2 in upper and lower respiratory specimens. To monitor specimen quality control, the assay also detects the Homo sapiens (human) RNase P (RP) gene. Because the Flu SC2 Multiplex Assay can test 93 samples in a 96-well plate, this technology improves the throughput of SARS-CoV-2 testing by 3-fold compared with the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel.[7] The Flu SC2 Multiplex Assay also simultaneously detects influenza A and B viruses, thereby reducing the overall strain on testing facilities, especially during influenza season. Continued testing and surveillance of influenza viruses during the COVID-19 pandemic provide critical guidance on selection of candidate vaccine strains; these processes also identify antiviral resistance genes and novel influenza viruses that have pandemic potential.[8]

We evaluated existing and novel SARS-CoV-2 primers and probes to identify the optimal SC2 assay components for this quadruplex rRT-PCR (Appendix Table 1, https://wwwnc.cdc.gov/EID/article/27/7/21-0462-App1.pdf). The SC2 assay components are selective for the 3′ region of the SARS-CoV-2 genome from the carboxy terminus of the nucleocapsid (N) gene into the 3′ untranslated region (UTR). The primer and probe sequences for the influenza A (InfA), influenza B (InfB), and RP targets are identical to those used in the singleplex assays of the US Food and Drug Administration (FDA)–approved CDC Human Influenza Virus Real-Time RT-PCR Detection and Characterization Panel [510(k) no. K200370].[9] The Flu SC2 Multiplex Assay is selective for the matrix (M) gene segment of the influenza A virus, the nonstructural (NS) gene segment of the influenza B virus, and the human ribonuclease P/MRP subunit P30 gene; the InfA assay is designed for universal detection of all influenza A viruses and InfB assay is designed for universal detection of all influenza B viruses.[10–14] The InfA assay was recently updated to address evolutionary changes and reactivity challenges; the updated CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel was cleared by FDA in 2020.[9] On July 2, 2020, FDA granted an emergency use authorization (EUA) for in vitro diagnostic use of the Flu SC2 Multiplex Assay.[15]

Multiplex detection of RNA from influenza A virus, influenza B virus, and SARS-CoV-2 can increase testing capacity and reduce use of reagents. The increased throughput can preserve staff resources and reduce turnaround time. The Flu SC2 Multiplex Assay and similar panels identify co-infections or alternative causes of influenza-like and COVID-19–like illnesses. The Flu SC2 Multiplex Assay can enable collection of critical data on influenza A and B viruses and SARS-CoV-2, as well as the prevalence of co-infection among these respiratory viruses.

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