Design and performance of a real-time RT-PCR assay for detection of influenza C viruses

Influenza C virus (ICV) usually causes a mild upper respiratory tract infection in children and those infected are frequently co-infected with other respiratory viruses. However, there have only been a few hundred documented cases of ICV infection in humans as of the end of 2024. To better understand the epidemiology and clinical impact of ICVs, we developed an influenza C real-time RT-PCR (InfC rRT-PCR) assay that targets a highly conserved region of the matrix gene segment of ICVs. The analytical sensitivity evaluation demonstrated that the InfC rRT-PCR assay was highly sensitive, as it was able to detect as few as five RNA copies per PCR reaction and had robust reactivity over a range of viral RNAs from historical and recent ICVs. The analytical specificity evaluation confirmed the assay did not cross-react with any influenza A or B viruses tested, including several animal-origin viruses, or other common non-influenza respiratory viruses. The performance evaluation on clinical specimens demonstrated the assay was highly sensitive and specific for the detection of ICVs. • Universal Detection : The real-time RT-PCR assay was developed targeting a highly conserved region of the matrix gene to allow universal detection of influenza C viruses. • Standardized Conditions : The influenza C real-time RT-PCR reaction and thermocycling conditions are consistent with the current CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel for the qualitative detection and characterization of influenza virus types and subtypes. • High Performance : The influenza C real-time RT-PCR assay exhibits high sensitivity and specificity, ensuring reliable detection of influenza C viruses. • Effective method : The influenza C real-time RT-PCR primers and probes have been provided to many laboratories to support several influenza C virus investigations and surveillance projects national and internationally.