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

J Clin Virol. 2025 Dec:181:105874. doi: 10.1016/j.jcv.2025.105874. Epub 2025 Sep 25.

Abstract

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.

Keywords: Diagnostic assay; Influenza C virus; Real-time RT-PCR.

Publication types

  • Evaluation Study

MeSH terms

  • Child
  • Child, Preschool
  • Gammainfluenzavirus* / genetics
  • Gammainfluenzavirus* / isolation & purification
  • Humans
  • Influenza, Human* / diagnosis
  • Influenza, Human* / virology
  • Molecular Diagnostic Techniques* / methods
  • RNA, Viral / genetics
  • Real-Time Polymerase Chain Reaction* / methods
  • Reverse Transcriptase Polymerase Chain Reaction* / methods
  • Sensitivity and Specificity
  • Viral Matrix Proteins / genetics

Substances

  • RNA, Viral
  • Viral Matrix Proteins