A novel real-time RT-PCR assay for influenza C tested in Peruvian children

Background: Influenza C virus (ICV) is associated with acute respiratory illness. Yet ICV remains under recognized, with most previous studies using only culture to identify cases. Objectives: To develop a sensitive and specific real-time RT-PCR assay for ICV that allows for rapid and accurate detection in a clinical or research setting. Study design: Multiple ICV sequences obtained from GenBank were analyzed, including 141 hemagglutininesterase (HE), 106 matrix (M), and 97 nucleoprotein (NP) sequences. Primers and probes were designed based on conserved regions. Multiple primer-probe sets were tested against multiple ICV strains. Results: The ICV M and NP genes offered the most conserved sequence regions. Primers and probes based on newer sequence data offered enhanced detection of ICV, especially for low titer specimens. An NP-targeted assay yielded the best performance and was capable of detecting 10-100 RNA copies per reaction. The NP assay detected multiple clinical isolates of ICV collected in a field epidemiology study conducted in Peru. Conclusions: We report a new real-time RT-PCR assay for ICV with high sensitivity and specificity.