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

Howard, Leigh M.; Johnson, Monika; Gil, Ana I.; Pekosz, Andrew; Griffin, Marie R.; Edwards, Kathryn M.; Lanata, Claudio F.; Grijalva, Carlos G.; Williams, John V.

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 hemagglutinin-esterase (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.