Sensitive detection and differentiation of rhinoviruses and enteroviruses by a nested real-time RT-PCR assay

Rhinoviruses (RVs) and enteroviruses (EVs) are important respiratory pathogens. Although numerous molecular assays have been developed for detection of RVs and EVs, their genetic similarities pose challenges for molecular differentiation. In this study, we described a real-time nested reverse transcription (RT)-PCR assay using SYBR green and RV- and EV-specific reverse primers to differentially detect RVs and EVs. The primers were designed so that the numbers and locations of mismatches should be the most adequate for objective viruses and the least adequate for opposite viruses using all EV and RV sequences in GenBank. The assay was validated using nasopharyngeal swab specimens from pediatric patients who have fever and/or respiratory symptoms at Keio University Hospital from November 2021 to January 2023 and tested positive for Human Rhinovirus/Enterovirus by the FilmArray Respiratory Panel 2.1. The species and serotypes were identified by analyzing sequences of PCR products using the BLAST program. The results of the present RT-PCR assay and the BLAST analysis were completely consistent with each other. Furthermore, the current assay revealed the cases of dual infections of RV and EV. No significant differences were observed in patient demographics or clinical courses among viral species. The assay presented here may be the most suitable for routine diagnosis and surveillance of RV and EV infections. IMPORTANCE: We describe a real-time nested reverse transcription-PCR assay that enables us to differentially detect rhinoviruses and enteroviruses. Differential diagnosis of rhinovirus and enterovirus infections has not been succeeded because of their genetic diversities and similarities. We resolved this problem by using specific PCR primers that were designed by in silico analysis of all rhinovirus and enterovirus sequences obtained from GenBank. The developed method was validated by applying to more than 100 nasopharyngeal swab specimens from pediatric patients in Keio University Hospital in Japan and analyzing with the BLAST algorithm. The assay may be suitable for routine diagnosis and surveillance of rhinovirus and enterovirus infections.