Abstract
Background
Parechoviruses (PeV-As), which constitute a new genus within the family Picornaviridae, have been associated with numerous localized outbreaks of serious diseases, such as coryza, pneumonia, maculopapular exanthem, and conjunctivitis. However, only a few laboratories worldwide routinely conduct tests for the identification of this group of viruses; presently, to the best of our knowledge, no laboratory in China conducts routine test for PeV-A identification. Therefore, in this study, we aimed to develop and validate a real-time RT-PCR assay for the identification of PeV-As.
Methods
To designed and validate a real-time PCR primer-probe targeting the 5′-UTR region of PeV-As, the 5′-UTR sequences of PeV-As available in GenBank were aligned using the MUSCLE algorithm in MEGA v7.0. Thereafter, the highly conserved 5′-UTR region was selected, and its primer-probe sequence was designed using Primer Premier v5.0. This primer-probe sequence was then evaluated for specificity, sensitivity, and repeatability, and for its validation, it was tested using fecal samples from 728 healthy children living in Beijing (China).
Results
The PeV-A real-time RT-PCR assay detected only the RNA-positive standards of PeV-A genotypes (1–8, 14, 17, and 18), whereas 72 serotypes of non-PeV-A EV viruses were undetected. In addition, the VP1 region of these 11 PeV-A genotypes that tested positive were amplified using the primers designed in this study. Typing results indicated that eight, one, and two strains of the 11 were PeV-A1, PeV-A4, and PeV-A6, respectively. We also determined and presented the genetic characterization and phylogenetic analyses results corresponding to these 11 VP1 region sequences. Furthermore, the real-time RT-PCR assay showed good repeatability, reproducibility, and sensitivity.
Conclusion
Our findings suggested that the real-time RT-PCR assay developed in this study can be applied for routine PeV-A identification. Additionally, we believe that our results will serve as a foundation for further studies on PeV-As and facilitate the expansion of the gene sequence information available in GenBank.