Related Experiment Video
Updated: Oct 12, 2025
Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses
Published on: November 10, 2023
Development of a real-time RT-PCR assay for the detection of pan-human parechoviruses
Huanhuan Lu1, Jinbo Xiao1, Keyi Zhang1
1WHO WPRO Regional Polio Reference Laboratory and National Health Commission Key Laboratory of Biosafety, National Health Commission Key Laboratory of Medical Virology, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People's Republic of China.
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, to the best of our knowledge, only a few laboratories worldwide conduct tests for the identification of this group of viruses. Therefore, in this study, we aimed to develop and validate a real-time RT-PCR assay for the identification of PeV-As.
Methods:
To design 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, real-time RT-PCR assay showed good sensitivity with LOD of 102 copies/μL. Positive results in eight parallel experiments at each concentration gradient from 107 copies/μL to 102 copies/μL, indicating good repeatability.
Conclusion:
Our findings suggested that the real-time RT-PCR assay developed in this study can be applied for routine PeV-A identification. We detected PeV-A1, 4 and 6 genotypes in the 728 faecal samples using this method. 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.
Insights
A new real-time RT-PCR assay effectively identifies Parechoviruses A (PeV-As), a genus linked to serious diseases. This validated method detected PeV-A genotypes 1, 4, and 6 in fecal samples, aiding routine diagnostics.
Area of Science:
- Virology
- Molecular Biology
- Public Health
Background:
- Parechoviruses (PeV-As) are a genus within Picornaviridae associated with severe illnesses like coryza, pneumonia, and conjunctivitis.
- Limited global laboratory capacity exists for PeV-A identification, hindering disease surveillance and management.
- This study addresses the need for a reliable diagnostic tool for PeV-As.
Purpose of the Study:
- To develop and validate a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for accurate PeV-A identification.
- To establish a sensitive and specific method for detecting PeV-As in clinical samples.
Main Methods:
- Designed and validated a real-time PCR primer-probe targeting the highly conserved 5'-UTR region of PeV-As.
- Utilized MUSCLE algorithm for sequence alignment and Primer Premier v5.0 for primer-probe design.
- Tested the assay's specificity, sensitivity, and repeatability using RNA standards and 728 fecal samples from healthy children in Beijing.
Main Results:
- The developed real-time RT-PCR assay specifically detected PeV-A genotypes (1-8, 14, 17, 18) while excluding 72 non-PeV-A enterovirus serotypes.
- Identified PeV-A genotypes 1, 4, and 6 in 11 positive fecal samples, with subsequent VP1 region sequencing confirming the typing.
- Demonstrated good assay sensitivity with a limit of detection (LOD) of 10^2 copies/μL and excellent repeatability across multiple experiments.
Conclusions:
- The validated real-time RT-PCR assay is suitable for routine identification of PeV-As.
- The study successfully detected PeV-A genotypes 1, 4, and 6 in a cohort of healthy children.
- Findings provide a foundation for future PeV-A research and expand available genetic sequence data.
