Elsevier

Journal of Virological Methods

Volume 208, November 2014, Pages 125-128
Journal of Virological Methods

Short communication
Design and validation of a real-time RT-PCR for the simultaneous detection of enteroviruses and parechoviruses in clinical samples

https://doi.org/10.1016/j.jviromet.2014.08.008Get rights and content

Highlights

  • We developed a real-time RT-PCR for simultaneous EV/HPeV detection in clinical samples.

  • Validation with different EV/HPeV types indicates high amplification efficiency.

  • Reliable tool for rapid diagnosis of paediatric neurological and systemic infections.

Abstract

Human enteroviruses (EVs) and parechoviruses (HPeVs) are important etiological agents causing infections such as meningitis, encephalitis and sepsis-like disease in neonates and young children. We have developed a real-time RT-PCR for simultaneous detection of EV and HPeV in clinical samples.

Primers and probe sets were designed from the conserved 5′-noncoding region of the genomes. The sensitivity, specificity and reproducibility of the technique were measured using a set of 25 EV and 6 HPeV types. All EVs but no HPeVs were detected with the EV primers–probe set. The HPeV primers–probe set detected only the 6 HPeV types. The lower detection limit was found to be 4 and 40 CCID50/ml for HPeV and EV respectively, demonstrating high sensitivity of the technique for both viruses. The threshold cycle values were highly reproducible on repeat testing of positive controls among assay runs.

The assay was evaluated in 53 clinical samples of suspected meningitis, sepsis or febrile syndromes from children under 3 years. In 11 of these (21%) EVs were detected, while 4, i.e. 7.5%, were HPeV positive. Molecular typing was carried out for 73% of the viruses.

In summary, the RT-PCR method developed demonstrated effectively both EV and HPeV detection, which can cause similar clinical symptoms in infants.

Section snippets

Competing interests

None declared.

Authors’ contribution

Study concept and design: M. Cabrerizo, C. Calvo, N. Rabella, C. Muñoz-Almagro, E. del Amo, M. Pérez-Ruiz, S. Sanbonmatsu, A. Moreno-Docón, M.L.G. Trallero, and the study group of “Enterovirus and parechovirus infections in children under 3 y, Spain” PI12-00904 (see Appendix A). Contribution of samples and clinical data: C. Calvo, N. Rabella, C. Muñoz-Almagro, E. del Amo, M. Pérez-Ruiz, S. Sanbonmatsu, A. Moreno-Docón, and the study group of “Enterovirus and parechovirus infections in children

Ethical approval

An informed consent form was obtained from the parents at the time of each child's hospitalisation. Samples were anonymized and archived according to the research project protocol PI12-00904 approved by the Research Ethics and Animal Welfare Committee of National Health Institute “Carlos III”.

Acknowledgements

We thank Isidoro Bustillo, Hortensia del Pozo and Puri Higueras for their technical assistance and Ingrid M. Outschoorn for English editing.

The project was supported from a grant from the Health Research System (AES; PI12-00904).

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    1

    The members of the study group are listed in Appendix A.

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