title: Design and validation of a real-time RT-PCR for the simultaneous detection of enteroviruses and parechoviruses in clinical samples.
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 40CCID50/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.
authors.affiliation: Enterovirus Unit, National Centre for Microbiology, Health Institute "Carlos III", Madrid, Spain. Electronic address: mcabrerizo@isciii.es.
authors.forename: María
authors.initials: M
authors.lastname: Cabrerizo
authors.affiliation: Hospital Severo Ochoa, Leganés, Madrid, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Calvo
authors.affiliation: Hospital Santa Creu i Sant Pau, Barcelona, Spain.
authors.forename: Nuria
authors.initials: N
authors.lastname: Rabella
authors.affiliation: Hospital San Joan de Deu, Barcelona, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Muñoz-Almagro
authors.affiliation: Hospital San Joan de Deu, Barcelona, Spain.
authors.forename: Eva
authors.initials: E
authors.lastname: del Amo
authors.affiliation: Hospital Virgen de las Nieves, Granada, Spain.
authors.forename: Mercedes
authors.initials: M
authors.lastname: Pérez-Ruiz
authors.affiliation: Hospital Virgen de las Nieves, Granada, Spain.
authors.forename: Sara
authors.initials: S
authors.lastname: Sanbonmatsu-Gámez
authors.affiliation: Hospital Virgen de la Arrixaca, Murcia, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Moreno-Docón
authors.affiliation: Enterovirus Unit, National Centre for Microbiology, Health Institute "Carlos III", Madrid, Spain.
authors.forename: Almudena
authors.initials: A
authors.lastname: Otero
authors.affiliation: Enterovirus Unit, National Centre for Microbiology, Health Institute "Carlos III", Madrid, Spain.
authors.forename: Gloria
authors.initials: G
authors.lastname: Trallero
mesh_terms: D002675:Child, Preschool; D017931:DNA Primers / Q000235:genetics; D004770:Enterovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007223:Infant; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D030061:Parechovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D010850:Picornaviridae Infections / Q000175:diagnosis* / Q000821:virology; D022661:RNA, Untranslated / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D015345:Oligonucleotide Probes; D022661:RNA, Untranslated; D012367:RNA, Viral
keywords: Clinical samples; Enterovirus; Parechovirus; Real-time RT-PCR; Young children