title: Development of duplex real-time RT-PCR based on Taqman technology for detecting simultaneously the genome of pan-enterovirus and enterovirus 71.
abstract: Human enterovirus (EV) 71 is the main etiological agent of hand, foot, and mouth disease (HFMD). It is associated with neurological complications, and caused fatalities during recent outbreaks in the Asia-Pacific region. Infections caused by EV71 could lead to many complications, ranging from brainstem encephalitis to pulmonary oedema, resulting in high mortality. In this study, a duplex real-time RT-PCR assay was developed in order to simultaneously detect pan-EV and EV71. EV71-specific primers and probes were designed based on the highly conserved VP1 region of EV71. Five EV71 strains were detected as positive, and no positive fluorescence signal was observed in the duplex real-time RT-PCR for other viral RNA, which showed 100% specificity for the selected panel, and no cross-reactions were observed in this duplex real-time RT-PCR. The EV71-specific duplex real-time RT-PCR was more sensitive than conventional RT-PCR, and detected viral titers that were 10-fold lower than those measured by the latter. Of the 381 HFMD clinical specimens, 196 (51.4%) cases were pan-EV-positive, of which 170 (86.7%) were EV71-positive when tested by pan-EV and EV71-specific duplex real-time RT-PCR. EV71-specific duplex real-time RT-PCR offers a rapid and sensitive method to detect EV71 from clinical specimens, and will allow quarantine measures to be taken more effectively during outbreaks.
authors.affiliation: Division of Vaccine Research, Center for Infectious Diseases, National Institute of Health, Korea Centers for Disease Control and Prevention, Chungcheongbuk-do, Korea.
authors.forename: Seoyeon
authors.initials: S
authors.lastname: Hwang
authors.forename: Byunghak
authors.initials: B
authors.lastname: Kang
authors.forename: Jiyoung
authors.initials: J
authors.lastname: Hong
authors.forename: Ahyoun
authors.initials: A
authors.lastname: Kim
authors.forename: Hyejin
authors.initials: H
authors.lastname: Kim
authors.forename: Kisang
authors.initials: K
authors.lastname: Kim
authors.forename: Doo-Sung
authors.initials: DS
authors.lastname: Cheon
mesh_terms: D000818:Animals; D002648:Child; D002675:Child, Preschool; D029821:Enterovirus A, Human / Q000235:genetics / Q000302:isolation & purification*; D006232:Hand, Foot and Mouth Disease / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014773:Virology / Q000379:methods*
keywords: EV71; duplex real-time RT-PCR; enterovirus; hand, foot, and mouth disease