title: Development of a highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus 71 in hand, foot, and mouth disease.
abstract: Enterovirus 71 (EV71) is one of the major causative agents of outbreaks of hand, foot, and mouth disease (HFMD). A commercial TaqMan probe-based real-time PCR assay has been widely used for the differential detection of EV71 despite its relatively high cost and failure to detect samples with a low viral load (Ct value > 35). In this study, a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of EV71 in HFMD was developed. The sensitivity and specificity of this assay were evaluated using a reference EV71 stock and a panel of controls consisting of coxsackievirus A16 (CVA16) and common respiratory viruses, respectively. The clinical performance of this assay was evaluated and compared with those of a commercial TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional two-step nested RT-PCR assay. The limit of detection for the RTN RT-PCR assay was 0.01 TCID50/ml, with a Ct value of 38.3, which was the same as that of the traditional two-step nested RT-PCR assay and approximately tenfold lower than that of the qRT-PCR assay. When testing the reference strain EV71, this assay showed favorable detection reproducibility and no obvious cross-reactivity. The testing results of 100 clinical throat swabs from HFMD-suspected patients revealed that 41 samples were positive for EV71 by both RTN RT-PCR and traditional two-step nested RT-PCR assays, whereas only 29 were EV71 positive by qRT-PCR assay.
authors.affiliation: Key Laboratory for Medical Virology, National Health and Family Planning Commission, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People's Republic of China.
authors.forename: Peihua
authors.initials: P
authors.lastname: Niu
authors.affiliation: Institute for Viral Disease Control and Prevention, Center for Disease Control and Prevention of Hebei, Shijiazhuang, Hebei, People's Republic of China.
authors.forename: Shunxiang
authors.initials: S
authors.lastname: Qi
authors.affiliation: Department of Laboratory Medicine, Shengli Oil Field Central Hospital, Jinan Road, Dongying, Shandong, People's Republic of China.
authors.forename: Benzhang
authors.initials: B
authors.lastname: Yu
authors.affiliation: Key Laboratory for Medical Virology, National Health and Family Planning Commission, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People's Republic of China.
authors.forename: Chen
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Key Laboratory for Medical Virology, National Health and Family Planning Commission, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People's Republic of China.
authors.forename: Ji
authors.initials: J
authors.lastname: Wang
authors.affiliation: Institute for Viral Disease Control and Prevention, Center for Disease Control and Prevention of Hebei, Shijiazhuang, Hebei, People's Republic of China. liqinew@aliyun.com.
authors.forename: Qi
authors.initials: Q
authors.lastname: Li
authors.affiliation: Key Laboratory for Medical Virology, National Health and Family Planning Commission, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People's Republic of China. maxj@ivdc.chinacdc.cn.
authors.forename: Xuejun
authors.initials: X
authors.lastname: Ma
mesh_terms: 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*; D016133:Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity