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

• Published in: Archives of virology 2016-11, Vol.161 (11), p.3003-3010
• Main Authors: Niu, Peihua, Qi, Shunxiang, Yu, Benzhang, Zhang, Chen, Wang, Ji, Li, Qi, Ma, Xuejun
• Format: Article
• Language: English
• Subjects: Biomedical and Life Sciences; Biomedicine; Coronaviruses; Disease control; Disease prevention; Enterovirus; Enterovirus A, Human - genetics; Enterovirus A, Human - isolation & purification; Epidemics; Families & family life; Hand, Foot and Mouth Disease - diagnosis; Hand, Foot and Mouth Disease - virology; Humans; Infections; Infectious Diseases; Laboratories; Medical Microbiology; Molecular Diagnostic Techniques - methods; Original; Original Article; Pathogens; Picornaviridae; Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - methods; Reproducibility of Results; Sensitivity and Specificity; Virology; Viruses; Yeast
• ISSN: 0304-8608; 1432-8798
• DOI: 10.1007/s00705-016-2985-6

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 TCID 50 /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.