Hu, X; Zhang, Y; Zhou, X; Xu, B; Yang, M; Wang, M; Zhang, C; Li, J; Bai, R; Xu, W; Ma, X
Hand, foot and mouth disease (HFMD) is a contagious enteroviral disease occurring primarily in young children caused by enterovirus 71 (EV71), coxsackievirus A16 (CVA16) and other serotypes of coxsackievirus and echovirus. In this study, a GeXP analyzer-based multiplex RT-PCR assay (GeXP assay) consisting of chimeric primer-based fluorescently labeled PCR amplification and capillary electrophoresis separation was developed to simultaneously identify nine serotypes of enteroviruses associated with HFMD in China, including EV71, CVA16, CVA4, 5, 9, 10, CVB1, 3 and 5. The RNAs extracted from cell cultures of viral isolates and synthetic RNAs via in vitro transcription were used to analyze the specificity and sensitivity of the assay. The GeXP assay detected as few as 0.03 TCID(50) of EV71 and CVA16, 10 copies of pan-enterovirus, EV71, CVA16, CVB1, CVB5, and 100 copies of ten (including pan-enterovirus) pre-mixed RNAs templates. A total of 180 stool specimens collected from HFMD patients and suspects were adopted to evaluate the clinical performance. In comparison with the results of conventional methods, the sensitivities of the GeXP assay for detection of pan-enterovirus, EV71 and CVA16 were 98.79% (163/165), 91.67% (44/48) and 91.67% (33/36), respectively, and the specificities were 80.00% (12/15), 98.48% (130/132) and 100% (144/144), respectively. The accordance of typing other 7 serotypes of enteroviruses with the results of conventional methods was 92.59% (25/27). In conclusion, the GeXP assay is a rapid, cost-effective and high throughput method for typing nine serotypes of HFMD associated enteroviruses.