Development and evaluation of a real-time method of simultaneous amplification and testing of enterovirus 71 incorporating a RNA internal control system

Human enterovirus 71 (EV71) is a primary etiological agent of hand, foot, and mouth disease (HFMD). The associated infections have posed a serious threat to the public health. In the present study, a real-time simultaneous amplification and testing (SAT) technology was developed for detecting EV71 (SAT-EV71). The RNA of EV71 and an internal control (IC) were amplified and analyzed simultaneously by isothermal amplification and real-time detection of fluorescence using routine real-time PCR. Furthermore, this SAT-EV71 method with IC was evaluated by analyzing 256 clinical specimens including 60 enterovirus-positive ones confirmed by the virus-cell culture method. The other 196 putative ones were further analyzed by a PCR-fluorescence probing assay. The results showed that SAT-EV71 can detect the EV71 VP1 with a minimum of 10 copies per reaction at an optimal concentration of IC (5000 copies per reaction) with a high sensitivity and specificity. Meanwhile, the use of IC can prevent false negatives effectively by monitoring the processes of nucleic acid extraction and amplification. Overall, this easy-to-perform SAT-EV71 with IC can detect EV71 sensitively and specifically. It might be used in the molecular diagnosis of EV71 in putative cases of HFMD.