title: Development and evaluation of a real-time method of simultaneous amplification and testing of enterovirus 71 incorporating a RNA internal control system.
abstract: 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.
authors.affiliation: Pediatrics Research Institute, Nanjing Children's Hospital Affiliated to Nanjing Medical University, No. 72 Guangzhou Road, Nanjing 210008, China. Electronic address: chenqian97@163.com.
authors.forename: Qian
authors.initials: Q
authors.lastname: Chen
authors.affiliation: Pediatrics Research Institute, Nanjing Children's Hospital Affiliated to Nanjing Medical University, No. 72 Guangzhou Road, Nanjing 210008, China.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Hu
authors.affiliation: Pediatrics Research Institute, Nanjing Children's Hospital Affiliated to Nanjing Medical University, No. 72 Guangzhou Road, Nanjing 210008, China.
authors.forename: Qihua
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Shanghai Rendu Biotechnology Co., Ltd., No. 590 Ruiqing Road, East Area Zhangjiang High-Tech Park, Shanghai 201201, China.
authors.forename: Minghui
authors.initials: M
authors.lastname: Yu
mesh_terms: D029821:Enterovirus A, Human / Q000302:isolation & purification*; D006232:Hand, Foot and Mouth Disease / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods* / Q000592:standards*; D012015:Reference Standards; D012680:Sensitivity and Specificity; D013696:Temperature
keywords: Enterovirus 71; Isothermal amplification; PCR-fluorescence probing assay