title: Improved detection limit in rapid detection of human enterovirus 71 and coxsackievirus A16 by a novel reverse transcription-isothermal multiple-self-matching-initiated amplification assay.
abstract: Rapid detection of human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) is important in the early phase of hand-foot-and-mouth disease (HFMD). In this study, we developed and evaluated a novel reverse transcription-isothermal multiple-self-matching-initiated amplification (RT-IMSA) assay for the rapid detection of EV71 and CVA16 by use of reverse transcriptase, together with a strand displacement DNA polymerase. Real-time RT-IMSA assays using a turbidimeter and visual RT-IMSA assays to detect EV71 and CVA16 were established and completed in 1 h, and the reported corresponding real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assays targeting the same regions of the VP1 gene were adopted as parallel tests. Through testing VP1 RNAs transcribed in vitro, the real-time RT-IMSA assays exhibited better linearity of quantification, with R(2) values of 0.952 (for EV71) and 0.967 (for CVA16), than the real-time RT-LAMP assays, which had R(2) values of 0.803 (for EV71) and 0.904 (for CVA16). Additionally, the detection limits of the real-time RT-IMSA assays (approximately 937 for EV71 and 67 for CVA16 copies/reaction) were higher than those of real-time RT-LAMP assays (approximately 3,266 for EV71 and 430 for CVA16 copies/reaction), and similar results were observed in the visual RT-IMSA assays. The new approaches also possess high specificities for the corresponding targets, with no cross-reactivity observed. In clinical assessment, compared to commercial reverse transcription-quantitative PCR (qRT-PCR) kits, the diagnostic sensitivities of the real-time RT-IMSA assays (96.4% for EV71 and 94.6% for CVA16) were higher than those of the real-time RT-LAMP assays (91.1% for EV71 and 90.8% for CVA16). The visual RT-IMSA assays also exhibited the same results. In conclusion, this proof-of-concept study suggests that the novel RT-IMSA assay is superior to the RT-LAMP assay in terms of detection limit and has the potential to rapidly detect EV71 and CVA16 viruses.
authors.affiliation: School of Light Industry and Food Sciences, South China University of Technology, Guangzhou city, Guangdong, China Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping district, Beijing, China.
authors.forename: Xiong
authors.initials: X
authors.lastname: Ding
authors.affiliation: Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping district, Beijing, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Nie
authors.affiliation: School of Light Industry and Food Sciences, South China University of Technology, Guangzhou city, Guangdong, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
authors.affiliation: Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping district, Beijing, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping district, Beijing, China.
authors.forename: Li
authors.initials: L
authors.lastname: Guan
authors.affiliation: Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping district, Beijing, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Institute for Viral Disease Control and Prevention, Hebei Center for Disease Control and Prevention, Shijiazhuang, Hebei, China hbcdc999@126.com maxj@ivdc.chinacdc.cn.
authors.forename: Shunxiang
authors.initials: S
authors.lastname: Qi
authors.affiliation: Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping district, Beijing, China hbcdc999@126.com maxj@ivdc.chinacdc.cn.
authors.forename: Xuejun
authors.initials: X
authors.lastname: Ma
mesh_terms: D002648:Child; D002675:Child, Preschool; D004770:Enterovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D029821:Enterovirus A, Human / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D006232:Hand, Foot and Mouth Disease / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D007223:Infant; D057230:Limit of Detection; D008297:Male; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D048348:Reverse Transcription; D013997:Time Factors