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

• Published in: Journal of clinical microbiology 2014-06, Vol.52 (6), p.1862-1870
• Main Authors: Ding, Xiong, Nie, Kai, Shi, Lei, Zhang, Yong, Guan, Li, Zhang, Dan, Qi, Shunxiang, Ma, Xuejun
• Format: Article
• Language: English
• Subjects: Child; Child, Preschool; Coxsackievirus; Enterovirus - classification; Enterovirus - genetics; Enterovirus - isolation & purification; Enterovirus A, Human - classification; Enterovirus A, Human - genetics; Enterovirus A, Human - isolation & purification; Female; Hand, Foot and Mouth Disease - diagnosis; Hand, Foot and Mouth Disease - virology; Human enterovirus 71; Humans; Infant; Limit of Detection; Male; Molecular Diagnostic Techniques - methods; Nucleic Acid Amplification Techniques - methods; Reverse Transcription; Time Factors; Virology
• ISSN: 0095-1137; 1098-660X
• DOI: 10.1128/JCM.03298-13

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.