The novel RT-IMSA assay demonstrated higher diagnostic sensitivity for EV71 (96.4% vs 91.1%) and CVA16 (94.6% vs 90.8%) compared to RT-LAMP, with improved detection limits.
Does the novel RT-IMSA assay improve the detection limit and diagnostic sensitivity for EV71 and CVA16 compared to RT-LAMP and qRT-PCR?
The novel RT-IMSA assay provides a more sensitive and rapid detection method for EV71 and CVA16 compared to existing RT-LAMP assays.
Absolute Event Rate: 96.4% vs 91.1%
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.
Ding et al. (Thu,) conducted a other in Hand-foot-and-mouth disease (EV71 and CVA16 infection). Reverse transcription-isothermal multiple-self-matching-initiated amplification (RT-IMSA) assay vs. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was evaluated on Diagnostic sensitivity for EV71. The novel RT-IMSA assay demonstrated higher diagnostic sensitivity for EV71 (96.4% vs 91.1%) and CVA16 (94.6% vs 90.8%) compared to RT-LAMP, with improved detection limits.