A CRISPR/Cas12a-based reverse transcription multiple cross displacement amplification technique for sensitive, rapid, and highly specific detection of human enterovirus A71 in clinical application.

Enterovirus A71 (EVA71) is a major pathogen responsible for hand, foot, and mouth disease (HFMD) outbreaks and poses a considerable threat to public health. In this work, we established a molecular detection system that integrates reverse transcription multiple cross displacement amplification (RT-MCDA) with CRISPR/Cas12a technology, targeting the VP1 gene region for EVA71 identification. This platform, termed EVA71-RT-MCDA-CRISPR, utilizes RT-MCDA to achieve efficient pre-amplification of the target sequence, followed by sequence-specific recognition mediated by CRISPR/Cas12a. The detection results can be observed both through real-time fluorescence analysis and visual inspection under blue light illumination. The assay exhibits outstanding sensitivity, achieving a limit of detection as low as 3.36 copies per reaction, while retaining high specificity against non-EVA71 templates. Validation using clinical specimens further confirmed the diagnostic reliability of this RT-MCDA-CRISPR platform. The successful establishment of the EVA71-RT-MCDA-CRISPR system demonstrates its potential as a rapid, user-friendly, highly sensitive and specific method for on-site EVA71 detection, serving as a valuable tool for clinical diagnosis and epidemiological surveillance.