Highly Sensitive and Specific Diagnosis of Enterovirus A71 by Reverse Transcription Multiple Cross-Displacement Amplification-Labeled Nanoparticles Biosensor

Enterovirus A71 (EVA71) is a leading causative agent of hand, foot, and mouth disease, posing a significant threat to the health of young children, particularly in the Asia-Pacific region. Currently, there is no specific antiviral drug for EVA71 infection; therefore, early and rapid diagnosis is critical for disease prevention and control. Here, we report the development of a simple, rapid, and sensitive detection method for EVA71 infection using reverse transcription-multiple cross displacement amplification (RT-MCDA) combined with nanoparticle-based lateral flow biosensors (LFB). In the RT-MCDA system, a set of 10 primers was designed to target the highly conserved region of the VP1 gene of EVA71 and amplify the genes in an isothermal amplification device. The RT-MCDA amplification reaction products could then be identified by visual detection reagent (VDR) and LFB without the need for specialized equipment. The results demonstrated that the optimal reaction condition for the EVA71-RT-MCDA assay was 65℃ for 40 min. The EVA71-RT-MCDA assay could detect as low as 40 copies of plasmid and 50 copies of pseudotyped virus in a reaction. No cross-reaction was found between EVA71 strains and non-EVA71 strains. For 125 clinical anal swab samples, with EVA71-RT-MCDA assay, 30 samples were positive, which was in consistent with the the conventional real-time quantitative reverse transcription polymerase chain reaction assays. The entire procedure, including a 15-min specimen processing step, a 40-min MCDA reaction, and result reporting within 2 min, was completed in less than 60 min. In conclusion, the EVA71-RT-MCDA-LFB assay targeting the VP1 gene is a rapid, highly sensitive, simple, and specific test that could be widely applied in point-of-care settings and basic medical facilities in rural areas.