A method of detecting severe disease-associated mutations in an enterovirus 71 (EV-A71), comprising: performing an assay on a test sample containing an EV-A71 genomic RNA, a fragment thereof or an amplicon thereof, or an EV-A71 VP1 protein or fragment thereof to detect one or more severe disease-associated mutations in the EV-A71 genomic RNA, the fragment thereof or the amplicon thereof, or the EV-A71 protein or fragment thereof, wherein the one or more mutations are selected from mutations at positions corresponding to 5′ UTR nucleotide positions C580, A707, and C709 in an EV-A71 5′ UTR nucleic acid sequence and at residues corresponding to A280 and E145 in an EV-A71 VP1 protein sequence.