Sensitive, Seminested PCR Amplification of VP1 Sequences for Direct Identification of All Enterovirus Serotypes from Original Clinical Specimens

Schematic representation of the locations of the primers used in the CODEHOP VP1 RT-snPCR. (A) Similarity plot of the aligned capsid amino acid sequences of 64 enterovirus prototype strains. Sequence identity scores were calculated within each 6-residue window, and the window was progressively moved across the alignment in 1-residue increments, with the identity score plotted versus the position at the center of the window. The positions of the four mature EV capsid proteins, VP4, VP2, VP3, and VP1, are shown at the top. The orientations and approximate positions of the cDNA primers (open arrowheads) and PCR primers (filled arrowheads) are shown directly above the plot. (B) Amino acid motifs used in primer design and schematic representation of the steps in the CODEHOP VP1 RT-snPCR assay. Consensus amino acid motifs are shown. Asterisks indicate that the residue directly above the asterisk is present at that position in at least 90% of EV prototype strains; when only a single residue is shown, it is present in all prototype strains. Primer sequences are shown directly below the amino acid motif sequences. Ambiguity codes are as follows: R, A or G; Y, C or T; W, A or T; N, A, C, G, or T; I, inosine.