Sensitive, seminested PCR amplification of VP1 sequences for direct identification of all enterovirus serotypes from original clinical specimens

Abstract

A reverse transcription-seminested PCR (RT-snPCR) assay was developed for the detection and identification of enterovirus (EV) RNA in clinical specimens. Three conserved protein motifs were identified by aligning the VP3 and VP1 sequences of prototype EV strains. Consensus degenerate primers were designed from a conserved VP3 motif and a distal VP1 motif for the first PCR. Consensus-degenerate hybrid oligonucleotide primers were designed from an internal VP1 motif and used with the same distal VP1 motif for the second, seminested PCR step. The primers were designed for broad target specificity and amplified all recognized and proposed EV serotypes and other antigenic variant strains tested. The VP1 RT-snPCR assay was slightly more sensitive than our in-house EV 5' nontranslated region RT-snPCR assay, detecting as few as 10 RNA copies per reaction. As an example application, the VP1 RT-snPCR assay was used to identify EVs in clinical specimens. A product of the expected size was successfully amplified and sequenced from cerebrospinal fluid; serum; stool suspensions; and nasopharyngeal, eye, and rectal swab specimens, allowing unambiguous identification of the infecting virus in all cases. The VP1 sequences derived from the RT-snPCR products allow rapid phylogenetic and molecular epidemiologic analysis of strains circulating during the EV season and comparison with EV sequences from past seasons or from different locations around the world.

FIG. 1.

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.

FIG. 2.

Sensitivity of VP1 RT-snPCR and comparison of the sensitivity with that of the 5′ nontranslated region RT-snPCR. (A) Amplification of RNA extracted from 10-fold serial dilutions of an EV68 virus stock; (B) amplification of 10-fold serial dilutions of VP3-VP1 sRNA; (C) comparison of VP1 RT-snPCR (top) with 5′-NTR RT-snPCR (bottom) using 10-fold serial dilutions.

FIG. 3.

VP1 RT-snPCR amplification of RNA extracted directly from original clinical specimens. For each reaction, 50 μl of each seminested PCR2 product was analyzed and gel purified by electrophoresis on a 1.5% agarose gel containing 0.5 μg ethidium bromide per milliliter. The specimens tested were cerebrospinal fluid (CSF), stool, rectal swab (RS), nasopharyngeal swab (NPS), eye (conjunctival) swab (ES), serum, and postmortem liver tissue.