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J Gen Virol. 2006 Jan;87(Pt 1):119-28.

Species-specific RT-PCR amplification of human enteroviruses: a tool for rapid species identification of uncharacterized enteroviruses.

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Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.


The 65 serotypes of human enteroviruses are classified into four species, Human enterovirus (HEV) A to D, based largely on phylogenetic relationships in multiple genome regions. The 3'-non-translated region of enteroviruses is highly conserved within a species but highly divergent between species. From this information, species-specific RT-PCR primers were developed that can be used to rapidly screen collections of enterovirus isolates to identify species of interest. The four primer pairs were 100 % specific when tested against enterovirus prototype strains and panels of isolates of known serotype (a total of 193 isolates). For evaluation in a typical application, the species-specific primers were used to screen 186 previously uncharacterized non-polio enterovirus isolates. The HEV-B primers amplified 68.3 % of isolates, while the HEV-A and HEV-C primers accounted for 9.7 and 11.3 % of isolates, respectively; no isolates were amplified with the HEV-D primers. Twelve isolates (6.5 %) were amplified by more than one primer set and eight isolates (4.3 %) were not amplified by any of the four primer pairs. Serotypes were identified by partial sequencing of the VP1 capsid gene, and in every case sequencing confirmed that the species-specific PCR result was correct; the isolates that were amplified by more than one species-specific primer pair were mixtures of two (11 isolates) or three (one isolate) species of viruses. The eight isolates that were not amplified by the species-specific primers comprised four new serotypes (EV76, EV89, EV90 and EV91) that appear to be unique members of HEV-A based on VP1, 3D and 3'-non-translated region sequences.

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