Species-specific RT-PCR amplification of human enteroviruses: a tool for rapid species identification of uncharacterized enteroviruses.
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.
Oberste
M Steven
MS
Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Mailstop G-17, Atlanta, GA 30333, USA.
Maher
Kaija
K
Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Mailstop G-17, Atlanta, GA 30333, USA.
Williams
Alford J
AJ
Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Mailstop G-17, Atlanta, GA 30333, USA.
Dybdahl-Sissoko
Naomi
N
Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Mailstop G-17, Atlanta, GA 30333, USA.
Brown
Betty A
BA
Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Mailstop G-17, Atlanta, GA 30333, USA.
Gookin
Michelle S
MS
Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Mailstop G-17, Atlanta, GA 30333, USA.
Peñaranda
Silvia
S
Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Mailstop G-17, Atlanta, GA 30333, USA.
Mishrik
Nada
N
Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Mailstop G-17, Atlanta, GA 30333, USA.
Uddin
Moyez
M
Institute of Public Health, Dhaka, Bangladesh.
Pallansch
Mark A
MA
Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Mailstop G-17, Atlanta, GA 30333, USA.
D020413:3' Untranslated Regions / Q000032:analysis* / Q000235:genetics; D000595:Amino Acid Sequence; D002213:Capsid / Q000737:chemistry; D036022:Capsid Proteins / Q000235:genetics*; D017931:DNA Primers; D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification*; D016679:Genome, Viral; D006801:Humans; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D020413:3' Untranslated Regions; D036022:Capsid Proteins; D017931:DNA Primers