Serotype-Specific Identification of Polioviruses by PCR Using Primers Containing Mixed-Base or Deoxyinosine Residues at Positions of Codon Degeneracy

Abstract

We have developed a method for determining the serotypes of poliovirus isolates by PCR. Three sets of serotype-specific antisense PCR-initiating primers (primers seroPV1A, seroPV2A, and seroPV3A) were designed to pair with codons of VP1 amino acid sequences that are conserved within but that differ across serotypes. The sense polarity primers (primers seroPV1S, seroPV2S, and seroPV3S) matched codons of more conserved capsid sequences. The primers contain mixed-base and deoxyinosine residues to compensate for the high rate of degeneracy of the targeted codons. The serotypes of all polioviruses tested (48 vaccine-related isolates and 110 diverse wild isolates) were correctly identified by PCR with the serotype-specific primers. None of the genomic sequences of 49 nonpolio enterovirus reference strains were amplified under equivalent reaction conditions with any of the three primer sets. These primers are useful for the rapid screening of poliovirus isolates and for determining the compositions of cultures containing mixtures of poliovirus serotypes.

Molecular detection and identification of an enterovirus during an outbreak of aseptic meningitis

“…For this reason, the RT-PCR assay was supplemented with the molecular genotyping methods that are described in this study: RFLP and SSCP analysis. Most published data on biological/taxonomic studies and clinical investigations have described the use of genomic regions within the 5′-UTR of enteroviruses (21,23,27,32,35,39,40, and others), as was done in the present study, although other genomic regions of the enteroviruses have also been used (22,26,31,41).…”

“…It is therefore necessary to supplement RT-PCR with methods for the assessment of differences in the sequence of the PCR products, including Restriction Fragment Length Polymorphism (RFLP) analysis (26)(27)(28), hybridization with type-specific probes (29), or single-strand conformational polymorphism (SSCP) (30). Nevertheless, serotype-specific, or serotypegroup-specific RT-PCR have been described (31,32). Nucleotide sequences of RT-PCR products would also be quite helpful (33), at least for research purposes concerning the evolution and epidemiology of the viruses, although not for routine diagnosis of clinical isolates.…”

Specific detection of echoviruses 22 and 23 in cell culture supernatants by RT-PCR

“…Using cell culture to separate cultivable from noncultivable enteroviruses and, coupled with available pan-EV and pan-polio PCR primers [Yang et al, 1992;Kilpatrick et al, 1996], our E22/E23 RT-PCR method is an additional step toward molecular typing of enteroviruses by RT-PCR, allowing classification of an enterovirus isolate as "polio," "E22/E23," or "other cultivable enterovirus." Our method takes advantage of the fact that E22 and E23, while similar to other enteroviruses in their disease spectrum, are distinct in nucleotide sequence yet remaining very similar to one another [Hyypiä et al, 1992;Stanway et al, 1994;Oberste et al, 1998].…”

Polio Eradication Initiative (PEI) contribution in strengthening public health laboratories systems in the African region

“…Both laboratories have undergone trainings organized by the regional office on different methods to determine poliovirus (PV) serotypes and whether the virus was wild or related to vaccine strains (known as intratypic differentiation or ITD) [5][6][7][8][9][10][11]. Any wild poliovirus is further characterized to determine if it is an importation or indigenous virus [12,13].…”