title: Molecular Properties of Poliovirus Isolates: Nucleotide Sequence Analysis, Typing by PCR and Real-Time RT-PCR.
abstract: Virologic surveillance is essential to the success of the World Health Organization initiative to eradicate poliomyelitis. Molecular methods have been used to detect polioviruses in tissue culture isolates derived from stool samples obtained through surveillance for acute flaccid paralysis. This chapter describes the use of realtime PCR assays to identify and serotype polioviruses. In particular, a degenerate, inosine-containing, panpoliovirus (panPV) PCR primer set is used to distinguish polioviruses from NPEVs. The high degree of nucleotide sequence diversity among polioviruses presents a challenge to the systematic design of nucleic acid-based reagents. To accommodate the wide variability and rapid evolution of poliovirus genomes, degenerate codon positions on the template were matched to mixed-base or deoxyinosine residues on both the primers and the TaqMan™ probes. Additional assays distinguish between Sabin vaccine strains and non-Sabin strains. This chapter also describes the use of generic poliovirus specific primers, along with degenerate and inosine-containing primers, for routine VP1 sequencing of poliovirus isolates. These primers, along with nondegenerate serotype-specific Sabin primers, can also be used to sequence individual polioviruses in mixtures.
authors.affiliation: Polio and Picornavirus Laboratory Branch, Division of Viral Diseases, National Center for Immunization and Respiratory Disease, Centers for Disease Control and Prevention, 1600 Clifton Rd., NE MS G-10, Atlanta, GA, 30333, USA. cburns@cdc.gov.
authors.forename: Cara C
authors.initials: CC
authors.lastname: Burns
authors.affiliation: Polio and Picornavirus Laboratory Branch, Division of Viral Diseases, National Center for Immunizationand Respiratory Disease, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: David R
authors.initials: DR
authors.lastname: Kilpatrick
authors.affiliation: Polio and Picornavirus Laboratory Branch, Division of Viral Diseases, National Center for Immunizationand Respiratory Disease, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: Jane C
authors.initials: JC
authors.lastname: Iber
authors.affiliation: Polio and Picornavirus Laboratory Branch, Division of Viral Diseases, National Center for Immunizationand Respiratory Disease, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: Qi
authors.initials: Q
authors.lastname: Chen
authors.affiliation: Polio and Picornavirus Laboratory Branch, Division of Viral Diseases, National Center for Immunizationand Respiratory Disease, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: Olen M
authors.initials: OM
authors.lastname: Kew
mesh_terms: D001483:Base Sequence; D006801:Humans; D011051:Poliomyelitis / Q000175:diagnosis* / Q000821:virology; D017955:Poliovirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D017423:Sequence Analysis, RNA / Q000379:methods
chemical_list: D012367:RNA, Viral
keywords: Molecular; Molecular serotyping; Poliomyelitis; Poliovirus; Polymerase chain reaction; Sanger; Sequencing