title: Allele-specific PCR for quantitative analysis of mutants in live viral vaccines.
abstract: Monitoring consistency of genetic composition of oral polio vaccine (OPV) is a part of its quality control. It is performed by mutant analysis by PCR and restriction enzyme cleavage (MAPREC) used to quantify neurovirulent revertants in the viral genome. Here an alternative method based on quantitative PCR is proposed. Allele-specific quantitative polymerase chain reaction (asqPCR) uses a "tethered" oligonucleotide primer consisting of two specific parts connected by a polyinosine stretch. Homogeneous DNA from plasmids containing wild Leon/37 and attenuated Sabin 3 sequences with 100% 472(C) and 100% 472(T) could only be amplified using homologous primers. Real-time implementation of the allele-specific PCR resulted in sensitive detection of 472(C) revertants with the limit of quantitation of less than 0.05%. Monovalent vaccine batches and international viral references for MAPREC test were used to validate the method. asqPCR performed with the WHO references and monovalent batches of vaccine showed that the new method could measure accurately and reproducibly the content of revertants producing values comparable to MAPREC results. This suggests that asqPCR could be used as an alternative to MAPREC for lot release of OPV. The method could also be used for the quantitation of other mutants in populations of microorganisms.
authors.affiliation: Center for Biologics Evaluation and Research, US Food and Drug Administration, 1401 Rockville Pike, HFM 470, Rockville, MD 20852, USA.
authors.forename: Bella
authors.initials: B
authors.lastname: Bidzhieva
authors.affiliation: Center for Biologics Evaluation and Research, US Food and Drug Administration, 1401 Rockville Pike, HFM 470, Rockville, MD 20852, USA.
authors.forename: Majid
authors.initials: M
authors.lastname: Laassri
authors.affiliation: Center for Biologics Evaluation and Research, US Food and Drug Administration, 1401 Rockville Pike, HFM 470, Rockville, MD 20852, USA. Electronic address: konstantin.chumakov@fda.hhs.gov.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Chumakov
mesh_terms: D000483:Alleles; D006801:Humans; D017354:Point Mutation; D011055:Poliovirus Vaccine, Oral / Q000592:standards*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011786:Quality Control; D014613:Vaccines, Attenuated / Q000592:standards; D014780:Viruses / Q000235:genetics*
chemical_list: D011055:Poliovirus Vaccine, Oral; D014613:Vaccines, Attenuated
keywords: Alternative methods; Consistency monitoring; MAPREC; Oral Polio Vaccine; Virulent revertants