Live-attenuated influenza virus vaccines can be generated by reassortment of gene segments between an attenuated donor strain and a virulent wild-type virus. The annual production schedule for the seasonal influenza vaccine necessitates rapid and efficient genotyping of the reassorted progeny to identify the desired vaccine strains. This study describes a multiplex RT-PCR system capable of identifying each gene segment from the cold-adapted attenuated donor virus, B/Lee/40ca. The specificity of the amplification system was optimized by testing various wild-type influenza B viruses. The resulting RT-PCR method is sensitive and efficient enough for routine identification of reassortant clones to identify the desired gene constellation, consisting of six segments from the attenuated donor virus and the H and N genes from the wild-type virus. By providing a more rapid and efficient means of genotyping the candidate reassortant strains, this method could be implemented to expedite the generation of each component strain and allow more time to culture and process the final seasonal influenza vaccine.
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