Determination of the Genome Composition of Influenza Virus Reassortants Using Multiplex Reverse Transcription-Polymerase Chain Reaction Followed by Fluorescent Single-Strand Conformation Polymorphism Analysis☆

摘要: The influenza virus genome is composed of eight negative-strand RNA segments. In cells coinfected with two or more strains, the genomic RNAs reassort at random, resulting in progeny viruses (reassortants) that contain genes derived from each parent. Genetic reassortment among occurs naturally and plays an important role viral epidemiology pathogenicity. process also utilized for annual production vaccines. Each year, gene segments encode major surface antigens current virulent, wild-type are reassorted remaining six a laboratory-derived vaccine "master donor" strain. As appears to be identifying desired constellation can labor-intensive. We developed streamlined, cost-effective method genotype combines multiplex reverse transcription-polymerase chain reaction (RT-PCR) fluorescent single-strand conformation polymorphism (SSCP) analysis. This utilizes oligonucleotide primers labeled one three dyes generate RT-PCR products segment configuration. reassortants, wild-type, master donor then electrophoresed under SSCP conditions. origin identified by fluorescence mobility shift patterns corresponding products. demonstrate utility this differentiating strain, several viruses, reassortants.