Standardization of denaturing gradient gel electrophoresis for mutant screening of influenza A (H3N2) virus samples.
Because of the extensive genetic variability of the influenza viruses, new virus mutants arise worldwide. In the human population, some strains may become potentially epidemic after evading the immune response of the host. At present, molecular methods have made it possible to identify these variants. However, if a large number of samples need to be analyzed the identification of randomly mutated nucleotides cannot be achieved by sequencing analysis or restriction fragment length polymorphism (RFLP). In order to improve this process, a denaturing gradient gel electrophoresis (DGGE) protocol capable of discriminating between reference strains representative of different influenza seasons, some mutant strains, and five clinical isolates was standardized Ribonudeic acid (RNA) was isolated and submitted to a one-step RT-PCR that amplified the region codifying for the globular domain of the Haemagglutinin (HA) molecule. The amplicons were analyzed by electrophoresis in 6% polyacrylamide gel at 60 degreeC/150 V for 8 h, using a 31--41% urea--formamide gradient. This method was able to distinguish between closely related nucleotide sequences, confirming its suitability as screening methodology for the analysis of influenza virus epidemiology, by allowing a faster and more extensive evaluation of a large number of the variant strains detected in a specific region of the world.
Motta
F C
FC
Depto. de Virologia, Lab. de Virologia Molecular I, Instituto de Microbiologia Prof. Paulo de Goes, Universidade Federal do Rio de Janeiro, 21.941-590, RJ, Rio de Janeiro, Brazil.
Rosado
A S
AS
Couceiro
J N S S
JN
D000595:Amino Acid Sequence; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel / Q000592:standards*; D014644:Genetic Variation; D006388:Hemagglutinins / Q000737:chemistry; D006801:Humans; D053122:Influenza A Virus, H3N2 Subtype; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000453:epidemiology; D008969:Molecular Sequence Data; D009154:Mutation; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000032:analysis*; D016415:Sequence Alignment
D006388:Hemagglutinins; D012367:RNA, Viral