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Genotyping of GII.4 and GIIb norovirus RT-PCR amplicons by RFLP analysis

  • Autori: Ramirez, S.; Giammanco, G.; DE GRAZIA, S.; Colomba, C.; Martella, V.; Arista, S.
  • Anno di pubblicazione: 2008
  • Tipologia: Articolo in rivista (Articolo in rivista)
  • Parole Chiave: GIIb/Hilversum strain GII.4 genotype Restriction fragment length polymorphism (RFLP)
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GII.4 and GIIb/Hilversum norovirus (NoV) strains appear to have a prominent epidemiological role in outbreaks or sporadic cases of human gastroenteritis. Sequence analysis, although laborious, is the reference method used for characterization of noroviruses. In this study a screening test is proposed to characterize GIIb and GII.4 NoVs based on restriction fragment length polymorphism (RFLP) analysis of amplicons obtained from the RNA-dependent RNA polymerase (RdRp) region. Virtual analysis of 793 RdRp sequences of GGI and GGII NoVs, retrieved from GenBank, and representative of global geographical origins on a long-time period, permitted the selection of four restriction enzymes, XmnI, AhdI, BstXI, and AcuI, suitable for correct identification of GIIb and GII.4 NoV genotypes. Experimental analysis by the RT-PCR RFLP analysis of 41 NoV strains detected in Palermo during the years 2002–2005 allowed to recognize all the Italian strains as belonging to GIIb/Hilversum or GII.4, and sequence analysis confirmed these results. The PCR-RFLP protocol developed in this study proved to be a simple and reliable proxy for sequence-based classification of the GIIb/Hilversum and GII.4 NoV variants displaying high specificity (100%) and sensitivity (94%).