RESUMO
HPV infection is considered one of the leading causes of cervical cancer in the world. To date, more than 180 types of HPV have been described and viral typing is critical for defining the prognosis of cancer. In this work, a seminested PCR which allow fast and inexpensively detection and typing of HPV is presented. The system is based on the amplification of a variable length region within the viral gene E1, using three primers that potentially anneal in all HPV genomes. The amplicons produced in the first step can be identified by high resolution electrophoresis or direct sequencing. The seminested step includes nine specific primers which can be used in multiplex or individual reactions to discriminate the main types of HPV by amplicon size differentiation using agarose electrophoresis, reducing the time spent and cost per analysis.
Assuntos
Genoma Viral/genética , Papillomaviridae/classificação , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase/métodos , Neoplasias do Colo do Útero/virologia , Proteínas Virais/genética , Sequência de Aminoácidos , Colo do Útero/virologia , Primers do DNA/genética , Feminino , Genótipo , Técnicas de Genotipagem , Células HeLa , Humanos , Papillomaviridae/genética , Papillomaviridae/isolamento & purificação , Sensibilidade e Especificidade , Alinhamento de SequênciaRESUMO
Analyzing the positions of 2A-like polypeptide cleavage sites in all available genomes of arthropod totiviruses we propose the limits of all ORF1 coding sequences and observed that two proteins previously predicted in infectious myonecrosis virus genome are unique in the arthropod totiviruses group. A putative protein cleavage site upstream the major capsid protein was also identified only in these genomes. In addition, protein models generated using ab initio and threading approaches revealed conserved structures possibly related to formation of viral protrusions and RNA packaging, clarifying the mechanisms involved in the extracellular transmission. These data appoints that the group formed by arthropod totiviruses are sufficient distinctive to be clustered in new genus belonging to the Totiviridae family, in agreement with previous phylogenetic analysis.