title: One-step protocol for amplification of near full-length cDNA of the rabies virus genome.
abstract: Full-length genome sequencing of the rabies virus is not a routine laboratory procedure. To understand fully the epidemiology, genetic variation and evolution of the rabies virus, full-length viral genomes need to be obtained. For rabies virus studies, cDNA synthesis is usually performed using nonspecific oligonucleotides followed by cloning. When specific primers are used, the cDNA obtained is only partial and is limited to the coding regions. Therefore, the development of methods for synthesizing long cDNA using rabies virus-specific primers is of fundamental importance. A new protocol for the synthesis of long cDNA and the development of 19 new primers are described in this study. This procedure allowed the efficient amplification of the full-length genome of the rabies virus variant maintained by hematophagous bat (Desmodus rotundus) populations following the synthesis of a complete long cDNA. Partial sequencing of the rabies virus genome was performed to confirm rabies-specific PCR amplification. Because degenerate primers were employed, this technique can be adapted easily to other variants. Importantly, this new method is faster and less expensive than cloning methods.
authors.affiliation: Núcleo de Pesquisas em Raiva do Laboratório de Virologia Clínica e Molecular da Universidade de São Paulo, CEP: 05508-900, São Paulo, Brazil. camposac@usp.br
authors.forename: Angélica Cristine de Almeida
authors.initials: AC
authors.lastname: Campos
authors.forename: Fernando Lucas
authors.initials: FL
authors.lastname: Melo
authors.forename: Camila Malta
authors.initials: CM
authors.lastname: Romano
authors.forename: Danielle Bastos
authors.initials: DB
authors.lastname: Araujo
authors.forename: Elenice Maria Sequetin
authors.initials: EM
authors.lastname: Cunha
authors.forename: Débora Regina Veiga
authors.initials: DR
authors.lastname: Sacramento
authors.forename: Paolo Marinho
authors.initials: PM
authors.lastname: de Andrade Zanotto
authors.forename: Edison Luiz
authors.initials: EL
authors.lastname: Durigon
authors.forename: Silvana Regina
authors.initials: SR
authors.lastname: Favoretto
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D016679:Genome, Viral; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics*; D011820:Rabies virus / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014773:Virology / Q000379:methods*
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D012367:RNA, Viral