One-step protocol for amplification of near full-length cDNA of the rabies virus genome
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Citações na Scopus
17
Tipo de produção
article
Data de publicação
2011
Título da Revista
ISSN da Revista
Título do Volume
Editora
ELSEVIER SCIENCE BV
Autores
CAMPOS, Angelica Cristine de Almeida
MELO, Fernando Lucas
ARAUJO, Danielle Bastos
CUNHA, Elenice Maria Sequetin
SACRAMENTO, Debora Regina Veiga
ZANOTTO, Paolo Marinho de Andrade
DURIGON, Edison Luiz
FAVORETTO, Silvana Regina
Citação
JOURNAL OF VIROLOGICAL METHODS, v.174, n.1-2, p.1-6, 2011
Resumo
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. Crown
Palavras-chave
Rabies virus, Long cDNA, Sequencing, Method development, Reverse-transcriptase polymerase chain reaction
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