One-step protocol for amplification of near full-length cDNA of the rabies virus genome

Author(s)

de Almeida Campos, Angelica Cristine ^[1] ; Melo, Fernando Lucas ^[2] ; Romano, Camila Malta ^{[2, 3]} ; Araujo, Danielle Bastos ^[1] ; Sequetin Cunha, Elenice Maria ^[4] ; Veiga Sacramento, Debora Regina ^{[1, 5]} ; de Andrade Zanotto, Paolo Marinho ^[2] ; Durigon, Edison Luiz ^[1] ; Favoretto, Silvana Regina ^{[6, 1]}

Affiliation

^[1] Univ Sao Paulo, Nucleo Pesquisas Raiva, Lab Virol Clin & Mol, BR-05508900 Sao Paulo - Brazil

^[2] Univ Sao Paulo, Lab Evolucao Mol & Bioinformat, Inst Ciencias Biomed, BR-05508900 Sao Paulo - Brazil

^[3] Univ Sao Paulo, Virol Lab, Inst Trop Med, Fac Med, BR-05403000 Sao Paulo - Brazil

^[4] Secretaria Agr Estado Sao Paulo, Agencia Paulista Tecnol, Inst Biol, Lab Encefalites, Ctr P&D Sanidade Anim, Sao Paulo - Brazil

^[5] Genom Engn Mol, BR-01332903 Sao Paulo - Brazil

^[6] Inst Pasteur, BR-01311000 Sao Paulo - Brazil

• Document type: Review article
• Source: Journal of Virological Methods; v. 174, n. 1-2, p. 1-6, JUN 2011.
• Web of Science Citations: 9

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. Crown Copyright (C) 2011 Published by Elsevier B.V. All rights reserved. (AU)

• FAPESP's process: 07/01843-0 - Genetic study of rabies virus variant maintained by hematophagous bats Desmodus rotundus by viral genome complete sequencing
• Grantee: Silvana Regina Favoretto
• Support Opportunities: Regular Research Grants