Núcleo de Pesquisas em Raiva do Laboratório de Virologia Clínica e Molecular da Universidade de São Paulo, CEP: 05508-900, São Paulo, Brazil.
J Virol Methods. 2011 Jun;174(1-2):1-6. doi: 10.1016/j.jviromet.2011.03.030. Epub 2011 Apr 5.
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.
全长基因组测序的狂犬病病毒不是一个常规的实验室程序。要充分了解狂犬病病毒的流行病学、遗传变异和进化，需要获得全长病毒基因组。对于狂犬病病毒的研究，通常使用非特异性寡核苷酸进行 cDNA 合成，然后进行克隆。当使用特异性引物时，获得的 cDNA 是部分的，仅限于编码区。因此，开发使用狂犬病病毒特异性引物合成长 cDNA 的方法至关重要。本研究描述了一种新的长 cDNA 合成方案和 19 个新引物的开发。该程序允许在合成完整的长 cDNA 后，有效地扩增由吸血蝙蝠（Desmodus rotundus）种群维持的狂犬病病毒变异体的全长基因组。对狂犬病病毒基因组的部分测序进行了确认，以确认狂犬病特异性 PCR 扩增。由于使用了简并引物，这种技术可以很容易地适应其他变体。重要的是，这种新方法比克隆方法更快、更便宜。
