Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, 8 College Rd., Singapore 169857, Singapore.
J Virol Methods. 2010 Oct;169(1):202-6. doi: 10.1016/j.jviromet.2010.06.013. Epub 2010 Jun 30.
The availability of whole genome sequencing has contributed to many aspects of dengue research, and its use in dengue virus (DENV) surveillance for early epidemic warning has been proposed. Methods to sequence the genomes of individual dengue serotypes have been described previously, but no single method is known to be applicable for all four serotypes. This report describes a method for sequencing the entire genome of all four DENV serotypes. Using tagged oligonucleotide primers designed for the 3' end, viral RNA was reverse transcribed into a cDNA spanning the entire genome of each of the four serotypes (DENV-1 to -4). This was followed by amplification of the entire cDNA in five overlapping amplicons. A sequence tag was added to the sense primer annealing to the 5' UTR sequence and the antisense primer annealing to the 3' UTR sequence to ensure no terminal nucleotides were omitted during PCR. Sixty-one virus isolates were sequenced: 58 DENV-2, one DENV-1, one DENV-4 and one DENV-3 published previously. The method described could be applied readily for viral biology studies and incorporated into proactive dengue virologic surveillance.
全基因组测序的出现促进了登革热研究的许多方面，有人提议将其用于登革热病毒（DENV）监测以实现早期疫情预警。先前已经描述了对个别登革热血清型进行基因组测序的方法，但没有一种方法被认为适用于所有四种血清型。本报告介绍了一种对所有四种 DENV 血清型进行全基因组测序的方法。使用针对 3' 末端设计的标记寡核苷酸引物，将病毒 RNA 反转录成 cDNA，该 cDNA 跨越了所有四种血清型（DENV-1 至 -4）的整个基因组。随后，通过五个重叠的扩增子对整个 cDNA 进行扩增。在与 5' UTR 序列退火的正链引物和与 3' UTR 序列退火的反链引物上添加一个序列标签，以确保在 PCR 过程中不会省略末端核苷酸。对 61 个病毒分离株进行了测序：58 个 DENV-2、1 个 DENV-1、1 个 DENV-4 和 1 个之前发表的 DENV-3。描述的方法可以很容易地应用于病毒生物学研究，并纳入主动登革热病毒学监测。
