Shanghai Public Health Clinical Center, Key Laboratory of Medical Molecular Virology of Ministry of Health, Shanghai Medical College, Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China.
Chin Med J (Engl). 2011 Jan;124(2):304-8.
Although it was widely accepted that full-length HIV genome sequences is important in studying virus genetic evolution and variation as well as developing vaccine candidate, to directly sequencing HIV-1 genome of virion RNA remains as a challenge worldwide. Up to date, no published genomic sequences from virion RNA are available for Chinese prevalent HIV-1 strains due to the absence of specialized protocol and appropriate lab equipments. In this study we developed a straightforward approach for amplifying and sequencing HIV virion RNA from plasma by modifying published protocols and further confirmed it is suitable to process Chinese samples.
The methods for viral RNA extraction and gene amplification was modified and optimized as could be widely used in most Chinese labs. Gene alignment of Chinese HIV-1 strains was employed for designing specialized primer sets for Thai-B and BC recombinant strains. Based on comprehensively consideration of high variable gene region and recombinant breakpoints in BC recombinant strains, a three-amplicon strategy (including 4.3-kb gag-pol, 2.9-kb pol-env and 2.7-kb env-nef) was developed. In addition, one amplicon (9 kb near full-length genome) was also used in 32 samples with varied viral loads. All amplicons were directly sequenced by DNA automated sequencer.
Twenty-five percent (8/32) amplification efficiency was achieved by the one-amplicon strategy and 65.6% (21/32) by three-amplicon strategy. For one amplicon strategy, none of complete near full-length genome sequences was obtained by DNA sequencing. For three-amplicon strategy, 75% sequences were achieved in DNA sequencing. Amplification efficiency but not sequencing efficiency was closely associated with viral loads.
Three-amplicon strategy covering all encoding regions of HIV-1 is suitable for Thai-B and BC recombinant strains and could be potentially employed in less-well equipped Chinese labs.
虽然广泛认为全长 HIV 基因组序列对于研究病毒遗传进化和变异以及开发疫苗候选物非常重要，但直接对病毒 RNA 进行 HIV-1 全基因组测序仍然是全球范围内的一个挑战。迄今为止，由于缺乏专门的方案和适当的实验室设备，无法获得中国流行的 HIV-1 毒株的病毒 RNA 基因组序列。在这项研究中，我们通过修改已发表的方案，开发了一种从血浆中扩增和测序 HIV 病毒 RNA 的简单方法，并进一步证实该方法适用于处理中国样本。
对病毒 RNA 提取和基因扩增方法进行了修改和优化，使其能够广泛应用于大多数中国实验室。根据中国 HIV-1 毒株的基因序列比对，设计了专门用于 Thai-B 和 BC 重组株的引物组。基于对 BC 重组株高变区和重组断点的综合考虑，开发了三扩增策略（包括 4.3kb gag-pol、2.9kb pol-env 和 2.7kb env-nef）。此外，还在 32 个具有不同病毒载量的样本中使用了一个扩增子（9kb 全长附近的基因组）。所有扩增子均直接通过 DNA 自动测序仪进行测序。
通过单扩增策略获得了 25%（8/32）的扩增效率，通过三扩增策略获得了 65.6%（21/32）的扩增效率。对于单扩增策略，通过 DNA 测序均未获得完整的全长近基因组序列。对于三扩增策略，通过 DNA 测序获得了 75%的序列。扩增效率而不是测序效率与病毒载量密切相关。
覆盖 HIV-1 所有编码区的三扩增策略适用于 Thai-B 和 BC 重组株，并且可能适用于设备较差的中国实验室。
