A new approach for sequencing virion genome of Chinese HIV-1 strains subtype B and BC from plasma
A new approach for sequencing virion genome of Chinese HIV-1 strains subtype B and BC from plasma
复制标题
血浆中中国HIV-1毒株B和BC亚型病毒体基因组测序的新方法
DOI：
10.3760/cma.j.issn.0366-6999.2011.02.028
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发表时间：
2011-01-20
影响因子：
6.1
通讯作者：
Shao Yi-ming
中科院分区：
文献类型：
--
作者：
Meng Zhe-feng;Zhang Xiao-yan;Shao Yi-ming
研究背景尽管HIV-1全基因组序列的测定对于研究病毒的遗传进化和变异以及开发候选疫苗具有重要意义，但直接测定HIV-1病毒体RNA的全基因组序列仍是一个挑战。由于缺乏专门的实验方法和合适的实验设备，目前还没有发表的中国HIV-1流行株的病毒体RNA基因组序列。本研究通过改进现有的HIV病毒RNA提取和测序方法，建立了一种简便的HIV病毒RNA提取和测序方法，并进一步证实了该方法适用于中国样本的处理。方法对HIV病毒RNA的提取和基因扩增方法进行了改进和优化，使其能够在中国大部分实验室广泛使用。利用中国HIV-1毒株的基因序列比对设计了Thai-B和BC重组毒株的特异性引物。在综合考虑BC重组菌株高变区基因和重组断裂点的基础上，提出了一种3个扩增子的策略（包括4.3-kb gag-pol、2.9-kb pol-env和2.7-kb env-nef）。此外，还在32份不同病毒载量的样本中使用了一个扩增子（接近全长基因组的9 kb）。结果一次扩增策略的扩增效率为25%（8/32），三次扩增策略的扩增效率为65.6%（21/32）。对于一种扩增子策略，通过DNA测序没有获得完整的近全长基因组序列。对于三扩增子策略，在DNA测序中实现了75%的序列。结论覆盖HIV-1所有编码区的三扩增子策略适用于Thai-B和BC重组株，可在设备较落后的中国实验室推广应用。中华医学杂志2011;124（2）：304-308
Background 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.Methods 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.Results 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.Conclusion 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. Chin Med J 2011;124(2):304-308