摘要
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-ne） 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.
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-ne） 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.
基金
This study was supported by the grants from the Natural Science Foundation of China （No. 30901257）, the Chinese National Grand Program on Major New Drugs （No. 2009ZX09303-006）, the China Postdoctoral Science Foundation （No. 20090460585）, and the Shanghai Postdoctoral Program （No. 10R21410600）.
