摘要
目的 :建立一种简易的HIV 1感染者和艾滋病人血浆病毒载量测定的方法。方法 :构建含保守的引物SK14 5 SK4 31扩增靶序列的重组质粒pSPgag737,重组质粒体外转录产物经系列稀释得到HIV 1定量测定的外参照 ,在同样条件下以引物SK14 5 SK4 31对外参照和待测样品进行RT PCR扩增 ,用电泳图谱分析系统分析经EB染色的扩增产物电泳图谱 ,作出外参照的初始加入拷贝数与光强度的标准曲线 ,获得相应的回归方程 ,进而计算出待测样品的HIV 1病毒载量。结果 :重组质粒pSPaga737经酶切和PCR鉴定 ,确认含有引物SK14 5 SK4 31的扩增靶序列 ,用重组质粒体外转录 ,得到了 1～ 10 5copies的 10倍系列稀释的外参照 ,外参照标准曲线有较好的线性关系 (r =0 96 )。该方法的测定范围为 5× 10 2 ～ 5× 10 7拷贝 ml血浆。对 10个样品进行 3次重复测定 ,平均变异系数较小 (CV =2 0 % )。结论 :所建立的HTV 1病毒载量定量测定方法 ,操作简单 ,价格便宜 ,特异性好 ,同时有较好的可重复性。
Objective:To develop a simple and practical method for the quantitation of plasma HIV-1 load in HIV-1 infected and AIDS patients.Methods:The recombinant plasmid pSPgag737 containing the target sequence of primers SK145/SK431 was constructed.The transcripted RNA in vitro from recombinant plasmid was serially diluted to obtain the external standard for the quantitation of HIV-1 load.All the standards and examined patient samples were amplified with RT-PCR USING primers SK145/SK431. The electrophoresis image with EB staining was analyzed with Kodak Digital Science Electrophoresis Documentation and Analysis System 120(EDAS120),and the standard curve of external standard was calculated.Thereafter, the HIV-1 loads of examined samples were computed.Results:The results of PCR and restriction endonuclease digestion showed the target sequence of primers SK145/SK431 was inserted into the recombinant plasmid pSPgag737.The detectable range of pSPgeg 737-transcripted external standard was 10～10 5 copies per PCR reaction.The correlation coefficient of external standard between the input copies and fluorescence intensity was 0.96.The higher intra-assay reproducibility( CV =20%)was demonstrated by triplicate test of 10 patient samples.The HIV-1 load which can be detected by this method ranged from 5×10 2 to 5×10 7 copies per ml plasma.Conclusion:A simple, practical and reproducible method for quantitation of HIV-1 load was established. It is an useful tool in basic research and clinical practice on AIDS.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2003年第11期779-782,共4页
Chinese Journal of Immunology
基金
云南省与中国科学院省院合作项目资助 (96S0 0 4)
