两种HIV-1耐药准种分析方法的比较  

Comparison of two methods of analysis of HIV-1 drug-resistant quasispecies

作　　者：焦丽燕[1] 刘永健[1] 郭东星[1] 王铮[1] 耿庆茂[1] 李林[1] 庄道民[1] 鲍作义[1] 刘思扬[1] 李韩平[1] 李敬云[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《军事医学科学院院刊》2010年第3期261-264,共4页Bulletin of the Academy of Military Medical Sciences

基　　金：国家自然科学基金(30800938);国家自然科学基金重点项目(30830088)

摘　　要：目的比较两种HIV-1耐药准种分析方法 ---克隆PCR产物测序法和单基因扩增法(单基因组测序法,single-genome sequencing,SGS)。方法以本室贮存的某接受高效抗逆转录病毒治疗(highly active antiretroviraltherapy,HAART)的艾滋病患者的外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)为样本,分别用克隆PCR产物序列测定法和SGS方法得到HIV-1pol区准种序列,对准种序列进行离散率、耐药相关突变组合构成比等分析。结果克隆方法得到19条pol区序列,SGS方法得到18条pol区序列。对两种方法的核苷酸和蛋白质序列的平均离散率进行成对样本均值分析,采用t检验,P值(单尾)为0.311,差异没有显著性意义;两种方法检测出各种突变组合构成比的2检验结果 ,P>0.25,差异也没有显著性意义。但是,准种中占较少比例的突变模式在两种方法检测的结果不一致,如T215Y/K103N/Y181C/H221Y以及M41L/F116L/T215Y/K103N/Y181C/H221Y两种突变组合模式只在克隆方法中检测到,而携带M41L/A62V/T69Si/T215Y/A98G/K103N/Y181C以及K103N突变的两种毒株仅在SGS方法中出现。结论两种分析HIV-1耐药突变准种方法检测该例样本的核苷酸序列和蛋白质准种序列无显著性差异,检出的主要突变组合的构成比也无显著性差异,对优势准种的检测中两种方法差异不大,但在劣势准种的检测中两种方法有一定差异。Objective To compare two methods for analyzing HIV-1 drug resistant quasispecies—clone PCR sequencing method and single-genome sequencing（SGS）method.Methods The peripheral blood mononuclear cells（PBMCs）were taken from one AIDS patient receiving highly active antiretroviral therapy（HAART）within a HIV-1 drug resistance cohort.HIV-1 pol DNA quasispecies sequences from the PBMCs were obtained by clone PCR sequencing and SGS respectively.Results Nineteen pol sequences were acquired by clone PCR sequencing and 18 by SGS.Mean dispersion and variance of nucleotide and protein sequences by the two methods were analyzed using paired-samples t test,P=0.311,which meant that the two methods had no statistical difference.The composing proportion of each HIV-1 drug resistant mutation pattern between the two methods was compared utilizing Chi-square test,P0.25,which indicated the two methods had no significant difference.However,the inferior position quasipecies were detected diversely by the two methods,for example,the viruses with T215Y/K103N/Y181C/H221Y and M41L/F116L/T215Y/K103N/Y181C/H221Y were only detected by clone PCR sequencing and the viruses with M41L/A62V/ T69Si/T215Y/A98G/K103N/Y181C and K103N were merely detected by SGS.Conclusion These results suggest that the two methods have no obvious difference in detecting quasispecies of this sample.The results of two methods are coincident in detecting HIV-1 drug resistance mutation pattern,especially in detecting predominance viruses.Nevertheless,there is discrepancy in detecting quasipecies of inferior position.

关 键 词：HIV-1准种 克隆测序 单基因扩增法 聚合酶链反应 

分 类 号：R512.91[医药卫生—内科学]