SYBR Green Ⅰ荧光定量PCR检测HIV-1前病毒方法的建立及应用  

Foundation and application of SYBR Green I fluorescent quantitative PCR method for the detection of HAV-1 provirus

作　　者：莫雪梅[1] 闫莉[1] 孙晗笑[1] 

机构地区：[1]暨南大学药学院基因组药物研究所,广州510632

出　　处：《山东医药》2010年第23期10-12,共3页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(30872221)

摘　　要：目的建立SYBR Green Ⅰ荧光定量PCR技术测定HIV-1前病毒载量方法,并以其观察相关病毒进入抑制剂对HIV-1前病毒的作用。方法选择HIV-1的gag和env保守区域设计引物,建立SYBR Green I荧光定量PCR测定HIV-1前病毒载量的体系,并以其观察6μg/ml^6 ng/ml的重组病毒巨噬细胞炎症蛋白(rvMIP)、逆转录酶抑制剂(AZT)、膜融合抑制剂(T-20)、rvMIP+AZT、rvMIP+T-20对HIV-1前病毒载量的影响。结果所建立的荧光定量PCR体系的检测下限为101Copies,其标准曲线的相关系数为0.998 9,斜率为-4.925,截距为35.70;除6ng/ml的rvMIP、AZT、T-20外,三种药物处理后HIV-1前病毒载量均显著减小(P<0.05),并呈质量浓度依赖性;联合用药者前病毒载量显著低于单用rvMIP者(P<0.05),HIV-1前病毒载量rvMIP+T-20(rvMIP+AZT(rvMIP(T-20(AZT。结论本研究建立的SYBR Green Ⅰ荧光定量PCR检测方法具有灵敏、快速、准确及成本低廉等优点;与T-20或AZT联用可增强rvMIP对HIV-1前病毒的抑制作用。Objective To set up a SYBR Green I fluorescent quantitative PCR method for the detection of HIV-I provirus viral load, and use this method to study the effect of virus entry inhibitors on the HIV-1 provirus. Methods The SYBR Green I fluorescent quantitative PCR method for the detection of HIV-1 provirus viral load were encompassed the conservative region of gag and env in the HIV-1, the effects to the HIV-1 proviras viral load by 6ug/ml - 6 ng/ml recombinant viral maerophage inflammation protein （ rvMIP）, reverse transcriptase inhibitor （AZT） , membrane infusion inhibitor （ T- 20）, rvMIP ＋ AZT or rvMIP ＋ %20 were detected respectively. Results The detection limit of the fluorescent quantitative PCR was 101 Copies, The correlation coefficient of the standard curves was 0.998 9, the slope was -4.925, and the intercept was 35.70;except for the rvMIP,AZT,T-20（6 ng/ml） ,the HIV-1 provirus viral load decreased after treated by the three drugs （P 〈 0.05 ） , and showed concentration-depended manner;the provirus viral load after treated by drug combination were lower than that treated by rvMIP only （ P 〈 0.05 ）, HIV-1 provirus viral load rvMIP ＋ T-20 〈 rvMIP ＋ AZT 〈 rvMIP 〈 T-20 〈 AZT. Conclusions The SYBR Green I fluorescent quantitative PCR detection method is sensitive, quick, accu- rate and cheap;rvMIP combined with T-20 or AZT can boost up the inhibition of HIV-1 provirus.

关 键 词：聚合酶链反应 实时性 重组病毒巨噬细胞炎症蛋白 人类免疫缺陷病毒-1前病毒载量 

分 类 号：R512.91[医药卫生—内科学]