检测HIV-1载量的荧光实时定量PCR技术的建立及其应用

Establishment of Fluorescent Real Time Quantitative PCR for Detecting HIV-1 and Its Application

准确测定HIV 1的前病毒载量和病毒载量的技术 ,在感染者预后和艾滋病患者药物治疗效果的评价以及艾滋病的其它研究方面 ,都具有十分重要的应用价值。以定量的HIV 1DNA和RNA为标准外参照 ,利用SYBRGreen荧光染料和GeneAmp5 70 0SequenceDetectionSystem (5 70 0系统 ) ,建立了测定HIV 1的前病毒载量和病毒载量的荧光实时定量PCR技术。以病毒感染细胞和培养上清为材料 ,测定了三种化合物 (AZT ,GL和WT)对细胞内的前病毒载量和培养上清中的病毒载量的抑制活性 ,并与合胞体形成抑制方法测定化合物抗病毒活性的结果进行了比较。根据病毒载量、前病毒载量和合胞体形成计算出的三种化合物的治疗指数均依次变小 ,提出以荧光实时定量PCR技术测定前病毒载量 ,会在评价药物在体内外根除或减少存在于CD4休止或记忆T淋巴细胞中的HIV 1前病毒方面有特别的价值。

Accurate determination of HIV 1 proviral burden and viral load is very useful in prognosis of HIV 1 infected patients and in assessment of drug for therapy of AIDS patients. In order to establish a quantitative method in detecting HIV 1 proviral burden and viral load, 8E5 cell line and a recombinant RNA constructs were used as the HIV 1 proviral DNA and viral RNA external references, respectively. The PCR products were labeled with the fluorescent DNA dye SYBR green. The amount of burden or load was measured by GeneAmp 5700 Sequence Detection System. Using this method, the HIV 1 proviral burdens in PBMC of patient and in cell suspension treated with the compounds AZT, GL and WT were measured. HIV 1 viral loads in supernatant of the cell culture treated with the above compounds were also determined. The therapeutic indices (TIs) of the compounds calculated based on the inhibition of virus induced syncytial formation, and inhibitionn of proviral burdens and viral loads were compared, and their TIs successively increased. The fluorescent real time quantitative PCR possesses very good specificity, sensitivity and duplication. TI value of a drug based on inhibition of proviral burden in cell culture, and the proviral burden in PBMC of patient may be useful in evaluating a drug on eradicating provirus from resting and memory CD4 T cells.