Establishment of Fluorescent Real Time Quantitative PCR for Detecting HIV-1 and Its Application / 中国病毒学

ABSTRACT

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.