2.4　Reverse Transcription（RT）and PCR

Total extracted RNA from 100 μl of plasma，as a precipitate with 2 μg of carrier tRNA，was dissolved in dH20.Aliquots of the solubilized RNA，corresponding to 1 to 100 μl of the original plasma.were reverse transcribed into cDNA in a total 20 μl reaction mixture containing 50 mmol/L Tris-HCl，pH 8.3，150 mmol/L KCl，10 mmol/L MgCl2，10 mmol/L DTT，200 μmol/L dNTPs，0.1 μmol/L random hexamer primers，20 units of RNasin and 5 units of AMV reverse transcriptase（Seikagaku America Inc.，St.Petersburg，FL）.After incubation，first at room temperature for 10 min，then at 37℃for 30 min，the reaction was terminated by heating at 95℃for 5 min and quick-cooling on ice.PCR was carried in a 30 μl reaction containing 50 mmol/L Tris-HCl，pH 8.5，50 mmol/L KCl，3.3 mmol/L MgCl2，200 μmol/L dNTPs，200 μg/ml gelatin，0.5 μmol/L primer pairs SK38/SK39（Ou et al.，1988），1 unit of Taq polymerase（Perkin-Elmer，Norwalk，CT）and 10 μl of the RT product.The amplification protocol consisted of 30 cycles of 95℃for 30 s，55℃for 30 s，and 72℃for 1 min in a Perkin-Elmer-Cetus thermocycler.Samples were run in duplicate，with negative and positive controls included in all experiments.For quantitation purposes，the HIV RNA was diluted so that the RT-PCR signal was within the range of HIV-1 RNA standard curve.Control experiments that lacked the reverse transcriptase indicated that no HIV DNA was detectable with the RNAzol plasma extracts from seropositive patients.(https://www.daowen.com)