Real-time PCR. Real-time PCR amplification of HIV-1 RNA was performed in a one-step, single-tube closed system of 32 sample format using the LC-32 Roche LightCycler (Roche, Indianapolis, IN). All the samples were tested in duplicate in a 20 ml total reaction volume containing 16 ml of PCR reaction mix (Quantitect SYBR Green RT-PCR kit [Qiagen, Valencia, CA]; 0.5 mM each of forward and reverse oligonucleotide primer pairs) and 4 ml of the template. The primers were specific to a conserved region of HIV-1 LTR: 59-GRAACCCACTGCTTAASSCTCAA-39 (LTR sense; position 506 of HxB2) and 59-TGTTCGGGCGCCACTGCTAGAGA-39 (LTR antisense; position 626 of HxB2). [27] The PCR reaction was performed according to the following cycling parameters: 1) Reverse-Transcription: 50uC 20 minutes, ramp 20uC/second; 2) Activation: 95uC 15 minutes, ramp 20uC/second; 3) Amplification: 50 cycles a) 94uC 10 seconds, ramp 20uC/second, b) 52uC 20 seconds, ramp 20uC/second, and c) 72uC 20 second, ramp 2uC/ second (single data collection); 4) Melting: a) 92uC 0 second, ramp 20uC/second, b) 57uC 15 seconds, ramp 20uC/second, and c) 92uC 0 second, ramp 0.1uC/second (continuous data collection); 5) Cooling: 40uC 30 seconds, ramp 20uC/second.