Polymerase chain reaction (PCR)

DNA was extracted from the cellular components of the blood using a Puregene kit (Puregene, Gentra Systems, Inc., USA) according to the manufacturer’s instructions. The nucleic acid was eluted and stored at −20 °C until use. Subsequently, DNA was submitted to a nested PCR to amplify part of the region of the HIV-1 RT and HIV-1 PR. The first PCR cycle was performed using 4 μg of extracted DNA, 125 mM of each dNTP (Perkin-Elmer, USA), 20 pmol/μL of each of the two external primers, 2.5 mM MgCl₂ and 10× buffer (Perkin-Elmer, USA) in a final volume of 50 μL. The reactions were performed in a thermocycler (Perkin-Elmer, USA) for 5 min at 94 °C followed by 35 cycles at 94 °C (40 s), 50 °C (40 s), and 72 °C (1 min) and a 10 min extension at 72 °C. Five microliters of the amplified product was used in the nested PCR along with a set of internal primers in a final volume of 50 μL. The same incubation times and temperatures of the first reaction were used. For the PR, DP10/DP11 were used as external primers, and DP16/DP17 were used as internal primers [8]. For amplification of the fragment of the RT gene, RT09/RT12 were used as external primers, and RT01/RT04 were used as internal primers [9]. The amplified products were analyzed by electrophoresis on a 2% agarose gel. After amplification, the fragments were purified using the QIA Quick Purification Kit (Qiagen, USA) prior to nucleotide sequencing.