Detection of Southern African human immunodeficiency virus type 1 subtypes by polymerase chain reaction: Evaluation of different primer pairs and conditions
The purpose of the study was to develop a specific and sensitive PCR protocol using env, gag and LTR primer pairs to detect HIV-1 subtypes present in the Western Cape, South Africa. Twenty-two virus strains, belonging to HIV-1 subtypes B, C and D, were randomly selected for PCR evaluation. Cell lysates prepared from these virus-infected cultured cells were tested using 5 different primer pairs: gag SK38/SK39; gag 22/SK39; gag a/b, gag c/d (nested); env SK68/SK69 and LTR SK29/SK30. Eight different PCR profiles were evaluated: one profile each for the 3 gag primer pairs, 3 profiles for the env and 2 profiles for the LTR primer pairs. The number of PCR cycles, time per cycle and/or annealing temperature were changed in each profile. The optimum PCR profile for a specific primer pair was defined as that which detected one copy of proviral plasmid DNA after dot-blot hybridisation. Gag primer pairs detected HIV-1 DNA in all 22 samples. With the env primer pair, suboptimal conditions failed to detect most of the HIV-1 subtype C samples. By increasing the number of cycles and time per cycle, a 100% sensitivity was achieved. With the LTR primer pair all samples were detected by decreasing the annealing temperature and increasing the individual cycle times. This confirms that once PCR conditions are optimised, all HIV-1 subtypes in our study could be detected using different PCR primer pairs.During 1984-92, in South Africa, virologists isolated HIV-1 from HIV/AIDS patients at hospitals in the Western Cape. Two virologists from the University of Stellenbosch Hospital in Tygerberg selected 22 virus strains, belonging to HIV-1 subtypes B, C, and D, to study in order to develop a specific and sensitive polymerase chain reaction (PCR) protocol using env, gag, and LTR primers. They used five different primer pairs to prepare cell lysates from the HIV-infected cultured cells: gag SK38/SK39, gag 22/SK39, gag a/b, gag c/d (nested), env SK68/SK69, and LTR SK29/SK30. The virologists evaluated eight different PCR profiles: one profile each for the three gag primer pairs, three profiles for the env, and two profiles for the LTR primer pairs. They changed the number of PCR cycles, time per cycle, and/or annealing temperature in each profile. The PCR profile for a specific primer pair that detected one copy of proviral plasmid DNA after dot-blot hybridization was considered the optimum PCR profile. Gag primer pairs detected HIV-1 DNA in all 22 samples. The env primer pair did not detect most HIV-1 subtype C samples. When the researchers increased the number of cycles and time per cycle, the env primer pair achieved 100% sensitivity. When they decreased the annealing temperature and increased the individual cycle times, the LTR primer pairs detected all samples. These findings support that optimization of a PCR assay is necessary to achieve high assay sensitivity, specificity, and reproducibility and that PCR sensitivity should be considered seriously when interpreting PCR results for HIV diagnosis.