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.
Engelbrecht
S
S
Department of Medical Virology, University of Stellenbosch, Tygerberg, South Africa.
van Rensburg
E J
EJ
D000386:AIDS-Related Complex / Q000821:virology; D000163:Acquired Immunodeficiency Syndrome / Q000821:virology; D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics; D005069:Evaluation Studies as Topic; D015752:Genes, env / Q000235:genetics*; D015750:Genes, gag / Q000235:genetics; D015497:HIV-1 / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D013019:South Africa; D013696:Temperature
D017931:DNA Primers; D004279:DNA, Viral