Rapid detection of the CCR2-V64I, CCR5-A59029G and SDF1-G801A polymorphisms by tetra-primer PCR

Objective: To develop tetra-primer PCR assays for detection of the CCR2-V64I, CCR5-A59029G and SDF1-G801A polymorphisms associated with HIV pathogenesis. Design and Methods: For each assay, two primers for the amplification of the gene locus are combined in one tube with two primers for the subsequent allele specific amplification (ASA). In the first set of cycles, pre-amplification of the gene region of interest is ensured by the gene specific primers. In the second set of cycles, lowering the annealing temperature allows ASA on the newly produced template. Results: Analysis of 90 DNA samples resulted in allele frequencies for CCR2-V64I, CCR5-A59029G and SDF1-G801A which are similar to other Caucasian cohorts. Furthermore, re-analysis of sequenced genomic DNA by tetra-primer PCR analysis (7–11 times) always showed identical results. Conclusion: Our set of single-tube assays allows rapid and reproducible genotyping of the CCR2-V64I, CCR5-A59029G and SDF1-G801A polymorphisms. These inexpensive but accurate assays are valuable for screening these polymorphisms in cohorts of HIV-infected patients.