An evaluation of the probe hydridization method for large scale population studies of HIV-1 strain variation in Uganda

ABSTRACT

A strategy was developed for a long-term; large scale study of HIV-1 genetic variation in Uganda. Our approach was based on the premisethat DNA sequencing is costly and time consiming with respect to screening largenumbers of specimens. We adopted a dot-blod hybridization method using oligonucleotide probes specific for HIV-1 subtypes A and D in the envC2V3 region to screen for HIV-1 subtypes in Uganda. Specicimens which could not be subtyped in this way were sequenced directly. Genomic DNA from 72 HIV-1 seropositive subjects were amplified by PCR in the envC2V3 region. The results of dot-blot hybridization indicated that 40 were subtype A and 29 were subtype D. The remaining three specimens could not be typed due to either non-binding to probes of both subtypes or non specific binding. Hybdridization results were compared with results obtained by sequence phylogenetic analysis. Sequences were generated from 50 DNA specimens and analysis in the C2V3 region showed that 29 were subtype A and 21 were subtype D. Sequences were also generated from the immuno-dominant env gp41 region (384bp fragment) from specimens for which C2V3 sequences were not available. Phylogenetic analysis of these sequences indicated that 8 were subtype A and 8 were subtype D. Regardless of the region of the HIV genome under study sequence data fully supports the subtype assignment derived from hybridization data. Our findings suggest that probe hybridization using A and D subtype specific probes will be effective for large scale screening og the HIV-infected populationin Uganda. Application of this method should lead to significant savings in cost and time for large; population-based investigations