Comparison of two commercial assays for the detection of insertion mutations of HIV-1 reverse transcriptase

Insertions in the beta3-beta4 fingers subdomain of HIV-1 reverse transcript ase (RT) confer cross-resistance to various nucleoside analogs. The detecti on of these rearrangements in the region of codons 67-70 of RT is of primar y importance for adapting and optimizing combination treatment regimen. Rec ent reports suggest that some genotyping techniques based on the hybridizat ion of oligonucleotide probes may fail to detect insertion mutants of HIV-1 RT. In the present study, we have evaluated the efficiency of two commerci al kits TruGene (based on Dye Primer sequencing) and Viroseq (Big Dye Termi nator technique) for the detection of insertion mutations. The data were co mpared with an in-house dRhodamine sequencing method. Overall, all these cy cle sequencing techniques were operative ill the detection of insertion mut ants. The best peak homogeneity in the electrophoregrams was observed with the Dye primer technique. However, specific compression artifacts were freq uently encountered with this technique, rendering ambiguous the interpretat ion of the electrophoregrams in several regions of the sequence. This short coming did not occur with dRhodamine Dye terminator or Bigdye terminator cy cle sequencing. In any case, a manual inspection of the electrophoregrams i s highly recommended, for all types of cycle sequencing techniques, especia lly for detecting new mutational patterns of the RT and protease genes. Fin ally, some specific problems were encountered with the softwares provided w ith both Trugene and Viroseq kits. (C) 2001 Elsevier Science B.V. All right s reserved.