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 transcriptase (RT) confer cross-resistance to various nucleoside analogs. The detection of these rearrangements in the region of codons 67-70 of RT is of primary importance for adapting and optimizing combination treatment regimen. Recent reports suggest that some genotyping techniques based on the hybridization of oligonucleotide probes may fail to detect insertion mutants of HIV-1 RT. In the present study, we have evaluated the efficiency of two commercial kits TruGene (based on Dye Primer sequencing) and Viroseq (Big Dye Terminator technique) for the detection of insertion mutations. The data were compared with an in-house dRhodamine sequencing method. Overall, all these cycle sequencing techniques were operative in the detection of insertion mutants. The best peak homogeneity in the electrophoregrams was observed with the Dye primer technique. However, specific compression artifacts were frequently encountered with this technique, rendering ambiguous the interpretation of the electrophoregrams in several regions of the sequence. This shortcoming did not occur with dRhodamine Dye terminator or Bigdye terminator cycle sequencing. In any case, a manual inspection of the electrophoregrams is highly recommended, for all types of cycle sequencing techniques, especially for detecting new mutational patterns of the RT and protease genes. Finally, some specific problems were encountered with the softwares provided with both Trugene and Viroseq kits.
Koch
N
N
Laboratoire de Virologie, CHRU La Timone, 264 rue st Pierre, 13005, Marseille, France.
Tamalet
C
C
Tivoli
N
N
Fantini
J
J
Yahi
N
N
D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000627:therapeutic use; D001483:Base Sequence; D003062:Codon; D018076:DNA, Complementary; D004586:Electrophoresis; D005838:Genotype; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011933:Reagent Kits, Diagnostic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*
D019380:Anti-HIV Agents; D003062:Codon; D018076:DNA, Complementary; D011933:Reagent Kits, Diagnostic; D054303:HIV Reverse Transcriptase