LINE PROBE ASSAY FOR RAPID DETECTION OF DRUG-SELECTED MUTATIONS IN THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE GENE

Upon prolonged treatment with various antiretroviral nucleoside analog s such as 3'-azido-3'-deoxythymidine, 2',3'-dideoxyinosine, 2',3'-dide oxycytidine, (-)-beta-L-2',3'-dideoxy-3'-thiacytidine, and 2',3'-dideh ydro-3'-deoxythymidine, selection of human immunodeficiency virus type 1 (HIV-1) strains with mutations in the reverse transcriptase (RT) ge ne has been reported. We designed a reverse hybridization line probe a ssay (LiPA) for the rapid and simultaneous characterization of the fol lowing variations in the RT gene: M41 or WI; T69, N69, A69, or D69; K7 0 or R70; L74 or V74; V75 or T75; M184, I184, or V184; T215, Y215, or F215; and K219, Q219, or E219. Nucleotide polymorphisms for codon L41 (TTG or CTG), T69 (ACT or ACA), V75 (GTA or GTG), T215 (ACC or ACT), a nd Y215 (TAG or TAT) could be detected. In addition to the codons ment ioned above, several third-letter polymorphisms in the direct vicinity of the target codons (E40, E42, K43, K73, D76, Q182, Y183, D185, G213 , F214, and L214) were found, and specific probes were selected. In to tal, 48 probes were designed and applied on the LiPA test strips and o ptimized with a well-characterized and representative reference panel. Plasma samples from 358 HIV-infected patients were analyzed with all 48 probes. The amino acid profiles could be deduced by LiPA hybridizat ion in an average of 92.7% of the samples for each individual codon. W hen combined with changes in viral load and CD4(+) T-tell count, this LiPA approach proved to be useful in studying genetic resistance in fo llow-up samples from antiretroviral agent-treated HIV-1-infected indiv iduals.