Oligonucleotide ligation assay for detecting mutations in the human immunodeficiency virus type 1 pol gene that are associated with resistance to zidovudine, didanosine, and lamivudine

Abstract

This report describes the detection of mutations in the pol gene of human immunodeficiency virus type 1 associated with resistance to zidovudine, didanosine, and lamivudine by genotyping by an oligonucleotide ligation assay specific codons in the pol gene amplified by PCR. Our studies demonstrate the sensitivity, simplicity, and specificity of this genotyping system.

FIG. 1

OLA plate demonstrating the detection of wild-type genotypes (a) and mutant genotypes (b) from randomly selected patients receiving nucleosides. Some wells contain mixtures of genotypes (e.g., row 2, codon 74; row 4, codons 41, 70, and 215), while others reveal only wild-type (e.g., rows 1 and 7, all codons) or mutant (e.g., rows 2 and 6, codons 41 and 215) genotypes. Positive results were easily determined visually by the development of a colored product. The digoxigenin reporter (wild type) was identified by a specific antibody coupled with horseradish peroxidase, which resulted in a brilliant blue color (a). After washing this microtiter plate, the presence of the fluorescein reporter (mutant) was assessed by the production of a deep magenta color due to the alkaline phosphatase labelling of the antibodies to fluorescein (b). The absence of either genotype was also quite clear, because no color was produced in the wells (a and b). A and B, mutants A and B.