Detection and quantification of multiple drug resistance mutations in HIV-1 reverse transcriptase by an oligonucleotide ligation assay.
OBJECTIVE
To develop an assay for the early detection and quantification of minor human immunodeficiency virus-1 populations bearing multiple drug resistance (MDR) mutations.


METHODS
The oligonucleotide ligation assay (OLA) is based on ligation of probe and detector oligonucleotides annealed to a polymerase chain reaction amplicon strand with detection by an enzyme immunoassay. In OLA-MDR, oligonucleotides were designed to detect MDR mutations. The method was validated with wild-type and MDR mutant clones mixed at different proportions.


RESULTS
K103N mutants were detected as minor populations (5%-30%) by OLA in 6 of 18 samples from patients treated with nonnucleoside reverse transcription inhibitors and classified as wild type by sequencing. In one patient, the kinetics of the increase of MDR mutants could be followed in sequential samples, with K103N being detected earlier by OLA than by sequencing. Q151M mutants were detected as minor populations (13%-24%) by OLA but not by sequencing in 4 samples.


CONCLUSIONS
Oligonucleotide ligation assay MDR exhibits higher sensitivity than sequencing for detection of minor MDR mutant populations.
Villahermosa
M L
ML
Centro Nacional Biologia Fundamental, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain. rafael.najera@isciii.es
Beck
I
I
Pérez-Alvarez
L
L
Contreras
G
G
Frenkel
L M
LM
Osmanov
S
S
de Parga
E V
EV
Delgado
E
E
Manjon
N
N
Cuevas
M T
MT
Thomson
M M
MM
Medrano
L
L
Najera
R
R
D024921:Drug Resistance, Multiple, Viral / Q000235:genetics*; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use
D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase