[Show abstract][Hide abstract] ABSTRACT: In order to survive prolonged treatment with antiretroviral nucleoside analogs, the human immunodeficiency virus type 1 (HIV-1)
is selectively forced to acquire mutations in the reverse transcriptase (RT) gene. Some of these mutations are more common
than others and have become markers for antiretroviral resistance. For the early detection of these markers, a novel MultiCode-RTx
one-step testing system to rapidly and simultaneously characterize mixtures of HIV-1 targets was designed. For cDNA, nucleotide
polymorphisms for codon M184V (ATG to GTG) and K65R (AAA to AGA) could be differentiated and quantified even when the population
mixture varied as much as 1 to 10,000. Standard mixed-population curves using 1 to 100% of the mutant or wild type generated
over 4 logs of total viral particle input did not affect the overall curves, making the method robust. The system was also
applied to a small set of samples extracted from infected individuals on nucleoside reverse transcriptase inhibitor therapy.
Of 13 samples tested, all were positive for HIV and 10 of the 13 genotypes determined were concordant with the line probe
assay. MultiCode-RTx could be applied to other drug-selected mutations in the viral genome or for applications where single-base
changes in DNA or RNA occur at frequencies reaching 0.01% to 1%, respectively.