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Antimicrobial Agents and Chemotherapy, August 2005, p. 3334-3340, Vol. 49, No. 8
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.8.3334-3340.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Quantifying Mixed Populations of Drug-Resistant Human Immunodeficiency Virus Type 1

Michael J. Moser,¹ Meta Ruckstuhl,² Christine A. Larsen,¹ Amanda J. Swearingen,¹ Miroslaw Kozlowski,² Leda Bassit,² Prem L. Sharma,^2, Raymond F. Schinazi,² and James R. Prudent^1*

EraGen Biosciences, 918 Deming Way, Madison, Wisconsin 53717,¹ Emory University School of Medicine/Veterans Affairs Medical Center, 1670 Clairmont Road, Decatur, GA 30033²

Received 30 December 2004/ Returned for modification 15 February 2005/ Accepted 25 April 2005

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.

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* Corresponding author. Mailing address: EraGen Biosciences, Inc., 918 Deming Way, Madison, WI 53717. Phone: (608) 662-9000, ext. 302. Fax: (608) 662-9004. E-mail: jprudent{at}eragen.com.

Present address: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322.

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Antimicrobial Agents and Chemotherapy, August 2005, p. 3334-3340, Vol. 49, No. 8
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.8.3334-3340.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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