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Accuracy of the TRUGENE HIV-1 Genotyping Kit

Robert M. Grant,^1,2* Daniel R. Kuritzkes,^3, Victoria A. Johnson,^4,5 John W. Mellors,⁶ John L. Sullivan,⁷ Ronald Swanstrom,⁸ Richard T. D'Aquila,^9, Mark Van Gorder,¹⁰ Mark Holodniy,¹¹ Robert M. Lloyd, Jr.,^12, Caroline Reid,¹² Gillian F. Morgan,¹² and Dean L. Winslow¹²

Gladstone Institute of Virology and Immunology,¹ University of California, San Francisco,² Consolidated Laboratories, Van Nuys,¹⁰ AIDS Research Center, VA Palo Alto Health Care System and Division of Infectious Diseases and Geographic Medicine, Stanford University, Palo Alto, California,¹¹ Harvard University Medical School,⁹ University of Massachusetts, Boston, Massachusetts,⁷ University of North Carolina at Chapel Hill, Chapel Hill, North Carolina,⁸ University of Alabama at Birmingham,⁴ Veteran's Administration Medical Center, Birmingham, Alabama,⁵ University of Pittsburgh, Pittsburgh, Pennsylvania,⁶ Division of Infectious Diseases, University of Colorado Health Sciences Center, Denver, Colorado,³ and Visible Genetics, Inc., Toronto, Ontario, Canada,¹²

Received 11 February 2002/ Returned for modification 13 October 2002/ Accepted 21 December 2002

Drug resistance and poor virological responses are associated with well-characterized mutations in the viral reading frames that encode the proteins that are targeted by currently available antiretroviral drugs. An integrated system was developed that includes target gene amplification, DNA sequencing chemistry (TRUGENE HIV-1 Genotyping Kit), and hardware and interpretative software (the OpenGene DNA Sequencing System) for detection of mutations in the human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase sequences. The integrated system incorporates reverse transcription-PCR from extracted HIV-1 RNA, a coupled amplification and sequencing step (CLIP), polyacrylamide gel electrophoresis, semiautomated analysis of data, and generation of an interpretative report. To assess the accuracy and robustness of the assay system, 270 coded plasma specimens derived from nine patients were sent to six laboratories for blinded analysis. All specimens contained HIV-1 subtype B viruses. Results of 270 independent assays were compared to "gold standard" consensus sequences of the virus populations determined by sequence analysis of 16 to 20 clones of viral DNA amplicons derived from two independent PCRs using primers not used in the kit. The accuracy of the integrated system for nucleotide base identification was 98.7%, and the accuracy for codon identification at 54 sites associated with drug resistance was 97.6%. In a separate analysis of plasma spiked with infectious molecular clones, the assay reproducibly detected all 72 different drug resistance mutations that were evaluated. There were no significant differences in accuracy between laboratories, between technologists, between kit lots, or between days. This integrated assay system for the detection of HIV-1 drug resistance mutations has a high degree of accuracy and reproducibility in several laboratories.

Present address: Partners AIDS Research Center, Brigham and Women’s Hospital, Cambridge, MA 02139.

Present address: Division of Infectious Disease, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232.

Present address: Research Think Tank, Alpharetta, GA 30004.

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