Wu KY, Yang Q, Johnston D, Miyada G, Ryder T, Kaplan P; International Conference on AIDS.

Int Conf AIDS. 1998; 12: 812 (abstract no. 42196).

Affymetrix Inc., Santa Clara, CA, USA.

BACKGROUND: By using strand-specific arrays, the current GeneChip assay interrogates nucleotide changes in a 1.1 kb sequence encoding protease codons 1-99 and reverse transcriptase codons 1-243. OBJECTIVE: To develop a new GeneChip HIV genotyping assay which is able to (a) extend the target range of genotyping; (b) increase assay throughput by using a single array for both strands; and (c) incorporate recently discovered drug-resistant genotypes into the array design. METHODS: To amplify 1.7 kb viral RNA of HIV-1, a simplified nested RT-PCR procedure combined RT and first run PCR in a single-tube format. The assay sensitivity was measured against a plasma panel of known viral titers. Amplified product was fragmented by DNase I, terminally labeled with biotinylated dideoxy nucleotide triphosphate, and subsequently stained with a streptavidin-phyco-erythrin conjugate after hybridization. Standard procedures were employed for hybridization, array scanning and data analysis. A highly divergent HIV clone was also studied for variance tolerance of array performance. Selected targets from clinical samples were analyzed to examine the extended interrogation region for drug-resistant mutation and polymorphorism. RESULTS: The nested amplification procedure detected viral titers in plasma samples > or = 500 per ml while the non-nested procedure detected viral titers > or = 1000 per ml. When using a HXB2 plasmid clone, the new assay illustrated a base calling accuracy of > 99.5% for 0.25 pmol of amplified target over the 1.5 kb nucleotide range coding for protease codon 1-99 and RT codon 1-400. Both the new assay and existing assay yielded comparable results in terms of sensitivity and interrogation accuracy for a divergent clone (C3C), which contains about 4% variation from clade B consensus sequences. CONCLUSION: The new assay provides higher throughput, a wider base interrogation range and more information about nucleotide polymorphorism and drug-resistance related mutations, while maintaining equivalent performance in detection sensitivity and interrogation accuracy.

Publication Types:

• Meeting Abstracts

Keywords:

• AIDS Vaccines
• Acquired Immunodeficiency Syndrome
• Biological Assay
• Codon
• Genotype
• HIV Infections
• HIV Seropositivity
• HIV-1
• Human Development
• Laboratory Techniques and Procedures
• Mutation
• Nucleic Acid Hybridization
• Oligonucleotide Array Sequence Analysis
• Polymerase Chain Reaction
• RNA, Viral
• RNA-Directed DNA Polymerase
• genetics
• reverse transcriptase, Human immunodeficiency virus 1

Other ID:

• 98403126

UI: 102230995

From Meeting Abstracts