Abstract
Thirteen laboratories evaluated the reproducibility of sequencing methods to detect drug resistance mutations in HIV-1 reverse transcriptase (RT). Blinded, cultured peripheral blood mononuclear cell pellets were distributed to each laboratory. Each laboratory used its preferred method for sequencing proviral DNA. Differences in protocols included: DNA purification; number of PCR amplifications; PCR product purification; sequence/location of PCR/sequencing primers; sequencing template; sequencing reaction label; sequencing polymerase; and use of manual versus automated methods to resolve sequencing reaction products. Five unknowns were evaluated. Thirteen laboratories submitted 39 043 nucleotide assignments spanning codons 10-256 of HIV-1 RT. A consensus nucleotide assignment (defined as agreement among ≥75% of laboratories) could be made in over 99% of nucleotide positions, and was more frequent in the three laboratory isolates. The overall rate of discrepant nucleotide assignments was 0.29%. A consensus nucleotide assignment could not be made at RT codon 41 in the clinical isolate tested. Clonal analysis revealed that this was due to the presence of a mixture of wild-type and mutant genotypes. These observations suggest that sequencing methodologies currently in use in ACTG laboratories to sequence HIV-1 RT yield highly concordant results for laboratory strains; however, more discrepancies among laboratories may occur when clinical isolates are tested. Copyright (C) 1998 Elsevier Science B.V.
Original language | English (US) |
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Pages (from-to) | 93-104 |
Number of pages | 12 |
Journal | Journal of Virological Methods |
Volume | 75 |
Issue number | 1 |
DOIs | |
State | Published - Nov 1998 |
Funding
This work was supported in part by grants from the National Institutes of Health (5UO1-AI-27658, AI-29193, AI-27659, UO1-AI-27675, 2UO1-AI-27559, RR-00044-34S2) and the Chicago Pediatric Faculty Foundation.
Keywords
- Drug resistance
- HIV-1
- Sequence analysis
ASJC Scopus subject areas
- Virology