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Tropism Testing Who needs a Phenotype Arevir Meeting May 2011 Swiss - - PowerPoint PPT Presentation
Tropism Testing Who needs a Phenotype Arevir Meeting May 2011 Swiss - - PowerPoint PPT Presentation
Tropism Testing Who needs a Phenotype Arevir Meeting May 2011 Swiss 3 step System Element 1 (TrackC): (ca. 85% of analyses) Element 2 (Geno2Pheno): -RNA-DNA conversion (cDNA); (ca. 25% of analyses) -PCR-amplification of V3 region
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Swiss 3‐step System
Element 1 (TrackC): (ca. 85% of analyses)
- RNA-DNA conversion (cDNA);
- PCR-amplification of V3 region by PCR;
- Hybridization to known DNA probes;
- Capillary-based nucleic acid duplex tracking
assay “TrackC”: Element 2 (Geno2Pheno): (ca. 25% of analyses)
- RNA-DNA conversion;
- DNA-amplification of V3 region by PCR;
- Sequencing of the env V3-region of the
viral genome;
- Sequence based interpretation of V3
Amino Acid information using Geno2Pheno
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Element 3 (PhenX-R): (ca. 10% of analyses)
- RNA-DNA conversion; amplification of env;
- Cloning into replicative HIV cassette (plasmid lacking env);
- Transfection into permissive human cell line;
- 4-days virus replication in presence of drug;
- Read-out: HIV tropism; virus fitness; dualtrop/mixes
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Viral RNA from Patient
Provirus-Cassette for the Env Gene (=> complete Virus!)
RT
Treatment in vitro w/ Corec.-Antagonists
RT
Env Env Env Env Env
Amplification
Env Env
“PhenX-R”
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Replicative phenotypic Tropism test
- Why?
– To verify ambiguous Geno‐results – To assess minority populations – To discriminate dualtropic and mixed viruses – To determine viral fitness
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rPhenotypic Tropism Testing
- When?