Download
1 / 27
280 likes | 479 Views
HIV Drug Resistance Training. Module 5: Sequencing Procedures: Commercial and In-House. Topics. Overview of Genotyping Procedures Comparison of Three Different Procedures Application of Concepts. Objectives. Identify individual lab needs for sequencing.
E N D
HIV Drug Resistance Training Module 5: Sequencing Procedures: Commercial and In-House
Topics • Overview of Genotyping Procedures • Comparison of Three Different Procedures • Application of Concepts
Objectives • Identify individual lab needs for sequencing. • Identify differences among the procedures. • Based on the differences among the procedures, identify the procedure that best meets the lab’s needs.
overview of genotyping procedures What systems are available for HIV DR genotyping? What do these systems require?
HIV DR Genotyping Systems • US FDA-approved HIV-1 genotyping systems: • ViroSeq™ HIV-1 genotyping system • TRUGENE® HIV-1 genotyping system • In-house, or “home-brew” genotyping systems
US FDA-approved Systems • Characteristics: • Approved for HIV-1 subtype B viral DR genotyping * • Labor-intensive and very expensive • Requirements: • Specific instruments • Specimen: EDTA plasma • Viral load level: ≥1000-2000 copies/ml of plasma • Extensive training for technicians • Laboratory Biosafety level II working environment for sample preparation * They have been demonstrated to work well for non-B subtypes as well, although with less certainty around performance characteristics.
comparison of three different procedures What are the procedures used by ViroSeq™, TruGene®, and in-house assays? What factors should be considered in choosing one procedure over another?
Option One: ViroSeq™ HIV-1 Genotyping • Detects HIV-1 subtype B viral resistance • in plasma samples • collected in EDTA tubes • with a viral load ranging from 2,000 to >750,000 copies/ml. • Genotypes the entire HIV-1 protease coding region (codons 1-99) and codons 1-335 of reverse transcriptase.
Option Two: TRUGENE® HIV-1 Genotyping • Semi-automated technique • Covers PR (codons 4-99) and RT (codons 38-248) • Viral RNA extracted from plasma • Plasma viral load ≥1,000 copies/ml • cDNA amplified in a single-tube RT-PCR reaction • Sequenced with a modified Sanger technique • Dye-primer (not dye-labeled ddNTPs) • PR and RT sequences non-contiguous • Semi-automated data analysis, alignment and interpretation • Specific chemical waste disposal required (acrylamide)
Home-brew/In-house Genotyping Assays • Many home-brew genotyping assays • Differences: • Detection of HIV-1 viral resistance for non-B subtypes and circulating recombinant forms (CRFs) • Specimen type: plasma, dried blood spots (DBS), or dried plasma/serum spots (DPS/DSS) • Boundaries of the protease and the reverse transcriptase regions of the pol gene that are sequenced • Sensitivity (viral load requirement) • Other performance characteristics (validation) • Cost and turn-around time
Summary of HIV-1 Genotyping Procedures • Two US FDA-approved genotyping systems • Many home-brew genotyping systems • All are conventional viral population-based sequencing assays with a minor viral population detection limit of ≥20% • Complex assays - need highly skilled staff, dedicated instrumentation and infrastructure • Test and instrument maintenance are expensive
application of concepts Which type of procedure is most appropriate for our national lab?
Discussion • What factors are most important in choosing a procedure? • Which procedure (if any) meets your needs best? • What issues do you need to resolve in order to obtain and use the procedure you have selected?
Reflection • Which procedure will we use (if any)? • What must we do as “next steps?”
Summary • Overview of Genotyping Procedures • Comparison of Three Different Procedures • Application of Concepts
