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Semelhante a Lab Management of HIV: NASBA and Molecular Beacons
Semelhante a Lab Management of HIV: NASBA and Molecular Beacons (20)
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Lab Management of HIV: NASBA and Molecular Beacons
- 1. Lab Management of HIV Boniface Mudenge FLOW CYTOMETRY CENTRE, SUITE 12A MEDICAL CENTRE 52 BAINES AVENUE, HARARE, ZIMBABWE www.flowcytolab.com
- 2. NASBA and Molecular Beacons Are the perfect match: •NASBA generates s/sRNA amplicons •Molecular beacons binds easily s/sRNA or s/sDNA molecules •NASBA is isothermal (41°C) •Beacons need low temperature to remain “closed” in the absence of complementary target sequence
- 3. The engine: What is Nasba ? (1/2) – NASBA stands for Nucleic Acid Sequence-Based Amplification – It is a target amplification technique that produces exponential amounts of RNA from RNA targets: sensitivity – It is powered by 3 enzymes: • a reverse-transcriptase (from avian myeloblastosis virus AMV) • a RNAse H (from E. coli) • a T7 RNA polymerase (from phage T7) – It is isothermal (41°C) and thus quick (30-60 min.): speed – 2 primers frame a 100-300 nts target region: specificity – A single melting step is required to allow annealing the primers to the target (65°C)
- 4. What is Nasba ? (2/2) – The use of a single T°C eliminates the need for special thermocycling equipment, allowing simple instrumentation better CVs, robustness – End-product is single-stranded RNA, which makes it an ideal target for probe- based detection methods, robustness – Reviewed in Deiman B et al., Molecular biotechnology, 2002, 20: 163
- 5. NASBA Real-Time detection Uses real-time reporter probes called “molecular beacons” sense RNase H RNA oligo P1 oligo P1 Reverse Transcriptase RT RT RNase H & oligo P2 oligo P2 Reverse T7 RNAP Transcriptase anti-sense T7 RNA RNA polymerase F Q Molecular F Q beacon
- 6. Real-time detection with beaconssignal Fluorescent NASBA reaction (real-time) 20 ul Normalized fluorescence 0 10 20 30 40 50 60 Time (minutes)
- 7. Nuclisens Easy Q system EasyQ Instrument & Reagents NucliSens EasyQ Incubator Desktop computer NucliSens EasyQ Strip centrifuge Analyzer
- 8. HIV PATHOGENESIS Typical Course of HIV-1 Infection
- 9. So far so good
- 10. Now let us cross the equator
- 12. CAN SCIENCE HELP THEM?
- 14. Can science help them?
- 15. Can science help them?
- 16. Urban transport
- 17. Boarding gate
- 18. EVALUATION OF DBS VL IN ZIMBABWE • SAMPLE SIZE 96 PAIRS VL SAMPLES • 96 EDTA PLASMA SAMPLES • 96 DBS SAMPLES • DBS STORED FOR 30 DAYS AT RT • STANDARD PROTOCOL FOR PLASMA
- 19. DBS PROTOCOL
- 23. As a team we can cross deep rivers together
- 25. Conclusion • good agreement between DBS and plasma VL • DBS & nuclisens EasyMag /EasyQ is a feasible and reliable method for virological monitoring of patients on ARVs in rural areas with transport facilities for referring samples to a central laboratory. • The fast processing times ,<3hours for 48 samples on the Nuclisens Easy Mag/EasyQ significantly improves turnaround time.
- 26. Acknowledgements • Ministry of Health • Biomerieux • Fima Enterprises • Flow Cytometry Centre Staff
- 27. THANK YOU FOR YOUR TIME