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Lab Management of HIV       Boniface MudengeFLOW CYTOMETRY CENTRE, SUITE 12A MEDICAL CENTRE52 BAINES AVENUE, HARARE, ZIMBA...
NASBA and Molecular BeaconsAre the perfect match: •NASBA generates s/sRNA amplicons •Molecular beacons binds easily s/sRNA...
The engine: What is Nasba ? (1/2)– NASBA stands for Nucleic Acid Sequence-Based Amplification– It is a target amplificatio...
What is Nasba ? (2/2)– The use of a single T°C eliminates the need for special thermocycling equipment,  allowing simple i...
NASBA Real-Time detection                               Uses real-time reporter probes                                call...
Real-time detection with                    beaconssignal                   FluorescentNASBA reaction                     ...
Nuclisens Easy Q system                                                    EasyQ Instrument &                             ...
HIV PATHOGENESISTypical Course of HIV-1        Infection
So far so good
Now let us cross the equator
KAMOTO MISSION ZAMBIA
CAN SCIENCE HELP THEM?
THE BEST RESTAURANT
Can science help them?
Can science help them?
Urban transport
Boarding gate
EVALUATION OF DBS VL IN          ZIMBABWE•   SAMPLE SIZE 96 PAIRS VL SAMPLES•   96 EDTA PLASMA SAMPLES•   96 DBS SAMPLES• ...
DBS PROTOCOL
As a team we can cross deep       rivers together
Conclusion• good agreement between DBS and plasma VL• DBS & nuclisens EasyMag /EasyQ is a  feasible and reliable method fo...
Acknowledgements•   Ministry of Health•   Biomerieux•   Fima Enterprises•   Flow Cytometry Centre Staff
THANK YOU FOR YOUR TIME
Zasco flowcytolab dbs viral load
Zasco flowcytolab dbs viral load
Zasco flowcytolab dbs viral load
Zasco flowcytolab dbs viral load
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Zasco flowcytolab dbs viral load

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Zasco flowcytolab dbs viral load

  1. 1. Lab Management of HIV Boniface MudengeFLOW CYTOMETRY CENTRE, SUITE 12A MEDICAL CENTRE52 BAINES AVENUE, HARARE, ZIMBABWEwww.flowcytolab.com
  2. 2. NASBA and Molecular BeaconsAre 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. 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. 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. 5. NASBA Real-Time detection Uses real-time reporter probes called “molecular beacons”sense RNase HRNAoligo P1 oligo P1Reverse Transcriptase RT RTRNase H &oligo P2 oligo P2Reverse T7 RNAPTranscriptase anti-senseT7 RNA RNApolymerase F Q Molecular F Q beacon
  6. 6. Real-time detection with beaconssignal FluorescentNASBA reaction (real-time) 20 ul Normalized fluorescence 0 10 20 30 40 50 60 Time (minutes)
  7. 7. Nuclisens Easy Q system EasyQ Instrument & Reagents NucliSens EasyQ IncubatorDesktop computer NucliSens EasyQ Strip centrifuge Analyzer
  8. 8. HIV PATHOGENESISTypical Course of HIV-1 Infection
  9. 9. So far so good
  10. 10. Now let us cross the equator
  11. 11. KAMOTO MISSION ZAMBIA
  12. 12. CAN SCIENCE HELP THEM?
  13. 13. THE BEST RESTAURANT
  14. 14. Can science help them?
  15. 15. Can science help them?
  16. 16. Urban transport
  17. 17. Boarding gate
  18. 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. 19. DBS PROTOCOL
  20. 20. As a team we can cross deep rivers together
  21. 21. 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.
  22. 22. Acknowledgements• Ministry of Health• Biomerieux• Fima Enterprises• Flow Cytometry Centre Staff
  23. 23. THANK YOU FOR YOUR TIME

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