Lab Management of HIV Boniface MudengeFLOW CYTOMETRY CENTRE, SUITE 12A MEDICAL CENTRE52 BAINES AVENUE, HARARE, ZIMBABWEwww.flowcytolab.com
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
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)
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
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
Real-time detection with beaconssignal FluorescentNASBA reaction (real-time) 20 ul Normalized fluorescence 0 10 20 30 40 50 60 Time (minutes)
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.
Acknowledgements• Ministry of Health• Biomerieux• Fima Enterprises• Flow Cytometry Centre Staff