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Create value for risk assessment: Quicker time to result: hours: Salmonella

Create value for risk assessment:Quicker time to result: hours: Salmonella / ListeriaQuicker time to result: weeks Legionella, viruses, fungiNo culture: direct on sample (must be liquified in a small volume)Simplify multiple testing: water, airInnovative solution for current bottlenecks:Sample prep, nucleic acid purification.

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Create value for risk assessment: Quicker time to result: hours: Salmonella

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    4. bioMrieux & NAD commitment since 1990 1994 First commercial introduction: NASBA HIV-1 QT 1998 N.A. extraction / purification reagents, silica-based Boom NucliSens Extractor automate 2002 Real-time generation (NucliSens EasyQ) EasyQ HIV-1 2004 Magnetic extraction reagents Minimag (semi-automated) EasyMag (full automation)

    5. The NucliSens EasyQ System

    6. The NucliSens EasyQ System

    9. Main benefits of Boom silica extraction technology Effective purification and concentration of nucleic acids Both RNA and DNA are purified Compatibility with various amplification technologies: PCR, NASBA High purity: amplification inhibitory factors are effectively removed from many difficult sample types eg. stools, sputum, food. Allow sequencing Reproducible isolation allowing quantification No degradation of purified nucleic acid Applicable to a broad range of samples types: clinical, microbiological, plants, animal. Flexibility of input and output volumes The Boom Silica Method has become the Gold Standard nucleic acid isolation technology in molecular diagnostics. Several other companies arrived at this conclusion and license the technology from bioMerieux (Roche, Qiagen, Promega,.).

    10. Applications in infectious disease diagnostics Een aantal toepassingen in de humane diagnostiekEen aantal toepassingen in de humane diagnostiek

    11. The EasyMag project

    14. The NucliSens EasyQ System

    15. 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 (41C) 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 (65C)

    16. What is Nasba ? (2/2) The use of a single TC 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

    17. NASBA scheme

    19. NASBA Real-Time detection

    20. NASBA and Molecular Beacons NASBA generates s/sRNA amplicons Molecular beacons binds easily s/sRNA or s/sDNA molecules NASBA is isothermal (41C) Beacons need low temperature to remain closed in the absence of complementary target sequence

    21. Real-time detection with beacons

    23. EasyQ system benefits High-throughput (n=48) Minimal hands-on-time (30 min for 48 samples) Fast-time to result (30 to 60 min.) for amplification / detection steps, sample prep. dependent on sample Internal control (also used as calibrator for QT) due to duplex amplification No post-amplification step, no carry-over contaminations (closed tube) Combining various applications in one run Allows panel approach based on clinical signs e.g. pneumonia on sample-related risks e.g. water, food

    25. NucliSens EasyQ HIV-1 Dynamic range & Reproducibility

    26. Nasba & sensitivity

    27. Homebrew Nasba NASBA is available as a research tool through the basic kit set of Nasba reagents used on the EasyQ instrument Contains all the components for isolation, amplification and detection, except for the specific primers and probes Increasing number of papers describing the use of Nasba: Clinical RNA viruses (HIV-1, HCV, Influenza, Enterovirus, Poliovirus, West Nile, Dengue, Rabies, ) Food / water related viruses Bacteria, fungi and parasites using various targets (rRNA, specific) that result in extremely sensitive tests

    28. Nasba detects viable organisms Detection of RNA as been proposed as a molecular diagnostic indicator of viability (Bej et al., 1991) mRNA have short half-life in viable cells, e.g.< 7 min. for EF-tu in cultured E.coli (Vaitinlingom et al., 1998), being rapidly degraded by RNAses Simpkins et al. (2000) showed that Nasba can selectively amplify Salmonella RNA in the presence of genomic DNA from culture. The Nasba signal became negative 15 min. after pasteurization Min et al. (2002) showed that sonicated, boiled, or chlorinated E. coli cells were no longer detected in water (s= 40 cells/ml) Van gemen et al. (1994) showed that Mycobacterium smegmatis culture and 16S Nasba testing both negatived after 3 days of exposure to antibiotics (RIF, OFL) whereas PCR remained positive after 7days NASBA ensures that only viable organisms are detected. This is important for many in-process testing where -organisms have been killed by various treatments: heating, pressure, salting etc.

    29. Conclusions

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