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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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4. bioMérieux & NADcommitment 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 (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)
16. 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
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 (41°C)
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 ľ-organisms have been killed by various treatments: heating, pressure, salting etc.
29. Conclusions