1 APPLICATION OF NAIMA-MA FOR FAST HIGH THROUGHPUT AND QUANTITATIVE GMO DETECTION Dany Morisset , David Dobnik, Tina Likar and Kristina Gruden Department of Biotechnology and Systems Biology National Institute of Biology Ljubljana, Slovenia [email protected]
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APPLICATION OF NAIMA-MA FOR FAST HIGH THROUGHPUT AND
QUANTITATIVE GMO DETECTION
Dany Morisset, David Dobnik, Tina Likar and Kristina GrudenDepartment of Biotechnology and Systems Biology
• Direct labelling of NAIMA products with dendrimers (15 oyster dyes/molecule)• Signal amplification• 60 min NAIMA
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Customized EAT dual-chip
Sense and anti-sense probes (cRNA and cDNA detection)
S. Hamels, S. Leimanis, M. Mazzara, G. Bellocchi, N. Foti, W. Moens, J. Remacle and G. Van den Eede. (2007) Microarray Method for the Screening of EU Approved GMOs by Identification of their Genetic Elements. EUR 22935 EN-Joint Research Centre. ISBN 978-92-7906989-5
Each spot is present in triplicate
Det Ctl Cy5 Det Ctl Cy3 Det Ctl Cy5 Det Ctl Cy3 Det Ctl Cy5 Det Ctl Cy3 Det Ctl Cy5
Hyb Ctl negative Det Ctl Det Ctl Cy3 Hyb Ctl Det Ctl Cy3 Det Ctl Cy5 Hyb Ctl Det Ctl Cy3
negative Det Ctl Hyb Ctl negative hyb ctl Hyb Ctl Det Ctl Cy5 Hyb Ctl
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On-chip detection: specificity
• Mon810 100% (w/w) DNA tested (Mon810 triplex)• 200 starting copies of IVR, 100 starting copies of P35S and Mon810: NAIMA
amplified, labelled, hybridized • Specific for probes• ss cDNA and cRNA detected Det Ctl Cy5 Det Ctl Cy3 Det Ctl Cy5 Det Ctl Cy3 Det Ctl Cy5 Det Ctl Cy3 Det Ctl Cy5
Hyb Ctl negative Det Ctl Det Ctl Cy3 Hyb Ctl Det Ctl Cy3 Det Ctl Cy5 Hyb Ctl Det Ctl Cy3
negative Det Ctl Hyb Ctl negative hyb ctl Hyb Ctl Det Ctl Cy5 Hyb Ctl
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• Mon863 10% (w/w) DNA tested (screening triplex)• 4 dilutions of DNA: NAIMA-amplified, labelled and hybridized
dilutions IVR copies tNOS copies P35S copies
4x 2690 134 269
16x 672 33 67
64x 168 8 17
256x 42 2 4
256x
High sensitivity
On-chip detection: sensitivity
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• Mon863 10% (w/w) DNA tested (screening triplex)• 4 dilutions of DNA NASBA-amplified, labelled and hybridized• Perfect correlation between starting copy number and net intensity• Broad linear range
Quantitative measurements on microarray after NAIMA amplification
On-chip detection: quantification
IVR
R2 = 0.9991
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P35S
R2 = 0.9934
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copy numberN
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tNOS
R2 = 0.9973
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Measures in triplicate for each amplicon
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– Fast amplification (25min)– Multiplex (tailed primers)– Quantitative (fit with 0.9% threshold, wide linear range)– Sensitivity of the amplification (detection traces)– Difficult matrices (processed food, traces, high DNA
background)– Application with microarray (no further purification,
identification/quantification) – high throughput– Target and signal amplification– Short hybridization (1H)– Sensitivity of microarray/dendrimer (< 20 copies)
Conclusion
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Improvements
• 6-plex under development • 4 screening elements (P35S, tNOS, NPTII, BAR)• 1 endogene (IVR)• 1 event-specific (MON810)
(2.7), PAT (0.5)– Good sensitivity: < 10 copies– Linearity: ok
• Next step: quantification, complex samples
IVR R2 = 0.9684
NPtII R2 = 0.9764
Mon 810 R2 = 0.8999
P35S R2 = 0.9471 tNOS R2 = 0.9455
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0 1 10 100 1000 10000
IVR
Mon810
NPtII
P35S
PAT
tNOS
Log. (IVR)
Log. (NPtII)
Log. (Mon810)
Log. (P35S)
Log. (tNOS)
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Improvements
• 6-plex under development • Higher multiplexing is aimed• New elements to be added: event vs screening• Quantification on microarray• Simplified procedure (isothermal, silver staining)• Pre-validation: technology transfer to 2nd lab
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Additional information
Literature:• Morisset, D. et al. (2008). Alternative DNA amplification
methods to PCR and their application in GMO detection: a review. Eur. Food Res.Techn. 227:1287-97
• Morisset, D. et al. (2008)., NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs. Nucl. Acids Res. (online).
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David Dobnik Tina Likar
Dr. Kristina Gruden Pr. Jana Žel
European Commission’s Sixth Framework Program throughthe integrated project Co-extra (contract no. 7158).
Acknowledgments
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Time and cost (6 screening targets)
• qPCR• 6 samples/ 0.5 working day (96 well-plate)• 40 euros/ sample
• NAIMA-MA (25 min NAIMA-60min MA)• 24 samples/ 0.5 working day• 6-plex: 34 euros/sample