NAIMA method
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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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Presentation at the International Biotechnology Society conference in Dalian, China. October 2008
Transcript of NAIMA method
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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
National Institute of BiologyLjubljana, Slovenia
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– mimics retroviral replication (1)
– avian myeloblastosis virus RT/DNA polymerase, T7 RNA polymerase and RNase H
– T7 promoter-labelled target-specific primer– diagnostics applications (clinical, veterinarian,
environmental…) (2)
(1). Compton J. Nucleic acid sequence-based amplification. Nature. 1991;350(6313):91-2
(2). bioMérieux is NASBA patent holder
Nucleic Acid Sequence-Based Amplification (NASBA)
3WP5 & & meeting11-12 MayLjubljana
ssRNA/ss cDNA
second specific primer
Reverse Transcription
RNAse H activity
T7-specific primer
100-1000 copies
NASBA principle
Primer extension
Transcription
T7-RNA polymerase promoter sequence + specific sequence
Second target specific sequence
3’
5’ RNA
3’ c DNA (-)
Reverse Transcription
5’
5’ ds cDNA3’ RNA pol
Primer extension
5’3’
3’5’
ss DNA 5’
5’
3’
RNA (-)3’
3’
3’
3’
3’
3’
3’
3’
3’
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– Goal: • Multiplexing and integration with microarrays• High throughput identification and quantification of GMOs
NAsba Integrated Multiplex Amplification (NAIMA)
NASBA for GMO detection
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Denaturation, specific “tailed” primers
Primer extension (no cycling)
NAIMA multiplex template synthesis
ds DNA
Host DNA Host DNAGene TermProm
5’3’
5’3’
T7-RNA polymerase promoter sequence + universal sequence
Second universal sequence
Target specific sequence
5’3’
5’3’
5’3’
5’3’
5’3’
5’3’
5’3’
6WP5 & & meeting11-12 MayLjubljana
ssRNA/ssDNA
second universal primer
Reverse Transcription
RNAse H activities
T7-universal primer
RNA (-)
100-1000 copies
NAIMA universal amplification
Primer extension
3’
5’
Transcription
ss DNA
T7-RNA polymerase promoter sequence + universal sequence
Second universal sequence
3’5’
3’5’
3’
3’
3’
3’
3’
3’
3’
RNA pol
5’3’
ds DNA
5’3’
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Optimisation of technology
• Singleplex qualitative and quantitative detection: IVR, 35S, tNOS, Mon810• Specific• High amplification (> 1.106 in singleplex)• 25 min amplification sufficient• Linear• Sensitive (< 20 copies)
• Linear range > 3Log10
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Triplex : sensitivity and linearity
• Screening (IVR*P35S*tNOS) and Mon810 (IVR*P35S*Mon810)• Assessed on serial dilutions
– Same linear range as for singleplex (>3 log10)
– Same sensitivity as for singleplex (down to 2 copies)
– Same amplification profile as for singleplex
Mon863 ctl IVR R2 = 0.9868
IVR R2 = 0.9353
ctl 35S R2 = 0.9984
35S R2 = 0.9029
tNOS R2 = 0.9548
ctl tNOS R2 = 0.9992
0.00
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45.00
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1 10 100 1000 10000
copy nb
Ct
ctl IVR
ctl 35S
ctl tNOS
IVR
35S
tNOS
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Triplex : trueness
• Two triplexes (screening and Mon810)• Feed and food samples with different GM contents assayed
– Easily detects from 0.1% to 100%– Samples quantified in parallel using qPCR and NAIMA
Results are the average of two independent experiments
Triplex platform
35S NAIMA 35S qPCR Cv% Mon810 NAIMA Mon810 qPCR Cv%
Mon810(IVR x 35S x Mon810)
20.0 ± 8.18.4 ± 6.07.4 ± 2.91.5 ± 0.70.9 ± 0.60.3 ± 0.2
25.7 ± 8.011.5 ± 3.25.1 ± 0.63.9 ± 1.12.6 ± 0.80.6 ± 0.2
15.519.232.644.145.735.6
11.5 ± 55.0 ±2.12.6 ± 0.81.4 ± 0.60.3 ± 0.20.14 ± 0.11
8.7 ± 1.05.0 ± 0.44.1 ± 0.41.2 ± 0.50.7 ± 0.30.13 ± 0.04
23.10.225.216.037.41.5
35S NAIMA 35S qPCR tNOS NAIMA tNOS qPCR
Screening(IVR x 35S x tNOS)
17.1 ± 5.912.6 ± 4.74.0 ± 1.80.9 ± 0.50.5 ± 0.2ND
13.4 ± 4.310.1 ± 1.76.2 ± 1.91.0 ± 0.40.4 ± 0.10.04*
20.017.724.46.121.0ND
10.9 ± 5.99.6 ± 2.93.5 ± 0.60.6 ± 0.20.13 ± 0.130.00*
9.2 ± 3.49.9 ± 1.03.4 ± 0.80.6 ± 0.20.3 ± 0.10.02*
12.92.33.72.433.0NR
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Integration of NAIMA to detection on microarrays
• 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
MON spec MON spec_B negative hyb ctl MON spec MON spec_B Det Ctl Cy3 MON spec MON spec_B
P35S tNOS Invertase_B P35S tNOS Invertase_B Det Ctl Cy5 P35S tNOS Invertase_B
P35S_B tNOS_B Invertase P35S_B tNOS_B Invertase Det Ctl Cy3 P35S_B tNOS_B Invertase
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
MON spec MON spec_B negative hyb ctl MON spec MON spec_B Det Ctl Cy3 MON spec MON spec_B
P35S tNOS Invertase_B P35S tNOS Invertase_B Det Ctl Cy5 P35S tNOS Invertase_B
P35S_B tNOS_B Invertase P35S_B tNOS_B Invertase Det Ctl Cy3 P35S_B tNOS_B Invertase
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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copy number
Net
in
ten
sity
P35S
R2 = 0.9934
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copy numberN
et i
nte
nsi
ty
tNOS
R2 = 0.9973
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10000
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20000
25000
30000
35000
40000
0 20 40 60 80 100 120 140
copy number
Net
in
ten
sity
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)
• Preliminary results:– IVR (100), P35 (6.8), NPTII (3.6), tNOS (3.6), 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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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
