Latest Developments in the Area. of Detection Methods

Transcription

1 Latest Developments in the Area of Detection Methods Enrico Noli LaRAS, University of Bologna

2 Global status of Commercialized Biotech Crop: 2007 Source: (ISAAA)

3 Who needs GMO detection methods Those who want GM varieties: Trait Purity Biotech producers, seed companies, farmers To assist variety conversion To track different products To assure compliance of biotech varieties to commercial purity standards Those who don t: Adventitious Presence Seed companies, farmers, food & feed industry, enforcement authorities, testing laboratories To monitor products after commercial release To assure segregation of products To check compliance with legislation (authorized vs non-autorized, labelling thresholds)

4 The view of ISTA on GMO testing (Position Paper 2001) ISTA seed testing laboratories are competence centres for varietal purity testing of GM varieties as well as for the detection, identification and quantification of GM seeds in conventional seed lots and provide uniform test results all over the world

5 Methods for GMO detection Analysis of the phenotype Biossays Protein-based methods ELISA lateral flow stick Analysis of the genotype DNA-based methods end-point PCR real-time PCR

6 Trait Purity testing Biossays for HT Soak test Spray test Usually individual seeds/seedlings Simple Reasonable cost (20-30 US$) Take time Difficult interpretation

7 Trait Purity testing Protein-based methods ELISA Immunochromatography (lateral flow strips) Usually individual seeds Relatively simple Results in hours Cost US$

8 AP testing Detection - Does the lot contain GMOs? Identification - If lot is positive, is/are the GMO(s) authorised? - Necessary for correct quantification Quantification - Does the lot comply with thresholds?

9 AP testing - Methods utilised Protein-based ELISA Immunostrips DNA-based (PCR) Test conducted on bulk samples

10 DNA-based methods end-point PCR Amplification Electrophoresis Detection Result: presence/absence

11 DNA-based methods real time PCR Amplification Real time monitoring Reference gene Transgene Data analysis Result: : qualitative/quantitative

12 Advantages of real time PCR Rapidity (no electrophoresis) Specificity (based on probe hybridisation) Sensitivity (automated optical detection) Low risk of contamination (no post-pcr manipulation) and, of course, possibility of quantification In the EU, all the assays validated by the Community Reference Laboratory are based on real time PCR

13 GM targets for PCR: Different levels of specificity Event specific targets HIGH Gene specific targets Construct specific targets specificity Screening targets LOW promoter plant promoter gene terminator plant

14 AP testing Detection problems Availability of common elements Number of events containing a given element (based on BIOTRADESTATUS and AGBIOS databases)

15 AP testing Identification problems Increasing number of transgenic events on the market Assays informativity How many assays are necessary for unambiguous identification?

16 + MON832 + T-35S - CBH-351 P-ract AP testing Dichotomic keys MS3, MS6 + neo - 3 MON801 MON802 + MON P-ract cry3bb1 NK603 + bar CP4-epsps MON pat - Bt P-35S MON863 + T-nos P-ract + GA T-PINII MIR TC1507 bar GM corn bla - + T-35S - T-35S DBT418 2 TC BT176 + bla pat T14 T25 B116-2 cry1fa DAS MON810

17 AP testing Strategies (Challenges) for improvement Simplicity Speed of testing Increase throughput

18 AP testing Increase throughput PCR - MULTIPLEXING Bt11 MON810 T25 GA21 Hernandez et al., 2005 J. Agric. Food Chem., 53 (9), , 2005

19 AP testing Problems with PCR-multiplexing MW % each event non GM Hernandez et al., 2005 J. Agric. Food Chem., 53 (9), , 2005

20 High throughput Sample MICROARRAY general concept DNA extraction PCR with labelled primers (multiplexing and/or multipooling) Capture probes spotted on slide (plant species-, genes-, or event-specific sequences) Hybridization Detection

21 Sample DNA Multiplex PCR with primers Amplified Sample targets 21 MICROARRAY general concept P35S invertase lectin T-nos lectin Hybridization and detection invertase P35S T-nos

22 GMO-Dual Chip Sample DNA extraction PCR (with biothynilated primers) PCR1: Tnos, P35S PCR2: Pnos/nptII, CaMV PCR3: Pat, Cry1Ab, EPSPS PCR4: maize, soybean, rapeseed, plant generic ARRAY GMO target elements - P35S - Tnos -Pat - Cry1Ab(Bt176), Cry1Ab(Bt11), Cry1Ab(Mon810) - CP4-EPSPS, m-epsps - Pnos/nptII Species specific targets - Invertase (maize) - Cruciferin (rapeseed) - Lectin (Soybean) - rbcl (plant universal) Control target -CaMV Hybridization Detection

23 GMO-Dual Chip 1. Specific binding of biothynilated products 2. Binding of antibiotin conjugated gold 3. Silver precipitation catalysed by gold

24 Rep.1 Rep.2 Rep.3 GMO-Dual Chip Detection control P35S, Tnos PCR control epsps cruciferin rbcl, lectin invertase Feed sample containing RR soybean, rapeseed and maize Orientation markers False positive rate very low False negative rate <5% down to 0.1% level for all GM targets, down to 0,045% for 7 of them

25 Quantification of AP presence Real-time PCR is the most precise method for gene quantification However quantification is strongly affected by sample properties DNA quantity DNA quality (extraction methods and matrices) Purity and integrity Amplification efficiency (DNA quality and PCR assay

26 Effect of DNA quality on amplification efficiency and quantification Ct log of target amount (ng) Sample efficiency (transgene and reference) GMO% estimate GMO% estimate (transgene efficiency=1) True GMO content = 5% Standard curve efficiency =1

27 How to check amplification efficiency Test efficiency on dilution series of each sample DNA both for GM and reference target Compare with efficiency of standard samples Accept quantitative estimate only if efficiencies are similar More controls needed 96-well plate 384-well plate

28 Chemistries for real time quantification Intercalating dye SYBR Green FRET-based quenching Probes TaqMan MGM (minor groove binding) LNA (locked nucleic acid) CPT (cycling probe technology) Molecular Beacon Hydrolysis Primers Scorpion D-LUX (light upon extension) Plexor

29 TaqMan reporter quencer Taq Taq

30 LNA (locked nucleic acid) H H Increased thermal stability of duplexes Shorter probes (8-mer vs 25-mer TaqMan) shorter amplicons ( bp) Facilitated assay design, especially for construct- or event-specific assays Easily adopted Costs comparable to ordinary TaqMan

31 Amplification methods alternative to PCR Real multiplexing possibilities Linear amplification Isothermal conditions Low sensitivity to inhibitors Amplification on microarray LDR (ligase detection reaction) NASBA (Nucleic Acid Sequence Based Amplification) RCA (Rolling Cycle Amplification)