ISO/9001:2000 Certified Organisation NABL Accredited (ISO 17025) ISO 14000 DETECTION OF GMOs (LMOs). LALITHA R. GOWDA DEPARTMENT OF PROTEIN CHEMISTRY AND TECHNOLOGY CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE MYSORE 570 020 lrg@cftri.res.in
Internationally referred centre of excellence for post graduate studies and research in foodcentric disciplines 53 Years of dedicated R&D Service to the nation Multi-disciplinary spread across 17 R&D departments covering every field of scientific investigation connected with foods and their relationship to humans, including the cutting edge area of food biotechnology Competitive and wide array of technologies for several commercially attractive, nutritionally superior and safe foods with humane touch State-of-the-art resource base comprehensive Ultra-sophisticated pilot plant A nationally referred food safety analytical quality control laboratory Sensory evaluation Package development Food Engineering services
Genetically Modified Organisms Plants or animals containing genes from other species to produce certain characteristics Pest resistance Herbicide resistance
Overview of GM-traits herbicide tolerance 646 male sterility/fertility restoration 149 Bt-derived insect resistance 139 virus resistance 130 fungal resistance 35 alteration of starch biosynthesis 31 modification of oil composition 29 improvement of processing quality 23 marker system 20 alteration of lignin biosynthesis 10 increased nutritional value 7 drought tolerance 5 alteration of ripening characteristics 4 bacterial resistance 4 nematode resistance 4
Gene construct for a GMO: Plant DNA Promoter Enhancer Gene Plant DNA Terminator
Some Common GMO Constructs
Who needs detection methods, and why? GMO producers To assure purity and segregation of products To be able to trace genetic modification in breeding Food & feed industry, seed companies To assure purity and segregation of products To assure compliance with legislation Competent (enforcement) authorities Product control, compliance with legislation To be able to retrieve specific products E.g. if marketing permission withdrawn Laboratories To provide services to society (incl. above)
Operational procedures involved in detection, identification and quantification of GMOs. Negative GMO test GMO detection Positive Sampling CRM. GMO identification Illegal No Authorised? Yes Assay individual ingredients No need for labelling Less than 1% Labelling required More than 1%
Requirements for GM-detection Target molecules must be present: DNA (template) or protein Capture molecules must be developed: For DNA: primers and probes For proteins: antibodies Reference materials Positive and negative controls Calibrants for quantitation
GMO DETECTION AND TESTING METHODS DNA Based methods Protein based methods Qualitative and Quantitative
DNA-BASED METHODS Detects a known DNA sequence by amplification Target sequence is duplicated billions of times Results obtained can be quantitative or qualitative Amplification is done in changing temperature cycles Enzyme = Thermophyllic TAQ polymerase Instrument = Thermocycler
Field of application Screening targets: false positives and false negatives possible! Not suited for identification and quantification Cost efficient Gene and construct specific targets: Positives GMO presence Event specific targets: Well suited for identification and quantification
Reference genes (for plants) A GMO belongs to a species Reference gene: Demonstrates presence of DNA from species Quantification of species DNA Reference for GMO quantification
GMO ANALYSIS FLOW CHART NON GM FOOD (-ve Control) STANDARD REFERENCE (+ve Control) GM FOOD MATERIAL DNA Extraction CTAB Method DNA Purity WIZARD TM Method PCR Amplification DNA Electrophoresis Interpretation & Documentation
POLYMERASE CHAIN REACTION - PCR A 'licence' to do molecular biology A key central technique that has revolutionised molecular and consequently cell biology devised by Kary Mullis c1983 http://www.nobel.se/chemistry/laureates/1993/mullis-autobio.html Mullis, K.B. (1990) The unusual origin of the polymerase chain reaction. Scientific American. 262 (4) 56-65.
DENATURATION 93 C - 95 C ANNEALING 37 C - 65 C PCR Cycle 25-35 CYCLES DENATURATION 93 C - 95 C EXTENSION 72 C
Polymerase Chain Reaction (PCR): Mg++ A C T G DENATURATION PRIMER ANNEALING POLYMERASE RECOGNITION TAAACTAGCGAAACTGCGTAGTGTGTCTAATCCGTGGACGACTTATT ATTTGATCGCTTTGACGCATCACACAGATTAGGCACCTGCTGAATAA Mg++ Mg++ TAAACTAGCGAAACTGCGTAGTGTGTCTAATCCGTGGACGACTTATT ATTTGATCGCTTTGACGCATCACACAGATTAGGCACCTGCTGAATAA MAGNESIUM COFACTOR EXTENSION Mg++ THE STRAND IS DUPLICATED A C T G
Theoretical DNA Amplification Cycle # of DNA copies 1 2 2 4 4 16 10 1,024 15 32,768 20 1,048,576 25 33,554,432 30 1,073,741,824
Agarose Gel Electrophoresis UV Photograph Polaroid Photograph
DETECTION OF REGULATORY GENES 1 2 3 4 5 6 Amplification of TNOS gene 1 2 3 4 5 6 1 2 3 4 5 6 7 118 bp 192 bp 180 bp Lane 1, Premise control, Lane 2, 100 bp DNA ladder, Lane 3, Control biscuit, Lane 4, GM-2, Lane 5, GM-1 and Lane 6, GM-Soya Different segments of the NOS gene using different primer pairs were amplifiable.
DETECTION OF CONSTRUCT SPECIFIC GENES OF RUR-SOYA CaMV 35S CTP EPSPS 1 2 3 4 5 6 EPSPS-TNOS 1 2 3 4 5 6 169 bp 210 bp Lane 1, Premise control, Lane 2, Control biscuit, Lane 3, 100 bp DNA ladder, Lane 4, GM-1, Lane 5, GM-2 Lane 6, GM Soya Lane 1, Premise control, Lane 2, 100 bp DNA ladder, Lane 3, Control biscuit, Lane 4, GM Soya, Lane 5, GM-1 and Lane 6, GM -2 The CaMV 35S promoter Petunia- EPSPS (169bp) and EPSPS-NOS (210 bp) are cross border segments of the transgene of RUR Soya
Detection of GM-Soya in soya milk 1 2 3 4 5 6 7 8 413 447 Lane 1: Premise control Lane 2: 100bp DNA ladder Lane 3: CP4 EPSPS-TNOSa Lane 4: CaMV 35S-CTP Lane 5: TNOS Lane 6: CaMV 35S Lane 7: Soya Lectin Lane 8: 35S-CP4 EPSPS 145 172192 195
PCR Quality Control/Quality Assurance Positive and negative controls Extraction controls (Blanks) Control reactions (i.e. Lectin gene), spikes, CRM s Validated primer systems All analysis performed at least in duplicate Participation in sponsored check sample programs/ collaborative studies Internal validation trials
A major drawback of conventional PCR is lack of quantitative information due to effect of amplification efficiency. Efficiency is not constant for each amplification cycle but varies between different reactions & within the same reaction particularly in the later cycles. Conventional PCR thus relies on endpoint measurements.
PCR based relative quantification At least two different types of assays Quantitative Competitive PCR Real-time PCR For relative GMO concentrations: Quantification of plant-specific reference gene to GM-specific gene
RELATIVE QUANTITIES 0.5%GM 1.5%GM
REAL TIME PCR Amplification of the target DNA sequence is followed during each cycle by monitoring the product formed. Detection strategies rely on continuous measurements of the increase in fluorescence
Most popular assay is the Taqman or 5 -exonclease assay Employs a fluorogenic probe in addition to gene specific primers Fluorogenic probe is an oligonucleotide with both a reporter and a quencher dye attached.
Denaturation
Annealing Due to its target specific sequence, the probe anneals specifically to the target DNA between the forward and reverse primer
Extension If hybridization occurs the 5 exonuclease activity of Taq polymerase cleaves the internal probe.
5 Exonuclease Activity Cleavage reduces the quenching effect and a fluoroscent signal of the reporter is produced.
Plant specific GMO-specific The number of PCR cycles necessary to generate a signal that is significantly and statistically above the noise level is called cycle threshold.
Different techniques to monitor the PCR product formation in real time using amplicon-specific probes.
COMPETITIVE PCR
Validation Sensitivity Specificity Primer systems tested against other species Primer systems tested against DNA dilutions in various percentages and/or samples with different weight-to weight ratios Accuracy/Precision Primer systems tested in replicates against various matrices of known composition Robustness Other labs able to duplicate results
PCR (Advantages) High species specificity High sensitivity. Detection limits 0.1% of DNA present. Semi-quantitative PCR can provide a rough estimate of the level of contamination Real-Time PCR providing quite accurate quantitation in the less than 10% range DNA highly stable
PCR (Limitations) Not all tissues contain the same amount of DNA Require relatively advanced lab facilities and instrumentation and highly trained staff Cost is fairly expensive ($75- $300) Time (usually >24 hrs for results) Special measures needed for avoidance of contamination and inhibition
Challenges in PCR detection Target molecules Degraded or absent target molecules undetectable! Effects of processing usually significant Important to improve methods to recover target Capture molecules must be developed: Impossible without (description of) target molecules Need material for method development / validation Reference materials Material for production of RMs difficult to obtain Materials must serve many different functions Contd..
Challenges in PCR detection Gene stacking Hybrids between GMOs Detection methods will detect GMO1 + GMO2 GMO1 X GMO2 GMO1 X GMO2 Unknown GMOs Not described in any available documents
Two-tiered approach for DNA detection 1. Detection + Identification (Microarray) 2. Quantitation (Real time PCR)
GMO CHIPS Based on classical DNA hybridization principle Many specific probes are attached to a solid surface Screen & identifies GMOs in raw material and processed food, currently available on soybean, maize, oilseed, rape, rice, CaMV, RR-soybean, Bt etc., Allows screening for all GMOs with the CaMV 35S promoter, Nosterminator, bar-gene and pat-gene
GMO DETECTION AND TESTING METHODS DNA Based methods Protein Based Methods Qualitative and Quantitative
PROTEIN BASED DETECTION
Different strategies to perform an ELISA Test
GMO protein specific to antigen Protein methods - ELISA Enzyme-labelled Antiantibody antibody Blocking protein Antigen Antibody-coated microwells; Quantitative, highly sensitive, economical, high throughput & ideal for laboratory analysis Standard of known GMO-protein well Less expensive than PCR and faster; Risk of false positive less Cannot discriminate difference expression pattern Denaturing protein food processing Separate test is needed for each trait Colour Response Concentration Dependent
Quicktest strip for Cry1Ab/Ac control test positive negative
Quick test strip for EPSPS 0% GM 0.1 % GM 1.0% GM
Captured PCR-ELISA
METHOD HARMONISATION Check that the method works Check that the method is reliable Check that other laboratories can use the method Performance Measurement Validation Proficiency Testing
Summary of detection methods for rdna products of GM foods Parameter Western blot ELISA Lateral flow strip Southern blot Qualitative PCR QC-PCR RT-PCR Ease of use Difficult moderate Simple Difficult Difficult Difficult Difficult Special equipment needed Yes Yes No Yes Yes Yes Yes Sensitivity High High High Moderate Very High High High Duration 2d 30-90 min 10 min 6h 1.5d 2d 1d Cost/sample 150 5 2 150 250 350 450 Quantitative No Yes No No No Yes Yes Field application No Yes Yes No No No No Where applied Academic lab Test facility Field Testing Academic lab Test facility Test facility Test facility Ahmed, 2002
CONCLUSIONS PCR with its various formats is currently the leading analytical technology employed in the qualitative and quantitative analysis of GMOs (LMOs) because of its unparalleled sensitivity and specificity. On the other hand immunoassays are tools for rapid field monitoring of the integrity of agricultural commodities, whereby non-specialized personnel can employ them in a cost effective manner.