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Detection of transgenic canola (Roundup Ready® - Monsanto)
 

Detection of transgenic canola (Roundup Ready® - Monsanto)

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Determination of a validation protocol, based on ...

Determination of a validation protocol, based on
established European Union methods, for the detection
of transgenic canola (Roundup Ready® - Monsanto) in
seed samples using molecular techniques

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  • Self introduction: Good morning/afternoon to everybody My name is Claudio Iannetta and I am a Master student in Biotechnology, with a major in Food Sc & Tech here at RMIT University My research project has been a collaboration between RMIT-University and NMI (National Measurement Institute), both placed in Melbourne. The supervision of the project has been guided by Dr. Saman Buddhadasa (NMI) and Dr Gregory Nugent (RMIT University).

Detection of transgenic canola (Roundup Ready® - Monsanto) Detection of transgenic canola (Roundup Ready® - Monsanto) Presentation Transcript

  • Claudio Iannetta (s3154021) Supervisor: Saman Buddhadasa Senior Food Scientist Food Metrology National Measurement Institute
  • PROJECT
    • Validation of the protocol for the detection of transgenic canola
    • (Roundup Ready ® - Monsanto)
    • in seed samples, using real-time PCR
    • Project based on established European Union method
    • (2007, European Commission)
  • AGENDA
    • About Canola
    • GM Canola Seed
    • Australian Legislation
    • Project Aims
    • Methods
    • Results & Data Analysis
    • Conclusion
  • About Canola
    • Brassica genus consists of about hundred species
    • B. napus commonly known as
    • oilseed rape or canola
    • Canola is grown for:
      • Oil extraction for human consumption
      • Seed-meal for animal feed
      • Biofuels (Codex, 2002)
    (www.whitemoldresearch.com)
  • GM Canola (1)
    • In canola cultivation, weed control is one of the major challenges
      • The broad spectrum herbicide, Roundup Ready ® (active ingredient glyphosate) is one of the most popular
    • Herbicide-tolerant canola is an effective and popular solution for farmers:
      • Increased crop yields
      • Increased crop quality
      • Helps the conservation tillage system (Biotechnology Australia, 2007)
    • GM Canola produced by Monsanto is tolerant to the glyphosate herbicide
      • Using T-DNA of Agrobacterium ,
      • two new genes have been inserted
      • into the canola line to encode
      • the GOX and CP4-EPSPS
      • enzymes
      • (Monsanto 2004)
    GM Canola (2) (www.biotechnologie.it)
  • GM Canola (3)
      • Transgenic plants are resistant because they bypass the block of amino acid biosynthesis caused by glyphosate,
      • which is a shikimic
      • acid pathway
      • inhibitor (Monsanto, 2004)
  • Health Risks Associated
    • Toxicological
      • Plants and other living organisms contain natural toxins and may give unexpected changes when subjected to GE
    • Nutritional
      • Genetic modifications can lead to loss of essential food components like phytoestrogen, which is an important hormone for human health
  • Health Risks Associated
    • Allergenic
      • New allergens could be inadvertently created
    • Gene Transfer
      • DNA fragments from GM foods can be transferred to human cells through:
      • GI microflora bacteria
      • Direct absorption of the somatic cells lining the intestinal tract
      • (WHO/FAO, 2004)
  • International Legislation
    • The Codex Alimentarius Commission (Codex) and FAO/WHO are international organizations involved in developing protocols for GMOs, to assure:
      • General rules for safety evaluations
      • Risk analysis
      • Labeling of GMO
      • Assessing nutritional aspects of GM food
      • Detection of specific protein and/or DNA in GM food
    • The Commonwealth Government Office of Gene Technology Regulator (OGTR)
    • has approved the cultivation
    • of GM canola by Monsanto,
    • under State Moratoria
    • In Victoria, the legislated
    • Moratorium on commercial
    • GM crops was removed in 2008
    • (FSANZ, 2007; NACMA, 2007 and ASF, 2007)
    Australian Legislation (1)
  • Australian Legislation (2)
    • Seed labeling is compulsory if the genetically modified traits in canola are over
    • 0.5% for seeds used
    • for sowing and over
    • 0.9% for grain
      • Cut-off for unintentional
      • contamination
      • ( NACMA, 2007 and ASF, 2007)
  • Detection Methods
    • DNA based methods to detect foreign DNA
      • Competitive PCR
      • Real time PCR
      • Microarray
    • Protein based methods to detect novel protein
      • ELISA
      • Strip (Gonzalez, 1999; Rajeevan, 2001)
  • DNA Based Methods
    • Highly sensitive
      • Can detect trace amounts of transgenic DNA
    • Work with most product types
      • Processed and unprocessed products
    • Can test simultaneously multiple GM varieties
  • DNA Based Methods
    • Takes a number of days to perform, typically 3-5 days
    • Requires highly skilled personnel and laboratory analysis
    • More expensive than Protein Based Methods
      • (Rajeevan, 2001)
  • Protein Based Methods
    • Relatively cheap to perform
    • Rapid: 24 hours (ELISA)
    • Not very sensitive
      • Concentration >1% of GM protein
    • Not appropriate for some GM varieties
    • (Mullis, 1987)
  • Why real-time PCR?
    • Recognised world-wide standard method for GMO quantification
      • High Sensitivity, Specificity and Reproducibility
      • Rapid cycling, from 30 to 120 min
      • Permits numerical quantification
      • Not influenced by non-specific amplification
      • No post-PCR processing of products
      • Amplification can be monitored in real time
      • Similar cost to conventional PCR (Wang, 2005)
  • Project Aims
    • Optimization of the DNA extraction method to provide suitable DNA quantity and quality
    • UV Spectrometer - DNA purity, DNA concentration
    • Bioanalyzer - DNA fragmentation
    • Conventional PCR - Presence of the target event
    • Real-time PCR - Quantification of the transgenic canola
    • Validation of the method - Australian legislation
  • Justifications
    • The method can be used:
      • In Australian forensic arena on Judicial/Legal matters
      • To ensure when the canola seed samples require specific GM labeling in compliance with legislation
      • To protect consumer rights giving them the possibility to make informed product choices
  • Project – Flow Chart ✔ ✔ ✔
  • Grinding of the Seed Samples
    • Samples
      • Certified Roundup Ready ® canola seeds
      • Certified wild type canola seeds
    • Ground canola seeds
      • Retsch 301 Mixer Mill
      • In this step it is important to
      • assure the homogenization of the sample (www.retsch.com)
  • DNA Isolation
    • DNA isolation of canola seeds
      • Incubation with Nucleic Lysis Solution
      • Centrifugation and incubation with Protein Precipitation
      • and RNase
      • DNA precipitation by Isopropanol
      • DNA resuspension/storage
      • in TE 0.1 buffer
    • To avoid any possible contamination to
    • the external environment, seed grinding,
    • DNA extraction and molecular
    • methods have been performed in the PC2 lab
      • (NMI Bioanalysis Group 2006)
  • DNA Concentration
    • UV Spectrophotometer
    • It is possible to quantify the amount of DNA in a sample by looking at its absorbance at the wavelength of 260 nm in the UV region
    • Pure double-stranded DNA sample has an absorbance of 1.0 at 260 nm UV, its DNA content is approximately of 50 µg/mL
    A 260 DNA Concentration 0.0122 37 ng/µL 0.0114
  • DNA Purity
    • UV Spectrophotometer
    • To estimate the purity of nucleic acid solution.
      • The absorbance of the solution has to be measured at two wavelengths: 260 nm and 280 nm (Sagdum, 2000)
    • The pure DNA has a Abs ratio of 1.8 at A 260 /A 280
    A 260 A 280 Average: A 260 /A 280 0.0122 0.0076 1.81 0.0114 0.0054
  • DNA Fragmentation
    • Agilent 2100 Bioanalyzer
    • Microfluidics-based platform
    • for the analysis of DNA
    • Charged DNA is electrophoretically
    • driven by a voltage gradient and
    • the molecules are separated by size
    • Detection by laser-induced
    • fluorescence
      • Canola DNA size: 1182 Mb
      • No smearing
  • Conventional PCR (1)
    • The polymerase chain reaction
    • is a molecular biology technique for isolating and exponentially amplifying a fragment of DNA, via enzymatic replication
    • DNA replication enzymes
    • use the primers as docking points and start doubling the target sequences
    • The process is repeated over and over again
    • the millions of identical fragments are then purified in a slab of gel and seen with UV light ( Mullis, 1987)
  • Conventional PCR (2)
    • Reagents
    • Polymerase enzyme
      • GoTaq – BioTaq - Accuzyme
    • Primers (RT73; FatA )
    • Buffer (10 x)
    • H 2 O
    • MgCl 2
    • dNTPs
    • Template
    • Dye
    Stage Cycle No Setting C 1 1 94 1 ' 2 40 94 1 ' 47 1 ' 72 30 '' 3 1 72 7 ' 4 1 4 ∞
  • PCR Agarose Gel Electrophoresis
    • Agarose Gel (1%)
    • Ethidium Bromide (Toxic)
    • Ladder (Fermentas - 1 Kb DNA)
    • Electricity 120 V (30 min)
  • Real-time PCR (1)
      • Applied Biosystem ® 7500 Fast
        • TaqMan Universal Master Mix
        • Endogenous Gene
        • Primers FatA
        • Probe FatA
        • New Gene
        • Primers RT73
        • Probe RT73
        • DNA Reference Templates
  • Real-time PCR (1)
      • Real-time allow accurate quantification of
      • starting amounts of DNA
      • TaqMan hydrolysis probe principles
        • The fluorescent reporter probe method is the most accurate and most reliable
        • The increase in DNA product during PCR leads to an increase in the intensity of fluorescence which is measured in each cycle
  • Real-time PCR (2)
    • 5’-3’ Exonuclease activity of the thermostable enzyme Taq DNA polymerase
    • Cleavage of the probes, separates
    • the detectable reporter fluorophore
    • (FAM) from a quencher (TAMRA)
    • Fluorescence emission activity
    • is proportional to the amount of
    • PCR product formed
  • Real-time PCR (3)
    • The amplification plot reflects the generation of the reporter dye during amplification and it’s directly related to the formation of PCR products
    • Threshold cycle (C T value) is the
    • cycle at which a significant increase
    • in fluorescence is first detected
    Stage Cycle No Setting C 1 1 1 1
    • 2 '
    • 95 10 '
    2 40 95 15 '' 60 1 ' 4 1 4 ∞
  • 96 Well PCR Plate
    • Blue: detection of total canola
    • amount and standards (pure wt)
      • Primers and Probe FatA
      • Dyes FAM and TAMRA
    • Green: detection of GM canola
    • amount and standards (pure GM)
      • Primers and Probe RT73
      • Dyes FAM and TAMRA
    The plate is split in two identical parts and the difference in some of the reagents enables the detection of the total amount of canola or the transgenic one
  • Standard Curve WT Canola
    • Six Dilutions (duplicates)
      • From 1/3 To 1/729
    • R 2 = 0.99
    • Efficiency
      • Slope= -3.45
    • E= (10 (-1/slope) -1)x100
    • E= 95%
  • Standard Curve GM Canola
    • Six Dilutions (duplicates)
      • From 1/3 To 1/729
    • R 2 = 0.95
    • Efficiency
      • Slope= -1.93
    • E= (10 (-1/slope) -1)x100
    • E= Odd Value!
    • WHY?
  • Problems with Standard Curve
    • C T values <29 cycles indicates
    • abundant target DNA in the sample
    • C T values between 38-40 cycles
    • indicates a minimal target DNA
    • interference with contamination
    • ISSUE:
    • The only available GM canola sample
    • had a concentration of 2%. The standard curve obtained
    • was hardly detectable and thus lead to unreliable results
  • Data Analysis
    • Relative Quantitation
    • The C T value is directly related to the amount of PCR product and therefore related to the initial amount of target DNA present in the PCR
    • Ratio = 2 (RT73 Ct - FatA Ct)
    • %GM = (1/Ratio)x100
    Expected % Determined % Mean Standard Deviation Coefficient Variation <25% 2 0.90 0.54% 0.005 92.1% 0.19 1 0.07 0.04% 0.004 106.1% 0.01
  • Some Good Results ( FatA )
    • Adopting the detection of the total canola amount
      • LOD=0.05%
      • LOQ=0.01%
    • Results:
    • triplicate samples
    • at two different
    • concentrations…
  • Data Analysis The consistency of the results is assured if Coefficient of Variation is below 25%, which is the recommended value from EU Network of GMO Laboratories Sample A Sample B E ( FatA ) R 2 ( FatA ) 1 2.18 x 10 3 917.891 95% 99% 2 2.26 x 10 3 840.949 3 2.24 x 10 3 879.143 Mean 2.23 x 10 3 879.327 SD 41.63 38.47 CV 1.87% 4.37%
  • Conclusions
    • Due to bureaucratic issues with Monsanto rules, I was not able to obtain important reference material which was fundamental to achieve suitable results for the validation protocol
    • In spite of the above issue and considering the consistency of the positive results on the canola samples analysed with the FatA reagents, it is possible to assume the reliability of the presented procedure with a good degree of confidence
    • Definitive results will be obtained if the standard curve for transgenic detection is obtained using 100% GM canola
  • References