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Real-Time PCR Validation of GM Canola Detection Method

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claudio iannetta
claudio iannetta

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

Technology
1 of 41
Claudio Iannetta (s3154021) Supervisor: Saman Buddhadasa Senior Food Scientist Food Metrology National Measurement Institute
PROJECT ,[object Object],[object Object],[object Object],[object Object],[object Object]
AGENDA ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
About Canola ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],(www.whitemoldresearch.com)
GM Canola (1) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],GM Canola (2) (www.biotechnologie.it)
GM Canola (3) ,[object Object],[object Object],[object Object],[object Object]
Health Risks Associated ,[object Object],[object Object],[object Object],[object Object]
Health Risks Associated ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
International Legislation ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Australian Legislation (1)
Australian Legislation (2) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Detection Methods ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
DNA Based Methods ,[object Object],[object Object],[object Object],[object Object],[object Object]
DNA Based Methods ,[object Object],[object Object],[object Object],[object Object]
Protein Based Methods ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Why real-time PCR? ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Project Aims ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Justifications ,[object Object],[object Object],[object Object],[object Object]
Project – Flow Chart ✔ ✔ ✔
Grinding of the Seed Samples ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
DNA Isolation ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
DNA Concentration ,[object Object],[object Object],[object Object],A 260 DNA Concentration 0.0122 37 ng/µL 0.0114
DNA Purity ,[object Object],[object Object],[object Object],[object Object],A 260 A 280 Average: A 260 /A 280 0.0122 0.0076 1.81 0.0114 0.0054
DNA Fragmentation ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Conventional PCR (1) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Conventional PCR (2) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],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 ,[object Object],[object Object],[object Object],[object Object]
Real-time PCR (1) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Real-time PCR (1) ,[object Object],[object Object],[object Object],[object Object],[object Object]
Real-time PCR (2) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Real-time PCR (3) ,[object Object],[object Object],[object Object],[object Object],Stage Cycle No Setting C 1 1 1 1 ,[object Object],[object Object],2 40 95 15 '' 60 1 ' 4 1 4 ∞
96 Well PCR Plate ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],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 ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Standard Curve GM Canola ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Problems with Standard Curve ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Data Analysis ,[object Object],[object Object],[object Object],[object Object],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 ) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
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 ,[object Object],[object Object],[object Object]
References

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Real-Time PCR Validation of GM Canola Detection Method

  • 1. Claudio Iannetta (s3154021) Supervisor: Saman Buddhadasa Senior Food Scientist Food Metrology National Measurement Institute
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  • 20. Project – Flow Chart ✔ ✔ ✔
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  • 39. 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%
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Editor's Notes

  1. 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 &amp; 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).