1. Techniques in Molecular Biology 2017 Fall - Lecture -8
Detection methods for GMOs/LMOs by
Jasmin Sutkovic , 14th December 2017

2. Definition
GMO / LMO
Any living organism that possesses a novel combination of genetic material obtained through the use of modern biotechnology
– Living organism means biological entity capable of transferring or replicating genetic material including sterile organisms, viruses and viriods

3. Transgenic plants
Characterized by the insertion of a new gene or sets of genes into their genome
The new genes translate and new protein expressed
This gives the plant new characteristic

4. Transgenic plants - Examples
Bt cotton
Cotton plants resistant to lepidopteran insects
Round-up ready soybean
Soybean resistant to glyphosate
Golden rice
Rice grains with beta-carotene

5. Detection Methods
GM crops are indistinguishable from non- GM crops to the naked eye ==

6. Detection Methods
Basis of detection is to exploit the differences between the unmodified variety and the transgenic plant
Testing methods need to look
for the genes (DNA) introduced into the crop
for the proteins produced in the plant by the introduced gene
Workshop on Safety Assessment and Regulation of GM crops; 2-3 August 2006

7. Detection methods – purpose
Producers of LMO
To assure purity and segregation of products
To be able to trace genetic modification in breeding
Food & feed industry, seed companies
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e.g. if marketing permission withdrawn
Workshop on Safety Assessment and Regulation of GM crops; 2-3 August 2006

8. How transgenic plants are made?
Identification of useful gene
The cloning of the gene into a suitable plasmid vector,
Delivery of the vector into plant cell (insertion and integration) followed by expression and inheritance of the foreign DNA encoding a polypeptide.
A gene construct consists typically of three elements:
The promoter functions as an on/off switch for when and where the inserted/modified gene is active in the recipient plant
The transgene encodes a specifically selected trait
The terminator functions as a stop signal for transcribing the inserted/altered gene
marker genes for distinguishing GM from non-GM cells during crop development
Workshop on Safety Assessment and Regulation of GM crops; 2-3 August 2006

9. How transgenic plants are made?
Promoter
Gene
Terminator
transformation
Plant DNA before transformation
Plant DNA after transformation
Workshop on Safety Assessment and Regulation of GM crops; 2-3 August 2006

11. General Procedure
Detection: to determine whether a product is GM or not. For this purpose, a general screening method can be used. The result is a positive/negative statement.
Identification: to find out which GM crop or product are present and whether they are authorized or not in the country.
Quantification: If a crop or its product has been shown to contain GM varieties, then it become necessary to assess compliance with the threshold Regulation by the determination of the amount of each of the GM variety present.

12. To detect novel protein
Detection methods
GM Detection
DNA Based methods
To detect foreign DNA
Protein based methods
To detect novel protein
Qualitative and Quantitative

13. Detection methods – requirements
Target molecules
DNA or protein
Capture molecules
For DNA: primers and probes
For proteins: antibodies
Reference materials
Positive and negative controls
Calibrants for quantitation

14. Detection methods – requirements
Target molecules
Degraded or absent target molecules undetectable!
Effects of processing usually significant
Important to improve methods to recover target
Capture molecules
Impossible without description of target molecules
Need material for method development / validation

15. DNA based methods Highly sensitive - can detect trace amounts GM- DNA
Work with most product types - both processed and unprocessed products
Can test for multiple GM varieties simultaneously
Takes a number of days to perform - typically days
Requires highly skilled personnel and laboratory analysis
More expensive than Protein Based Methods

16. Protein based methods Relatively cheap to perform
Rapid turnaround - 5 to 20 mins for strips; 24 hours for ELISA
Strips do not require trained personnel
Limited to one or a small number of varieties per test
Not appropriate for processed products
Not appropriate for some GM varieties - in certain crops the GM protein is only produced in the leaves or stems and not in the actual grain. Protein tests on the grain are therefore not informative.
Not very sensitive (~1% of GM protein)

17. Protein based methods
ELISA
Lateral flow strip

18. Protein based methods Enzyme-Linked Immunosorbent Assay (ELISA)
Enzyme immunoassay utilizing an enzyme-labelled immunoreactant (antigen or antibody) and immunosorbent (antigene or antibody bound to solid support)
Enzyme catalyses colour reaction
– more intense colour, more proteins present
Quantification based on standard curve (reference)

19. Protein based methods Enzyme-Linked Immunosorbent Assay (ELISA) 1.2.
Prepare a surface to which a known quantity of antibody is bound. Apply the antigen-containing sample to the plate. Wash the plate, so that unbound antigen is removed.
Apply the enzyme-linked antibodies which are also specific to the antigen. Wash the plate, so that unbound enzyme-linked antibodies are removed.
Apply a chemical which is converted by the enzyme into a fluorescent signal.
View the result: if it fluoresces, then the sample contained antigen 3. 4. 5.

20. Protein based methods
Enzyme-Linked Immunosorbent Assay (ELISA)

21. Protein based methods Enzyme-Linked Immunosorbent Assay (ELISA)
Specific Antibodies
Positive control
Negative control
Buffer control
Each sample in duplicate wells

22. Protein based methods
Lateral flow stick

23. Protein based methods Lateral flow stick
Collect leaf and extract sample
Place the strip into the extraction tube
The sample will travel up the strip
Allow the strip to develop for 10 minutes
To retain the strip, cut off and
Discard bottom section of the strip

24. Protein based methods
Lateral flow stick

25. Protein based methods Lateral flow stick positive negative control
test
positive
negative

26. Protein based methods Company Test Kit Test Format
EnviroLogix Inc., USA
Cry 1Ab & Cry1Ac in Bt corn and cotton
ELISA
Cry2A, Cry1C, Cry1F, Cry3B and Cry9C
Cry9C
Quickstix (1kernel in in 5 min.)
Neogen Corporation, USA
CP4
Lateral flow assay
(Soybeans 0.1%;
corn 0.125%)
(corn 0.125%)
Strategic Diagnostics Inc., USA
RUR Soya Meal, Soya Grain, Bt 1 Maize, Bt 9 Maize

27. Protein based methods Advantages of ELISA Reasonably sensitive
Less susceptible to ‘false positives’
Low per sample cost
Handles large number of samples
Can be subjected to automation
Detection kits available commercially

28. Protein based methods Disadvantages of ELISA
High development costs for the assays
GM products might be produced only during certain developmental stages or in certain plant parts and such GMs are difficult to be detected by ELISA
In processed foods the proteins denatures easily, which makes it difficult to use ELISA for processed food fractions

29. DNA based methods
DNA based methods are based on detection of the specific genes, or DNA genetically engineered into the crop.
Commercial testing is conducted using PCR technology
The PCR technique is based on multiplying a specific target DNA allowing the million or billion fold amplification by two synthetic oligonucleotide primers.

30. DNA based methods PCR based detection
Detects a known DNA sequence by amplification
Target sequence is duplicated billions of times
Results obtained can be quantitative or qualitative
Enzyme :Taq polymerase
Equipment : Thermocycler/PCR machine

31. DNA based methods Principles of PCR Denaturation primer annealing
amplification
30 cycles

32. DNA based methods Sample homogenization DNA extraction
Check for availability of Plant DNA
Plant DNA present
Plant DNA absent
Screening PCR
GMO present
GMO absent
GMO identification
GMO detection

33. DNA based methods Screening Construct specific-PCR Event-specific PCR
Insert DNA
Plant DNA
Plant DNA
Screening
Construct specific-PCR
Event-specific PCR
GMO Detection
GMO identification

34. DNA based methods Construct specific PCR gene x M P 1 2 3 gene y 1 2 3
1 2 3
gene z
1 2 3
Event 1
Event 2
Event 3

35. DNA based methods Positive and negative controls
PCR Quality Assurance
Positive and negative controls
Extraction controls (Blanks)
Validated primers
All analysis performed at least in duplicate
Internal/ external validation trials

36. DNA based methods
Advantages of PCR
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

37. DNA based methods Not all tissues contain the same amount of DNA
Limitations of PCR
Not all tissues contain the same amount of DNA
Require relatively advanced lab. facilities and instrumentation and highly trained staff
Cost is fairly high
Time (usually >24 hrs for results)
Special precautions need to be taken to avoid the contamination

38. DNA based methods grains, seeds Crudely processed material – pure
Types of analysis
Event specific quantification
Quantification
Identification Detection
Increasing complexity
1 2 3
grains, seeds
Crudely processed material – pure
Crudely processed material – mixed
Processed products 4
Types of samples

39. DNA based methods M 1 2 3 4 5 Multiplex PCR-based detection methods
500bp 400bp 300bp
200bp
356bp fragment of EPS synthase gene
195bp fragment of 35S promoter
100bp
Multiplex PCR for detection of 35S promoter and EPS synthase gene sequence in transgenic soybean

40. DNA based methods Quantitative PCR Micro-array
End point analysis (Competitive PCR)
Real time analysis
Micro-array
Automated rapid screening
Based on nucleic acid hybridisation
Specific probes are attached to the chip
LMO chip – GeneScan Europe