gmo testing: current trends - ministry of environment...
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GMO TESTING: Current trends
Amar Immunodiagnostics Pvt Ltd, Hyderabad
by
Dr. Jayant K. Bhanushali
What is GMO Testing !
Testing of (Seed, Leaf, Plant tissue,
or processed food) to determine
if it has been genetically modified
by recombinant DNA technology
to produce a desired trait.
What is GMO Food !
Food from crops that have been modified to produce a desired trait through genetic
engineering
Agricultural Biotechnology
• New Crops– New Traits– New DNA– New Protein
• Why Test?– Consumer Choice– Food Labeling Laws
• Finished foods• Thresholds based on %GMO
Label1
GMO Testing Different approaches
ELISA
ImmunoStrips
PCR
Immunoblot
Bioassay
Current Test Methods
• DNA - Polymerase Chain Reaction (PCR) – Qualitative Screen ( Event specific)– Quantitative – Promoter gene ( 35 S)– Marker gene ( Antibiotic marker)
• Protein - Immunoassay– Lab-Based ELISA for specific protein ( Cry1Ac,
Cry2A, Cry1Ab, Cry1F etc)– Field-Based Rapid Strip test for seed, leaf and grain
– BIOASSAY
GMO TESTINGAdvantages of Nucleic acid based tests (PCR)
• Confirmed Proof of transformation• Presence in all plant tissues• Very Sensitive• Small Sample Size• Can be tested at any time after
transformation
GMO TESTINGDisadvantages of Nucleic acid based tests (PCR)
• Expensive instruments and extensive laboratory setup
• Highly Skilled Manpower
• Expensive
• Farm testing not possible
• Processed food testing difficult
• Primarily a qualitative test
• Quality of sample is very important
• Error prone
Protein
Au
Au
Vial
Absorbent Pad
Test Line
Sample
Filter Pad
Gold Pad
Membrane
Control Line
Strip TestStrip Test
So many Traits, ... so little timeWhen does Multi-Trait testing
make sense ?• Seed Testing
– When traits are often stacked together– When detection criteria are similar– When common extraction can be used
• Grain Testing– When a common sample preparation is possible– When detection limits for each trait fit within
guidelines
Lateral Flow Strip Sensitivity
Event ProteinLateral Flow
Strip DetectionLimit
BT11 Cry1Ab 1 in 600MON810 Cry1Ab 1 in 125MON863 Cry3Bb 1 in 800TC1507 Cry1F 1 in 600CBH351 Cry9c 1 in 800NK603 CP4 EPSPS 1 in 800
T25 PAT(pat) 1 in 70
Note T25 and TC1507 require 10 minutesTC1507 requires extraction in buffer
0 5 10 15 20
Concentration (ng/ml)
0
0.5
1
1.5
2
2.5
OD
at 6
30 n
m
C838
Bt-Cry3A Response Curve
OD Conc.ng/ml
2.936 321.979 161.107 80.570 40.274 20.139 10.000 0
Bt-Cry3A
GMO TESTINGAdvantages of Immunoassay based testing
(Elisa/Strips/Immunoblots)
• Confirmed proof of presence of a gene product (protein)
• Farm testing possible• Processed food testing easy• Quick and easy• Inexpensive• Quantitative testing possible• Quality of sample is less critical
compared to PCR
GMO TESTINGDisadvantages of Immunoassay based testing
(Elisa/Strips/Immunoblots)
• Gene product (Protein) may be organ specific
• Quantity may vary depending upon environmental conditions
• Plants may need to grow for few days before testing
COST COMPARISION
PCR : Rs. 250 – RS. 500 /Test
Rapid Test : RS. 80 –Rs. 100 /Test
ELISA : RS. 20 - RS. 150 /Test
Factors Affecting Quantification of Proteins Using Immunoassays• Variability of protein expression levels
– Within an event (crop variety)– Between events expressing the same
protein (e.g. Cry1Ab)• Sampling variation• Sample processing effects on protein
conformation and antibody binding• Extraction efficiency• Calibration
Expression of Bt Cry1Ab in Corn
Event Form Seed (ug/g fw)
Leaf (ug/g fw)
MON810 Full length 0.31 9.35
BT11 Truncated 4.76 20.00
Event 176 Full length and truncated
<0.005 1.00
Source – USDA Petitions
Differences in Expression Levels Can Lead to Differences in %GMO
detection LimitsEvent Protein Average
Expression LevelBT11 Cry1Ab 1.4 ug/g
MON810 Cry1Ab 0.3 ug/gMON863 Cry3Bb 70 ug/gDBT418 Cry1Ac 1.6 ug/gTC1507 Cry1F 12 ug/gCBH351 Cry9c 19 ug/gNK603 CP4 EPSPS 11 ug/g
T25 PAT(pat) 0.12 ug/g
Appropriate Sensitivity
• Appropriate Sensitivity for– Breeding– Seed Quality– Grain Handling
• Avoid Contamination– Processing Equipment (samplers, grinders,
fingers )– Trucks, Harvesters, etc.– Other crops
Summary• Improper sampling leads to false, imprecise and
variable results• Sampling bulk loads
– Take a representative sample– Sample size is a balance between
• Sensitivity• Cost• Probability of false negative results• Buyer’s and Seller’s risks
• Subsampling– Qualitative and quantitative sampling plans are NOT
the same– Number of particles effects accuracy and precision– Lab samples of two different particle sizes are not
comparable
Laboratory Testing for GMO
• Sampling the Grain• Obtain a representative sample• Process further to allow sample size reduction
• Sampling the Meal• Obtain a representative sample• Prepare it for the quantitative test
• Sampling the DNA/protein solution• Obtain a representative sample
GMO Tests and Matrices
Corn Pesticide Bt-resistance to ECB
Cry9C protein – Starlink®
Cry1Ab protein – Bt11, MON810, Bt176
Herbicide tolerance: BAR / PAT protein - Liberty LinkCP4 EPSPS protein – NK 603
RoundUp Ready®
Soy Herbicide (Glyphosate) Resistance CP4 EPSPS - RoundUp Ready®
GMO Tests and Matrices
Cotton Pesticide Bt-resistance to ECB
Cry1Ac protein – Bollgard®
Cry2A protein – Bollgard II®
Cry1Ac/Cry1b- Chinese Bt
Potatoes Pesticide Bt-resistance to ECB
GM CROPS
Non FoodCotton - Bt (Bollgard)
FoodMustard - Barnase-BarstarSoyabean - Herbicide Glyphosate
(Roundup Ready)Corn - Bt (Starlink)Tomato - Bt, Flavar savarPotato - BtVegetables - BtPigeon Pea - BtRice - Vitamin A, Bt
• Seed companies
• Seed dealers
• Seed Growers - Producers
• Crop consultants
• Seed companies
• Seed dealers
• Seed Growers - Producers
• Crop consultants
SEED CUSTOMERS
• Growers
• Agricultural Extension Services
• Researchers / Breeders
• Government Agencies
• Grain elevators / processors
• Importers / Exporters
• Growers
• Agricultural Extension Services
• Researchers / Breeders
• Government Agencies
• Grain elevators / processors
• Importers / Exporters
Crop Testing Customers
Routine AnalysisAudit
Seed Company
PCRELISA
Lateral Flow
FarmerLateral Flow
ElevatorLateral Flow
PCR, ELISA, Lateral Flow
Ingredient Supplier
Retail
Food Processor PCR
Lateral Flow, ELISA
Basic Analytical Chain
Export Analytical Chain
Farmer
Routine AnalysisAudit
Seed Company
PCRELISA
Lateral Flow
Lateral FlowExport Elevator
Lateral Flow PCR, ELISA,
Lateral Flow
Border Control Customs
Retail
Ingredient SupplierPCR
ELISALateral Flow
Food ProcessorPCR
ELISALateral Flow, ELISA
GMO FOOD / NON GMO FOOD LABELLING
• Edible Oils (Soya, Mustard, Sunflower, Palm)
• Processed feed for Animals (Cattle, Poultry)
• Rice, Wheat, Pulses & Other food grains
• Processed Food & Snacks
• Food Processing Plants
• Milk and dairy Products
• Marine Food Supplies
• Merchant Traders…..
GMO TESTINGA. Trait Specific GMO detection : Starlink corn
Liberty Link SoyaB. Single - Species GMO detection : Total GMO in a
single species (Corn)
C. Cross-Species GMO detection : Multiple Ingredient product where source of GMO is not known. (Detection of CAMV 35s, Ubiquitinepromoter, Antibiotic Kanamycin / Hygromycin marker or Bar gene)
GMO Testing Labs
In order to comply with the
requirement of GMO / Non GMO
food labeling, licensing of GMO
testing labs will be necessary
in near future
What lies ahead inGM-Food?
“Transgenic Crops or Genetically Modified”(GM Corn = 1 gene in 60,000)
Generation I Technology: INPUT TRAITS: improve production yield and costs with benefits to Seed Company / FarmerGeneration II Technology: OUTPUT TRAITS: improve processing energy, storage or transportation costs with benefits to Food Processors / ConsumerGeneration III Technology: OUTPUT TRAITS: enhance nutritional value (Vitamin A) with benefits to Consumer
