GMO TESTING: Current trends - Ministry of Environment ...envfor.nic.in/divisions/csurv/biosafety/Gef2/T5/15 Dr.Jayant_GMO... · GMO TESTING: Current trends Amar Immunodiagnostics

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

Thank you

Email :[email protected]

[email protected]

Phone : 098490 16777