Detection methods for GMOS/LMOs

Gurinder Jit RandhawaSenior Scientist

NRC on DNA Fingerprinting,NBPGR, New Delhi

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GM

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non-GM ?

GM Detection is required by• GM Developer

• To assure purity and segregation of different events

• To trace the genetic modifications• Food & feed industry, seed companies

• To assure purity and segregation of products• To assure compliance with legislation

• Competent (enforcement) authorities• Compliance with legislation• To solve the legal dispute if they arise

• Laboratories• To provide service to the society

DNA Based methods

To detect recombinant DNA

Protein based methods

To detect recombinant protein

Qualitative and Quantitative

GM Detection

Detection MethodsDetection Methods

1. ImmunologicalBt-Express : InstantaneousELISA : Three hoursWestern / blot analysis : Six hours

2. DNAPCR :Confirm gene identity/ Locus specificity

For GM-detection

Target molecules must be presentDNA (template) or protein

Capture molecules must be developedFor DNA: primers and probesFor proteins: antibodies

Reference materialsPositive and negative controlsCalibrants for quantitation

BtBt--ExpressExpress

Quicktest strip for Cry1Ab/Ac

control

test

positive negative

0% GM 1.0% GM0.1 % GM

Enzyme-Linked Immunosorbent Assay(ELISA)

• Specific Antibodies

• Positive control

• Negative control

• Buffer control

• Each sample in duplicate wells

Principle of ELISA

Different ELISA based Kits are available

Company Test Kit Test Format

Cry9C Quickstix (1kernel in 800 in 5 min.)

CP4 Lateral f;ow assay(Soybeans 0.1%; corn 0.125%)

Neogen Corporation, USA

Cry9C Lateral flow assay(corn 0.125%)

Cry 1Ab & Cry1Ac in Bt corn and cotton

ELISA

Cry2A, Cry1C, Cry1F, Cry3B and Cry9C in Starlink Corn

ELISA

EnviroLogix Inc., USA

Strategic Diagnostics Inc., USA

RUR Soya Meal, Soya Grain, Bt 1 Maize, Bt 9 Maize

ELISA

ELISA-based Diagnostic Kits Available for GM Testing

Diagnostic Kits Available for GM Testing Company Test Kit Test Format

Bt- Cry 1Ab, -Cry1Ac, -Cry1c, -Cry2A, -Cry3A, -Cry9C

DAS-ELISA

Cry 1Ab (Corn Mon809, 810, Bt 11, E-176)

ELISA (about 0.15% detection level)

Cry1F (Corn Herculex) ELISA

Biogenetic Services Inc., USA

Cry9C (Corn CBH 351, Starlink)

ELISA (about 0.04% detection level)

Agdia Inc., USA

Bt- Cry 1Ab, -Cry1Ac Immunostrips

CP4 (EPSPS Soybean RR)

ELISA (about 0.05% detection level)

Company Test Kit Test FormatCP4 (EPSPS)- soybean RR, Corn RR (NK 603), Cotton RR

QE, ISA

Cry1Ab (Corn Mon809, 810, Bt 11, E-176)

QE, ISA

Cry9C (Corn CBH 351, Starlink) QE, ISAT-25 (Corn PAT, BAR, Liberty Link)

QE, ISA

Cry3Bb (Corn Mon 863) QE, ISACry1F (Corn Herculex) QE, ISACry1Ac (Cotton Bollgard I) QE, ISACry2A (Cotton Bollgard II) QE, ISACry3A (Potato New leaf, New leaf+, New leaf y)

QE

Biogenetic Services Inc., USA

QE = Qualitative ELISA; ISA: Immunostrip Assay

Diagnostic Kits Available for GM Testing

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

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

Limitations of ELISA

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

PCR BASED DETECTION

PCR - principle

Denaturation

primer annealing

amplification

electrophoresis

508 bp

TnosInsect-resistance orherbicide-resistance CaMV 35S

Promoter Coding region of trait Terminator

Screening Assay

TerminatorPromoter Coding region of trait

TerminatorPromoter Coding region of trait

Event -specific Assay

TerminatorPromoter Coding region of trait

Transgene- specific Assay

PCR-based Transgene Detection

Positive and negative controls

Extraction controls (Blanks)

Validated primers

All analysis performed at least in duplicate

Participation in sponsored check sample programs/ collaborative studies

Internal/ external validation trials

PCR Quality Assurance

STANDARD REFERENCE(+ve Control)

NON GM FOOD(-ve Control)

GM FOOD MATERIAL

DNA Extraction

DNA Purity

PCR Amplification

DNA Electrophoresis

Interpretation & Documentation

GM ANALYSIS FLOW CHART

Gel Electrophoresis and Photography

DNA Extraction

Procedure for PCR Detection of LMOs

Sample preparation Crushing in Liq. N2 Centrifugation

Electrophoresis Assemblyz Agarose gel loading Gel on UV-transilluminator Polaroid Gel Photography

PCR Analysis

High species specificity

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

Advantages 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 ($75- $300)

Time (usually >24 hrs for results)

Special precautions need to be taken to avoid the contamination

Limitations of PCR

COST COMPARISION

PCR : Rs. 250 – RS. 500 /Test

Rapid Test : RS. 80 –Rs. 100 /Test

ELISA : RS. 20 - RS. 150 /Test

COMPANY KIT NAME PRIMARY MATRICES

Applied Biosystems TaqManR GMO Maize 355 Detection Kit

Processed foods and ingredients, including flour, meal, seeds, grain, plant tissues and oils

Applied Biosystems TaqManR GMO Soy 355 Detection Kit

Processed foods and ingredients, including flour, meal, seeds, grain, plant tissues and oils

BioteCon Diagnostics GmbH Bt-176 Maize corn

BioteCon Diagnostics GmbH Round-up Ready Soya soy bean products

EnviroLogix QualiPlate Kit for Roundup Ready Corn, Event 603 and Cotton

Roundup Ready corn, Roundup Ready cotton, Soybean and soy flour

EnviroLogix QuickStix Kit for Roundup Ready Bulk Grain

Roundup Ready corn, soybeans, canola

EnviroLogix QuickStix Kit for Roundup Ready Leaf & Seed

Roundup Ready corn. soybean, cotton, canola

Hong Kong DNA Chips Limited GMO Watcher 1.0 (DNA extraction & quantitative GMO detection kit for unprocessed food)

genetically modified ingredients, unprocessed foods

Hong Kong DNA Chips Limited GMO Watcher 2.0 (DNA extraction & quantitative GMO detection kit for unprocessed food)

genetically modified ingredients, unprocessed foods

Investigen, Inc. CommodityCheck PCR Based GMO Detection Kit

soy, corn, potato, canola in raw, processed, intermediate food products

PCR-based Diagnostic Kits Available for GM Testing

COMPANY KIT NAME PRIMARY MATRICES

Perkin Elmer HyPure seed

Promega Corporation seed corn, soybeans, cornmeal, corn starch, soy flour, cornflakes, soy milk

Qualicon Wizard Magnetic DNA Purification System for Food

food

Qualicon corn, soybeans, cornmeal, corn starch, soy flour, cornflakes, soy milk

soy products

Scil Diagnostics / GeneScan BAX for Screening Qualitative GMO plant tissues, seeds, meals, flours, grits, processed and unprocessed foods / feeds, food additives, and unrefined oils

Scil Diagnostics / GeneScan food plant tissues, seeds, meals, flours, grits, processed and unprocessed foods / feeds, food additives, and unrefined oils

Scil Diagnostics / GeneScan BAX for GMO Quantification plant tissues, seeds, meals, flours, grits, processed and unprocessed foods / feeds, food additives, and unrefined oils

Scil Diagnostics / GeneScan soy products plant tissues, seeds, meals, flours, grits, processed and unprocessed foods / feeds, food additives, and unrefined oils

Scil Diagnostics / GeneScan GMOIdent Kit LibertyLinkTM T25 Corn plant tissues, seeds, meals, flours, grits, processed and unprocessed foods / feeds, food additives, and unrefined oils

Scil Diagnostics / GeneScan plant tissues, seeds, meals, flours, grits, processed and unprocessed foods / feeds, food additives, and unrefined oils

plant tissues, seeds, meals, flours, grits, processed and unprocessed foods / feeds, food additives, and unrefined oils

PCR-based Diagnostic Kits Available for GM Testing

Capacity Building in GM detection at NBPGR

National Bureau of Plant Genetic Resources, New Delhi

Nodal agency for• issue of Import Permit• quarantine processing

• issue of Phytosanitary CertificateFor germplasm/research material/ transgenic

planting material

DBT- ICAR projectNational Containment/ Quarantine Facility for Transgenic Planting Material

• Quarantine processing of transgenic planting material

»The Containment (Level - 4) Facility

• Development of probes/primers for testing Transgenes Check for the absence of terminator gene

• Human Resource Development

Transgenic material imported along with transgenes

Crop Transgenes Traits

B. juncea

B. napus

B. oleracea

barnase and barstar

osmad 1

cry9C

Male sterility and fertility restorationRelated to photoperiod insensitivityInsects resistance

Soybean cp4epsps Resistance to glyphosate herbicide

Maize Insects resistance Resistance to glyphosate herbicide

Cotton cp4epsps

CryX, vip-3, GFM Cry 1A, Cry 1F and Cry 1AC

Resistance to glyphosate herbicide

Insects resistance

Cry 1 Ab,cp4epsps

Ama1,cry 1Ac, cry 1ab, cry 19C, Xa 21, Cry 2a

Phytoenesynthase,Phytoene desaturase andlycopene cyclase

AFP-AG, AFP-RS, AFP-lactoferricin, AFP-chitinase

For improved nutritionInsects resistance

Synthesis of β-carotene

Anti-fungal proteins

Rice

Insertional mutagenesis ricelines for molecular characterization

Selection markers and reporter genes

Maize transposable element activator (AC)

Maize transposable DS element

Bar, hpt IGreen Fluorescent Protein (GFP

Insertional mutagenesis ricelines for molecular characterization

Chickpea Bean alpha A1 Against storage grain pests

Wheat human serum albumin

AFP-AG, AFP-RS, AFP-Lactoferricin, AFP- chitinase

Bar

Pharmaceutical product

Anti-fungal proteins

Selection marker for herbicide resistance

Tobacco For alternate oxidaseAox 9

GENE BPROMOTER B

Cre F Cre R

RECOMBINASE GENE

M 1 2 3 4 5 6 7

1031 bp

1500 bp1200 bp1000 bp900 bp

Agarose gel electrophoresis of PCR performed to detect terminator gene technology cassette in imported transgenic cotton samples (Lanes, 4-7). Lane 3, positive control

giving an amplicon of 1031 bp. Lane 1 and 2, negative control.

Detection of terminator technology in imported transgenic planting material

Terminator Cre-recombinaseCRE-F 5’ -CTAATCGCCATCTTCCAGCA -3’CRE-R 5’ –ATGTCCAATTTACTGA ……….

Interlinking Project

Department of Biotechnology (DBT)

Started in April 2002

Development of Analytical methods for rapid detection of Genetically Modified Foods and products derived

therefrom

Development of Analytical methods for rapid detection of Genetically Modified Foods and products derived there from

CDFD, Hyderabad

NIN, Hyderabad

ITRC, Lucknow

NBPGR, New Delhi

CFTRI, Mysore

PCR-based detection

NBPGR

CFTRI CDFDCross validation of detection protocols

DBT

ELISA-based detection

National Bureau of Plant Genetic Resources (NBPGR), New Delhi

Cotton,SoybeanMustard

Cry1AcESP synthase Barnase Barstar

Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad

Bt cottonBt Rice

Cry1A(b), Cry1A(c) Cry2A(b)

Central Food Technological Research Institute (CFTRI), Mysore

Processed soybean Maize

Cry1A(c) ESP-synthase

National Institute of Nutrition (NIN, Hyderabad)

GM products Cry9C, Cry1A(b)Cry1A(c)

Industrial Toxicology Research Centre, India (ITRC), Lucknow

GM products Cry1A(c) Cry1C

Interlinking laboratories were assigned to undertake study on

Detection of Cry1A(c) gene in Bt cotton using PCR

M, molecular weight marker (100 bp ladder); Lane 2-21, samples of transgenic Cotton with Cry1A(c) gene; Lane 22, Negative control.

M 2 3 4

195bp

Detection of 35S promoter gene in transgenic soybean

M, Molecular weight marker (50 bp ladder); Lane2-4, sample of transgenic soybean.

320bp

M 2 3 4 5 6

Detection of EPS synthase gene in transgenic soybeanusing PCR

M, Molecular weight marker (50 bp ladder); Lane 2-6, sample of transgenic soybean.

PCR assays standardized for detection of GM planting material

Multiplex PCR for detection of 35S promoter and EPS synthase gene sequence in transgenic soybean

500bp400bp300bp

200bp

100bp

356bp fragment of EPS synthase gene

195bp fragment of 35S promoter

M 1 2 3 4 5

Multiplex-PCR detection assay

Cross validation of PCR assays

NBPGR provided NBPGR received

CDFD,

Hyderabad

1. Bt cotton DNA (C1, C2, C3), primers for Cry1Ac gene and PCR protocol

2. Transgenic soybean DNA (A1, A2, A3), primers for 35Spromoter and EPS synthase gene, and PCR protocols .

Bt cotton DNA (A, B, C), primers for Cry1Ac, Cry2Ab and PCR protocols.

CFTRI,

Mysore

1. Bt cotton DNA (C1, C2, C3), primers for Cry1Ac gene and PCR protocol

2. Transgenic soybean DNA (A1, A2, A3), primers for 35Spromoter and EPS synthase gene, and PCR protocols .

1. GM soybean DNA (A1, A2, A3), primers for 35S promoter, TNOS terminator and EPSsynthase gene

2. GM maize DNA (B1, B2, B3), primers for Cry1Ab and PCR protocols.

Inter validation of PCR assays developed at CFTRI, Mysore

Detection of EPSPS gene cross border sequence in GM-soybean by PCR.

M-100bp ladder, Lane-1, Negative control without DNA, Lane-2, Non-GM Soya DNA, Lane-3, Positive control (GM Soya); Lane 4-6, GM soybean test samples

M -ve

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Sam

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A1

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A3

1 2 3 4 5 6

447 bp

Inter validation of PCR assays developedat CFTRI, Mysore

M -ve

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1 2 3 4 5 6 Detection of CaMV 35S promoter in GM-soybean by PCR.

M-100bp ladder, Lane-1, Negative control without DNA, Lane-2, Non-GM Soya DNA, Lane-3, Positive control (GM Soya); Lane 4-6, GM soybean test samples

123 bp

Inter validation of PCR assays developed at CFTRI, Mysore

Detection of TNOS in GM-soybean by PCR.

M-100bp ladder, Lane-1, Negative control without DNA, Lane-2, Non-GM Soya DNA, Lane-3, Positive control (GM Soya); Lane 4-6, GM soybean test samples

M -ve

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Sam

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A1

Sam

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A2

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A3

1 2 3 4 5 6

192 bp

Inter validation of PCR assays developed at CFTRI, Mysore

Detection of Cry1Ab gene in GM-maize by PCR.

M-100bp ladder, Lane-1, Negative control without DNA, Lane-2, Non-GM Maize DNA, Lane-3, Positive control (GM Maize); Lane 4-6, GM Maize test samples

M -ve

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Sam

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B1

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B2

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B3

1 2 3 4 5 6

204 bp

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M

Detection of Cry 1Ac (373bp) gene in Bt CottonLane-1,3,5,7,9,11,13,15 - GM-cotton DNA; Lane-2,4,6,8,10,12,14-Non-GM-cotton DNAM-100bp ladder

373 bp

Detection of Cry 1Ac (373bp) gene in Bt Cottonsamples to be sent to CFTRI and CDFD

Inter validation

Detection of Lectin (413bp) gene and CaMV 35S promoter (123bp) in GM-soya sent by CFTRI

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 M

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Detection of Lectin gene and CaMV 35S promoter in GM-soyaM-100bp ladder, Lane-1, Non-GM-soya DNA; Lane 2-16, GM-soya test samples

413 bp

CaMv 35S promoter (123bp) could not be detected

Inter validation

Detection of CP4EPSPS (447bp) & Lectin (118bp) gene in GM-maize sent by CFTRI

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Mar

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Detection of CP4EPSPS & Lectin genes in GM-maizeM-100bp ladder, Lane-1, Non-GM-soya DNA; Lane 2-16, GM-soya test samples

447 bp

118 bp

Inter validation

Detection of NOS (192) & Lectin (413) gene in GM-soya sent by CFTRI

Inter validation

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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Detection of NOS & Lectin gene in GM-soya M-100bp ladder, Lane-1, Non-GM-soya DNA;

Lane 2-16, GM-soya test samples

413 bp

192 bp

Second and third rounds of Inter validations

of PCR assays developed by these labs. were completed successfully

Implementation of Cartagena Protocol on Biosafety in India

GEF-World Bank Capacity Building Project

GEF-World Bank Capacity Building Project

Central Food Technology Research Institute, Mysore

Ministry of Environment and Forests,

Govt. of India GB Pant Agricultural University, Pant Nagar, Uttaranchal

National Bureau on Plant Genetic Resources, New Delhi

NRC on Plant Biotechnology, IARI, New Delhi

Biosafety Clearing House

Two hands-on training programmes onBiosafety Concerns of Transgenics and Detection of LMOs

Sponsored by Ministry of Environment and ForestsGovt. of India and GEF World Bank Project

Organised at NBPGR from 14th to 21st March, 200516th to 20th January, 2006