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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
Enzyme-Linked Immunosorbent Assay(ELISA)
• Specific Antibodies
• Positive control
• Negative control
• Buffer control
• Each sample in duplicate wells
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
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
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
with
out D
NA
Neg
ativ
e (w
ith
DN
A)
Pos
itive
con
trol
Sam
ple
A1
Sam
ple
A2
Sam
ple
A3
1 2 3 4 5 6
447 bp
Inter validation of PCR assays developedat CFTRI, Mysore
M -ve
with
out D
NA
Neg
ativ
e (w
ith
DN
A)
Pos
itive
con
trol
Sam
ple
A1
Sam
ple
A2
Sam
ple
A3
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
with
out D
NA
Neg
ativ
e (w
ith
DN
A)
Pos
itive
con
trol
Sam
ple
A1
Sam
ple
A2
Sam
ple
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
with
out D
NA
Neg
ativ
e (w
ith
DN
A)
Pos
itive
con
trol
Sam
ple
B1
Sam
ple
B2
Sam
ple
B3
1 2 3 4 5 6
204 bp
Sam
ple
S1
Sam
ple
S2Sa
mpl
e S3
Sam
ple
S4Sa
mpl
e S5
Sam
ple
S6
Sam
ple
S7Sa
mpl
e S8
Sam
ple
S9Sa
mpl
e S1
0Sa
mpl
e S1
1
Sam
ple
S12
Sam
ple
S13
Sam
ple
S14
Sam
ple
S15
Mar
ker
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
Mar
ker
Neg
ativ
e D
NA
Sam
ple
S1Sa
mpl
e S2
Sam
ple
S3
Sam
ple
S4
Sam
ple
S5Sa
mpl
e S6
Sam
ple
S7
Sam
ple
S8
Sam
ple
S9
Sam
ple
S10
Sam
ple
S11
Sam
ple
S12
Sam
ple
S13
Sam
ple
S14
Sam
ple
S15
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
ker
Neg
ativ
e D
NA
Sam
ple
S1Sa
mpl
e S2
Sam
ple
S3
Sam
ple
S4
Sam
ple
S5Sa
mpl
e S6
Sam
ple
S7
Sam
ple
S8
Sam
ple
S9
Sam
ple
S10
Sam
ple
S11
Sam
ple
S12
Sam
ple
S13
Sam
ple
S14
Sam
ple
S15
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
Mar
ker
Neg
ativ
e D
NA
Sam
ple
S1
Sam
ple
S2
Sam
ple
S3Sa
mpl
e S4
Sam
ple
S5Sa
mpl
e S6
Sam
ple
S7Sa
mpl
e S8
Sam
ple
S9Sa
mpl
e S1
0Sa
mpl
e S1
1Sa
mpl
e S1
2Sa
mpl
e S1
3Sa
mpl
e S1
4 Sa
mpl
e S1
5
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
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