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Qualitative and Quantitative Detection
Methods for GMO/LMO in Korea
Seong-Hun Lee, Ph.D.
National Agricultural Products Quality Management Service
2015 . 10. 15 .
Contents
Ⅰ
Ⅱ
Ⅲ
Ⅳ
Detection Methods
Reference Materials and Quantitative Analysis
Introduction
Ⅴ
GMO Testing
Recent Study
Ⅰ. Introduction
Authorization of GMO/LMO
use for Seed use for Food, Feed and Processing
GMO/LMO Labeling System for consumer’s right-to-know
GMO/LMO Management System by Government
Identification of GMO/LMO Events
Quantification of GMO/LMO Events
GMO Labeling System (for food)
Government Organs Targets Labeling Items Adventitious
Presence
Ministry of Food and Drug
Safety
(MFDS)
-Raw Materials
-General Processed
Foods
-Authorized GM crops events
-Processed foods made from
the authorized GM crops(the
top 5 ingredients used in terms
of blending ratio) events
3 %
National Agricultural Products
Quality Management Service
(NAQS)
Organic Processed
Foods - 0 %
LMO Management System (for feed)
MAFRA
Import Approval Border Inspection Management
For Feed & Processing
For Seed
N A Q S Q I A
For Unauthorized LMOs
Risk Assessment
Authorization for Seed, Feed & Processing
R D A
For Labeling and monitoring authorized LMOs
Notify
Notify Transfer
K S V S N A Q S K S V S
For Feed & Processing
For Seed
▣ MAFRA : Ministry of Agriculture, Food and Rural Affairs ▣ NAQS : National Agricultural Products Quality Management Service ▣ KSVS : Korea Seed and Variety Service ▣ QIA : Animal and Plant Quarantine Agency ▣ RDA : Rural Development Administration
Detection Method Development &
Verification
Protein based Method
- use on site (Lateral Flow Strip)
- rapid, convenient, low cost
- not event specific
- not effective for processed foods
Ⅱ. Detection Methods
DNA based Method
- use in laboratory (Qualitative PCR, Quantitative PCR)
- relatively slow, high cost
- event specific
- applicable for processed foods
Qualitative PCR
The response is either the presence or absence
of the target sequences in a sample
Design of Primers/Probes
Terminator Enhancer Hos
t
Gene Promoter Hos
t
Screening
Gene Specific
Construct
Specific
Event Specific
T E H G P H
T E H G P H
T E H G P H
P35S, tNOS
15/63 events
48/63 evnets
ami797E t35S ZmUbilnt pmi tNOS
E350-R E350-F
E101-P
LB
E101-F E112-R
GZein RB
(Ⅰ) Event3272
cordapA rAct1 int
L168-R L168-F
LB
L96-F L196-R
Glb1 pro RB mDHDPS CTP Glb1 UTR
L129-P
(Ⅱ) LY038
Specificity test
M NTC NG MON810 Bt11 E176 GA21 T25 NK603 TC1507 MON863 MIR604 MON88017 E3272 LY038 DAS-59122
(Event 3272)
(LY 038)
(SSⅡb) (bp)
◀ 114
◀ 350
◀ 168
The property of a method to respond exclusively to the
characteristic or analyte of interest
LOD(Limit of Detection) test
M 0 0.01 0.05 0.1 0.5 1 3 5 10 (%)
(Event 3272)
(LY 038)
(bp)
◀ 350
◀ 168
The lowest concentration or content of the analyte in a sample that can
be reliably detected
Qualitative PCR methods by NAQS
Crops Events Qualitative PCR product size(bp) Specificity
Soybean (2) GTS 40-3-2 121 Construct-
MON89788 245 Event-
Maize (14)
GA21 133 Construct-
Event 176 100 Construct-
Bt11 127 Construct-
T25 149 Construct-
Mon810 113 Construct-
NK603 231 Construct-
TC1507 251 Construct-
MON863 271 Construct-
MIR604 350 Event-
MON88017 151 Event-
Event3272 350 Event-
LY038 168 Event-
DAS-59122-7 185 Event-
MIR162 200 Event-
Cotton (5)
MON531 201 Construct-
MON1445 171 Event-
MON15985 116 Construct-
MON88913 120 Event-
LLcotton25 90 Event-
Canola (2)
GT73 115 Construct-
MS8/RF3 118 (MS8 marker) Construct-
113 (RF3 marker) Construct-
Ⅲ. Reference Materials & Quantitative Analysis
Materials Levels Strong & Weak Points Type
Certified
Reference
Material
IRMM
(Institute for
Reference
Materials and
Measurements)
0.1, 0.5, 1,
2, 5, 10 %
- Do not need to make standard levels
- One CRM for one GMO event
Most
Flour
AOCS
(American Oil
Chemists Society)
100 % - Need to make standard level
- One CRM for one GMO event
Reference
Material
GMO Company
RM 100 %
-GMO company submitted RM with detection method
- For all authorized GMO events in Korea
- One RM for one GMO event
- Need to make standard levels
Standard Plasmid
DNA
10, 20, 125,
1.5k, 20k,
250k copies
- Do not need to make standard levels
- Unlimited supply and consistency of quality
- One RM for several GMO events
- 9 plasmids for 4 crops 23 events
- Complicated construction and need conversion factor
Plasmid
DNA
Method using Flour type (C)RM
The response is the quantity of the target DNA sequence in
a sample using reference materials.
Level S1 S2 S3 S4 S5
Plant genome
arbitrary copies
20000
(100)
2000
(10)
400
(2)
100
(0.5)
10
(0.05)
GMO DNA
arbitrary copies
20000
(100)
2000
(10)
400
(2)
100
(0.5)
10
(0.05)
Standard levels from GMO Company RM (100%)
GMO standard levels from Flour type RM
cf. Standard levels from JRC TC1507 RM (10%) in EURL validation report
Level S1 S2 S3 S4
Maize genome
absolute copies
73,394
(100)
14,679
(20)
2,936
(4)
587
(0.8)
TC1507 GM DNA
absolute copies
7,339
(10)
1,468
(2)
294
(0.4)
59
(0.08)
20000
2000
400
100
10
20000
2000
400
100
10
= Copy numbers of Target-Specific DNA sequence
Copy numbers of Taxon-Specific DNA sequence
GMO content(%) by GMO Company RM
X 100
Th
Method using Plasmid type RM
Synthesis of Standard Plasmid
ⅵ)
ligation
ⅰ)
ⅱ)
A B
B′ A′
2nd PCR
1st PCR
mix
A5′
B3′
cA3′+B5′
A3′+cB5′
A5′
B3′
A B Ⅳ)
ⅲ) A′ B′
Using the method by Kuribara et al. (J. AOAC Int.,
85, 1077-1089, 2002)
pTOP TA V2
Ampicillin
F1 ori
Kanamycin
pUC ori
P lac lacZ
p3238
zSSⅡb Event3272 LY038
zSSⅡb Event3272 LY038
Standard Plasmid RM used in NAQS
Hmg GT73 RF3
p35S zSSⅡb NK603 TC1507 MON863
zSSⅡb MIR604 MON88017 DAS-59122-7 Adh1
zSSⅡb Event3272 LY038 MIR162 Adh1
fsACP MON531 MON1445 MON15985 MON88913 LLcotton25
Hmg GT73 MS8
Maize (3)
Cotton (1)
Canola (2)
Plasmids developed by Japan
Soybean (1)
Maize (1)
Plasmids developed by NAQS
Le1 MON 89788 Soybean (1)
GMO standard levels from Plasmid type RM
Level S1 S2 S3 S4 S5
Plant endogenous
DNA plasmid
absolute copies
250K 20K 1.5K 125 20
GMO DNA plasmid
absolute copies 250K 20K 1.5K 125 20
Standard levels of Plasmid DNA RM
plasmid
Endogenous DNA GMO DNA
250K
20K
1.5K
125
20 Th
250K
20K
1.5K
125
20
X 100 Cf ÷ = Copy numbers of Target-Specific DNA sequence
Copy numbers of Taxon-Specific DNA sequence
GMO content(%) by Standard Plasmid DNA
Conversion Factor(Cf)
Conversion Factor(Cf)
Copy Numbers of GMO Target-Specific DNA sequence
Copy Numbers of Plant Taxon-Specific DNA sequence =
Homozygous GM Soybean F1 Seed Heterozygous GM Maize F1 Seed
GM Maize Mean of Cf SD RSD
Event 3272 0.63 0.05 7.94
LY 038 0.44 0.02 4.55
Cf = 1 / 2 = 0.5
GMO 100% by mass /mass
GMO 50% by DNA copy/copy
50% by copy ÷ Cf = 100% by mass
Cf = 2 / 2 = 1.0
GMO 100% by mass /mass
GMO 100% by DNA copy/copy
100% by copy ÷ Cf = 100% by mass
AB7900
Conversion Factor(Cf) of AB7900 by NAQS
Crops Events Conversion Factors
Soybean (2) GTS 40-3-2 1.04
MON89788 1.32
Maize (14)
GA21 1.99
Event 176 2.02
Bt11 0.40
T25 0.34
Mon810 0.36
NK603 0.54
TC1507 0.30
MON863 0.46
MIR604 0.46
MON88017 0.30
Event3272 0.64
LY038 0.47
DAS-59122-7 0.41
MIR162 0.53
Cotton (5)
MON531 0.40
MON1445 0.98
MON15985 1.10
MON88913 1.00
LLcotton25 1.06
Canola (2)
GT73 0.85
MS8/RF3 0.18 (MS8 marker)
0.30 (RF3 marker)
In-house / Inter-laboratory Validation
In-house validation
- for validation of NAQS developed methods & verification of GMO
company submitted methods
- with three times repeat by one operator in one laboratory
- on five test samples (0.1, 0.5, 1, 3, 10%) using Standard plasmid RM or
GMO company RM with five standard levels
Inter-laboratory validation
- for validation of NAQS developed methods
- with one time by five operators in five laboratories ( 1 central ERI + 4
branch GMO laboratories in NAQS)
Accuracy: Closeness of agreement between a test result and the accepted reference
value. → differences ≤ 30%
Precision: The RSD(relative standard deviation) of test results obtained under
repeatability conditions. Repeatability conditions are conditions where test results
are obtained with the same method, on identical test items, in the same laboratory,
by the same operator, using the same equipment (AB7900)
→ Repeatability Standard Deviation (RSDr) ≤ 30%
LOQ(limit of quantification): The lowest amount or concentration of analyte in a
sample which can be reliably quantified with acceptable level of accuracy and
precision → 0.1 or 0.5%
Accuracy Precision
Below
20 copies
Mean Bias
GMO events True value
(%)
Experimental
Value (%)
True value
(%) SD RSD
Event 3272
0.1
0.5
1.0
3.0
5.0
10.0
0.11
0.58
1.16
3.12
5.07
9.23
10.0
16.0
16.0
4.0
1.4
-7.7
0.03
0.05
0.14
0.30
0.30
0.48
27.3
8.6
12.1
9.6
5.9
5.2
0/3
0/3
0/3
0/3
0/3
0/3
LY 038
0.1
0.5
1.0
3.0
5.0
10.0
0.09
0.44
0.99
2.85
4.24
8.98
-10.0
-12.0
-1.0
-5.0
-15.2
-10.2
0.02
0.06
0.21
0.23
0.44
0.52
22.2
13.6
21.2
8.1
10.4
5.8
0/3
0/3
0/3
0/3
0/3
0/3
Ⅳ. GMO Testing
Qualitative PCR
No Detection
Detection
Taxon-Specific
Screening (P35S, tNOS)
Construct -/ Event- Specific
Decision of Result
-/- Nullity
+/+ -/- -/- No Detection with event name
+/+ +/+, +/- -/- No Detection with event name
+/+ -/- +/+ or +/- Detection with event name
+/+ +/+ or +/- +/+ or +/- Detection with event name
Quantitative PCR
Non-GMO IP for Non-GMO
Quantitative PCR
Exemption IP for Non-GMO
3% < GMO Label
0 < ≤ 3%
Target Adventitious Presence
Result
General Food/Feed Authorized G(L)MOs 3% with event name(%)
Organic Processed Food/Feed All G(L)MOs 0%
General Food Unauthorized G(L)MOs 0%
General Feed Unauthorized G(L)MOs (pending assessment)
0.5% with event name(%)
X GMO Label
Detectable GMO events(5/63) by NAQS
Crops Events
Soybean
(16)
GTS40-3-2, MON89788, A2704-12, DP-356043-5, DP-305423-1, A5547-127, CV127, MON87701,
FG72, MON87769, MON87705, DAS-68416-4, MON87708, DAS-44406-6, SYHTOH2, DAS-
81419-2
Maize
(24)
Bt11, MON810, Bt176, T25, GA21, NK603, TC1507, MON863, MIR604, MON88017,
Event3272, LY038, DAS-59122-7, MIR162, MON89034, Bt10, DP-098140-6, MON87460, DAS-
40278-9, 5307, MON87427, DP-004114-3, MON87411, VCO-01981-5
Cotton
(13)
MON531, MON1445, MON15985, MON88913, LLcotton25, 757, 281/3006, GHB614, COT67B,
T304-40xGHB119, GHB119, COT102, MON88701
Canola
(8) GT73, MS8xRF3, T45, MS1xRF1, MS1xRF2 , TOPAS19/2, MON88302, DP-073496-4
Alfalfa
(2) J101/J163, KK179
Red letter: events analyzed by standard plasmid RM method. 4 GM crops 23 events
Blue letter: events analyzed by GMO company RM method. 5 GM crops 40 events
Ⅴ. Recent Study-Multiplex Qualitative PCR
Target Name Specificity Length
(bp)
Multiplex Event-Specific
MIR 604 M 500-F
M 500-R
tNOS
maize genome 500
Event 3272 M 500-F
E 135-R
tNOS
maize genome 341
LY 038 L 248-F
L248-R
GLb1
maize genome 248
MON 88017 M8-151-F
M8-151-R
maize genome
T-DNA 151
DAS 59122-7 D 81-F
D 81-R
T-DNA
maize genome 81
M MIR604 E3272 LY038 M88017 59122 M-Plex
M 0 0.1 0.5 1.0 5.0 (%)
(bp)
◀ 500
◀ 341
◀ 248
◀ 151
◀ 81
Duplex Quantitative PCR
Adh1-VIC
LY038-FAM
Adh1-VIC / LY038-FAM
GMO chip
Soybean Soybean negative Maize Maize
EPSPS1 EPSPS1 Position EPSPS2 EPSPS2
Cry1Ac Cry1Ac Pmi Cry3B Cry3B
Pat Pat Pmi Bar Bar
P35S P35S Position tNOS tNOS
Cotton Cotton negative Canola Canola
MON531
MON1445
Soybean Soybean negative Maize Maize
EPSPS1 EPSPS1 Position EPSPS2 EPSPS2
Cry1Ac Cry1Ac Pmi Cry3B Cry3B
Pat Pat Pmi Bar Bar
P35S P35S Position tNOS tNOS
Cotton Cotton negative Canola Canola
Pending Issue
1) How to detect Unauthorized GMOs or Unknown GMOs
2) How to quantify Stacked variety GMO
NK603 TC1507 NK603
X
TC1507
GMO content: 66.6% Test result: 66.6% =33.3+33.3
NK603
X
TC1507
NK603 TC1507
GMO content: 33.3% Test result: 66.6% =33.3+33.3
GMO content: 100.0% Test result: 133.2% =33.3+33.3+33.3+33.3
Non-
GMO
Non-
GMO
Non-
GMO A B C
3) How to harmonize several GMO detection methods
(Correct) (Incorrect) (Incorrect)
