qualitative and quantitative detection methods for gmo/lmo...

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)

SEONG-HUN LEE, Ph.D.

starslee7@hanmail.net

starlee65@korea.kr

Thank you for your attention