Litao Yang, Dabing Zhang, zhen Li

College of Life Science and Biotechnology

Activities of GMOs analysis in China

China-UK Bilateral Workshop on Modern Biology & its Social Impact

Authorized AgGMOs in the world and China

AgGMOs regulation and labeling in China

GMOs analysis:

-Detection labs of GMOs in China

-Development of GMOs analysis methods in China

-Reference molecules

-Chinese standards

-Database of GMOs analysis

-Challenges on GMOs analysis in China

-International cooperation.

Global Area of Growing GMOs (2006) (Total 102.0 Million Hectares)

Chinese GM-plants approved for commercialization

Plant Trait

Tomato (CMV) resistant

Tomato shelf-life altered

Sweet Pepper virus resistant

Cotton (2) insect resistant

Petunia color altered

papaya virus resistant

21 GM crops from foreign countries approved for

importing as processed materials in China

1 soybean: GTS 40-3-2 (for processed use only, 3 years )

9 corn varieties: Bt-corn Mon810, Bt11,Bt176,Mon 863,NK603,

GA21, T25, MON59122, TC1507 (for processed use only, 3 years )

7 canola varieties: Ms1Rf1 、 Ms1Rf2 、 Ms8Rf3 、 GT73 、 T45 、 Oxy235 、 Tapos 19/ 2 (for

processed use only, 3 years )

4 cotton varieties: MON531, MON1445, MON15985, LLcotton 25

(used for 3 years) (for processed use, 5 years )

MOA 2007

－－ A science-based regulatory approach

AgGMOs regulation and labeling in China

Regulatory regime

Legislation systems

Countries/Regions Type of Policy GM Content Threshold

Australia/New Zealand Mandatory 1%

China Mandatory n/a

Czech Mandatory 1%

European Union/UK Mandatory 0.9%

Hong Kong Mandatory 5%

Japan Mandatory 5%

Russia Mandatory 5%

Switzerland Mandatory2% or 3% depending on

different situations

Brazil (draft) Mandatory n/a

India (draft) Mandatory n/a

Israel (draft) Mandatory 1%

Malaysia (draft) Mandatory 3%

Korea (draft) Mandatory 3%

Taiwan (draft) Mandatory 5%

Thailand (draft) Mandatory 5%Canada Voluntary 5%USA Voluntary 5%

* Sources: International Service for the Acquisition of Agri-biotech Applications

(http://www.isaaa.org) and the United State Department of Agriculture

(http://www.fas.usda.gov)

Labelling Policies

17 kinds of GM products should be labeled

Soybean seed, soybean, flour, oil, soy meal

Corn seed, corn, flour, oil, corn meal

Rapeseed seed, rapeseed, oil, rapeseed meal

Cotton seed

Tomato seed, fresh tomato, tomato sauce

Three labeling methods:

GM raw materials: seeds

GM products: containing detectable GM materials

GM products: containing non-detectable GM materials

Almost of the oil has been labeled with GM.Why consumer accept GMOs? Price of imported soybean is lower!

30 million tons of GM soybean to be imported in 2007

-Detection labs of GMOs

-Development of GMOs analysis methods

-Reference molecules

-Chinese standards

-Platform construction for GMOs analysis

-International cooperation

GMOs analysis in China

Detection labs of GMOs in China

Entry-Exit Inspection and Quarantine Bureaus (23): Entry-Exit Inspection

(1 lab involved in MOA system)

Ministry of Agriculture (MOA) (49) : Domestic Inspection and Detection

Official

Non-official: Companies (GeneScan, SGS, etc)

GM-plants and derived foods detection procedure

Samples

Tested Samples Saved Samples

Protein Detection Methods

Nucleic Acids Detection Methods

ELISA Lateral Flow StripDNA Extraction

Conventional PCR

Contained GM contents

No GM contents

Quantitative PCR

GM Contents (xx%)

Sampling

Negative Positive

Samples

Tested Samples Saved Samples

Protein Detection Methods

Nucleic Acids Detection Methods

ELISA Lateral Flow StripDNA Extraction

Conventional PCR

Contained GM contents

No GM contents

Quantitative PCR

GM Contents (xx%)

Sampling

Negative Positive

Development of GMOs analysis methods in China

Endogenous reference genes

Screening PCR method ：CaMV 35S promoter; NOS terminator; nptII marker gene .

Gene-specific PCR method:

Cp4-epsps 、 Cry1Ac 、 Cry1Ab 、 Cry9c

Construct-specific PCR method:

Huafan No.1 、 GK19 、 SGK321 、 MON531 、 MON810 、 GA21 etc.

Event-specific PCR method: Roundup Ready soybean

Nine GM maize lines (GA21, Bt11, Bt176, MON810, T25, Mon863, TC1507, NK603, and CBH351)

Seven GM canola lines (Oxy235, T45, Ms1Rf1, Ms1Rf2, Ms8Rf3, GT73, and Topas 19/2)

Two GM cotton lines (Bt-cotton531 、 RR-cotton 1445 )

R&D Reference molecules

－－ PCR based methods (qualitative and quantitative)

The importance of a reference gene

Real Time quantitation of GM-event is based upon the notion of species-specific reference gene

An optimal reference gene should be:

1. single (or low and known) copy number

2. low allelic variation in cultivars

3. species specificity

Ideally, reference gene should be proposed by the scientific community as golden standard for a given taxon

Endogenous reference genes

How many reference genes?

Maize

Cotton

Rice

Canola

Soybean

Sugar beet

Potato

Tomato

Hmg (MON810), adh-70bp (NK603, MON863), adh-136bp (T25), Adh-136 (Bt11)”

SAH7 (281-24-236x3006-210-23), Adh (LL25), Acp1 (Mon1445), Sad1 (L. Yang et al)

Phospholipase D, SPS(J.Ding et al)

Cruciferin (Ms1, Ms8, Rf1,2,3), Fat A (GT73), BnACCg8 HMGI (H. Wen et al)

Lectin 100bp (A2704-12), lectin 72bp (40-3-2)

Glutamine synthetase

UGP-ase

Lat 52 (L.Yang et al)

From CRL

Journal of AOAC International, 2005, 88 (2): 577-584

13 pg DNA (about 10 haploid genomes) 1.3 pg DNA (about 1 haploid genome).

no allelic variation among the tested cultivars of B. napus.

Sample

GM Rapesee

d %

HMG I/Y Ct

(Mean)

GOXCt

(Mean)∆Ct

CalculatedGM

Rapeseed %

S1 1 23.71 28.14 4.43 1.1

S2 10 24.36 25.56 1.20 9.81

99bp

Huyou

-12

Huyou

-50

An-43

7 Su-

9912

46Su-

97-2

126

Suyou

No.

1

Zhes

huan

g-75

8

Jiax

ing-

9308

Zhon

gshu

ang

No. 6

Hunon

g No.

1

PGCP-1

5PG

CP-23

PGCP-5

5PG

CP-57

PGCP-5

9

13 n

g1.

3 ng

130

pg13

pg

1.3

pg

0.13

pg

2.0 kb

Huyou

-12

Huyou

-50

An-43

7Su-

9912

46Suy

ou N

o. 1

Zhes

huan

g-75

8

PGCP-1

5PG

CP-23

PGCP-5

5PG

CP-57

Canola endogenous reference gene: HMG I/Y

Rice endogenous reference gene SPS (Sucrose-Phosphate Synthase)

055 5’ TTGCGCCTGAACGGATATCTTTCAGTTTGTAACCACCGGATGACGCACGGACGGCTCGGAGACGCACGGACGGCTCGGA(probe)TCATCCCGAAAAGAT CAACCG 3’ 1135

•No allelic variation among rice cultivars

•Species specific

•One copy

J.Agr.Food.Chem.2004 52(11) ： 3372-3377 gramineous varieties

dicotyledon

species C43

βg94

-1

81bp

Hanfe

ng Nongh

u No.

695

20 Xiush

ui04

Shenx

iang

jin N

o.6

Taih

unuo

2056

6

D5

Hei

heai

hui

C26 P

R106

D20 B

aSm

ati

C21 Z

hong

yuza

o18

D57

kho

lt m

arsh

iP

2C1

Hua

yuyi

hao

xian

gqin

g

sunf

low

er

arbu

tus

eggp

lant

toba

cco

cotto

n

A.th

alia

na

rice

tom

ato

pepp

er

soyb

ean m

ung

bean

rape

seed

carr

ot

rice

barle

yw

heat

mai

ze

Tomato seeds Ketchup tomato juice

Journal of Agriculture Food Chemistry 2005,50(2):122-125

Southern blot analysis confirmed that this gene was single copy in the tested varieties

Allelic variation analysis

Tomato endogenous reference gene: Lat52

Xifen

No. 3

nega

tive

cont

rol

Dongn

ong

No. 7

04

Shenf

en N

o. 2

Mao

hong

No.

1

Jifa

n No.

4Za

okua

i

Lich

unZa

ofen

Zhon

gsu

No. 5

Hongz

a No.

10

R144

Nongy

ou N

o. 3

01

Shuan

gfu

Shenn

ong

No. 2

Jiaf

en N

o. 1

Yashu

No.

6

Cotton endogenous reference gene: SadI

(Gossypium hirsutum) Stearoyl-Acyl Carrier Protein Desaturase

Allelic variation analysis of 15 Gossypium hirsutum

Plant Cell Reporter, 2005, 23: 759-763

107 bp

Zhon

gmia

nsuo

27

Coker

312

Sumia

n 14

Sumia

n 15

Xuzho

u 14

2

Numia

n 11

Chuan

mia

n 24

3

Chouy

ou 1

Yumia

n 22

Jim

ian

17 Wan

mia

n 13

Jinm

ian

16

Simai

n 3 Li

aom

ian

14

Xiang

zam

ian

2

G. barbadense,G. arboretum and G. herbaceum,

Inter-laboratory study on developed

endogenous reference genes

Validation of endogenous reference gene, LAT52, used for GM

tomato detection (8 countries/14 labs, finished)

Validation of endogenous reference gene, SPS, used for GM rice

detection (Samples have been sent to each participants)

Validation of endogenous reference gene, Sad1, used for GM cotton

detection (In preparing)

Validation of endogenous reference gene, HMG I/Y, used for GM

canola detection

Development of Chinese GMOs

analysis methods using these new

endogenous reference genes

plant genomic DNA plant genomic DNApromoter geneintron terminator

Screening targets

Gene specific targets

Construct specific

Event specific targets

Levels of specificity – GM targets (DNA)

LOW

HIGHArne HA et al., Euro Food Res Tech, 2007

Screen and construct specific detection of transgenic Huafan No.1 tomato

CaMV35s promoter

Anti-EFE

NOS terminator

EcoR I Hind III

conc= 10^(-0.308*CT + 10.235)R=0.9984

conc= 10^(-0.290*CT + 8.125)R=0.9997

conc= 10^(-0.309*CT + 9.283)R=0.9986

Journal of the Science of Food and Agriculture 85:2159–2166 (2005)

PCaMV35S

Nos Terminator

Huafan construct specific fragment

A

B

C

195bp

180bp

153bp

mix

ed tr

ansg

enic

mix

ed s

eeds

non-

tran

sgen

ic

no te

mpl

ate

cont

rol

Identification and quantification of three Genetically Modified insect resistant cotton

lines using conventional and TaqMan real-time PCR methods

J.Agr.Food.Chem.2005, 53, 6222-6229Multiplex PCR

non-

GM

mai

ze

SadI

non-

GM

GK

19

SGK

321

Mon5

31

non-

GM

soy

bean

NTC

CpTI (SGK321)

Cry1A(c)-NOS(GK19,SGK321)

Cry1A(c)-NOS- Mon531

CryIA(c)

GK19

Mon

531

SGK32

1m

ixtu

re

non-

GM

cot

ton

non-

GM

soy

bean

NTC

Event-specific PCR Detection of Genetically ModifiedEvent-specific PCR Detection of Genetically Modified MON1445 MON1445 and MON531and MON531

5%5% 3%3% 1%1% 0.5%

0.5%

0.1%

0.1%

0.05

%0.

05%

0.01

%0.

01%

Multiple PCRMultiple PCRSingle PCRSingle PCR

M 1 2 3 4 5 6 7 8 9

NTC

NTC

Non

-GM

Non

-GM

MO

N53

1

MO

N53

1

SG

K32

1

SG

K32

1

MO

N14

45

MO

N14

45

GK

19G

K19

RR

SR

RS

Bt1

1B

t11

2 co

tton

mix

ture

2 co

tton

mix

ture

5%5% 3%3% 1%1% 0.5%

0.5%

0.1%

0.1%

0.05

%0.

05%

0.01

%0.

01%

NTC

NTC Non

-GM

Non

-GM

MO

N53

1

MO

N53

1

SG

K32

1

SG

K32

1

MO

N14

45

MO

N14

45

GK

19G

K19

RR

SR

RS

Bt1

1B

t11

Quantitative Quantitative PCRPCR

LOD : 10 copiesLOD : 10 copies ，， LOQ :10 LOQ :10 copiescopies

MON1445

MON531

Coefficient Values of the Coefficient Values of the reference reference moleculemolecule of MON1445of MON1445 and MON531 were and MON531 were 0.47 and 0.44, 0.47 and 0.44, respectivelyrespectively 。。

Transgenic Research, 2005, 14, 817-831

M 1 2 3 4 5 6 7 8

Specific analysisSpecific analysis

M 1 2 3 4 5 6 7 8 9

Event-specific PCR Detection of Genetically Modified Event-specific PCR Detection of Genetically Modified Soybean Soybean GTS 40-3-2GTS 40-3-2

NTC

NTCNon

-GM

Non

-GM

MO

N53

1

MO

N53

1

GA

21G

A21

MO

N14

45

MO

N14

45R

T73

RT7

3

NK

603

NK

603

GTS

40-

3-2

GTS

40-

3-2

5%5% 3%3% 1%1% 0.5%

0.5%

0.1%

0.1% 0.05

%0.

05%

0.01

%0.

01%

NTC

NTC

Conc= 10^(-0.323*CT + 12.302)R^2 ＝ 0.9993

Sensitivity analysisSensitivity analysis

LOD:2 copiesLOD:2 copies ，， LOQ: 20 copiesLOQ: 20 copies 。。Standard deviation repeatability andStandard deviation repeatability and

reproducibility were less than reproducibility were less than 0.20.2Coefficient Values of the Coefficient Values of the reference moleculereference molecule was was 0.92 0.92 sample sample

analysisanalysis

Event-specific PCR Detection of Nine Genetically Modified MaizesEvent-specific PCR Detection of Nine Genetically Modified Maizes （（ MON810MON810 、、 MON863MON863 、、 T25T25 、、 BT11BT11 、、 TC1507TC1507 、、 GA21GA21 、、 176176 、、 CBH351CBH351 and and

NK603NK603 ））

Standard Reference MoleculeStandard Reference Molecule for Genetically for Genetically Modified MaizesModified Maizes

LOD: 2LOD: 2 ～～ 20 copies20 copies ，， LOQ:20 copiesLOQ:20 copies

Maize Events

BT11 176 GA21 MON810 MON863 NK603 CBH351 TC1507 T25

LOD(Copies)

2 20 20 2 20 20 20 20 20

LOQ(Copies)

20 20 20 20 20 20 20 20 20

Coefficient Values of the Coefficient Values of the reference moleculereference molecule

EventMean CVs

SD RSD

BT11 1.23 0.10 8.28

176 0.88 0.08 9.30

GA21 0.78 0.07 8.41

MON810 1.12 0.09 8.08

MON863 1.08 0.20 18.00

NK603 0.98 0.15 15.37

CBH351 0.87 0.13 15.07

TC1507 1.10 0.15 13.44

T25 0.82 0.11 12.86

Based on the Based on the CVs, quantitative estimates of quantitative estimates of the maize samples were calculated the maize samples were calculated

J Agric Food Chem. 2005, 53, 9312-9318J Cereal Sci. 2006, 43, 250 － 257. J Agric Food Chem. 2007, 55:15-24.

Event-specific PCR detection of two GM-rapeseed Event-specific PCR detection of two GM-rapeseed （（ RT73 and T45RT73 and T45 ））

Specific analysisSpecific analysis

（（ AA ）） T45T45 ；（；（ BB ）） RT73RT73

LOD andLOD and LOQ LOQ inin event-specific quantitative event-specific quantitative

PCR detection of event PCR detection of event T45 T45 were were 11 copy copy andand

10 10 copiescopies respectively;respectively;For For RT73,RT73, LOD and LOD and

LOQ LOQ werewere 10 10 copiescopies and 100 and 100 copies copies

respectivelyrespectively 。。

。。

NTC

NTC Non

-GM

Non

-GM

T45

T45 Ms3

Rf8

Ms3

Rf8

176

176

RT7

3R

T73

Ms1

Rf2

Ms1

Rf2

RR

SR

RS

AA

BB

Sensitivity analysisSensitivity analysis

5%5% 3%3% 1%1% 0.5%

0.5%

0.1%

0.1%

0.05

%0.

05%

0.01

%0.

01%

NTC

NTC

AA

BB

BB

Based on the standard curve , rapeseed Based on the standard curve , rapeseed samples were analysised by quantitative samples were analysised by quantitative PCR.PCR.

Several CRMs were developed by IRMM, GIPSA, and ACOS, etc. Ground seed materials Genomic DNA Plasmids

R&D Reference molecules

- The certified reference materials for GMOs analysis is not easily obtained.

- Several CRMs have been R&D by IRMM, and series of CRMs might be

purchased from Fluka. However, the price is very expensive, and the

catalogue of CRMs did not contain the GMOs developed by China.

- Reference molecule can used in GMOs analysis instead of CRMs

-Novel reference molecules are being constructed for GMOs detection in

China, such as 9 GM maize lines, 7 GM GM canola lines, 4 GM cotton

lines,.

Several RMs for GM soybean, maize and canola detection

RMs for Bt cottons detection

Yang et al., 2005

RMs for MON1445 and MON531 cottons

Yang et al., 2005

RMs for RRS detection

RMs for 9 GM maize lines detection

Yang et al., 2007

Standards for GMOs detection in China

Based on the gene-specific and event-specific PCR detection methods,

respectively.

Published standards (more than 20):

NY/T 672-2003 Detection of Genetically Modified Plant Organisms and

Derived Products －－ General Requirements

NY/T 673-2003 Detection of Genetically Modified Plant Organisms and

Derived Products －－ Sampling

NY/T 674-2003 Detection of Genetically Modified Plant Organisms and

Derived Products －－ DNA Extraction and Purification

NY/T 675-2003 Detection of Genetically Modified Plant Organisms and

Derived Products －－ Qualitative PCR Methods for Soybeans

Detection of Genetically Modified Plant Organisms and Derived Products

—— Qualitative PCR method for Bt cotton

Detection of Genetically Modified Plant Organisms and Derived Products

—— Qualitative PCR method for Bt Rice

Detection of genetically modified plants and derived products ——

Qualitative PCR method for insect-resistant and herbicide-tolerant maize

event 176 and its derivate

Detection of genetically modified plants and derived products ——

Qualitative PCR method for insect-resistant and herbicide-tolerant maize

event NK603 and its derivate

Detection of genetically modified plants and derived products ——

Qualitative PCR method for herbicide-tolerant canola event GT73 and its

derivate

GMO information exchange platform

GMO biosafety database GMO detection method database GMO detection, new method validation, and

training service GMO public education

GMO Detection Method Database (GMDD) －－ http://gmdd.shgmo.org

Search Interface:

Inserted DNA Sequences

Diagram of Inserted DNA

Nucleic acid-based methods of GTS-40-3-2

soybean

4 types of PCR methods

Validation Information

PCR Program

Reference

GA21

Protein-based methods of GTS-40-3-2

CRMs

Submit interface

GMDD is an open format database

Ongoing International cooperationEU: Official ENGL Meeting, RIKILT Institute of Food SafetyUSA: The Grain Inspection, Packers and Stockyards

Administration (GIPSA),Proficiency Program Japan: NFRI Singapore: Agri-Food and Veterinary Authority Korea: Experiment Research Institute of National Agricultural

Products Quality Management Service Kyung Hee University

Validation of the novel endogenous reference genes of

Tomato, Canola , rice and cotton;

Development and optimization of PCR detection method

for GM soybean, corn, tomato, cotton, canola.

Screening, Gene-specific, Construct-specific, Event-

specific detection methods are developing.

Novel cotton 、 maize and soybean event-specific

standard reference molecules for GMOs detection have

been constructed instead of reference material.

GMO information exchange platform

Summary

Challenges in further GMOs analysisChallenges in further GMOs analysis

GMOs labeling threshold and its utilized detection method?

The accuracy of GM contents based on Quantitative RT-PCR?

Validation of CRMs and RMs for approved GMOs in China

Qualitative and quantitative detection for GMOs with stacked-

gene?

Communication of GMOs analysis

Develop the GMO detection training course in China

Acknowledgement Collaborators

Litao Yang 杨立桃 Xin Liu 刘 信

Aihu Pan 潘爱虎 Ning Li 李 宁

Aibo Wu 武爱波 Yufa Peng 彭于发

Zheng yuan 袁政 Shirong Jia 贾士荣

Wanqi Liang 梁婉琪 Liangwen Pan 潘良文

Supported by the Funds from

MOA, MOST, and NSFC project

Thank you!