USDA/GIPSA Proficiency Program

Testing for the Presence of Biotechnology Events in Corn and Soybeans

April 2004 Sample Distribution Results

 

Purpose of USDA/GIPSA Proficiency Program

Through the USDA/GIPSA Proficiency Program, USDA seeks to improve the overall performance of testing for biotechnology-derived grains and oil seeds.  The USDA/GIPSA Proficiency Program helps organizations identify areas of concern and take corrective actions to improve testing accuracy, capability and reliability.

 

Program Description

In February 2003, USDA/GIPSA’s Technical Services Division expanded the program to offer samples for qualitative or quantitative analysis.  Participants could request samples for qualitative analysis or quantitative analysis.  In this round of the USDA/GIPSA Proficiency Program one set of samples was used for both qualitative and quantitative analyses.  The samples were fortified with various combinations and concentrations of transgenic events, and participants had the choice of providing qualitative or quantitative results.  Scoring of the participant’s results was done by computing the “percentage of correctly reported transgenic events” in the samples.  Two new biotechnology corn events commercialized in the U.S. in 2003 were included in this round of samples:  MON863 (Monsanto event) and TC1507 (Dow AgroScience/Pioneer Dupont event)

 

Sample Composition  

The corn samples contained various combinations and concentrations of the following transgenic events: T25, CBH351, MON810, GA21, E176, Bt11, NK603, TC1507, and MON863; or, no events (i.e., negative corn sample).  The various transgenic concentration levels were produced on a percentage weight-weight basis (%w/w). A calculated amount of ground transgenic corn was mixed with a calculated amount of non-transgenic corn to produce concentrations of 0.1 %, 0.4 % and 0.8 % of the event.  The soybean samples were either non-transgenic soybeans, or fortified soybeans samples containing 0.1 % or 1.5 % of the transgenic glyphosate-tolerant soybeans (RoundUp Ready).   
Each participant received six corn samples and three soybean samples.  Each sample contained approximately 20 grams of ground material.

 

Program Participants

Participants included organizations from Africa, Asia, Europe, North America, and South America.  Each participant received a study description and a data report form by electronic mail, and with the samples.  Participants submitted results by electronic mail, FAX, or regular mail.  No analytical methodologies were specified, and organizations used both DNA- and protein-based testing technologies.  Fifty organizations participated in the April 2004 round of proficiency testing.

 

 

·                    Twenty-one participants submitted qualitative results only,

 

·                    Five participants submitted quantitative results only, and

 

·                    Twenty-four participants submitted a combination of qualitative and quantitative results. 

 

In this report, participating organizations are identified by either a confidential “Participant Identification Number”, or by name.  Appendix I identifies those organizations who gave GIPSA permission to list them as participants in the USDA/GIPSA Proficiency Program.

 

Data Summary Results

Data submitted by the participants are summarized in this report primarily in tables and figures.  Participants reported their results on a qualitative basis, quantitative basis, or a combination of both qualitative and quantitative bases.  Qualitative results were reported as the presence or absence of a particular event in each sample.  Quantitative results were reported as the concentration of a particular event in the sample.   Due to the complexity of the data, this report summarizes the data as follows:

 

Qualitative Data Summaries.  This section summarizes qualitative sample analysis data:

 

·             Table 1:  Percentage correct scores for all participants by event (DNA-based assays).

 

·             Figure 1:  Summary data of all participants for each event combined with the number of results submitted for that particular event (DNA-based assays).

 

·             Table 2:  Percentage correct scores for all individual participants by event (Lateral Flow Strip testing; Protein-based assays).

 

·             Table 3:  Percentage correct scores for all individual participants by event (Enzyme-Linked Immunosorbent Assay testing; Protein-based assays).

 

Quantitative Data Summaries.  This section summarizes quantitative sample analysis data:

 

·        Table 4: Percentage correct scores for all participants by event (DNA-based assays).

 

·        Figure 2:  Summary data of all participants for each event combined with the number of results submitted for that particular event (DNA-based assays).

 

·        Table 5:  Descriptive statistics for participant’s results relative to GIPSA’s fortification level.

 

·        Figures 3 A – D:  Percentage relative error analysis of participant’s reported quantifications.

 

·        Table Five:  Summary (table) of participant’s results relative to GIPSA’s fortification level.

 

·        Table Six: Percentage correct scores for all individual participants by event (Enzyme-Linked Immunosorbent Assay testing; Protein-based assays).

 

·        Table Seven. Percentage of false positive and false negative results in Qualitative and Quantitative reports.

 

Table 1.  Percentage correct scores for all participants reporting Qualitative results using DNA-based testing.  Events labeled as 35S through MON863 were assayed in corn samples.  The transgenic soybean samples contained the glyphosate tolerant event. 

NR = Participants who did not report a result

QUANT = Participants who reported a quantitative value (refer to Table Four for those results).

 

 

 

 

 

 

Figure 1. Group average of percentage correct for Qualitative reports on each event combined with the total number of results reported using DNA-based testing.  Events labeled as 35S through MON863 were assayed in corn samples.  The soybean samples contained the glyphosate tolerant event (RoundUp Ready/RUR) producing the CP4 EPSPS protein.  Numbers embedded in the histogram represent the total number of reported results for that event. Data are shown on a composite basis (i.e., all participants results combined)

 

 

Tables 2 and 3 show the percentage correct scores for all participants reporting Qualitative results using Lateral Flow Strip (LFS) testing and Enzyme-Linked Immunosorbent Assay (ELISA) testing (protein-based testing).  Events labeled as 35S through MON863 were assayed in corn samples.  The transgenic soybean samples contained the glyphosate tolerant event producing the CP4 EPSPS protein. 

 

Table 2. Lateral Flow Strip (LFS) Testing (Protein-based testing)

 

 

 

 

 

 

 

Table 3.  Enzyme-Lined Immunosorbent Assay (ELISA) Testing (Protein-based testing)

 

 

 

 

Table 4.  Percentage correct scores for all participants reporting Quantitative results using DNA-based testing.  Events labeled as 35S through MON863 were assayed in corn samples.  The transgenic soybean samples contained the glyphosate tolerant event. 

NR = Participants who did not report a result

QUAL = Participants who reported a qualitative (positive or negative) results (refer to Table One for those results).

 

 

 

 

Figure 2. Group average of percentage correct for Quantitative reports on each event combined with the total number of results reported using DNA-based testing.  Events labeled as 35S through MON863 were assayed in corn samples.  The soybean samples contained the glyphosate tolerant event (RoundUp Ready/RUR) producing the CP4 EPSPS protein.  Numbers embedded in the histogram represent the total number of reported results for that event. Data are shown on a composite basis (i.e., all participants results combined).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 5.  Descriptive statistics for participant’s reported quantifications relative to GIPSA fortification levels.  N = total number of quantitative results reported; Reported Mean = average of all reported quantitations; CV% = coefficient of variation [standard deviation/mean value x 100%] for the reported means; %R.E. = percentage relative error between the fortified and reported levels [reported value – fortification value / fortification value x 100].

Event: Fortification (%w/w)

N-results

Reported Mean (%w/w)

CV %

% R.E.

T25: 0.1%

20

0.88

216

783

T25: 0.4%

20

1.49

256

272

 

 

 

 

 

CBH351 : 0.1%

12

0.23

107

129

CBH351 : 0.4%

10

0.27

38.2

-32

 

 

 

 

 

MON810 : 0.1%

16

0.13

66.9

33

MON810 : 0.4%

17

0.26

50.7

-35

MON810 : 0.8%

34

0.82

55.9

2.5

 

 

 

 

 

GA21 : 0.1%

20

0.12

83.3

20

GA21 : 0.4%

25

0.4

47.5

1.5

 

 

 

 

 

Ev176 : 0.1%

29

0.23

192

132

Ev176 : 0.4%

32

0.38

55.7

-4.5

 

 

 

 

 

Bt-11 : 0.1%

23

0.24

100

143

Bt-11 : 0.4%

22

0.67

91.7

69

 

 

 

 

 

NK603 : 0.1%

8

0.12

57.6

25

NK603 : 0.4%

7

0.43

43.2

9.25

NK603 : 0.8%

18

0.66

38.1

-17.1

 

 

 

 

 

TC1507 : 0.1%

3

0.13

42.8

33

TC1507 : 0.4%

4

0.63

43.6

58.7

TC1507 : 0.8%

6

0.74

41

-2.1

 

 

 

 

 

MON863 : 0.1%

7

0.13

85

34

MON863 : 0.4%

7

0.5

84

26

MON863 : 0 .8%

14

0.98

103

22.5

 

 

 

 

 

RUR : 0.1%

31

0.13

60

29

RUR : 1.5%

22

2.39

56.9

59.3

 

 

 

 

 

 

Figure 3 A.  Comparison of relative errors in reported quantifications of T-25 in corn proficiency test samples.  Fortification levels were 0.1% and 0.4% (%w/w).

 

 

 

 

 

 

 

 

 

Figure 3 B.  Comparison of relative errors in reported quantifications of CBH 351, MON810, GA21, and Event 176 in corn proficiency test samples.  Fortification levels were 0.1%, 0.4%, (%w/w); only MON810 was fortified at the 0.8% level in addition to the 0.1 and 0.4 levels.

 

 

 

Figure 3 C.  Comparison of relative errors in reported quantifications of Bt-11, NK603, TC1507, and MON863 in corn proficiency test samples.  Fortification levels were 0.1%, 0.4%, and 0.8% (%w/w); only Bt-11 was not fortified at the 0.8% level.

 

 

 

Figure 3 D.  Comparison of relative errors in reported quantifications of soybean fortified with the RoundUp Ready genetic insert in proficiency test samples.  Fortification levels were 0.1%, 1.5%, (%w/w).

 

 

Table 7. Percentage of false positive and false negative results in Qualitative and Quantitative reports (samples fortified at 0.0% transgenic event)

 

Transgenic Event

Qualitative Reports

Quantitative Reports

 

% False Positive

% False Negative

% False Positive

% False Negative

35S

1.4

0.4

0

0

NOS

0.5

3.9

0

0

T 25

2.6

4.6

0

0

CBH 351

0.6

10.6

3.9

3.3

MON 810

2.7

5.5

0

1.9

GA 21

0.75

1.5

0

0

Event 176

0.69

5.5

0

0

Bt 11

1.2

4.9

0

0

NK 603

0

1

0

0

TC 1507

0

1.2

0

0

MON 863

0

0.8

0

0

RUR

0

0

0

0


Summary of Findings

 

Qualitative Sample Analysis

 

1)      As evidenced by the “percentage correct scores” in Table 1 and Figure 1, participants were able to correctly identify most of the transgenic events in the corn test samples with 90% to 100% accuracy through the use of conventional PCR.  The best performance was observed in the detection of MON863 and the least accurate detection was observed for CBH 351.

2)      Detecting the presence or absence of the protein product of the various transgenes was done through the use of either lateral flow strips (Table 2) or ELISA (Table 3).  Detection by lateral flow strips displayed only moderate overall accuracy.  In most cases, a correct determination was made on four of the six corn test samples (note that most of the performance scores were 66% correct).  However, in the three soybean test samples most participants were able to detect the gene product of the RoundUp Ready insert with 100% accuracy.  When ELISA was used, the performance results were quite variable for the corn test samples, but were uniformly accurate for detecting the transgenic soybean.

3)      The transgenic events that proved difficult to detect were CBH351, MON810, and Event 176 as evidenced by these events having the highest percentage of false negatives (Table 7).  Only MON810 displayed any noticeable tendency towards being falsely positive in this round of test samples.

 

Quantitative Sample Analysis

 

1)      As evidenced by the “percentage correct scores” in Table 4 and Figure 2, participants were able to identify with 100% accuracy all of the transgenic events–with the exception of MON 810 and RoundUp Ready.

2)       There was a characteristic inverse relationship between precision (CV%) of reported quantifications and event fortification level, such that the reported quantifications were highly variable for the least fortified samples (0.1%) while being less variable for the highly fortified samples (0.4% and 0.8%) (Table 5).  The reported quantifications that displayed the best precision (i.e., lowest CV%) were: CBH351 fortified at 0.4%; NK603 fortified at 0.8%, and TC1507 fortified at 0.8%, respectively.  There was noticeable difficulty in obtaining reproducible quantifications at the 0.4% fortification level, perhaps indicating that better standardization of real-time PCR methods needs to be developed by testing laboratories.

3)      There was a characteristic inverse relationship between accuracy (% relative error) and event fortification level, such that the participant’s reported quantifications were highly inaccurate for the least fortified samples.  In contrast, the reported quantifications were moderately accurate when samples were fortified at the 0.4% and 0.8% levels.  The reported quantifications that displayed the best accuracy (i.e., lowest % Relative Error) were:  GA21 fortified at 0.4%, TC1507 fortified at 0.8%, and Ev176 fortified at 0.4%.  Many laboratories have difficulty obtaining accurate quantifications at ≤ 0.4% fortification level, perhaps indicating that better standardization of real-time PCR methods needs to be developed.

4)      This trend of inverse relationships between fortification levels, accuracy, and precision has been observed in previous proficiency test sample rounds; however, in each round there were different event/concentration types that displayed the best or worst accuracy and precision.  This indicates that from round to round, there is still inconsistency in the reported quantifications for a particular transgene; furthermore, that at the ≤ 0.4% fortification level this effect is most pronounced.

 

 

 

 

Note:  It is important to understand that there are no internationally recognized standard reference materials for all transgenic events.   The transgenic seed or grain used to prepare these samples was made available to GIPSA by the Life Science Organizations.  Care was taken to ensure the transgenic material was either essentially 100 % positive for the event, or adjusted accordingly.  The fortified samples were prepared using a process that has been verified to produce homogenous mixes, and representative samples were analyzed to ensure proper fortification and homogeneity.

 

To obtain additional information on the USDA/GIPSA Proficiency Program, contact Dr. Ron Jenkins, USDA/GIPSA Proficiency Program Manager, at US 816-891-04442, or by e-mail at biotech-lab@usda.gov.

     

Appendix I.  . List of organizations who wished to be identified as a participant in the April GIPSA 2004 Proficiency Program.

 

A. Bio. C – Molecular Biology Division

                        Route de Samadet

                        64410 ARZACQ        

                        France

Contact            Dr. F. Bois

Telephone        33 5 59 04 49 20

Fax                  33 5 59 04 49 30

E-mail              bio.moleculaire@labo-abioc.fr

 

AINIA (Instituto Tecnologico Agroalimentario)     

           

                        Benjamin Franklin 5-11

                        Parque Tecnologico

                        46980 Paterna

                        Valencia

                        Spain

Contact            David Tomas

Telephone        +34961366090

Fax                  +34961318008

E-mail              dtomas@ainia.es

 

 

Biolytix AG

                        Benkenstrasse 254

                        CH-4108 Witterswil

                        Switzerland

Contact            Peter Brodmann

Telephone        41 (0)61 723 20 70

Fax                  41 (0)61 723 20 71

E-mail              peter.brodmann@biolytix.ch

 

 

 

Bureau of Food and Drug Analysis (BFDA), DOH, Taiwan

161-2, Kuen Yang Street

Nankang         

Taipei, Taiwan 

Contact            Dr. Lih-Ching Chiueh

Telephone        02-26531273

Fax                  02-26531268

E-mail              clc1025@nlfd.gov.tw

 

 

Bureau of Quality and Safety of Food

Dept. of Medical Sciences

88/7 Tiwanon Road

Amphur Muang

Nonthaburi 11000 Thailand

 

Canadian Grain Commission

                        1404-303 Main Street

                        Winnipeg, Manitoba

                        R3C 3G8,  Canada

Contact            Tigst Demeke

Telephone        204-984-4582

Fax                  204-983-0724

E-mail              tdemeke@grainscanada.gc.ca

 

CONGEN Biotechnology GmbH

                        CONGEN Biotechnology GmbH

Robert Roessle Str. 10

13125 Berlin, Germany

Telephone        Fon +49-(0)30-9489 3506

Fax                   +49-(0)30-9489 3510

E-Mail  l.grohmann@congen.de

 

 

 

CNTA-Laboratorio del Ebro

                        Ctra N-134 km 50

                        31570 San Adrian

                        Navarra

                        Spain

Contact            Blanca Jauregui, Ph.D.

Telephone        34 948 670159

Fax                  34 948 696127

E-mail              bjauregui@ctncv.es

 

DNA Technology Laboratory

                        Kamphaengsaean, Nakorn Pathon 73140

                        Thailand

Contact            Apiwan Yoojinda

Telephone        66-34-355192-4

Fax                  66-34-355196-7

E-mail              apiwan@dnatec.kps.ku.ac.th

 

Eurofins Scientific, Woodson-Tenent Laboratories Division         

3507 Delaware

                        Des Moines, IA  50313

Contact            David Pinero

Telephone        515-265-1461

Fax                  515-266-5453

E-mail              davidpinero@eurofinsus.com

 

European Commission - JRC

Via Fermi 1

I-21020 Ispra (VA)

Italy

Contact            Marco Mazzara

Telephone        0039 0332 785773

Fax                  0039 0332 789333

E-mail              marco.mazzaro@jrc.it

 

FASMAC CO., LTD

                        5-1-3 Midorigaoka, Atsugi-shi

                        Kanagawa 243-0041 

JAPAN

Contact            Dr. Satoshi Futo

Telephone        +81 46-295-8787

Fax                  +81 46-294-3738

E-mail              sfuto@fasmac.co.jp

 

 

GeneScan Analytics GmbH, Freiburg

Engesserstr. 4

79108 Freiburg i. Br.

                        Germany

Contact            Dr. Castor Menendez

Telephone        +49-(0)761-5038

Fax                  +49-(0)761-5038-111

E-mail              gmoanalytics@genescan.com

 

Genetic ID NA

                        501 Dimick Drive        

                        Fairfield, Iowa  52557

Contact            Jane Pappin/Bernd Schoel

Telephone        641-472-9979, ext 124

Fax                  641-472-9198

E-mail              jpappin@genetic-id.com/bshoel@genetic-id.com

 

GeneScan do Brasil Ltda

                        Gerente de Qualidade

                        Avenida Antonia Gazzola, 1001

3 andar

                        13.301-245  ITU - SP - Brazil

Contact            Flavia Machado

Telephone        +55 11 4023 0522

Fax                  +55 11 4023 0625

 

GeneScan USA, Inc.

                        101 Woodland Highway

                        Belle Chasse, LA  70037

Contact            Dr. Frank Spiegelhalter

Telephone        504-398-0940

Fax                  504-398-0945

E-mail              fspiegel@gmotesting.com

 

Institute of Food Chemistry & Technology

Graz, University of Technology

Petergasse 12/2

8010 Graz Austria

 

JenaGen GmbH

                        JenaGen Diagnostik-Gentechnik-Biotechnologie

Loebstedter Str. 78

D-07749 Jena

Germany

Contact            Dr. Reinhard Baier

Telephone:        +49(0)3641-464913

Fax:                  +49(0)3641-464991

E-mail:              r.baier@jenagen.de

 

Laboratorio CHMICO 

Via Vettingmiglia 165

10127 Turin, Italy

 

Laboratorio COOP ITALIA

Via del Lavoro 6/8

40033 Casalecchio di Reno

Bologna, Italy

 

Landesuntersuchungsanstalt fur das Gesundheits-und Veterinarwesen Sachsen

                        Sitz Dresden    

                        Amtliche Lebensmitteluberwachung      

                        Fachgebiet 6.6

Postfach 2002744

D – 01192 Dresden

Germany

Contact            Dr. Gerda Hempel

Telephone        +49-0351-8144-474

Fax                  +49-0351-8144-497

 

LAV Sachsen-Anhalt

Freiimfelder Str. 66/68

D-061112 Halle

Germany

 

Mid-West Seed Services

                        236 32nd Avenue         

                        Brookings, SD  57006

Contact            Kalyn Brix-Davis

Telephone        605-692-7611

Fax                  605-692-7617

E-mail              kalynb@mwseed.com

 

National Food Institute Thailand

                        2008 Soi Charansanitwong 40

                        Charansanitwong Road, Bangyeekhan, Bangphlad

                        Bangkok, Thailand 10700

Contact            Phattraphorn Choo-in

Telephone        66(0)2886-8088

Fax                  66(0)28868106-7

E-mail              phattrapornc12@hotmail.com

 

National Institute of Chemical Physics & Biophysics

Laboratory of Molecular Genetics

Akadeemia tee 23

Tallin 12618, Estonia

 

National Testing Center of GMO

No. 6 Xixinghua Street

Gongzhuling City, Jilin Province, PR

China. Post code 136100

 

Oregon Dept. of Agriculture

Export Service Center/Laboratory Services

1207 NW Naito Parkway, Suite 204

Portland, OR  97209

 

Reading Scientific Services Ltd.                                          

The Lord Zuckerman Research Centre

Whiteknights

Reading RG66LA

United Kingdom

Contact            Andrew P Tingey, PhD.

Telephone        +44 (0)118 986 8541

Fax                  +44 (0)118 986 8932

E-mail              andrew.p.tingey@rssl.com

 

ScanGene AB

P.O.Box 166

SE-230 53 Alnarp

Sweden

Contact            PeO Gummeson/Anders Dahlqvist

Tel:                   +46 40415321

Fax:                  +46 40415321

Mobile:             +46 702966603

e-mail: per.olov.gummeson@scangene.se

 

Silliker, Inc.

                        405 8th Ave SE                       

                        Cedar Rapids, IA  52401                    

Contact            Dr. Daniel Wetsch

Telephone        319-366-3570

Fax                  319-366-4018

E-mail              daniel.wetsch@silliker.com

 

Sistemas Genomicos

Valencia Technology Parck

C/Benjamin Franklin Avenue, 12

E-46980 Paterna Valencia

Spain

 

Staaliches Untersuchungsamt Hessen

Standort Kassel

Fachgbiet 2.6; Gentechnische Veranderungen

Druseltalstr. 67

34131 Kassel Germany

 

 

Staaliches Veterinaeruntersuchungsamt Krefeld

Deutshcher Ring 100

D-47798 Krefeld

Germany

 

Sygenta Seeds Ltda

BR 452 KM 142

Uberlandia-MG

Brazil 38405-232

 

TECAM                                 

                        Rua Fabia, 59                                                              

                        Sao Paulo – SP – CEP:  05051-030

                        Brazil

Contact            Dr. Daniela Contri

Telephone        55 11 3873 2553

Fax                  55 11 3862 8954

E-mail              biomol@tecam.com.br