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
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
·
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
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
Contact David Tomas
Telephone +34961366090
Fax +34961318008
E-mail dtomas@ainia.es
Biolytix AG
Benkenstrasse 254
CH-4108
Witterswil
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,
161-2,
Nankang
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
Amphur Muang
Nonthaburi 11000
Canadian Grain Commission
R3C
3G8,
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
Telephone Fon +49-(0)30-9489 3506
Fax +49-(0)30-9489 3510
E-Mail l.grohmann@congen.de
CNTA-Laboratorio
Ctra
N-134 km 50
31570
San Adrian
Navarra
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
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)
Contact Marco Mazzara
Telephone 0039 0332 785773
Fax 0039 0332 789333
E-mail marco.mazzaro@jrc.it
FASMAC CO., LTD
Kanagawa
243-0041
Contact Dr. Satoshi Futo
Telephone +81 46-295-8787
Fax +81 46-294-3738
E-mail sfuto@fasmac.co.jp
GeneScan Analytics GmbH,
Engesserstr. 4
79108
Contact Dr. Castor Menendez
Telephone +49-(0)761-5038
Fax +49-(0)761-5038-111
E-mail gmoanalytics@genescan.com
Genetic ID NA
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 -
Contact Flavia Machado
Telephone +55 11 4023 0522
Fax +55 11 4023 0625
GeneScan USA, Inc.
Belle
Contact Dr. Frank Spiegelhalter
Telephone 504-398-0940
Fax 504-398-0945
E-mail fspiegel@gmotesting.com
Petergasse 12/2
8010
JenaGen GmbH
JenaGen Diagnostik-Gentechnik-Biotechnologie
Loebstedter Str. 78
D-07749
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
Laboratorio COOP ITALIA
Via del Lavoro
6/8
40033 Casalecchio di Reno
Landesuntersuchungsanstalt fur das Gesundheits-und Veterinarwesen Sachsen
Sitz Dresden
Amtliche
Lebensmitteluberwachung
Fachgebiet 6.6
Postfach 2002744
D – 01192
Contact Dr. Gerda Hempel
Telephone +49-0351-8144-474
Fax +49-0351-8144-497
LAV Sachsen-Anhalt
Freiimfelder Str. 66/68
D-061112
Mid-West Seed Services
Contact Kalyn Brix-Davis
Telephone 605-692-7611
Fax 605-692-7617
E-mail kalynb@mwseed.com
National Food Institute
2008
Soi Charansanitwong 40
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
National Testing
Export Service
Center/Laboratory Services
Reading Scientific Services
Ltd.
The Lord Zuckerman Research Centre
Whiteknights
Reading RG66LA
Contact Andrew P Tingey, PhD.
Telephone +44 (0)118 986 8541
Fax +44 (0)118 986 8932
E-mail andrew.p.tingey@rssl.com
SE-230 53 Alnarp
Contact PeO Gummeson/Anders Dahlqvist
Tel: +46
40415321
Fax: +46
40415321
e-mail: per.olov.gummeson@scangene.se
Silliker, Inc.
Contact Dr. Daniel Wetsch
Telephone 319-366-3570
Fax 319-366-4018
E-mail daniel.wetsch@silliker.com
Sistemas Genomicos
E-46980 Paterna
Staaliches Untersuchungsamt Hessen
Standort Kassel
Fachgbiet 2.6; Gentechnische Veranderungen
Druseltalstr.
67
34131
Staaliches Veterinaeruntersuchungsamt
Deutshcher Ring 100
D-47798
Sygenta Seeds Ltda
BR 452 KM 142
Uberlandia-MG
TECAM
Rua Fabia, 59
Sao
Paulo – SP – CEP: 05051-030
Contact Dr. Daniela Contri
Telephone 55 11 3873 2553
Fax 55 11 3862 8954
E-mail biomol@tecam.com.br