USDA/GIPSA Proficiency Program

Testing for the Presence of Biotechnology Events in Corn and Soybeans

September 2003 Sample Distribution

 

Date of Report:  January 27, 2004

 

 

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 specifically request samples for either qualitative or quantitative analysis.  GIPSA then mails the participant the samples they requested.  The samples for qualitative analyses contain different combinations of biotechnology events at a single concentration.  The samples for quantitative analyses contain combinations of biotechnology events at various concentrations. 

 

Qualitative Sample Analysis.  Samples for qualitative analysis are prepared gravimetrically using non-transgenic corn and biotechnology-derived corn, and the events present in the sample are at approximately 0.1 % (weight/weight).  The participants report the samples as either positive or negative for each of the events. The corn samples contain various combinations of the events T25, CBH351, MON810, GA21, E176, Bt11 and NK603.   The soybean samples are either non-transgenic soybeans or non-transgenic soybeans fortified with Roundup ReadyTM soybeans at the 0.1 % level.  Each participant receives twelve (12) corn and three (3) soybean samples containing approximately 20 grams of ground grain.

 

Quantitative Sample Analysis.  Samples for quantitative analysis are prepared gravimetrically using non-transgenic corn and biotechnology-derived corn, and the events present in the samples vary in concentration from 0.1 % to 5.0 %.  The corn samples contain various combinations of the events T25, CBH351, MON810, GA21, E176, Bt11 and NK603.   The soybean samples are either non-transgenic soybeans or non-transgenic soybeans fortified with Roundup ReadyTM soybeans at the 0.1 % or 3.0 % level.  For quantitative sample results, the participants are asked to submit results in weight % of the biotechnology event, or in accordance with their standard reporting procedures.  Each participant receives six (6) corn and three (3) soybean samples containing approximately 20 grams of ground grain.

 

Program Participants

GIPSA mailed samples to fifty-nine (59) participants.   Thirty-four (34) participants requested samples for qualitative analysis, twenty-three (23) participants requested samples for quantitative analysis, and two (2) participants requested samples for qualitative and quantitative analysis.  Participants included organizations from Africa (1), Asia (7), Europe (23), North America (21) and South America (7).  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 methodologies were specified, and organizations used both DNA- and protein-based testing technologies. 

 

Fifty-two (52) of the participating organizations returned results by the deadline date.  Thirty (30) participants submitted results on samples for qualitative analysis, twenty-one (21) participants submitted results on samples for quantitative analysis, and one (1) participant submitted results on samples for both qualitative and quantitative analysis. 

 

Those organizations giving GIPSA permission are listed by name in an attachment to this report (Attachment).  Those organizations giving GIPSA permission are also listed by name in the Proficiency Program May 2003 Summary Tables (Tables 1, 2, 3 and 4).  All other organizations were assigned a Participant Identification Number.

 

Data Summary Results

Data submitted by the participants are summarized in this report, including tables and charts.  The data are summarized to reflect the number of participants who submitted results, the number of participants who classified all samples correctly as either positive or negative for the event, and the percentage of samples that were classified correctly by all participants. 

 

Participants were given the option of receiving samples for qualitative or quantitative analysis.  Some participants requested samples for quantitative analysis, but did not have quantitative methods for all events, and therefore reported results for some events on a qualitative basis.  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 includes a summary of the following sample results:

·             Samples for qualitative analysis:  DNA-based results

·             Samples for quantitative analysis:  DNA-based results

·             Samples for qualitative analysis:  Protein-based results

·             Samples for quantitative analysis:  Protein-based results

 

Quantitative Data Summaries.  This section provides summaries of DNA-based quantitative analysis data submitted by participants who received samples for quantitative analysis.  The data are presented on a composite basis and do not reflect any individual participant data assessment.  The following information is presented in Table 5:

·             Average analytical results reported for each fortification level and event combination (corn and soybean)

·             Coefficients of variation (CVs) for the analytical results reported for each fortification level for each event (corn and soybean)

 

Qualitative Data Summaries

Samples for Qualitative analysis:  DNA-Based Results

The data submitted by the participants who received samples for qualitative analysis were assessed as follows: 

·             The result was correct if the participant reported the sample positive for an event which the sample did contain. 

·             The result was correct if the participant reported the sample negative for an event which the sample did not contain. 

·             The result was incorrect if the participant reported the sample negative for an event which the sample did contain (i.e., false negative result).

·             The result was incorrect if the participant reported sample positive for an event which the sample did not contain (i.e., false positive result).  

 

 

Table 1 shows results reported by each participating organization on qualitative samples analyzed using DNA-based procedures.  Participants are either listed by name or Participant Identification Number. 

 

The data are also summarized on a composite basis for each grain (corn and soybean) event in Graph 1 (Summary of Qualitative Sample Analyses) showing the percentage of correct responses and number of sample analytical results submitted for each transgenic event in corn and soybeans.

 

Table 1.  Samples for Qualitative Analysis:  DNA-Based Results by Participant

 

 

Graph 1.  Summary of Qualitative Sample Analyses

 

 

Samples for Quantitative analysis:  DNA-Based Results

Participants were evaluated on their ability to detect the biotech event contained in the samples for quantitative analysis using the same process as described in the section Samples for Qualitative Analysis:  DNA-Based Results. 

 

Table 2 shows results reported by each participating organization on quantitative samples analyzed using DNA-based procedures.  Participants are either listed by name or Participant Identification Number. 

 

Graph 2 (Summary of Quantitative Sample Analyses) shows the percentage of correct responses and number of sample analytical results submitted for each transgenic event in corn and soybeans on a composite basis (i.e., all participants combined).

 

Table 2.  Samples for Quantitative Analysis:  DNA-based Results by Participants

 

 

Graph 2.  Summary of Quantitative Sample Analyses

Qualitative Data Summaries

Samples for Qualitative analysis:  Protein-Based Results

Table 3 shows the results reported by each participating organization on qualitative samples analyzed using protein-based procedures.  This table presents data from both Lateral Flow Strip and ELISA technologies.  Participants are either listed by name or by a Participant Identification Number. 

 

Table 3.  Samples for Qualitative Analysis:  Protein-Based Results by Participant

 

 

Quantitative Data Summaries

Samples for Quantitative analysis:  Protein-Based Results

Table 4 shows the results for the Cry1Ab protein (MON810 and/or BT11 corn events) and the CP4 EPSPS protein (soybean event) using the ELISA Plate tests reported by this participant.

 

Table 4.  Samples for Quantitative Analysis:  Protein-Based Results by Participant

 

Quantitative Data Summaries

Organizations that requested samples for quantitative analysis received six corn samples and three soybean samples.  The six corn samples consisted of duplicate samples from three different formulations.  This resulted in a varying number of reported results for each event/concentration combination.  Table 5 summarizes all samples results reported on a quantitative basis.

 

Table 5.  Summary Quantitative Sample Analyses

 

Fortification                                                                 Standard                            Results

Level                Mean                Range                       Deviation         CV            Reported

Event T25 (Corn)

0.0 %                0.0 %                0.0 % - 0.05 %          NA                    NA                    22

0.1 %                0.19 %              0.0 % – 1.1 %            0.28                   147 %               22

1.5 %                1.34%               0.6 % - 2.3 %            0.45                   33.7 %              22    

Event CBH351 (Corn)

0.0 %                0.0 %                 NA                            NA                    NA                    10

0.5 %                0.54 %              0.0 % - 0.8 %            0.25                   45.8 %              10

3.0 %                3.2 %                 0.8 % - 5.2 %            1.3                     40.1 %              10

Event MON810 (Corn)

0.1 %                0.26 %              0.0 % - 1.36 %          0.35                   134 %               32    

1.5 %                1.3 %                0.1 % - 2.9 %            0.81                   61.8 %              32

5.0 %                3.1 %                0.72 % - 5.0 %           1.4                     45.2 %              32    

Event GA21 (Corn)

0.0 %                0.04 %              0.0 % - 0.51 %          0.14                   353 %               26

0.5 %                0.37 %              0.1 % - 0.7 %            0.16                   42.7 %              26

1.5 %                1.1 %                0.1 % - 2.1 %             0.51                   45.9 %              26

Event 176 (Corn)

0.1%                          0.06 %              0.0 % - 0.1 %            0.036                 57.3 %              30            

0.5%                          0.46 %              0.2 % - 1.5 %             0.34                   73.9 %              30

5.0 %                3.9 %                0.93 % - 6.4 %          1.39                   35.9 %              30

Event BT11 (Corn)

0 %                   0 %                   NA                            NA                    NA                    24

0.5 %                0.48 %              0.01 % - 0.7 %           0.14                   30.1 %              24

1.5 %                1.5 %                0.01 % - 3.2 %          0.68                   46.8 %              24

Event NK603 (Corn)

0.5 %                0.38 %              0.2 % - 0.5 %            0.11                   29.5 %              16

1.5 %                1.2 %                0.6 % - 1.6 %            0.33                   27.4 %              16

3.0 %                2.4 %                1.2 % - 3.2 %            0.56                   22.8 %              16

Roundup ReadyTM (Soybean)

0 %                   0.0 %                NA                            NA                    NA                    14

0.1 %                0.19 %              0.0 % – 0.53 %          0.18                   94.7 %              18

3.0 %                3.6 %                1.5 % - 5.4 %            1.2                     34.1 %              13

 

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.

 

 

USDA/GIPSA Proficiency Program for September 2003:  Observations

Qualitative Analysis Data

It is interesting to note that the organizations analyzing samples for qualitative analysis (Table 1 and Graph 1) and organizations analyzing samples for quantitative analysis (Table 2 and Graph 2) differed in their capability to detect the presence of biotechnology events.  Table 6 shows the performance by event for the organizations receiving the different samples.  For every event except NK603, the organizations analyzing samples for qualitative analysis scored lower in their ability to correctly identify the presence of the events relative to the organizations analyzing samples for quantitative analysis. 

 

Table 6.  Comparison of Performance (Mean % for each event)

 

                                           CBH         MON       

Sample Type      T25           351           810           GA21        E176         Bt11          NK603     RR-Soy

Qualitative 88.4 %      90.6 %      93.5 %      93.8 %      91.7 %      93.2 %      100 %       88.2 %

Quantitative        98.9 %      98.5 %      97.2 %      100 %       96.1 %      100 %       100 %       95.0 %

Difference  +10.5 %    +7.9 %      +3.7 %      +6.2 %      +4.4 %      +6.8 %      +0 %         +6.8 %

 

 

Possible explanations for this observation:

 

1.                  This Program continues to grow, with new and probably less experienced organizations being added.  These less experienced organizations typically request samples for qualitative analysis, and therefore, probably have a higher error rate.  In addition, the samples distributed to these organizations are fortified to contain 0.1 % of the event, a level that approaches the limit of detection reported by many organizations.

 

2.                  The organizations requesting samples for quantitative analysis are typically the more experienced organizations.  In addition, the samples distributed to these organizations are fortified at levels ranging from 0.1 % to 5.0 %.  Typically, higher concentrations are easier to detect, and therefore, the error rate should be lower. 

 

Quantitative Analysis Data

Overall, the data from this sample distribution show improvement in both accuracy and precision:

 

1.                  The mean results for corn events T25, CBH351, GA21, E176, Bt11 and NK603 were generally within +25 % of the target fortification level (except the 0.1 % fortification level).  Overall, this is a slight improvement from the previous cycle (May 2003).

 

2.                  The mean results for the samples fortified with 3.0 % transgenic soybean event were consistently higher than the fortification level, but within 25 % of the target fortification level.

 

3.                  The majority of the Coefficients of Variation (CV’s) were below 50 % for all event/concentration combinations except the 0.1 % fortification level.  The CV’s for the majority of event/concentration combinations were lower for this cycle as compared to May 2003. 

 

 

January 2004 Sample Distribution

Several changes are planned for the January 2004 sample distribution:

 

1.                  Samples distributed in January 2004 will include two new biotechnology corn events commercialized in the U.S. in 2003:  MON863 (Monsanto event) and TC1507 (Dow AgroScience/Pioneer Dupont event).  

 

2.                  All participants will receive six corn samples and three soybeans samples. 

 

3.                  The same fortified samples will be used for both qualitative and quantitative analysis.

 

4.                  Individual participant performance on the quantitative analysis of samples will be posted on GIPSA’s biotechnology web page. 

 

To obtain additional information on the USDA/GIPSA Proficiency Program, contact Donald C. Kendall, Biotechnology Program Manager, at US 816-891-0463, or by e-mail at biotech-lab@usda.gov.