CORN, CANOLA, & ALFALFA BULK GRAIN

Transcription

1 CORN, CANOLA, & ALFALFA BULK GRAIN AgraStrip RUR-HS Bulk Grain Strip Test Part Number Intended Use The intended use of the kit is the qualitative (yes/no) determination of the CP4 EPSPS protein in Roundup Ready bulk seed and grain samples. The AgraStrip RUR-HS Bulk Grain Strip Test - Trait has a detection limit of one (1) Roundup Ready (CP4 EPSPS) corn kernel in 800 non-rur corn kernels in 5 minutes, one (1) Roundup Ready canola seed in 1000 non-rur canola seeds in 5 minutes, and one (1) Roundup Ready alfalfa seed in 600 non-rur alfalfa seeds in 5 minutes Note: This application is intended for the detection of NK603 Roundup Ready corn (RUR NK603). It does not detect genetic event GA21 Roundup Ready corn. Product Description The AgraStrip RUR-HS Bulk Grain Strip Test - Trait Test Kit detects the CP4 EPSPS protein produced by a gene, derived from Agrobacterium sp. strain CP4. This gene has been incorporated into herbicide-resistant crops including Roundup Ready and Roundup Ready 2 brands (RUR) from Monsanto and other companies. The lateral flow strips and other kit components are sufficient to detect the presence or absence of the CP4 EPSPS protein in both field and laboratory environments. Different protocols are required for leaf, seed, and bulk grain detection. Principle of the Assay The assay uses a double antibody sandwich format. Antibodies specific to the CP4 EPSPS protein are coupled to a color reagent and incorporated into the lateral flow strip. When the lateral flow strip is placed in a small amount of an extract from plant tissue that contains CP4 EPSPS protein, binding occurs between the coupled antibody and the protein. A sandwich is formed with some, but not all the antibody that is coupled to the color reagent. The membrane contains two capture zones, one captures the bound CP4 EPSPS protein and the other captures color reagent. These capture zones display a reddish color when the sandwich and/or unreacted colored reagents are captured in the specific zones on the membrane. The presence of only one line (control line) on the membrane indicates a negative sample and the presence of two lines indicates a positive sample. Contents of Kit Quantity AgraStrip RUR-HS Bulk Grain Strip Test- Trait 100 Conical microtubes, 1.5mL (COOLS2005) 100 Transfer pipettes (COOLS2010) 100 AS-GMO-RURHSBULKGRAINTC PI GB Finalized 1 Oct

2 Materials Required but not Supplied: Conical microtube rack (EQOLE1215) Timer (EQOLE1301) Graduated Cylinder, 50mL (EQOLE1051) Balance, 400g (EQOLE1010) Blender (110V: EQOLE1020; 220V: EQOLE1027) Blender blade with gasket set (EQUOLE1026) Storage and Preparation of Reagents The AgraStrip RUR-HS Bulk Grain Strip Test - Trait can be stored at room temperature or refrigerated (2-25ºC). Do not freeze. Once opened, the AgraStrip RUR-HS Bulk Grain Strip Test - Trait lateral flow test strips must be stored in the closed canister with the moisture-indicating card. The moisture-indicating card must be blue in color. If the moisture-indicating card is pink, contact Romer Labs, Inc. Technical Service. Storage conditions higher than room temperature may adversely affect performance. Purpose The AgraStrip RUR-HS Bulk Grain Strip Test - Trait Test Kit has been designed to screen bulk grain samples for the presence of the CP4 EPSPS protein. Sample preparation procedures are outlined below. Please refer to the instructions to be sure that all of the necessary materials are available prior to testing. Sampling The samples used for the AgraStrip RUR-HS Bulk Grain Strip Test - Trait Corn Grain Test Kit can be sub-samples of representative samples collected following GIPSA sampling procedures from trucks, railcars, barges, etc. for other tests. The size of the sub-samples to be used for the AgraStrip RUR-HS Bulk Grain Strip Test - Trait will depend on the percent RUR screening level desired and an acceptable level of risk that the RUR level is close to the screening level. The number and size of the sub-samples will be discussed in more detail in the Principle of the Screening Application section. For canola, the size of the sub-sample used for the AgraStrip RUR-HS Bulk Grain Strip Test - Trait has been optimized for 1000 canola seeds. For alfalfa seed, the size of the sub-sample used for the AgraStrip RUR-HS Bulk Grain Strip Test - Trait has been optimized for 600 alfalfa seeds. Note: It is assumed that the samples collected are representative of the contents of the truck or container and are sufficiently mixed to contain a random distribution of the sample contents. Sample Preparation: Weighing the Sample The statistical sampling plan (see Principle of the Screening Application) is dependent on the number of seeds or kernels used. However, it is more practical for routine testing to weigh seeds or kernels instead of counting to obtain the desired number. The average weight of seeds depends on the variety and environmental conditions. AS-GMO-RURHSBULKGRAINTC PI GB Finalized 1 Oct

3 It is recommended that the weight-to-seed/kernel ratio for each variety be determined as follows: 1. Count 100 kernels of the variety to be tested. 2. Weigh the 100 seeds to the nearest 0.01 gram. 3. Divide the weight of the seeds by 100 to get the average grams per seed. 4. Multiply this average weight by the desired number of seeds in the sub-samples to determine the weight for the sub-samples. 5. Construct a weight-to-seed ratio table for each variety for the different subsample sizes to be used. Example: One hundred (100) corn kernels of Variety X weigh grams. corn kernel then weighs 0.25 grams. Multiply the 0.25-gram per corn kernel times the number of corn kernels in each sample size to get the following table. Table A: Example: Weight-to-Seed/Kernel Ratio Grams per Sample of X No. Corn Kernels (a) Sample Weight (g) No. Canola Seeds Sample Weight (g) This average weight is then used to obtain the number of corn kernels for this corn variety. Sample Preparation The sample is ground and then extracted with water in a polypropylene Mason -type jar. The sample preparation is important for the proper function of the test, especially the ratio of water to the weight of the seed sample. The size of Mason jar required and the grinding time depends on the sample size to be analyzed. Table B lists those parameters. Corn: Sample Weight (g) x 1.25 = Water Volume (ml) Canola: Sample Weight (g) x 3.0 = Water Volume (ml) Alfalfa Seed: Sample Weight (g) x 10.0 = Water Volume (ml) Table B: Parameters for Preparing Samples Number of Kernels in Sample Jar Size (oz.) Grind Time (sec) Corn: Corn: Corn: Corn: > Canola: Alfalfa Seed: The processing parameters were determined using the laboratory grade Waring Model 31BL91 food processor with the standard blade. Other food processors may require different parameters. All kernels should be ground. AS-GMO-RURHSBULKGRAINTC PI GB Finalized 1 Oct

4 Sample Processing 1. Weigh sub-samples from each truck or container. 2. Place each sub-sample in a clean, dry polypropylene Mason jar of the appropriate size. See Table B. 3. Attach the jar adapter and clean, dry cutting blades. 4. Place the jar onto the food processor, place a shield over the jar if desired, and grind the sub-sample on high speed for the time indicated in Table B. Caution: If using glass Mason jars, it is recommended to shield the jars during grinding with a tri-cornered 1-liter plastic beaker. 5. Remove the adapter and cutting blades. Note: A coffee grinder may also be used to grind small sample sizes (i.e. alfalfa). If a coffee grinder is used, transfer the sample to a clean container for extraction. 6. Add the volume of water (see below) to the ground seed in the jar, place a lid on the jar and shake the jar until all the ground corn is well wetted (about sec.) For Corn: Sample Weight (g) X 1.25 = Water Volume (ml) For Canola: Sample Weight (g) X 3.00 = Water Volume (ml) For Alfalfa Seed: Sample Weight (g) X = Water Volume (ml) Note: The sample will have a thick consistency but should contain some free liquid after a short settling time. There should be no whole seeds remaining. 7. Use this free liquid as sample in the Test Procedure. Test Procedure 1. Transfer some liquid from the prepared sample to a conical microtube by squeezing the bulb of the transfer pipette and inserting it in the free liquid in the top of the sample jar. 2. Release the bulb to pull up the sample. Add the sample from the pipette to the sample tube by squeezing the bulb. This should be approximately 0.5 ml. The sample tube has a 0.5-mL indicator at the top of the tapered section. 3. Place one AgraStrip Cry1F Bulk Grain Strip Test - Trait Test Strip into the sample tube. Let sit for 5 minutes. 4. The appearance of one line (control) on the strip indicates a negative result. 5. The appearance of two lines on the strip indicates a positive result. Interpreting the Lateral Flow Strip Test Check the test strips 5 minutes after inserting the strip. At least one line, the Control Line, should always develop approximately one (1) cm up from the bottom sample pad. AS-GMO-RURHSBULKGRAINTC PI GB Finalized 1 Oct

5 A red line in this position indicates that the strip is functioning properly. A red line appearing below the Control Line is the RUR Test Line and indicates a positive result for CP4 EPSPS protein. If the test strip displays two (2) red lines, the test is complete and the sample is positive for the RUR trait. If at 5 minutes the test strip only shows a clearly visible Control Line, then the sample is negative for the RUR transgenic trait. If no control line develops, the result is inconclusive and needs to be repeated. Note: Test strip results should be interpreted after 5 minutes. Test strips interpreted after 60 minutes are invalid. Illustration of Lateral Flow Strips Example of an unreacted, negative (1- line) and positive (2-lines) test strip Archiving Test Strips If it is desired to archive test strip results, cut off the bottom and top strip pads as illustrated to the left within one (1) hour of test completion. Equipment Cleaning and Drying Caution: It is important to clean and dry the jars and cutting blades and/or coffee grinder between samples. AS-GMO-RURHSBULKGRAINTC PI GB Finalized 1 Oct

6 1. The Mason jar should be emptied, rinsed thoroughly with water and completely dried with a paper towel between uses. 2. The cutting blades for the blender should be rinsed with water until all ground seed is removed, washed using standard household liquid soap, rinsed well and carefully dried. If available, spraying or rinsing with methanol or isopropyl (rubbing) alcohol will assist drying. 3. Coffee grinders should be emptied, wiped down with moist towels thoroughly and completely dried with paper towels between uses. Special attention should be paid to the area at the base of the blades. It is not advisable to put water directly into the grinder. Be sure to unplug the grinder when cleaning. Principle of the Screening Application Screening at Very Low GM Levels Screening grain at very low GM levels can be accomplished by using a sufficiently large sample size that tests negative for the GM trait. Lateral flow strips can be used by testing multiple sub-samples, the size of which do not exceed the sensitivity of the strip test. The AgraStrip RUR-HS Bulk Grain Strip Test - Trait sensitivity is at least one RUR NK603 corn kernel in 800, one RUR canola seed in 1000, and one RUR alfalfa seed in 600. The AgraStrip RUR-HS Bulk Grain Strip Test - Trait provides a yes/no answer for the presence or absence of RUR seed in a given sample. Testing multiple statistically selected sub-samples allows an estimate of the percent of RUR seed. The test results provide information about the probability of the percent RUR seed in the sample. Note: The test protocol does not determine the exact percent of RUR seed. It determines the probability that a sample contains greater or less than a specified threshold concentration. Statistical Interpretation The following tables provide information at five confidence levels with the use of multiple samples of corn (400 kernels, 600 kernels, or 800 kernels each), canola (1000 seeds each) and alfalfa (600 seeds each). The tables provide the maximum percent GM levels that would be expected in the sample if all test-samples provide negative results. The appropriate table can be used depending on the desired screening level and how the samples will be processed. Table C: 400 Kernel Sub-Samples (Corn) (All Sub-Samples Must be Negative) 400 Kernels Maximum Percent RUR Kernels, Using Sub-Sample Sizes of 400 Kernels at Five Different Confidence Levels (%) AS-GMO-RURHSBULKGRAINTC PI GB Finalized 1 Oct

7 Table D: 600 Kernel Sub-Samples (Corn) (All Sub-Samples Must be Negative) 600 Kernels Maximum Percent RUR Kernels, Using Sub-Sample Sizes of 600 Kernels at Five Different Confidence Levels (%) Table E: 800 Kernel Sub-Samples (All Sub-Samples Must be Negative) 800 Kernels Maximum Percent RUR Kernels, Using Sub-Sample Sizes of 800 Kernels at Five Different Confidence Levels (%) Table F: 1000 Seed Sub-Samples (Canola) (With Some Sub-Samples Positive) 1000 Seeds No. of Positive Subsamples Maximum Percent RUR Kernels, Using Sub-Sample Sizes of 1000 Seeds at Three Different Confidence Levels (%) AS-GMO-RURHSBULKGRAINTC PI GB Finalized 1 Oct

8 Table G: 600 Seed Sub-Samples (Alfalfa) (With Some Sub-Samples Positive) 600 Seeds No. of Positive Subsamples Maximum Percent RUR Kernels, Using Sub-Sample Sizes of 600 Seeds at Five Different Confidence Levels (%) *All values determined using SeedCalc5 Exclusion of Warranty Statement The user assumes all risk in using Romer Labs, Inc. products and services. Romer Labs, Inc. will warrant that its products and services meet all quality control standards set by Romer Labs, Inc., and Romer Labs, Inc. will, at its option, repair or replace any product, components, or repeat services which prove to be defective in workmanship or material within product specific warranty periods or expiration dates and which our examination shall disclose to our satisfaction to be defective as such. This warranty is expressly in lieu of all other warranties, expressed or implied, as to description, quality, merchantability, fitness for any particular purpose, productiveness, or any other matter. Romer Labs, Inc. shall be in no way responsible for the proper use of its products. Romer Labs, Inc. hereby disclaims all other remedies, warranties, guarantees or liabilities, expressed or implied, arising by law or otherwise, and it shall have no liability for any lost profits or damage, direct, indirect or otherwise, to person or property, in connection with the use of any of its products or services. This warranty shall not be extended, altered or varied except by a written instrument signed by an authorized representative of Romer Labs, Inc. For Technical Service Call: Romer Labs, Inc Stylemaster Dr. Union, MO Phone: (636) Fax: (636) AS-GMO-RURHSBULKGRAINTC PI GB Finalized 1 Oct