SEMI-QUANTITATIVE Low-LOD CORN COMB BULK GRAIN AgraStrip Semi-Quantitative Low-LOD Corn Comb Order Number: 7880621 Semi-Quantitative Procedure to Detect CP4 EPSPS, Cry1, Cry3Bb, Cry1F, PAT, Cry34Ab1 and/or VIP3A Proteins in Corn Grain Using Multi-Trait Combs Application Description Transgenic products have been developed for insect-resistant corn including YieldGard, YieldGard Rootworm, Genuity, Herculex I, and Herculex RW brands, and herbicide-resistant corn including the Roundup Ready brand and LibertyLink. The intended use of this application is the semi-quantitative determination of transgenic protein(s) in corn grain samples using a single 800-kernel sample extracted with water. This application is based on the USDA GIPSA Directive 9181.2 which recommends 2400 kernel sub-samples be screened for qualitative detection (e.g. 3 sub-samples of 800 kernels). This application can screen for Genuity VT Triple Pro (CP4 EPSPS, Cry1A.105, Cry3Bb1), Herculex I (Cry1F), LibertyLink (PAT), or Herculex RW/XTRA, (Cry34Ab1) corn grain at specified levels. The strip(s) will also detect YieldGard (Cry1Ab), YieldGard Rootworm (Cry3Bb1), and Roundup Ready (CP4 EPSPS). Contents of Kit Quantity AgraStrip SemiQuantitative Corn Comb Bulk Grain Strip Test- Trait 20 combs Sample Cups 20 Transfer pipettes (COOLS2010) 20 Note: The AgraStrip Semi-Quantitative Corn Comb Bulk Grain Strip Test Trait kit contains 7 test strips to detect the following transgenic proteins: Protein Detected CP4 EPSPS (blue strip) Cry1A.105) (green strip) Cry3Bb (silver strip) Cry1F (orange strip) PAT (purple strip) Cry34Ab1 (black strip) VIP3A (red strip on end) GMO Variety Genuity VT Triple Pro (MON88017 x MON89034) Genuity VT Triple Pro (MON88017 x MON89034) Genuity VT Triple Pro (MON88017 x MON89034) Herculex I TC1507 LibertyLink T25 Herculex RW DAS-59122-7 Detection Level # GM Kernels in 800 0.1% 1 0.5% 4 0.25% 2 0.25% 2 0.5% 4 0.1% 1 AgriSure Viptera TM 3110 0.1% 1 Finalized 25 April 2014 1
Materials Required but not Supplied: SemiQuantitative GMO Buffer Concentrate (7000103, makes 1L; 7000104, makes 4L, or 7000105, makes 20L). 1L Nalgene bottle, for use with 7000103 (COSST5080) 4L Cubitainer, for use with 7000104 (COSST5081) 20L Cubitainer, for use with 7000105 (COSST5096) Timer (EQOLE1301) Graduated Cylinder, 50mL (EQOLE1051) Balance, 400g (EQOLE1010) Blender (110V: EQOLE1020; 220V: EQOLE1027) Blender blade with gasket set (EQOLE1026) AgraVision Reader (EQASR1007) and GMO Comb Tray (EQASR1033) Storage and Preparation of Reagents The AgraStrip Semi-Quantitative Corn Comb Bulk Grain Strip Test - Trait can be stored at room temperature or refrigerated (2-25ºC). Do not freeze. Once opened, the AgraStrip Semi-Quantitative Corn Comb Bulk Grain Strip Test Bulk Grain - Trait lateral flow test strips must be stored in the closed foil bag with desiccant. The desiccant must be blue in color. If the desiccant is pink, contact Romer Labs, Inc. Technical Service. Storage conditions higher than room temperature may adversely affect performance. Preparation of Semi-Quantitative Extraction Buffer: The Semi-Quantitative extraction buffer should be prepared according to the instructions on the bottle. For 7000103, the contents of the bottle are added to 1L of water in a 1L Nalgene bottle. For 7000104, the contents of the bottle are added to 4L of water in a 4L Cubitainer. For 7000105, the contents of the bottle are added to 20L of water in a 20L Cubitainer. Sampling The samples used for the AgraStrip Semi-Quantitative Corn Comb Bulk Grain Strip Test - Trait 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 Semi-Quantitative Corn Comb Bulk Grain Strip Test - Trait will depend on the percent GMO screening level desired and an acceptable level of risk that the GMO level is close to the screening level. 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 The corn sample is ground and then extracted with Semi-Quantitative GMO Buffer in a polypropylene Mason -type jar. The sample preparation is important for the proper function of the test, especially the ratio of buffer to the weight of the corn sample. The volume of buffer in milliliters should be 2.0 times the weight of the corn sample in grams (g). Corn: Sample Weight (g) x 2.0 = Buffer Volume (ml) Finalized 25 April 2014 2
The size of the Mason jar required and the grinding time depends on the sample size to be analyzed. For 800-kernel samples, a 32-oz. Mason jar is used. 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. 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. 6. Add the volume of semiquantitative GMO buffer (see below) to the ground corn in the jar, place a lid on the jar and shake the jar until all the ground corn is well wetted (about 15 sec.) Sample Weight (g) X 2.0 = Buffer 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 kernels remaining. 7. Use this free liquid as sample in the Test Procedure. Test Procedure 1. Transfer ~10mL of the liquid from each sample prepared above into separate sample cups. Note: This is approximately ¼ fill volume of the sample cup. 2. For each sub-samle, place one AgraStrip Semi-quantitative Corn Comb - Trait Test Comb into the sample cup. Note: The sample liquid level in the cup should be between the arrows and the strip name on the filter cover. 3. Let the strip comb sit for 5 minutes. 4. The appearance of one line (control) on the strip indicates a negative result. 5. The appearance of two lines or more on the strip indicates a positive result. 6. Cut off the colored section at the bottom of each strip on the comb. 7. Insert the strips from the comb into the AgraVision Reader. A maximum of 4 strips can be read at once. The comb must be cut in half on the dotted line (between the Cry1F and LL strips) to allow reading in the AgraVision reader. Note that the right half (3 strips) includes a blank spacer on the card to aid in positioning in the tray. 8. Select the respective methods for each half of the comb from the menu on the AgraVision reader. These are listed as Low LOD Corn Comb 1 of 2 and Low LOD Corn Comb 2 of 2. Each method is for the respectively-labeled section of the comb (RUR strip starts 1 of 2; LL strip starts 2 of 2). 9. Results will appear in semi-quantitative ranges (i.e. <0.1%, 0.1 0.5%, 0.5 1.0%, 1.0-5.0%, >5%). Finalized 25 April 2014 3
Interpreting the Lateral Flow Strip Test Check the result window at five (5) minutes after inserting the strip. At least one line, the Control Line, should always develop approximately one (1) cm down from the Reservoir Pad. A red line in this position indicates that the device is functioning properly. A red line appearing below the Control Line is the Test Line and indicates a positive result. If the test strip displays two (2) red lines, the test is complete and the sample is positive for GMO corn. If at 5 minutes the test strip only shows a clearly visible Control Line, then the sample is negative for GMO corn. Use the AgraVision Reader to Semi-Quantitatively interpret the results. Note: Test strip results should be interpreted after 5 minutes. Test strips interpreted after 10 minutes are invalid. Limit of Detection and Quantitation Range: Strip LOD Range of Test CP4 EPSPS 0.1% 0.1 5.0% Bt-Cry1A.105 0.5% 0.5 5.0% Bt-Cry3Bb1 0.25% 0.25 5.0% Bt-Cry1F 0.25% 0.25 5.0% PAT 0.5% 0.5 5.0% Bt-Cry34Ab1 0.1% 0.1 5.0% VIP3A 0.1% 0.1 5.0% Illustration of Lateral Flow Strips Cut Here Finalized 25 April 2014 4
Example of an unreacted, negative (1- line) and positive (2-lines) test strip Equipment Cleaning and Drying Caution: It is important to clean and dry the jars and cutting blades between samples. 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 corn 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. Finalized 25 April 2014 5
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. 1301 Stylemaster Dr. Union, MO 63084 Phone: (636) 583-8600 Fax: (636) 583-2340 Finalized 25 April 2014 6