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AS-GMO-RURHSBULKGRAINTC-7000011-PI-000387-9-GB Finalized 28 July 2014 1
AgraStrip®
RUR-HS Bulk Grain
Strip Test –
Qualitative Method
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Order Number: 7000011
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, and alfalfa hay 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. It has a detection limit of 5% in alfalfa hay (ground or
cored). 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.
AS-GMO-RURHSBULKGRAINTC-7000011-PI-000387-9-GB Finalized 30 July 2014 4
Romer Labs Methods
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 10-20
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 10.00 = Water Volume (mL)
For Alfalfa Hay (Ground): Sample Weight (g) X 20.00 = Water Volume (mL)
(i.e. – use 5g/100mL)
For Alfalfa Hay (Cored): Sample Weight (g) X 15.00 = Water Volume (mL)
(i.e. – use 5g/75mL)
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.
3. Place one AgraStrip® RUR-HS 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.
AS-GMO-RURHSBULKGRAINTC-7000011-PI-000387-9-GB Finalized 30 July 2014 5
Romer Labs Methods
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.
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
AS-GMO-RURHSBULKGRAINTC-7000011-PI-000387-9-GB Finalized 30 July 2014 9
Romer Labs Methods
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AgraStrip® RUR-HS Bulk Grain
Strip Test – Semi-Quantitative
Method Part Number 7000011 Intended Use
The intended use of the kit is the semi-quantitative etermination of the CP4 EPSPS
protein in Roundup Ready® bulk corn and canola grain samples. The AgraStrip®
RUR-HS Bulk Grain Strip Test - Trait has a detection limit of one (1) Roundup
Ready® (CP4 EPSPS) corn kernel or canola seed in 1000 non-RUR corn kernels or
canola seeds in 5 minutes. Note: This application is intended for the
detection of NK603 Roundup Ready® corn (RUR NK603) and Roundup
Ready® canola. 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. This product application can semi-quantitatively screen for CP4
EPSPS grain.
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. The intensity of
the second line can be used to semi-quantitatively measure the level of CP4 EPSPS
AS-GMO-RURHSBULKGRAINTC-7000011-PI-000387-9-GB Finalized 30 July 2014 10
Romer Labs Methods
Materials Required but not Supplied:
Semi-Quantitative Extraction Buffer (7000103, makes 1L; 700104, 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)
Conical microtube rack (EQOLE1215)
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 Tray (EQASR1023)
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.
For Semi-Quantitative Bulk Grain Corn and Canola Testing:
If the semi-quantitative bulk corn grain method is to be run, 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.
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.
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
AS-GMO-RURHSBULKGRAINTC-7000011-PI-000387-9-GB Finalized 30 July 2014 11
Romer Labs Methods
Sample Preparation
The sample is ground and then extracted with 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 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 2.0 = Buffer Volume (mL)
Canola: Sample Weight (g) x 3.0 = Buffer Volume (mL)
Table B: Parameters for Preparing Samples
Number of Kernels in
Sample Jar Size (oz.) Grind Time (sec)
Corn: 25 – 125 4 10 – 20
Corn: 125 – 250 8 15 – 25
Corn: 250 – 500 16 20 – 35
Corn: > 500 32 45 – 60
Canola: 1000 4 10 – 15
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.
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.
canola). If a coffee grinder is used, transfer the sample to a clean container
for extraction.
6. Add the volume of buffer (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 10-20
sec.)
For Corn: Sample Weight (g) X 2.0 = Buffer Volume (mL)
For Canola: Sample Weight (g) X 3.0 = 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 seeds remaining.
7. Use this free liquid as sample in the Test Procedure.
AS-GMO-RURHSBULKGRAINTC-7000011-PI-000387-9-GB Finalized 30 July 2014 12
Romer Labs Methods
Test Procedure – Semi-Quantitative Bulk Grain Corn and Canola
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.
3. Place one AgraStrip® RUR-HS 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.
6. Cut off the bottom, colored portion of the strip using scissors.
Discard this portion.
7. Place the top white-colored strip into the AgraVision Reader for
semi-quantitative analysis. The cut end of the strip points towards the
number printed on the tray.
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. A red line in this position indicates that the strip is functioning properly. The strip
must be left in the extract for the entire 5 minute incubation period if it is to be read
for semi-quantitative analysis. For semi-quantitative analysis, insert the strip into the
AgraVision Reader and read. The AgraVision Reader will display the result as a range
of %GMO (i.e. <0.5%, 0.1 – 0.5%, 0.5 – 1.0%, 1.0 – 5.0%, or >5%). 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
AS-GMO-RURHSBULKGRAINTC-7000011-PI-000387-9-GB Finalized 30 July 2014 13
Romer Labs Methods
Equipment Cleaning and Drying
Caution: It is important to clean and dry the jars and cutting blades and/or coffee
grinder 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
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.
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