agrastrip ll bulk grain strip test qualitative method ain...©2013 romer labs®, inc. all rights...

©2013 Romer Labs®, Inc. All Rights Reserved. This document is the property of Romer Labs®, Inc.

AS-GMO-LLBULKGRAINTC-7000043-PI-000397-6-GB Finalized 06 September 2013 1

AgraStrip® LL Bulk

Grain Strip Test –

Qualitative Method

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Order Number: 7000043

Intended Use

The intended use of the kit is the qualitative (yes/no) determination of the PAT

protein in corn, canola, sugarbeet, and soybean (LL55 only) grain samples. The AgraStrip® LL Bulk Grain Strip Test - Trait test strip has a detection limit of one

(1) LibertyLink (PAT) corn kernel in 111 (0.9%) non-LL corn kernels or sugarbeet

seeds, one (1) LL55 soybean seed in 200 non-LL55 soybean seeds, and one (1)

LibertyLink (PAT) canola seed in 50 non-LL canola seeds .

Product Description

The AgraStrip® LL Bulk Grain Strip Test - Trait detects the PAT protein produced

by genes derived from Streptomyces hygroscopicus or S. viridochromogenes.

These genes have been incorporated into herbicide-resistant corn, cotton canola

and other crops including LibertyLink® brands from Bayer CropScience and other

companies. The test will detect both the PAT proteins produced by both the pat

and bar genes found in Liberty® resistant brands (LibertyLink®, StarLink®, Bt-

Xtra™, etc.) . The lateral flow strips and other kit components are sufficient to

detect the presence or absence of the PAT protein in both field and laboratory

environments. Different application protocols are required for leaf, seed and bulk grain detection. This product can screen LibertyLink (T25) corn, LibertyLink

(Invigor) canola, and LibertyLink soybean (LL55 ) grain at specified levels.

Principle of the Assay

The assay uses a double antibody sandwich format. Antibodies specific to the PAT 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 PAT 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 PAT 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® LL Bulk Grain Strip Test- Trait 100

Conical microtubes, 1.5mL (COOLS2005) 100 Transfer pipettes (COOLS2010) 100



Romer Labs Methods

Materials Required but not Supplied:

Trait Sample Buffer Concentrate (for sugarbeet seed testing) (7000006)

Conical microtube rack (EQOLE1215)

Timer (EQOLE1301)

Graduated Cylinder, 50mL (EQOLE1051)

Balance, 400g (EQOLE1010)

Blender (110V: EQOLE1020; 220V: EQOLE1027)

Blender blade with gasket set (EQOLE1026)

Storage and Preparation of Reagents

The AgraStrip® LL Strip Test Bulk Grain - Trait can be stored at room temperature or

refrigerated (2 - 25ºC). Do not freeze. Once opened, the AgraStrip® LL Strip Test Bulk

Grain - 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 Sugarbeet Seed Testing:

The Trait Sample Buffer is shipped as a concentrate. Follow the procedure below to

prepare the sugarbeet seed sample buffer.

1. Pour the contents of a one (1) liter bottle of Trait Sample Buffer

Concentrate (P/N 7000006) into a 10-liter cubitainer or other suitable

container.

2. Add four (4) liters of water to the sample buffer.

3. Mix well and label as Trait Sample Buffer. Label buffer expiration as thirty

(3) days from date of preparation. Store at room temperature.

Purpose

The AgraStrip® LL Bulk Grain Strip Test - Trait Test Kit has been designed to screen

bulk grain samples for the presence of the PAT 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® LL Bulk Grain Strip Test - Trait 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® LL Bulk Grain Strip Test - Trait will depend on

the percent LL screening level desired and an acceptable level of risk that the Cry1A

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.

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.



Romer Labs Methods

Sample Preparation: Weighing the Sample

The statistical sampling plan (see Principle of the Screening Application) is dependent on the number of seeds used. However, it is more practical for routine

testing to weigh seeds instead of counting to obtain the desired number of seeds. The

average weight of the seeds depends on the variety and environmental conditions.

It is recommended that the weight-to-seed ratio for each variety be determined as

follows:

1. Count 100seeds 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 sub-

sample sizes to be used.

Example: One hundred (100) corn kernels of Variety X weigh 25.00 grams. Each 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-Kernel Ratio

Grams per Sample of X

No. Corn Kernels 40 50 70 100

Sample Weight (g) 10 15 17.5 25

This average weight is then used to obtain the number of corn kernels for this corn

variety.

Sample Preparation – Corn

The samples 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 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)

Sugarbeet Seeds: Sample Weight (G) X 5.00 = Water Volume (mL)

Table B: Parameters for Preparing Samples

Number of Seeds in

Sample Jar Size (oz.) Grind Time (sec)

25 – 125 4 10 – 20

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.



Romer Labs Methods

Sample Processing – Corn, Soybean, and Sugarbeet

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 water (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 10-20

sec.)

Corn: Sample Weight (g) X 1.25 = Water Volume (mL)

Sugarbeet Seeds: Sample Weight (g) X 5.00 = Buffer Volume (mL)

Soybean: Sample Weight (g) X 6.00 = 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.

Sample Processing - Canola

1. Weigh 50-seed sub-samples from each truck or container.

2. Place each sub-sample in a clean, dry plastic bag.

3. Place the bag on a hard surface and grind the dry seeds by pounding the bag with

the rubber mallet (or pestle) until all the seeds are broken. Note: The seed coats

will not be fully ground.

4. Add 1.0mL of water and mix the seed sample with the mallet until homogeneous

slurry is formed (approx. 1 min). See example below. 5. Use this free liquid as sample in the Test Procedure.


after a short settling time. There should be no whole seeds remaining. Alternative

methods of grinding the seed may also be used as long as all seeds are broken open

and relatively homogeneous slurry is formed.



Romer Labs Methods

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® LL 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.

A red line in this position indicates that the strip is functioning properly. A red line

appearing below the Control Line is the LL Test Line and indicates a positive result for

PAT protein. If the test strip displays two (2) red lines, the test is complete and the

sample is positive for the LL trait. If at 5 minutes the test strip only shows a clearly

visible Control Line, then the sample is negative for the LL 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 30 minutes are invalid.

Illustration of Lateral Flow Strips

Example of an unreacted, negative (1-line) and positive (2-lines) test strip

The sample tube has a 0.5-mL

indicator at the top of the tapered

section.



Romer Labs Methods

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 30 minutes of test completion.

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 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.

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® LL Bulk Grain Strip Test - Trait sensitivity is at least one

LL corn kernel or sugarbeet seed in 111 (0.9%), and one LL canola seed in 50

(2%).

The AgraStrip® LL Bulk Grain Strip Test - Trait provides a yes/no answer for the

presence or absence of LL seed in a given sample. Testing multiple statistically selected

sub-samples allows an estimate of the percent of LL seed. The test results provide

information about the probability of the percent LL seed in the sample.

Note: The test protocol does not determine the exact percent of LL seeds. 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 (40 kernels, 60 kernels, or 70 kernels 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.



Romer Labs Methods

Table C: 40 Seed Sub-Samples (All Sub-Samples Must be Negative)

No. of Sub-

Samples of

40 Seeds

Each

Maximum Percent LL Corn Kernels, Using Sub-Sample Sizes of 40

Seeds at Five Different Confidence Levels (%)

50 75 90 95 99

1 1.7 3.4 5.6 7.2 10.9

2 0.86 1.7 2.8 3.7 5.6

3 0.58 1.1 1.9 2.5 3.7

4 0.43 0.86 1.4 1.9 2.8

5 0.35 0.69 1.1 1.5 2.3

6 0.29 0.58 0.95 1.2 1.9

7 0.25 0.49 0.82 1.1 1.6

8 0.22 0.43 0.72 0.93 1.4

Table D: 60 Seed Sub-Samples (All Sub-Samples Must be Negative)

No. of Sub-

Samples of

60 Kernels

Each

Maximum Percent LL Corn, Using Sub-Sample Sizes of 60 Seeds at Five

Different Confidence Levels (%)

50 75 90 95 99

1 1.1 2.3 3.8 4.9 7.4

2 0.58 1.1 1.9 2.5 3.8

3 0.38 0.77 1.3 1.7 2.5

4 0.29 0.58 0.95 1.2 1.9

5 0.23 0.46 0.76 0.99 1.5

6 0.19 0.38 0.64 0.83 1.3

7 0.16 0.33 0.55 0.72 1.1

8 0.14 0.29 0.48 0.62 0.95

Table E: 70 Seed Sub-Samples (All Sub-Samples Must be Negative)

No. of Sub-

Samples of

70 Kernels

Each

Maximum Percent LL Corn, Using Sub-Sample Sizes of 70 Kernels at

Five Different Confidence Levels (%)

50 75 90 95 99

1 0.99 2.0 3.2 4.2 6.4

2 0.49 0.99 1.6 2.1 3.2

3 0.33 0.66 1.1 1.4 2.2

4 0.25 0.49 0.82 1.1 1.6

5 0.20 0.40 0.6 0.85 1.3

6 0.16 0.33 0.55 0.71 1.1

7 0.14 0.28 0.47 0.61 0.94

8 0.12 0.25 0.41 0.53 0.82



Romer Labs Methods

Table F: 50 Seed Sub-Samples (Canola) (All Sub-Samples Must be Negative)

No. of Sub-

Samples of

50 Seeds

Each

Maximum Percent GM, Using Sub-Sample Sizes of 50 Seeds at Five

Different Confidence Levels (%)

50 75 90 95 99

1 1.19 2.77 4.60 6.00 9.20

2 0.69 1.40 2.20 3.00 5.00

3 0.46 0.92 1.53 2.00 3.00

4 0.35 0.69 1.15 1.50 2.30

5 0.28 0.56 0.92 1.20 1.80

6 0.23 0.46 0.77 1.00 1.50

*All values determined using SeedCalc5



Romer Labs Methods

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AgraStrip® LL Bulk Grain

Strip Test – Semi-Quantitative

Method Part Number 7000043

Intended Use

The intended use of the kit is the semi-quantitative determination of the PAT protein in corn and canola grain samples. The AgraStrip® LL Bulk Grain Strip Test -

Trait test strip has a detection limit of one (1) LibertyLink (PAT) corn kernel or

canola seed in 200 (0.5%) non-LL corn kernels or canola seeds.

Product Description

The AgraStrip® LL Bulk Grain Strip Test - Trait detects the PAT protein produced

by genes derived from Streptomyces hygroscopicus or S. viridochromogenes.

These genes have been incorporated into herbicide-resistant corn, cotton canola

and other crops including LibertyLink® brands from Bayer CropScience and other companies. The test will detect both the PAT proteins produced by both the pat

and bar genes found in Liberty® resistant brands (LibertyLink®, StarLink®, Bt-

Xtra™, etc.) . The lateral flow strips and other kit components are sufficient to

detect the presence or absence of the PAT protein in both field and laboratory

environments. This product is validated for the semiquantitative analysis of LL corn

grain at specified levels.

Principle of the Assay

The assay uses a double antibody sandwich format. Antibodies specific to the PAT

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 PAT 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 PAT 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® LL Bulk Grain Strip Test- Trait 100

Conical microtubes, 1.5mL (COOLS2005) 100

Transfer pipettes (COOLS2010) 100



Romer Labs Methods

Materials Required but not Supplied:

Semi-Quantitative Extraction Buffer (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)

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® LL Strip Test Bulk Grain - Trait can be stored at room temperature or

refrigerated (2 - 25ºC). Do not freeze. Once opened, the AgraStrip® LL Strip Test Bulk Grain - 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 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® LL Bulk Grain Strip Test - Trait Test Kit has been designed to screen

bulk grain samples for the presence of the PAT 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® LL Bulk Grain Strip Test - Trait 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® LL Bulk Grain Strip Test - Trait will depend on

the percent LL screening level desired and an acceptable level of risk that the Cry1A

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.



Romer Labs Methods

Sample Preparation

The samples 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 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 Seeds in

Sample Jar Size (oz.) Grind Time (sec)

Corn: 25 – 125 4 10 – 20

Corn: 100 – 200 8 15 – 25

Corn: 200 – 300 16 20 - 35

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.

6. Add the volume of water (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 10-20 sec.)

Corn: Sample Weight (g) X 2.0 = Buffer Volume (mL)

Soybean: Sample Weight (g) X 4.0 = Buffer Volume (mL)

Canola: Sample Weight (g) X 3.0 = Buffer Volume (mL)


after a short settling time. There should be no whole kernels remaining.

7. Use this free liquid as sample in the Test Procedure.



Romer Labs Methods

Test Procedure – Semi-Quantitative Bulk Grain Corn

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® LL Bulk Grain Strip Test - Trait Test Strip into the sample

tube. Let sit for 5 minutes. For canola, let sit for 10 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 faces 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. 4%). 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 30 minutes are invalid.

The sample tube has a 0.5-mL

indicator at the top of the tapered

section.



Romer Labs Methods

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

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.

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

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