WO2001044779A2 - Method of processing and testing powdered samples using immunochromatographic strip tests - Google Patents
Method of processing and testing powdered samples using immunochromatographic strip tests Download PDFInfo
- Publication number
- WO2001044779A2 WO2001044779A2 PCT/US2000/033774 US0033774W WO0144779A2 WO 2001044779 A2 WO2001044779 A2 WO 2001044779A2 US 0033774 W US0033774 W US 0033774W WO 0144779 A2 WO0144779 A2 WO 0144779A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sample
- com
- strip
- test
- protein
- Prior art date
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/32—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Bacillus (G)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
Definitions
- Field of the Invention This relates to the field of immunology and more specifically relates to a method for the analysis of a powdered sample.
- Immunoassays have many of these characteristics.
- One type of immunoassay referred to as an immunochromatographic or lateral flow strip test, has been successfully developed into 'one-step' tests and employed for on-site analysis.
- the over-the-counter home pregnancy test is an example of a simple, one-step immunochromatographic strip test.
- Immunoassays have been used to detect substances in many different kinds of samples in many different markets, including the agriculture market. Modern biotechnology methods are being used to genetically modify plants. These genetically modified plants, and the seeds, grain and food derived from them all may contain novel or recombinant proteins. It is important to determine the presence of such proteins for regulatory, environmental, safety, and world trade issues. Very large amounts of grain and seed are harvested, transported, mixed, stored, distributed and traded throughout the world, and it is important to have tests that can detect these novel proteins in order to address these issues. It is desirable to have a rapid, simple and inexpensive method that can be used to test grain samples at many points along the distribution channel including trucks, elevators, barges, ships, etc. A strip test is ideally suited for this purpose. For example, recently, an immunochromatographic strip test (strip test or lateral flow device, LFD) that detects a specific protein in genetically modified soybeans was developed.
- strip test strip test or lateral flow device, LFD
- Strip tests are comprised of multiple porous components, membranes and filters, through which liquid sample is drawn by capillary action. Analyte in the sample reacts with the test reagents contained within the test strip as it traverses the length of the strip.
- an analyte such as a protein or mycotoxin
- grain or seed e.g., corn, soybean, rice, wheat, etc.
- What is needed is a means for preparing and testing a finely ground powdered sample that maximizes extraction efficiency, sensitivity, speed and reliability of the strip test while minimizing the complexity and cost.
- a method and kit for processing and testing powdered samples are provided herein.
- the powdered sample is combined with a fluid to produce a slurry or liquid suspension, and the slurry is contacted with an immunochromatographic test strip containing detectable reagents that are immunoreactive with an analyte to be detected in the sample.
- the method is particularly useful for the analysis of agricultural products, crops, or food fractions, such as the identification of recombinant grains.
- Figure 1 is a table showing chromatographic strip test results for the analysis of corn kernels using the method described herein.
- the sample is provided in powdered form or is processed from a solid or semi-solid sample such as a slice, granule, seed, grain, particle, or the like, to produce a powder.
- the powdered sample is then mixed with a fluid, such as an aqueous solution, to form a slurry or liquid suspension.
- the slurry is then contacted with an immunochromatographic strip.
- the method and kit are useful for the detection of a wide variety of analytes including, but not limited to, agricultural products, biological and environmental contaminants or additives, industrial chemicals, toxins (particularly mycotoxins), and biological analytes, such as antigenic determinants of proteins, polysaccharides, glycoproteins, lipoproteins, nucleic acids and hormones, of organisms such as viruses, bacteria, fungi, parasites, plants and animals, including large and small molecules, polymers and haptens.
- the method is particularly useful for the identification or quantification of proteins or peptides in genetically modified plants or recombinant agricultural products, such as grains.
- the method is useful for the detection of the Bacillus thuringiensis CrylAb or Cry 1 Ac proteins in genetically modified plants, such as corn, as described below in the examples. Definitions
- analyte refers to a drug, hormone, chemical, toxin, compound, receptor, nucleic acid molecule or other molecule and fragments thereof to be measured by the method described herein.
- sample is collected or obtained using methods well known to those skilled in the art.
- the sample containing the analyte to be detected may be obtained from any biological or environmental source.
- the sample may be any plant tissue or extract including root, stem, leaf, or seed.
- the sample may be diluted, purified, concentrated, filtered, or otherwise manipulated prior to being mixed with the fluid to create a slurry or liquid suspension to optimize the assay results.
- Analytes to be detected using the immunoassay method described herein include, but are not limited to, the following analytes: molecules, such as organic and inorganic molecules, peptides, proteins, glycoproteins, carbohydrates, nucleic acids, lipids, toxins, and the like. Analytes also include but are not limited to neuro transmitters, hormones, growth factors, antineoplastic agents, cytokines, monokines, lymphokines, nutrients, enzymes, receptors, antibacterial agents, antiviral agents, and antifungal agents.
- molecules such as organic and inorganic molecules, peptides, proteins, glycoproteins, carbohydrates, nucleic acids, lipids, toxins, and the like.
- Analytes also include but are not limited to neuro transmitters, hormones, growth factors, antineoplastic agents, cytokines, monokines, lymphokines, nutrients, enzymes, receptors, antibacterial agents, antiviral agents, and antifungal agents.
- analyte also means detectable components of structured elements such as cells, including all animal and plant cells, and microorganisms, such as fungi, viruses, bacteria including but not limited to all gram positive and gram negative bacteria, and protozoa.
- structured elements such as cells, including all animal and plant cells, and microorganisms, such as fungi, viruses, bacteria including but not limited to all gram positive and gram negative bacteria, and protozoa.
- microorganisms such as fungi, viruses, bacteria including but not limited to all gram positive and gram negative bacteria, and protozoa.
- analyte also means detectable components of organelles and cells.
- the sample is ground, homogenized, chopped, blended, or otherwise manipulated to create a powder.
- the size and texture of the powder or fine granules can be adjusted by modifying the technique or increasing the amount of manipulation time, such as grinding or blending, as needed.
- the fluid with which the powdered sample is combined or mixed is one capable of forming a slurry or liquid suspension.
- the preferred fluid is an aqueous solution, such as a buffer that is compatible with the immunochromatographic strip.
- the immunochromatographic strip or detection membrane, is a membrane or strip having reagents deposited in zones along the longitudinal length of the membrane. Immunochromatographic strips are readily available from commercial suppliers or can be customized by laboratory personnel skilled in the art, or a commercial immunodiagnostic supplier, to include immunoreagents specific for the analyte to be detected.
- the immunoassay reactions conducted on the strip may be in competitive or sandwich immunoassay formats
- the reagents suspended or immobilized on the membrane provide means for detecting analyte, preferably by visually detecting a labeled substance or substances, such as colloidal gold, that have been bound to analyte.
- the reagents may detect the absence of labeled substance, and the label may be detected using instrumentation know to those skilled in the art such as a spectrophotometer or fluorescence detector.
- the reagents on the membrane may be immobilized or may be diffusible but contained on the membrane in a solid or semi -solid state that, when contacted with the sample, becomes mobile and moves with the sample toward the distal end of the membrane.
- Reagents may also be included on the immunochromatographic strip to enhance or clarify the signal produced by the label being detected. Additional reagents are optionally incorporated in zones on the detection membrane for calibration.
- the membrane is preferably a non-woven substrate upon which the reagents can be immobilized or deposited, and which is capable of conveying sample in a fluid flow direction generally parallel to the longitudinal length of the chromatographic strip.
- Desirable chromatographic strips are composed of a fluid- conducting material including, but not limited to, nylon, polyethylene, glass fiber, nitrocellulose, cellulose, and other common membrane matrices or bibulous materials.
- the preferred chromatographic strip is composed of nitrocellulose.
- the membrane of the chromatographic strip is optionally backed with, or laminated to, another material. Desirable backing or laminating material is polyethylene or vinyl, although other suitable materials known in the art may be used.
- a reagent sink is optionally included at the distal end of the immunochromatographic membrane or strip for enhancing the flow of the fluids, including reagents and sample, along the longitudinal length of the strip.
- the sink may be composed of an absorbent material, such as blotting paper.
- an end of test indicator for indicating completion of the assay.
- the end of test indicator may be included in the reagent sink described above or may be a separate component located at the distal end of the immunochromatographic strip or reagent sink, if present.
- the end of test indicator is preferably composed of an absorbent material containing an indicator, or dye, that travels with the liquid to indicate that the liquid has traveled to the distal end of the strip and that the strip is ready for analysis. Analysis can be conducted either by visual inspection or with the aid of instrumentation. Preferably, movement of the dye through the absorbent material to a predetermined location is visually detectable.
- An immunoassay kit for the detection of analyte in a sample contains a fluid, such as a buffer to be combined with the powdered sample to form a slurry or liquid suspension, and an immunochromatographic strip or membrane.
- the kit may optionally contain one or more antibodies or other reagents to combine with the sample prior to contacting the sample slurry with the membrane.
- the kit may additionally contain additional reagents or buffers, equipment for obtaining or collecting the sample, a vessel for containing the sample and reagents, a timing means, and a colorimeter, reflectometer, or standard against which a color change may be measured.
- additional reagents or buffers equipment for obtaining or collecting the sample
- a vessel for containing the sample and reagents a timing means
- a colorimeter, reflectometer or standard against which a color change may be measured.
- a simple, inexpensive reflectometer is preferred.
- the reagents, including any antibody reagents may be lyophilized, in a single vessel or in individual vessels. Addition of the slurry sample to the vessel results in solubilization of the lyophilized reagents, causing them to react.
- This example illustrates the use of the method described herein to detect the CrylAb protein in a genetically modified, insect-resistant corn sample.
- a 70 mm blender base and blade assembly (Eberbach Co., Ann Arbor, MI) was attached.
- the MASONTM jar was placed onto the laboratory grade blender (Waring Model 31 BL91 ) and covered with a safety shield. The sample was ground on high speed for 30 seconds to produce powdered corn. The safety shield, adapter and blade were removed.
- MASONTM jar For the test tube procedure, 60 kernel samples were combined with 18.0 mL of buffer. The 125 kernel samples required 40.0 mL of buffer to produce a slurry or liquid suspension. For samples run directly in the MASONTM jar, 60 kernel samples used 16.0 mL of buffer, and 125 kernel samples required only 31.0 mL of buffer.
- the MASONTM jar lid was attached to the MASONTM jar and the contents shaken for approximately 30 seconds, until all the ground com was evenly and thoroughly wet. For samples run in the test tube, the MASONTM jar lid was removed, and the sample allowed to settle approximately 30 seconds. A 500 ⁇ L aliquot of extract was pipetted off and placed in a test tube. A chromatographic test strip, upon which was immobilized antibody having immunospecificity for the Cryl Ab protein of Bacillus thuringiensis, was inserted into the extract until it reached the bottom of the test tube.
- the MASONTM jar lid was removed and the chromatographic strip inserted into the com mixture. The strip was immersed until the check mark ( f) on the filter cover was reached (approximately 16 mm).
- Each strip was allowed to run for 10 minutes at room temperature. Various readings were taken, including time until control line was visible, time until test line was visible, times for control and test, readings at 3 minutes and 10 minutes, and time until the reagents reached the sink.
- kits are used to detect the CrylAb protein produced by a gene derived from Bacillus thuringiensis. This gene has been incorporated into insect-resistant com including YIELDGARDTM brands from both Monsanto and Novartis.
- the intended uses of the kits include the qualitative (yes/no) determination of the Cryl Ab protein in plant leaf and seed tissue and com grain samples.
- the lateral flow strips and other components provided in the kit are sufficient to make qualitative determinations for the presence of absence of the CrylAb protein in both field and laboratory' environments. Different application protocols are required for leal " , seed and bulk grain determinations.
- Principle of the Assay The assay uses a double antibody sandwich format.
- Antibodies specific to the CrylAb protein are coupled to a color reagent and incorporated into the lateral flow strip.
- the lateral flow strip When the lateral flow strip is placed in a small amount of an extract from plant tissue that contains CrylAb 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 zone, one captures the bound CrylAb protein and the other captures unreacted antibodies coupled to the 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 100 Bacillus thuringiensis (Btl) chromatographic Test Strips
- TraitsTM also referred to as "Traitcheck”
- sample buffer (Strategic
- a shield should be used over the blender jars while grinding. Safety glasses should be used.
- TraitVTM sample extraction buffer for grain is shipped as a concentrate.
- the Trait- ⁇ /TM Com Sample Buffer may be stored at room temperature.
- Btl Com Grain Test Kit User Guide
- the Lateral Flow Test Kit should be stored at room temperature.
- the Btl Test Strips must be kept in the foil pouch with desiccant. Storage conditions higher than room temperature may adversely affect performance. Sampling
- the samples used for the Btl Com Grain Test Kit can be sub-samples of those "representative samples" collected from trucks, railcars, barges, etc. for other tests.
- the size of the sub- samples to be used for the test will depend on the level of genetically modified com kernels at which the screening is being conducted and an acceptable level of risk that the genetically modified level is close to the screening level. The number and size of the sub-samples is discussed in more detail in the application protocol section below.
- 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.
- the table below is a guideline for the weight to com kernel number ration for seed corn.
- This average weight is then used to determine the weight of com kernels that will be used to represent the number of com kernels (for this variety) selected in the Probability Tables.
- the preparation of the sample is very important for the proper function of the test, especially the ratio of extraction buffer to the weight of the com sample.
- the following table contains recommendations for the amount of TraitVTM Com Sample Buffer for extraction based on the sample weight.
- a 4 oz MASONTM jar and a grinding time of 15 seconds on high speed is used for all the sample sizes (40, 60, 70, and 125 kernels) used in this example.
- the processing parameters were determined using the laboratory grade Waring Model 31BL91 food processor. Other food processors or extreme sized com kernels will require further validation.
- the MASONTM jar should be emptied, rinsed well with water and completely dried with a paper towel between uses.
- the cutting blades for the blender should be rinsed, wiped clean, sprayed or rinsed with methanol and dried with a paper towel between uses.
- Test Procedure 1 With a wooden spatula from the kit, transfer sufficient sample to the sample cup to fill about level full and smooth the top surface of the sample.
- top line is clearly visible then determine if the test line is visible. This may require 5-8 minutes.
- test trip contains a test strip top (reservoir pad where sample is contacted), a result window below the test strip top, and a test strip bottom (labeled filter cover).
- the control line should always develop approximately 1 cm down from the reservoir pad.
- the control line contains an antibody specifically immunoreactive with a common com protein, but not a Bt protein.
- 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, which contains monoclonal antibodies specifically immunoreactive with the Bt protein, and, if visible, indicates a positive results. If the test strip displays two red lines, the test is complete and the sample is positive for CrylAb Bt com kernels. If, after approximately eight minutes, the test strip only shows a clearly visible control line, then the sample is negative for CrylAb Bt com.
- the test strip contains immobilized immunologic (antibody) reagents that react with the recombinant Bt proteins present in genetically modified corn that has been engineered to include one or more genes for Bt proteins.
- immobilized immunologic (antibody) reagents that react with the recombinant Bt proteins present in genetically modified corn that has been engineered to include one or more genes for Bt proteins.
- the sample migrates down the chromatographic strip, it reacts with the immobilized antibodies and generates a visible, colorimetric signal, thereby providing a yes/no answer for the presence or absence of CrylAb Bt com kernels in a given sample.
- One red line at the top of the test strip indicates a negative result and two red lines, the second line lower on the test strip, indicates a positive result for the presence of
- CrylAb com kernels Testing two statistically selected sub- samples allows an estimate of the percent of CrylAb Bt com kernels. Te test results can provide information about the probability of the percent in the representative sample being within certain ranges. (Note: this test protocol will not specifically determine the percent of genetically modified com kernels.)
- the statistical model used for this method is the Poisson Probability Distribution which provides good approximations to binomial (yes/no) probabilities when the number of items tested (i.e. com kernels) is large but the probability of a positive result is expected to be small (i.e. low level of genetically modified com kernels).
- the probability of having zero genetically modified corn kernels in a random sample of a given size (number of com kernels) at a given percent genetically modified com For example, a random sub-sample of 100 com kernels selected from a larger sample at one percent genetically modified com has a 36.8% probability of containing no genetically modified com kernels. The probability of a 75 com kernel sub-sample (at one percent genetically modified) containing zero genetically modified com kernels is 47.2%.
- a single negative test result provides the information that the representative sample has a 60.7% probability of having 0.5% or less genetically modified co kernels, 36.8% probability of 1 % or less genetically modified corn kernels and 13.5% probability of having 2% or less genetically modified com kernels.
- the application protocols developed for screening com by using this test kit use two sub-samples of equal number of com kernels. The size of the two sub-samples is determined by the desired screening level and the level of risk tolerance. If screening at 1% genetically modified level or higher, a passing results is achieved if one or both sub-samples are negative for CrylAb Bt com kernels. If screening at 0.5% (or lower) the decision criterion is changed so that both sub-samples must be negative to pass the sample.
- probability tables provide guidelines for selection of the two sub-sample sizes for screening at different genetically modified (GM) com kernel levels. Probability Tables Screening at 3.0%> genetically modified (GM) com: Pass is one or two negative results
- the 0.5% screening level requires both of the two sub- samples to be negative to pass the sample.
- the recommended size of the two sub-samples is 60 com kernels.
- the two sub-samples of 60 com kernels each are tested. The test can determine if either zero corn kernels (negative) or one or more genetically modified co kernels (positive) are in each sub-sample.
- the test cannot determine if a positive sub-sample has only one genetically modified com kernel or several genetically modified com kernels.
- the "fail" criterion is when both sub-samples are positive.
- samples at the following genetically modified com kernel levels will have the indicated statistical probabilities of passing the test:
- a 60-corn kernel sub-sample for a 2% screening level has the risks associated with the above statistical probabilities. If a lower risk of accepting a "more contaminated" sample is desired, then a 75 -com kernel sub-sample can be chosen for the 2% screen.
- the use of two sub-samples is recommended for screening at all genetically modified levels.
- the recommended sub-sample size has been chosen to provide about a 50/50 probability of passing a representative sample at the screening level.
- those probabilities can be changed.
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Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002394196A CA2394196A1 (en) | 1999-12-14 | 2000-12-14 | Method of processing and testing powdered samples using immunochromatographic strip tests |
BR0016406-2A BR0016406A (en) | 1999-12-14 | 2000-12-14 | Processing and testing method of spray samples using immunochromatographic tape tests |
US10/149,866 US7354776B2 (en) | 2000-12-14 | 2000-12-14 | Method of processing and testing powdered samples using immunochromatographic strip tests |
AU29077/01A AU2907701A (en) | 1999-12-14 | 2000-12-14 | Method of processing and testing powdered samples using immunochromatographic strip tests |
EP00993440A EP1240515A4 (en) | 1999-12-14 | 2000-12-14 | Method of processing and testing powdered samples using immunochromatographic strip tests |
US11/931,383 US20080108087A1 (en) | 2000-12-14 | 2007-10-31 | Method of Processing and Testing Powdered Samples Using Immunochromatographic Strip Tests |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17099699P | 1999-12-14 | 1999-12-14 | |
US60/170,996 | 1999-12-14 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/931,383 Continuation US20080108087A1 (en) | 2000-12-14 | 2007-10-31 | Method of Processing and Testing Powdered Samples Using Immunochromatographic Strip Tests |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2001044779A2 true WO2001044779A2 (en) | 2001-06-21 |
WO2001044779A3 WO2001044779A3 (en) | 2001-11-01 |
Family
ID=22622114
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2000/033774 WO2001044779A2 (en) | 1999-12-14 | 2000-12-14 | Method of processing and testing powdered samples using immunochromatographic strip tests |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1240515A4 (en) |
AU (1) | AU2907701A (en) |
BR (1) | BR0016406A (en) |
CA (1) | CA2394196A1 (en) |
WO (1) | WO2001044779A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003102208A2 (en) * | 2002-05-31 | 2003-12-11 | Indian Council Of Agricultural Research | Rapid detection of bt-cry toxins |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5358690A (en) * | 1989-01-10 | 1994-10-25 | Lamina, Ltd. | Environmental sample collection and membrane testing device |
US5508468A (en) * | 1990-01-22 | 1996-04-16 | Dekalb Genetics Corporation | Fertile transgenic corn plants |
US5874662A (en) * | 1987-11-18 | 1999-02-23 | Mycogen Corporation | Method for producing somoclonal variant cotton plants |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2436201A (en) * | 1999-12-15 | 2001-06-25 | Strategic Diagnostics, Inc. | Reagents, methods and kits for detecting bacillus thuringiensis proteins |
-
2000
- 2000-12-14 WO PCT/US2000/033774 patent/WO2001044779A2/en not_active Application Discontinuation
- 2000-12-14 BR BR0016406-2A patent/BR0016406A/en not_active Application Discontinuation
- 2000-12-14 AU AU29077/01A patent/AU2907701A/en not_active Abandoned
- 2000-12-14 CA CA002394196A patent/CA2394196A1/en not_active Abandoned
- 2000-12-14 EP EP00993440A patent/EP1240515A4/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5874662A (en) * | 1987-11-18 | 1999-02-23 | Mycogen Corporation | Method for producing somoclonal variant cotton plants |
US5358690A (en) * | 1989-01-10 | 1994-10-25 | Lamina, Ltd. | Environmental sample collection and membrane testing device |
US5508468A (en) * | 1990-01-22 | 1996-04-16 | Dekalb Genetics Corporation | Fertile transgenic corn plants |
Non-Patent Citations (4)
Title |
---|
SAXENA ET AL.: 'Insecticidal toxin from bacillus thuringiensis is relased from roots of transgenic Bt corn in vitro and in situ' FEMS MICROBIOL. ECOLOGY vol. 33, 2000, pages 35 - 39, XP002940178 * |
SAXENA ET AL.: 'Insecticidal toxin in root exudates from Bt corn' NATURE vol. 402, 02 December 1999, page 480, XP002940179 * |
See also references of EP1240515A2 * |
WRAIGHT ET AL.: 'Absence of toxicity of bacillus thuringiensis pollen to black swallowtails under field condition' PROC. NATL. ACAD. SCI. USA vol. 97, no. 14, 05 July 2000, pages 7700 - 7703, XP002940180 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003102208A2 (en) * | 2002-05-31 | 2003-12-11 | Indian Council Of Agricultural Research | Rapid detection of bt-cry toxins |
WO2003102208A3 (en) * | 2002-05-31 | 2004-03-25 | Indian Agricultural Council | Rapid detection of bt-cry toxins |
CN100403028C (en) * | 2002-05-31 | 2008-07-16 | 印度农业研究委员会 | Rapid detection of bt-cry toxins |
Also Published As
Publication number | Publication date |
---|---|
AU2907701A (en) | 2001-06-25 |
EP1240515A4 (en) | 2004-07-14 |
WO2001044779A3 (en) | 2001-11-01 |
EP1240515A2 (en) | 2002-09-18 |
CA2394196A1 (en) | 2001-06-21 |
BR0016406A (en) | 2004-06-29 |
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