CN110133292A - A kind of colloidal gold immunochromatographimethod quick measuring card of herbicide resistant protein Bar - Google Patents
A kind of colloidal gold immunochromatographimethod quick measuring card of herbicide resistant protein Bar Download PDFInfo
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- CN110133292A CN110133292A CN201910482631.4A CN201910482631A CN110133292A CN 110133292 A CN110133292 A CN 110133292A CN 201910482631 A CN201910482631 A CN 201910482631A CN 110133292 A CN110133292 A CN 110133292A
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- colloidal gold
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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention discloses a kind of colloidal gold immunochromatographimethod quick measuring cards and its application method for transgenic paddy rice herbicide resistant protein Bar for belonging to technical field of bioengineering, the colloidal gold immunochromatographimethod quick measuring card includes the interior test strips that get stuck and get stuck, the test strips include bottom plate and adhere to the sample pad being sequentially connected on bottom plate, gold-labelled pad, reaction film and water absorption pad, and the gold-labelled pad is coated with herbicide resistant protein Bar monoclonal antibody-colloidal gold conjugate.The quick measuring card high specificity, high sensitivity, also, easy to operate, quick, accurate, the field quick detection and safety evaluatio of herbicide resistant protein Bar suitable for transgenic paddy rice and products thereof.
Description
Technical field
The present invention relates to technical field of bioengineering, and in particular to one kind is used for transgenic paddy rice herbicide resistant protein Bar
Colloidal gold immunochromatographimethod quick measuring card.
Background technique
Turn the plantation for having anti-herbicide gene crop, make to become effective management of weeds mode in field sprinkling herbicide,
Herbicide resistance is thus set to become one of the character of most advantage in agricultural production.Turn currently, Resistant Herbicide Crops account for the whole world
80% or more of gene crops.From development trend, the crop ratio of transgenosis is still being continuously increased.
The albumen of Bar gene expression can be catalyzed in the presence of acetyl coenzyme A in streptomycete (streptomyces)
Acetyl coenzyme A makes L-glufosinate-ammonium become acetyl-L-glufosinate-ammonium in conjunction with the free amine group of herbicide glufosinate-ammonium, to make weeding
Agent inactivation, assigns genetically modified crops Tolerance To Herbicides.Bar gene is widely used in base as the marker gene in genetic transformation
Because in Engineering Breeding.
While bringing tremendous economic interests with the rapid development of genetically modified crops, people also increasingly pay close attention to transgenosis
The possible not expected edible safety of crop and environmental safety problem.Therefore suitable method is established to eat transgenosis
Product transgenic ingredient is identified and is detected, and can promote the safety management of agriculture genetically modified organism, ensure people, animal and
The safety of microorganism can preserve the ecological environment.It is quick in order to be carried out to Bar albumen in transgenic paddy rice or its derivative
Quantitative or qualitative analysis, is studied and the monoclonal antibody for obtaining antiweed Bar albumen has very big meaning.
Immune colloidal gold technique (Immune colloidal gold technique) is using colloidal gold as tracer label
Object is applied to a kind of novel immunolabelling technique of antigen-antibody.Colloidal gold be by gold chloride (HAuCL4) reducing agent such as
Under the effects of ascorbic acid, sodium citrate, polymerization becomes the gold particle of particular size, and since electrostatic interaction becomes a kind of stabilization
Colloidal state, referred to as colloidal gold.Colloidal gold is negatively charged under mild alkaline conditions, can be with the positive charge group shape of protein molecule
It is combined at firm, since this combination is electrostatical binding, so not influencing the biological nature of protein.Colloidal gold-labeled method
It is the core technology of immunochromatography quick diagnosis, it is applied to antigen-antibody reaction using colloidal gold as trace labelling object, in fact
Matter is the coating process that the macromolecules such as protein are adsorbed to colloid gold particle surface.According to some physical behaviors of colloidal gold,
Such as high electron density, granular size, shape and color reaction, in addition the immune and biological characteristics of conjugate, thus make colloid
Gold is widely used in the fields such as immunology, histology, pathology and cell biology.
Summary of the invention
The purpose of the present invention is to provide a kind of colloidal gold immunochromatographimethods for transgenic paddy rice herbicide resistant protein Bar
Quick measuring card and its application method, thus the scene of the herbicide resistant protein Bar quickly inspection suitable for transgenic paddy rice and products thereof
Survey and safety evaluatio.
To achieve the above object, the present invention provides a kind of colloidal gold immunochromatographimethod quick measuring cards, including get stuck and get stuck and is interior
Test strips, the test strips include bottom plate and adhere to the sample pad being sequentially connected on bottom plate, gold-labelled pad, reaction film and suction
Water cushion, the gold-labelled pad are coated with herbicide resistant protein Bar monoclonal antibody-colloidal gold conjugate.
Above-mentioned colloidal gold immunochromatographimethod quick measuring card in another embodiment, herbicide resistant protein Bar monoclonal antibody
It secretes to obtain by the Bar monoclonal antibody hybridoma cell strain PA1-18030030/1B8 3D6 that deposit number is CGMCC No.17083.
In another embodiment, reaction film is equipped with coating antiweed egg to above-mentioned colloidal gold immunochromatographimethod quick measuring card
The detection line of white Bar monoclonal antibody and the nature controlling line of coating sheep anti-mouse igg.
In another embodiment, the coating that the reaction film is equipped with is anti-to be removed above-mentioned colloidal gold immunochromatographimethod quick measuring card
The Bar monoclonal antibody hybridoma cell strain PA1- that careless agent protein B ar monoclonal antibody is CGMCC No.17084 by deposit number
18030030/6F5 1D2 secretes to obtain.
Above-mentioned colloidal gold immunochromatographimethod quick measuring card in another embodiment, detection line and nature controlling line interval 0.5-
1.0cm。
In another embodiment, described get stuck is equipped with well and observation to above-mentioned colloidal gold immunochromatographimethod quick measuring card
Window, the well are set to above sample pad, and the observation window is set to above reaction film.
Above-mentioned colloidal gold immunochromatographimethod quick measuring card in another embodiment, the sample pad and gold-labelled pad, gold-labelled pad
The overlay region of 1-2mm is equipped between reaction film, reaction film and water absorption pad.
In another embodiment, the bottom plate is PVC (polyvinyl chloride) plate to above-mentioned colloidal gold immunochromatographimethod quick measuring card,
The reaction film is nitrocellulose filter.
In another embodiment, the sample pad is by glass fibre membrane or filter for above-mentioned colloidal gold immunochromatographimethod quick measuring card
Paper is made, and the water absorption pad is made of filter paper.
The present invention also provides the detection methods of above-mentioned colloidal gold immunochromatographimethod quick measuring card, comprising the following steps:
1) if sample to be tested is the liquid substances such as plant tissue extracting solution, 40-60 μ can directly sample to be tested pre-treatment: be taken
The detection of L sample to be tested takes sample to be tested 90-110mg if sample to be tested is the non-liquid matters such as plant leaf blade, stem, seed
It grinds, 0.5-1.5mL sample treatment liquid is added and mixes well, take supernatant 40-60 μ L to detect after standing 25-35min on ice.Institute
The formula for stating sample treatment liquid is as shown in table 1:
1 sample treatment formula of liquid of table
2) it detects: sample to be tested is instilled in well, act on 5-10min at room temperature, observe result；
3) Analysis of test results:
Positive: band is presented in nature controlling line, detection line, illustrates in sample to be tested with the presence of Bar albumen；
Negative: there is not band in detection line, and band is presented in nature controlling line, illustrates do not have the presence of Bar albumen in sample to be tested；
Failure: if detection line, nature controlling line do not occur band or only band occurs in detection line, illustrate that test strips are lost
Effect.
Bar monoclonal antibody hybridoma cell strain (PA1-18030030/1B8 3D6) according to the present invention and the hybridization of Bar monoclonal antibody
Tumor cell strain (PA1-18030030/6F5 1D2) is successively preserved in Chinese microorganism strain preservation on December 21st, 2018
Administration committee's common micro-organisms center, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.Postcode: 100101, it protects
Hiding number is followed successively by CGMCC No.17083 and CGMCC No.17084.
Compared with prior art, resist the invention has the following beneficial effects: one kind of the application for transgenic paddy rice and remove
Careless agent protein B ar colloidal gold immunochromatographimethod quick measuring card high specificity, high sensitivity is easy to operate, quick, accurate, is suitable for turning
The field quick detection and safety evaluatio of herbicide resistant protein Bar in trans-genetic hybrid rice and products thereof.
Detailed description of the invention
Fig. 1 is the interior test strips structure schematic diagram that gets stuck and get stuck of colloidal gold immunochromatographimethod quick measuring card of the invention.
Fig. 2 is the testing result schematic diagram of colloidal gold immunochromatographimethod quick measuring card of the invention；The positive, nature controlling line (i.e. C line)
Colour developing, detection line (i.e. T line) naked eyes are as it can be seen that no matter shade is judged to the positive；Feminine gender, the colour developing of C line, T line do not develop the color, sentence
For feminine gender；In vain, C line does not develop the color, and T line is regardless of whether colour developing is invalid.
Fig. 3 is that colloidal gold immunochromatographimethod quick measuring card of the invention detects transgenic paddy rice positive leaf sample at room temperature
Result figure.
Specific embodiment
With reference to the accompanying drawing, specific embodiments of the present invention will be described in detail, it is to be understood that guarantor of the invention
Shield range is not limited by the specific implementation.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples, it is unless otherwise specified, commercially commercially available.
1 colloidal gold immunochromatographimethod quick measuring card of embodiment detects transgenic paddy rice blade
Using colloidal gold immunochromatographimethod quick measuring card of the invention, interior test strips structure schematic diagram such as Fig. 1 institute of getting stuck and get stuck
Show.The testing result of the colloidal gold immunochromatographimethod quick measuring card will appear three kinds of situations, as shown in Figure 2: positive, nature controlling line (i.e. C
Line) colour developing, detection line (i.e. T line) naked eyes are as it can be seen that no matter shade is judged to the positive；Feminine gender, the colour developing of C line, T line do not develop the color,
It is judged to feminine gender；In vain, C line does not develop the color, and T line is regardless of whether colour developing is invalid.
Transgenic paddy rice blade 100mg is taken, through liquid nitrogen grinding, is sufficiently mixed with sample extraction liquid, on ice after static 30min
50 μ L of supernatant is taken to detect.5min observes testing result at room temperature.Concrete outcome is detailed in Fig. 3, as shown in Figure 3, contained in stoste
Bar albumen can the colloidal gold immunochromatographimethod quick measuring card specific detection involved in the application arrive.
The inspection of 2 colloidal gold immunochromatographimethod quick measuring card of embodiment
1. preparation: (sampling amount is not less than 20 parts, for examining；It rechecks, keep sample and other special circumstances can separately take out
It takes)
1) instrument involved in the inspection project or relevant device checked, adjusted, checked and approved when necessary.
2) whether verification examines the information such as finished product title, lot number, batch consistent with instruction；
2. appearance: whether reagent (box) each component is complete, whether complete, liquid is leaked whether there is or not miserable；Whether label is clear easily to be known
Not.
3. film width: using 2 test paper of vernier caliper (precision 0.02mm) random detection, calculate 2 measurement results
Average value, as a result should meet " Bar colloidal gold final product quality standard " requirement.
4. liquid movement speed: being operated to specifications, start timing after sample is added, record sample is from being loaded onto
Flow completely through the time (t) of detection window；Window is loaded to the upper along distance (L) of detection window with vernier caliper measurement, calculates L/t i.e.
For liquid movement speed, " Bar colloidal gold final product quality standard " requirement as a result should be met.
5. net content: taking Sample dilution component in kit, using general gage measuring, testing result should meet " Bar
Colloidal gold final product quality standard " it requires.
6. performance: detection enterprise's reference material should meet following standard:
1) minimum detectability: retest minimum detectability reference material 10 times, calculated result.As a result it should meet " Bar colloid
Golden final product quality standard " it requires；
Calculation method:
LLD=X+3×S
In formula: X-- Series Measurement value；
XMeasure mean value；
N-- measures number；
S-- standard deviation；
LLD-- minimum detectability
2) accuracy: with 40 various concentration samples within the measurement range are no less than, with specified analysis system, manufacturer
System is used as comparison method, and every part of sample is measured respectively by test agent (box) operating method and comparison method.
Calculation method:
In formula: Bias-- relative deviation；
Measure mean value；
X-- calibration object, standard items or reference material definite value.
3) repeated: to detect repeated reference material CV1 and CV2, every part of repetition detection is not less than 10 times, calculates separately each
The horizontal coefficient of variation (CV) as a result should meet " Bar colloidal gold final product quality standard " requirement:
Calculation method:
In formula: X-- Series Measurement value；
Measure mean value；
N-- measures number；
S-- standard deviation；
CV-- variation within batch coefficient.
By above-mentioned inspection, the obtained colloidal gold immunochromatographimethod quick measuring card detection limit of the application: 5ppb (ng/mL).
Disclosed above is only specific embodiments of the present invention, and still, the present invention is not limited to this, any this field
What technical staff can think variation should all fall into protection scope of the present invention.
Claims (10)
1. a kind of colloidal gold immunochromatographimethod quick measuring card for transgenic paddy rice herbicide resistant protein Bar, including get stuck and get stuck
Interior test strips, test strips include bottom plate and adhere to the sample pad being sequentially connected on bottom plate, gold-labelled pad, reaction film and water suction
Pad, which is characterized in that the gold-labelled pad is coated with herbicide resistant protein Bar monoclonal antibody-colloidal gold conjugate.
2. it is used for the colloidal gold immunochromatographimethod quick measuring card of transgenic paddy rice herbicide resistant protein Bar according to claim 1,
It is characterized in that, the Bar monoclonal antibody hybridoma that the herbicide resistant protein Bar monoclonal antibody is CGMCC No.17083 by deposit number
Cell strain PA1-18030030/1B8 3D6 secretes to obtain.
3. it is used for the colloidal gold immunochromatographimethod quick measuring card of transgenic paddy rice herbicide resistant protein Bar according to claim 1,
It is characterized in that, the reaction film is equipped with the detection line and coating sheep anti-mouse igg of coating herbicide resistant protein Bar monoclonal antibody
Nature controlling line.
4. it is used for the colloidal gold immunochromatographimethod quick measuring card of transgenic paddy rice herbicide resistant protein Bar according to claim 3,
It is characterized in that, the coating herbicide resistant protein Bar monoclonal antibody that the reaction film is equipped with is CGMCC by deposit number
The Bar monoclonal antibody hybridoma cell strain PA1-18030030/6F5 1D2 of No.17084 secretes to obtain.
5. it is used for the colloidal gold immunochromatographimethod quick measuring card of transgenic paddy rice herbicide resistant protein Bar according to claim 3,
It is characterized in that, the detection line and nature controlling line interval 0.5-1.0cm.
6. it is used for the colloidal gold immunochromatographimethod quick measuring card of transgenic paddy rice herbicide resistant protein Bar according to claim 1,
It is characterized in that, described get stuck is equipped with well and observation window, and the well is set to above sample pad, and the observation window is set to
Above reaction film.
7. it is used for the colloidal gold immunochromatographimethod quick measuring card of transgenic paddy rice herbicide resistant protein Bar according to claim 1,
It is characterized in that, the weight of 1-2mm is respectively provided between the sample pad and gold-labelled pad, gold-labelled pad and reaction film, reaction film and water absorption pad
Folded area.
8. it is used for the colloidal gold immunochromatographimethod quick measuring card of transgenic paddy rice herbicide resistant protein Bar according to claim 1,
It is characterized in that, the bottom plate is polyvinyl chloride panel, and the reaction film is nitrocellulose filter.
9. it is used for the colloidal gold immunochromatographimethod quick measuring card of transgenic paddy rice herbicide resistant protein Bar according to claim 1,
It is characterized in that, the sample pad is made of glass fibre membrane or filter paper, and the water absorption pad is made of filter paper.
10. it is used for the colloidal gold immunochromatographimethod quick measuring card of transgenic paddy rice herbicide resistant protein Bar according to claim 1,
Characterized by comprising the following steps:
1) sample to be tested pre-treatment: if sample to be tested is liquid substance, 40-60 μ L sample to be tested is directly taken to detect, if to be measured
Sample is non-liquid matter, then sample to be tested 90-110mg is taken to grind, and 0.5-1.5mL sample treatment liquid is added and mixes, quiet on ice
Supernatant 40-60 μ L is taken to detect after setting 25-35min；
The sample treatment liquid are as follows: 1M Tris, pH 7.5 500uL, 1M NaCL1.5mL, 0.5M EDTA 20uL, 50% is sweet
Oily 2mL, 10%SDS 1mL are made into 10mL with distilled water, the 1mM benzene first of 1 protease inhibitors of addition and 50uL when use
Base sulfuryl fluoride；
2) it detects: sample to be tested is instilled in well, act on 5-10min at room temperature, observe result；
3) Analysis of test results:
Positive: band is presented in nature controlling line, detection line, illustrates in sample to be tested with the presence of Bar albumen；
Negative: there is not band in detection line, and band is presented in nature controlling line, illustrates do not have the presence of Bar albumen in sample to be tested；
Failure: if detection line, nature controlling line do not occur band or only band occurs in detection line, illustrate that test strips fail.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110205300A (en) * | 2019-06-04 | 2019-09-06 | 中国农业科学院生物技术研究所 | Bar monoclonal antibody hybridoma cell strain, antibody of generation and preparation method thereof |
| CN112798793A (en) * | 2020-12-30 | 2021-05-14 | 中国农业科学院油料作物研究所 | Test strip and test card for detecting PAT/bar protein, and preparation method and application thereof |
| CN117169516A (en) * | 2023-11-01 | 2023-12-05 | 中国农业科学院生物技术研究所 | Insect-resistant protein Cry1Ah colloidal gold immunochromatography rapid test strip and detection method thereof |
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| CN107513521A (en) * | 2017-09-02 | 2017-12-26 | 中国农业科学院生物技术研究所 | Hybridoma cell strain, secreted monoclonal antibody and its application in bar/PAT albumen is detected |
| CN108051599A (en) * | 2017-12-13 | 2018-05-18 | 中国农业科学院生物技术研究所 | For the colloidal gold immunochromatographimethod quick measuring card and its application method of transgenic corns insect resistance protein Vip3Aa20 |
| CN208224281U (en) * | 2018-05-25 | 2018-12-11 | 上海市农业科学院 | A kind of colloidal gold immunochromatographimethod quick measuring card detecting Cowpea Trypsin Inhibitor |
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2019
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Patent Citations (3)
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| CN107513521A (en) * | 2017-09-02 | 2017-12-26 | 中国农业科学院生物技术研究所 | Hybridoma cell strain, secreted monoclonal antibody and its application in bar/PAT albumen is detected |
| CN108051599A (en) * | 2017-12-13 | 2018-05-18 | 中国农业科学院生物技术研究所 | For the colloidal gold immunochromatographimethod quick measuring card and its application method of transgenic corns insect resistance protein Vip3Aa20 |
| CN208224281U (en) * | 2018-05-25 | 2018-12-11 | 上海市农业科学院 | A kind of colloidal gold immunochromatographimethod quick measuring card detecting Cowpea Trypsin Inhibitor |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110205300A (en) * | 2019-06-04 | 2019-09-06 | 中国农业科学院生物技术研究所 | Bar monoclonal antibody hybridoma cell strain, antibody of generation and preparation method thereof |
| CN112798793A (en) * | 2020-12-30 | 2021-05-14 | 中国农业科学院油料作物研究所 | Test strip and test card for detecting PAT/bar protein, and preparation method and application thereof |
| CN117169516A (en) * | 2023-11-01 | 2023-12-05 | 中国农业科学院生物技术研究所 | Insect-resistant protein Cry1Ah colloidal gold immunochromatography rapid test strip and detection method thereof |
| CN117169516B (en) * | 2023-11-01 | 2024-02-23 | 中国农业科学院生物技术研究所 | Insect-resistant protein Cry1Ah colloidal gold immunochromatography rapid test strip and detection method thereof |
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