CN201796033U - Gold-labeled kit capable of quickly detecting glyphosate-resistant G2-EPSPS protein - Google Patents
Gold-labeled kit capable of quickly detecting glyphosate-resistant G2-EPSPS protein Download PDFInfo
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- CN201796033U CN201796033U CN2010201072466U CN201020107246U CN201796033U CN 201796033 U CN201796033 U CN 201796033U CN 2010201072466 U CN2010201072466 U CN 2010201072466U CN 201020107246 U CN201020107246 U CN 201020107246U CN 201796033 U CN201796033 U CN 201796033U
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- resistant
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Abstract
The utility model relates to a gold-labeled kit capable of quickly detecting a glyphosate-resistant G2-EPSPS protein, which comprises a kit body, a measurement band and an upper cover, wherein the upper cover covers the kit body to form a kit. The gold-labeled kit capable of quickly detecting a glyphosate-resistant G2-EPSPS protein is characterized in that the measurement band is placed on the inner surface of the box body, and the upper cover is provided with an observation window and a sample adding hole. The gold-labeled kit can provide direct proofs for quickly detecting if glyphosate-resistant G2-EPSPS protein gene ingredients are contained in plant seeds, leaves and processed products without requirements for additional instruments or devices. The gold-labeled kit is easy and convenient to operate and vivid, accurate and quick in detection result. In addition, through only dripping extracted solution of a submitted article into the sample adding hole of the kit, a user can directly observe accurate detection results without the aid of any instruments in five to ten minutes.
Description
Technical field
The utility model relates to a kind of pick-up unit of biological sample, especially the gold-labeled kit of fast detecting resistance glyphosate G2-EPSPS albumen.
Background technology
In the developments of genetically modified plants or transgenic product is being screened or during safety evaluatio, often needing the allogenic gene that changes over to is carried out qualitative or semiquantitative determination.
G2-EPSPS protein coding gene clone autofluorescence pseudomonad G2 genomic library, this bacterium screening has high glyphosate tolerant from the glyphosate contaminated soil.The albumen of this gene code belongs to Class I type by analysis, and enzyme activity determination shows that this albumen has high substrate affinity and low glyphosate affinity, therefore can give the high glyphosate herbicide tolerant of host power.
Existing pick-up unit, as adopt enzyme to exempt from, put the device of methods such as exempting from, be subjected to the restriction of factors such as instrument (microplate reader, put and exempt from scintiloscope, hydro-extractor etc.), place, and detection time is long, and (the inspection-free survey time of enzyme needs 20hr, put inspection-free survey needs 30min～1hr), the detection cost is higher, is difficult to apply.Therefore, but need G2-EPSPS albumen in a kind of fast detecting genetically modified plants in the practice, and easy and simple to handle, result accurately and reliably, device fast.
Summary of the invention
In order to overcome the long deficiency of existing apparatus complicated operation, detection time, the utility model provides the immunity detection reagent of a kind of fast detecting transgene component (G2-EPSPS), and is not only easy and simple to handle, and the result accurately and reliably.
The technical scheme that its technical matters that solves the utility model adopts is:
A kind of gold-labeled kit of fast detecting G2-EPSPS albumen, comprise box body, calibration tape and loam cake, tray cover is formed kit after going into loam cake, it is characterized in that described loam cake is provided with an observation window and a well, and the shape of described box body and loam cake is a hexagon.
The degree of depth of described box body is not limit, and its upper surface can be provided with a groove that can embed described calibration tape.
Described box body or loam cake can have draw-in groove, to guarantee box body and loam cake fastening.
Described observation window is rectangle preferably, and observation window is divided into test section (T) and Quality Control district (C) two parts, can indicate last covering with letter C, T.
Described calibration tape is shaped as the rectangle sheet, is used to detect G2-EPSPS albumen.Described calibration tape is a kind of test material with detection system body membrane reaction system function, is made by multilayer materials such as macromolecular fibre film, plastics.Calibration tape is divided into Quality Control district and detection zone, and the Quality Control district can wrap by sheep anti-mouse igg or rabbit anti-mouse igg polyclonal antibody; The sample detection district bag of G2-EPSPS albumen calibration tape is by exceptional function EPSPS monoclonal antibody or polyclonal antibody.
After foreign gene changes expression of plants over to, will produce destination protein (EPSPS), can determine whether this gene exists by whether containing this albumen in the immune response test sample.If foreign gene changes plant over to, the producer silence, no destination protein produces, and (resistance glyphosate) can not accomplish the end in view.
The purpose of this utility model is achieved in that after tray cover is gone into loam cake the corresponding calibration tape of observation window and well can carry out application of sample and observe testing result by well on the lid and observation window.
During detection, the extract of inspecting article by ready samples is dripped in the well of kit, after 5-10 minute, just can observe directly testing result accurately by the observation window of loam cake by naked eyes.
For each observation window, judge testing result with the following methods:
1, negative (-):
Reaction condition: an aubergine band appears in observation window Quality Control district (C).
Testing result: do not contain gene to be checked or its content in the sample to be checked at it below threshold value.
2, positive (+):
Reaction condition: an aubergine band appears in observation window Quality Control district (C), occurs the aubergine band simultaneously in test section (T).Testing result: gene content to be checked is more than threshold value.
3, invalid:
Reaction condition: the aubergine band does not appear in Quality Control district (C),
Testing result: show the rotten damage of incorrect operating process or kit.
The advantage of kit described in the utility model is:
Economical and efficient: use cost is cheap, and it is convenient to preserve;
Easy to be quick: as to detect the operation of transgenosis composition single stage method, do not need any optional equipment.Directly analyte sample fluid is dripped in well, can go out the result in 5-10 minute;
Visual result: antigen is not detected, with the naked eye can be observed the result by any instrument;
Accurately and reliably: highly sensitive, high specificity;
Be widely used: blade, seed and the converted products that can detect all kinds of crops such as corn, cotton, paddy rice, wheat, tobacco, soybean.
The utility model can carry out EPSPS to plant sample and analyze, detect quick, special, responsive, convenient, be specially adapted to the development of transgenic product and the rapid screening of transgenic product, can be used for departments such as laboratory, field, frontier defense, customs's plant inspection, inspection and quarantine bureau, food safety detection and seed operation sale suspicious sample scene, rapid screening.
Description of drawings:
Below in conjunction with drawings and Examples the utility model is further specified.
Fig. 1 and Fig. 2 are the structural representations of a kind of kit of the utility model, and wherein Fig. 1 is the vertical view of box body, and Fig. 2 is the vertical view of kit loam cake;
1 is well among the figure, the 2nd, and observation window, the 3rd, calibration tape, C are the Quality Control districts, T is the test section.
Embodiment:
The preparation of embodiment 1 kit
One, preparation calibration tape
The preparation of EPSPS odd contradictive hydroperitoneum
Mouse peritoneal injecting fluid paraffin 0.5mL.After 1 week, EPSPS monoclonal antibody hybridoma is injected in above BALB/C mice abdominal cavity of anticipating, every injected in mice hybridoma 1～3 * 10
6Gather in the crops ascites after 10 days, above process is all finished under aseptic condition.
1.EPSPS Purification of Monoclonal Antibodies
1) with DEAE ion-exchange chromatography and Sephacryl S-300 molecular sieve monoclonal antibody is carried out purifying; The SDS-PAGE disk electrophoresis detects the purity of purified monoclonal antibody; The ELISA method is measured the monoclonal antibody activity;
2) purge process: get mouse odd contradictive hydroperitoneum → 3,000 * g is centrifugal, and 15 minutes → supernatant adds 50% ammonium sulfate precipitation, spend the night → 3, it is centrifugal that centrifugal 20 minutes of 000 * g → precipitation is dissolved in 0.01M phosphate buffer (pH 7.2) → S-300 gel column → DEAE Blue chromatographic column → 0-800mM NaCl gradient elution → ultrafiltration, concentrates monoclonal antibody;
3) monoclonal antibody purity testing: conventional SDS-PAGE electrophoresis, the purity of the monoclonal antibody that said method is purified are all more than 95%;
4) determination of protein concentration: ultraviolet spectrophotometer is measured the OD value at 279nm place, calculates protein content: OD as follows
279/1.4=mg/mL (purified monoclonal antibody).With the monoclonal antibody concentration adjustment is about 1mg/mL;
5) monoclonal antibody determination of activity: select the antigen coated elisa plate of EPSPS (1 μ g/ hole) for use, and sheep anti-mouse igg-HRP compound, measure each monoclonal antibody tire (greater than 1: 5000).
2. goat-anti DOA
THeight tire immune sero-fast preparation and purifying
1) above-mentioned monoclonal antibody+Freund's complete adjuvant that purifying is good → immune goat → booster immunization secondary each strengthens getting in back about 10 days blood → centrifuging and taking serum → ammonium sulfate precipitation → DEAE ion-exchange chromatography purifying January → second time at interval;
2) titration: the ELISA sandwich method, the antibody titer dilutability should be greater than 1: 256;
3) determination of protein concentration: ultraviolet spectrometry is measured OD
279, calculate protein concentration.
3. collaurum and golden labeling antibody preparation
1) preparation of collaurum: the collaurum for preparing 40-60nm with the citrate reducing process.With 0.01%HauCl
4Be heated to and boil, add a certain amount of 1% trisodium citrate (Na
3C
6H
5O
72H
2O), continued heated and boiled 5 minutes, treat the colloidal gold solution color, after stablizing, cool off and get final product by orchid → purple → red.Colloidal gold solution should be limpid transparent, as the need long preservation, can add 0.02%NaN
3
2) collaurum liquid 500mL is transferred pH8.4 with 0.1MNaOH, slowly add monoclonal antibody 2mL under the magnetic agitation, stirred 20 minutes.The centrifugal 30Min of 5,500 * g removes unconjugated protein in the supernatant.Colloid gold label antibody precipitation is drawn in centrifugal back, and precipitation is dissolved in 10mL and preserves in the liquid, with 0.45 μ m filtering with microporous membrane.Sampling is examined and determine, and all the other put 4 ℃ of preservations.
4. the preparation of film reaction system M1
1) sheep anti-mouse igg with EPSPS antibody and purifying dilutes with the 0.1M phosphate buffer, and final concentration is about 0.2-2mg/mL.
2) sheep anti-mouse igg of purifying is dissolved in the 0.1M phosphate buffer, and concentration is 2.0mg/mL;
3) nitrocellulose filter size: 10cm * 27cm, every film can spray the detection of long 25cm and control each 4 on band;
4) above two kinds of solution are added respectively in two shower nozzle storage bottle of Biodot XYZ3000 flush coater;
5) spray speed being set is 2 μ L/cm, and the speed of dividing a word with a hyphen at the end of a line of NC film is 50mm/s.Wrap on the NC film by the reaction detection line with monoclonal antibody EPSPAD2 (2mg/mL), reacted control line with 1.0mg/mL rabbit anti-mouse igg bag, the distance of control line and detection line is 4-4.5mm;
6) finish bag by after, put 37 ℃ of drying boxes 24 hours, handle half an hour with the sealing of BB confining liquid, take out with the WB washing lotion and wash once;
7) 37 ℃ of drying box drying for standby;
8) nitrocellulose filter got ready of cutting: 1.8cm * 27cm/ bar, put into the thin aluminum bag hermetically drying and preserve.It is standby to put room temperature preservation.
5. the preparation of film reaction system M2a, M2b and M3
The water-absorption fiber film is soaked in respectively in M2a, M2b, the M3 solution, and after soaking into, taking-up is dried, in the polybag of packing into, and room temperature preservation.
1) M2a preparation: the glass fibre that makes is cut 27cm * 1.2cm/ bar;
2) M2b preparation: the glass fibre that makes is cut 27cm * 1.8cm/ bar;
3) M3 preparation: the glass fibre that makes is cut 27cm * 1.2cm/ bar.
6. the preparation of film reaction system M4
1) get collaurum-monoclonal antibody compound and add dilution, mixing is made into working concentration;
2) solution is added in the Airjet shower nozzle storage bottle of flush coater Biodot;
3) set pressure is 15PSI, and the translational speed of glass fibre membrane is 50mm/s;
4) spray specification is 0.5-1.5cm * 25cm/ bar, and every sprays 2 times;
5) in 37 ℃ of oven dry 12 hours, put into thin aluminum bag, add drying agent, heat sealing, room temperature preservation.
7. reaction body assembling
1) on the M6 plastic base, pastes two of double sticky tape M7;
2) in the middle of M7, paste reaction film M1 (18mm), from the about 22mm of M6 upper limb;
3) on M7, paste M5 (22mm), align with the M6 upper limb and hand over 1mm with the M1 upper limb;
4) on M7, paste M2a (12mm), join with the M1 lower edge;
5) on M2, paste M4 (10mm), push down M1 lower edge 0.5mm;
6) paste M3 (12mm) on M7, upper limb is pushed down M4 about 2/3;
7) paste M2b (18mm) on M7, it is about 2/3 that upper limb is pushed down M4, and lower edge aligns with the lower edge of M7;
8) paste transparency protected adhesive tape M8 on M4 and M7, upper limb is pushed down M4 fully, and pushes down the about 1.5mm of M1;
9) paste colour-coded adhesive tape M9 on M5, hand over 1mm with the M1 upper limb, the other end climbs over the M6 upper limb, is affixed on the M6 back side;
10) the full-automatic cutting cutter of reaction body and function with assembled formation is cut into the 3.5mm specification.
Two, kit assembling
The calibration tape 3 of the above-mentioned detection G2-EPSPS albumen for preparing is embedded in the groove of Fig. 1 box bodys, then with loam cake and box body fastening, kit.
After kit, dropper and the encapsulation of operation instructions dress polybag, get final product.
Embodiment 2 kit uses
1. detect sample process and preparation
Vegetable seeds, leaf, seedling equal samples need through grinding before detection, and with distilled water extracting and dilution.For obtaining the optimum detection effect, variant sample should after finishing grinding and dilution, with the sample mixing, leave standstill according to the dilution proportion in the following table, gets supernatant as test sample.
2. detect and the result
To detect box and keep flat, and with joining plastic suction pipe absorption sample liquid, drip 4-5 and drip in the well that detects box, absorbent material slowly moves sample to be checked, and the film reaction system is activated.No matter whether testing gene is present in the plant sample, and an aubergine band all can appear at (C) in the Quality Control district.The aubergine band that (C) manifested in the Quality Control district is to judge whether enough plant sample liquid is arranged, and whether normal chromatography process standard simultaneously also as the inner quality standard of reagent.
3. the result judges:
Whether observation window detects gene to be checked and exists.
1) if do not contain specific G2-EPSPS albumen in the plant sample to be measured, or its concentration is lower than detection sensitivity, colloidal gold antibody can not be fixed on the monoclonal antibody immunity that detects on the film in the band in the chromatography process, thereby an aubergine detection band can not appear in (T) in the test section, show negative (-) result, an aubergine band promptly only in Quality Control district (C), occurs.
When 2) being higher than its detection threshold as if G2-EPSPS protein concentration in the plant sample to be measured, antigen immunity gold with detect with on another monoclonal antibody combine, another aubergine detection band also will appear in (T) in the test section, show positive (+) result, promptly in Quality Control district (C) and detection zone (T), an aubergine band respectively occurs.Attention: the aubergine band in test section (T) can show the phenomenon of shade.But, in the observing time of regulation, no matter this colour band shade all should be judged to be positive findings.
3) if the aubergine band does not appear in kit Quality Control district (C), then testing result is invalid, shows the rotten damage of incorrect operating process or kit.
Claims (3)
1. the gold-labeled kit of a fast detecting resistance glyphosate G2-EPSPS albumen, comprise box body, calibration tape and loam cake, tray cover is formed kit after going into loam cake, it is characterized in that described calibration tape places described cartridge inner surface, and described loam cake is provided with an observation window and a well; The shape of described box body and loam cake is a hexagon.
2. the kit of fast detecting resistance glyphosate G2-EPSPS albumen according to claim 1 is characterized in that described cartridge inner surface is provided with the groove that can embed described calibration tape.
3. the kit of detection resistance glyphosate G2-EPSPS albumen according to claim 1 and 2 is characterized in that described box body or loam cake have draw-in groove.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010201072466U CN201796033U (en) | 2010-02-02 | 2010-02-02 | Gold-labeled kit capable of quickly detecting glyphosate-resistant G2-EPSPS protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010201072466U CN201796033U (en) | 2010-02-02 | 2010-02-02 | Gold-labeled kit capable of quickly detecting glyphosate-resistant G2-EPSPS protein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN201796033U true CN201796033U (en) | 2011-04-13 |
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ID=43850939
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010201072466U Expired - Lifetime CN201796033U (en) | 2010-02-02 | 2010-02-02 | Gold-labeled kit capable of quickly detecting glyphosate-resistant G2-EPSPS protein |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102707044A (en) * | 2012-05-31 | 2012-10-03 | 李云峰 | Vertical western blot antibody incubation box |
| CN103792370A (en) * | 2014-02-13 | 2014-05-14 | 中国检验检疫科学研究院 | Method for detecting CP4-EPSPs (5-enolpyruvylshikimate-3-phosphate synthase) protein by using QCM (Quartz Crystal Microbalance) sensor and special gold piece |
| CN105154408A (en) * | 2015-09-18 | 2015-12-16 | 中国农业科学院生物技术研究所 | Monoclonal antibody for detecting herbicide-resistant glyphosate protein and application of monoclonal antibody |
| CN105158475B (en) * | 2015-09-18 | 2016-10-05 | 中国农业科学院生物技术研究所 | For detect the monoclonal antibody of transgenic crop to and Double-antibody sandwich enzymelinked immunosorbent detection kit |
-
2010
- 2010-02-02 CN CN2010201072466U patent/CN201796033U/en not_active Expired - Lifetime
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102707044A (en) * | 2012-05-31 | 2012-10-03 | 李云峰 | Vertical western blot antibody incubation box |
| CN103792370A (en) * | 2014-02-13 | 2014-05-14 | 中国检验检疫科学研究院 | Method for detecting CP4-EPSPs (5-enolpyruvylshikimate-3-phosphate synthase) protein by using QCM (Quartz Crystal Microbalance) sensor and special gold piece |
| CN105154408A (en) * | 2015-09-18 | 2015-12-16 | 中国农业科学院生物技术研究所 | Monoclonal antibody for detecting herbicide-resistant glyphosate protein and application of monoclonal antibody |
| CN105158475B (en) * | 2015-09-18 | 2016-10-05 | 中国农业科学院生物技术研究所 | For detect the monoclonal antibody of transgenic crop to and Double-antibody sandwich enzymelinked immunosorbent detection kit |
| CN105154408B (en) * | 2015-09-18 | 2018-10-09 | 中国农业科学院生物技术研究所 | A kind of monoclonal antibody and application thereof of detection antiweed glyphosate albumen |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CX01 | Expiry of patent term |
Granted publication date: 20110413 |
|
| CX01 | Expiry of patent term |