CN202443018U - Gold-labeled kit for rapid joint detection of salt-resistant HIN protein and IrrE protein - Google Patents
Gold-labeled kit for rapid joint detection of salt-resistant HIN protein and IrrE protein Download PDFInfo
- Publication number
- CN202443018U CN202443018U CN2012200794500U CN201220079450U CN202443018U CN 202443018 U CN202443018 U CN 202443018U CN 2012200794500 U CN2012200794500 U CN 2012200794500U CN 201220079450 U CN201220079450 U CN 201220079450U CN 202443018 U CN202443018 U CN 202443018U
- Authority
- CN
- China
- Prior art keywords
- kit
- irre
- hin
- protein
- gold
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Disclosed is a gold-labeled kit for rapid joint detection of salt-resistant HIN protein and IrrE protein. The kit comprises a base plate and an upper cover, wherein the upper cover is covered by the base plate to form the kit. The kit is characterized in that detecting belts for detecting HIN protein and IrrE protein are respectively arranged on two sides of the upper surface of the base plate, and two sides of the upper cover are respectively provided with an observing window and sample adding holes. The kit is easy to operate, can detect results vividly, accurately and rapidly, extracting solution of articles to be detected is respectively dropwisely added into the two sample adding holes of the kit, after 3 minutes to 5 minutes, accurate detecting results can be observed directly by the naked eye through the observing window.
Description
Technical field:
The utility model relates to the gold-labeled kit of a kind of fast joint inspection salt tolerant HIN and IrrE albumen.
Background technology:
In the developments of genetically modified plants or transgenic product is being screened or during safety evaluatio, often need carry out qualitative or semiquantitative determination to the allogenic gene that changes over to.
All from the radiation hardness abnormal cocci R1 (Deinococcus radiodurans R1) of extreme radioresistance, HIN albumen belongs to the LEA2 family member for HIN albumen and IrrE albumen, and the osmotic stress of endosperm development and plant growth organ is had protective effect.IrrE protein coding gene (irrE) is the global regulation's factor among the radioresistant cocci R1, participates in the various reactions of coercing, and resists plant in the process of abiotic stress such as high salt and is bringing into play important effect.
Research shows that expression of gene all can significantly strengthen colibacillary salt resistance ability.With behind hin and the irrE gene transfered plant, obviously improved the salt resistance ability of plant respectively, the highest 350mmol salinity that tolerates.At present, the HIN protein coding gene has changed rape, corn over to; And the IrrE protein coding gene has changed crops such as corn, cotton, tobacco, rape over to.Therefore, often need carry out the detection of two kinds of genes in the practice to the genetically modified plants sample.
Existing detection technique such as enzyme exempt from, put method such as exempt from; Receive the restriction of factors such as instrument (ELIASA, put and exempt from scintiloscope, hydro-extractor etc.), place; And detection time long (the inspection-free survey time of enzyme needs 20h, puts and need to exempt from 30min-1h), it is higher to detect cost, is difficult to apply.
Though the method that existing test strips detects can partly overcome above deficiency, every kind of test strips can only detect foreign gene single in the genetically modified crops, is easy to generate error result in the practical operation.For example: if the tester only has the HIT test strip, and do not change the HIN gene in the detected object over to, but change the IrrE gene over to, draw the error result that there is not foreign gene in detected object so possibly, cause unnecessary loss.And when operation, need test strips be immersed in the liquid to be detected, operation is not very convenient.
Therefore, be sought after in the practice a kind ofly can detecting HIN albumen and IrrE albumen in the genetically modified plants simultaneously, and easy and simple to handle, result accurately and reliably, device fast.
Summary of the invention:
The purpose of the utility model is to set up a kind of easy and simple to handle, result accurately and reliably, fast, and can detect the immunity detection reagent of 2 kinds of different transgene components (HIN and IrrE albumen) simultaneously.
The technical scheme of the utility model is such:
The gold-labeled kit of a kind of fast joint inspection salt tolerant HIN and IrrE albumen comprises base plate and loam cake, and base plate cover is formed kit after going into loam cake, it is characterized in that the both sides of plate upper surface respectively are respectively arranged with the calibration tape that detects HIN albumen and IrrE albumen; Each has an observation window and a well respectively the both sides of loam cake.
The thickness of base plate is not limit, and its upper surface can be provided with 2 grooves, and every groove is embedded in 1 calibration tape.
Said base plate or loam cake can have draw-in groove, to guarantee base plate and loam cake fastening.
Said observation window is rectangle preferably.
Being shaped as of kit is flat box-like, and the shape of the upper surface of box is preferably two link together trapezoidal.
2 calibration tapes in the said base plate, one is used to detect HIN albumen, and another is used to detect IrrE albumen.Said calibration tape is a kind of test material with detection system body membrane reaction system function, is processed by multilayer materials such as macromolecular fibre film, plastics.The calibration tape of 2 kinds of albumen all can be divided into Quality Control district and detection zone, and the Quality Control district can encapsulate sheep anti-mouse igg or rabbit anti-mouse igg polyclonal antibody; The sample detection district of HIN albumen calibration tape encapsulates monoclonal antibody or the polyclonal antibody of exceptional function property HIN, and the sample detection district of IrrE albumen calibration tape encapsulates monoclonal antibody or the polyclonal antibody of IrrE.
When foreign gene changes plant over to, and in plant, express, will produce destination protein (HIN and IrrE), therefore confirm through whether containing above-mentioned two kinds of albumen in the test sample whether this gene exists.If foreign gene changes plant over to, producer is reticent, and no destination protein produces, and (salt tolerant) can not accomplish the end in view.
The purpose of the utility model is achieved in that
After base plate cover was gone into loam cake, each respectively corresponding calibration tape of each observation window and well can carry out application of sample and observe testing result through well on the lid and observation window, and each observation window can be checked a kind of gene respectively.
Each observation window is divided into test section (T) and Quality Control district (C) two parts, can indicate last covering with letter C, T;
During detection, same a kind of extract of inspecting article by ready samples is dripped respectively in 2 wells of kit, behind the 3-5min, just can observe directly testing result accurately through the observation window of loam cake through 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), and is same in test section (T)
The time aubergine band appears.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 the said kit of the utility model is:
Economical and efficient: a detection box can detect 2 kinds of different transgenosis compositions simultaneously, greatly reduces use cost;
Easy to be quick: 2 kinds of different transgenosis composition single stage method operations, detect simultaneously, do not need other any auxiliary instrumentation equipment.Directly analyte sample fluid is dripped in well, 3-5min can go out the result;
Testing result is directly perceived: not by any instrument, with the naked eye can be observed the result;
Be widely used: blade, seed and the converted products that can detect multiple kinds of crops such as cotton, wheat, corn, rape, tobacco.
Because the joint inspection kit that the utility model provides can carry out HIN to plant sample simultaneously and IrrE analyzes; 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 that laboratory, field, frontier defense, customs's plant inspection, inspection and quarantine bureau, food safety detection and seed are on-the-spot to suspicious sample through departments such as aspect of Marketing, rapid screening.
Description of drawings:
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 base plate, and Fig. 2 is the vertical view of kit loam cake;
3 is calibration tapes among Fig. 1, and 1 is well among Fig. 2, the 2nd, and observation window.
Embodiment:
The preparation of the calibration tape in the utility model kit brings with the test for preparing detection HIN and illustrates: embodiment 1 preparation calibration tape
1.HIN the preparation of odd contradictive hydroperitoneum
Mouse peritoneal injecting fluid paraffin 0.5mL.After 1 week, HIN 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 behind the 10d, above process is all accomplished under aseptic condition.
2.HIN 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; It is active that the ELISA method is measured monoclonal antibody;
2) purge process: get mouse odd contradictive hydroperitoneum → 3; Centrifugal 15min → the supernatant of 000 * g adds 50% ammonium sulfate precipitation; Spend the night → 3; It is centrifugal that the centrifugal 20min of 000 * g → deposition is dissolved in 0.01M phosphate buffer (pH 7.2) → S-300 gel column → DEAE Blue chromatographic column → 0-800mMNaCl 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 O.D. value at 279nm place, calculates protein content: OD by following formula
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 HIN (1 μ g/ hole) for use, and sheep anti-mouse igg-HRP compound, measure each monoclonal antibody tire (greater than 1: 5000).
3. 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 2 times, strengthen afterwards getting blood → centrifuging and taking serum → ammonium sulfate precipitation → DEAE ion-exchange chromatography purifying about 10d each 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.
4. 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), continue heated and boiled 5min, 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, like the need long preservation, can add 0.02%NaN
3
2) collaurum liquid 500mL is transferred pH 8.4 with 0.1M NaOH, slowly add monoclonal antibody 2mL under the magnetic agitation, stir 20min.The centrifugal 30min of 5,500 * g removes unconjugated protein in the supernatant.Colloid gold label antibody deposition is drawn in centrifugal back, and deposition 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.
5. the preparation of film reaction system M1
1) sheep anti-mouse igg with HIN 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 2 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.On the NC film, encapsulate the reaction detection line with monoclonal antibody HIN AD2 (2mg/mL), encapsulate the reaction control line with the 1.0mg/mL rabbit anti-mouse igg, the distance of control line and detection line is 4-4.5mm;
6) after completion encapsulates, put 37 ℃ of drying box 24h, handle 0.5h, take out with the WB washing lotion and wash 1 time with the sealing of BB confining liquid;
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 subsequent use to put room temperature preservation.
6. 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 spun glass that makes is cut 27cm * 1.2cm/ bar;
2) M2b preparation: the spun glass that makes is cut 27cm * 1.8cm/ bar;
3) M3 preparation: the spun glass that makes is cut 27cm * 1.2cm/ bar.
7. 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 12h, put into thin aluminum bag, add drying agent, heat sealing, room temperature preservation.
8. reagent assembling
1) on the M6 plastic base, pastes two 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) on M7, paste M3 (12mm), upper limb is pushed down M4 about 2/3;
7) on M7, paste M2b (18mm), it is about 2/3 that upper limb is pushed down M4, and lower edge aligns with the lower edge of M7;
8) on M4 and M7, paste transparency protected adhesive tape M8, upper limb is pushed down M4 fully, and pushes down the about 1.5mm of M1;
9) on M5, paste colour-coded adhesive tape M9, hand over 1mm with the M1 upper limb, the other end climbs over the M6 upper limb, is affixed on the M6 quilt cover;
10) reagent with assembled formation is cut into the 3.5mm specification with full-automatic cutting cutter.
The assembling of embodiment 2 kits
The detection HIN for preparing is embedded respectively in two grooves of base plate with two kinds of calibration tapes that detect IrrE albumen, then with loam cake and base plate fastening, kit.
After kit, dropper and the encapsulation of operation instructions dress polybag, get final product.
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 accomplishing 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, with joining plastic suction pipe draw sample liquid, Dropwise 5-6 is droplet in the well 1 that detects box, absorbent material slowly moves sample to be checked, 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 each observation window detects a kind of gene to be checked and exists.Below be the example explanation to detect HIN:
1) as not containing specific HIN albumen in the sample; 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, shows negative (-) result, an aubergine band promptly only in Quality Control district (C), occurs.
2) if when treating that the HIN protein concentration is higher than its detection threshold in the plant sample; Antigen immunity gold with detect with on another monoclonal antibody combine; Another aubergine detection band will also appear in (T) in the test section; Show positive (+) result, an aubergine band promptly in Quality Control district (C) and detection zone, 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) the aubergine band do not occur like Quality Control district (C), then testing result is invalid, shows the rotten damage of incorrect operating process or kit.
Claims (4)
1. the gold-labeled kit of fast joint inspection salt tolerant HIN and IrrE albumen comprises base plate and loam cake, and base plate cover is formed kit after going into loam cake, it is characterized in that the both sides of plate upper surface are respectively arranged with the calibration tape that detects HIN albumen and IrrE albumen; Each has an observation window and a well respectively the both sides of loam cake.
2. the gold-labeled kit of fast joint inspection salt tolerant HIN according to claim 1 and IrrE albumen is characterized in that the upper surface of said base plate is provided with two grooves that can embed calibration tape.
3. the gold-labeled kit of fast joint inspection salt tolerant HIN according to claim 1 and 2 and IrrE albumen is characterized in that said base plate or loam cake have draw-in groove.
4. the gold-labeled kit of fast joint inspection salt tolerant HIN according to claim 1 and 2 and IrrE albumen is characterized in that said observation window is a rectangle.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012200794500U CN202443018U (en) | 2012-03-05 | 2012-03-05 | Gold-labeled kit for rapid joint detection of salt-resistant HIN protein and IrrE protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012200794500U CN202443018U (en) | 2012-03-05 | 2012-03-05 | Gold-labeled kit for rapid joint detection of salt-resistant HIN protein and IrrE protein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN202443018U true CN202443018U (en) | 2012-09-19 |
Family
ID=46824462
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012200794500U Expired - Lifetime CN202443018U (en) | 2012-03-05 | 2012-03-05 | Gold-labeled kit for rapid joint detection of salt-resistant HIN protein and IrrE protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN202443018U (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016180300A1 (en) * | 2015-05-11 | 2016-11-17 | 中国农业科学院生物技术研究所 | Irre protein functional domain for improving anti-oxidation capability of cell and application thereof |
-
2012
- 2012-03-05 CN CN2012200794500U patent/CN202443018U/en not_active Expired - Lifetime
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016180300A1 (en) * | 2015-05-11 | 2016-11-17 | 中国农业科学院生物技术研究所 | Irre protein functional domain for improving anti-oxidation capability of cell and application thereof |
| US10738314B2 (en) | 2015-05-11 | 2020-08-11 | Biotechnology Research Institute, Chinese Academy Of Agricultural Sciences | IrrE protein functional domain for improving anti-oxidation capability of cell and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101226194B (en) | Malachite green vestigial ELISA detection kit and usage method thereof | |
| CN104849467A (en) | Fluorescent microsphere immunochromatography test paper strip for detecting clenbuterol hydrochloride residue, preparation method and applications thereof | |
| CN105759043B (en) | It is a kind of to detect carbendazim and the test paper and its application, preparation method of thiophanate-methyl residual in crops simultaneously | |
| CN202676705U (en) | Combined insect-resistant BT protein gold mark fast detection reagent kit | |
| CN201607443U (en) | Kit for quickly detecting salt resistant IrrE protein | |
| CN105259351A (en) | Test paper for detecting metalaxyl residue and application thereof | |
| CN201796033U (en) | Gold-labeled kit capable of quickly detecting glyphosate-resistant G2-EPSPS protein | |
| CN201600374U (en) | Quick joint inspection kit | |
| CN102879574A (en) | Bar/pat gene transfer plant and rapid test strip for derivative product of bar/pat transgenic plant | |
| CN201600373U (en) | Kit for joint inspection for IrrE protein and BT protein by one-step method | |
| CN109061146A (en) | A kind of test strips and its preparation method and application detecting Acetamiprid | |
| CN105717295B (en) | Test strip for rapidly detecting CP4-EPSPS transgenic plant and derivative thereof | |
| CN202033368U (en) | Reagent plate for detecting aflatoxins M1 in dairy | |
| CN202443018U (en) | Gold-labeled kit for rapid joint detection of salt-resistant HIN protein and IrrE protein | |
| CN203849263U (en) | Gold immnnochromatography rapid combined measuring kit of global regulation and control factors IrrE, Csp (cold shock protein) and CarD proteins | |
| CN205246668U (en) | Quick detect reagent strip of vibrio alginolyticus | |
| CN101706499A (en) | FLAG fusion tag colloidal gold test strip and preparation method thereof | |
| CN105334323A (en) | Method and test strip for detecting zilpaterol, and application of test strip | |
| CN1525173A (en) | Insecticidal crystallin BT-Cry1Ab/1Ac rapid detection test bar | |
| CN201600372U (en) | Reagent kit capable of fast performing combined detection to IrrE and G2-EPSPS protein | |
| CN103995107B (en) | A kind of method detecting lincomycin and special test paper thereof | |
| CN102520179A (en) | Quantitative detection method of fumonisins B1 | |
| CN105388286A (en) | Test strip for detecting iprodione and preparation method and application of test strip | |
| CN202443022U (en) | Quadruple detection kit for transgenic components | |
| CN202018460U (en) | Gold-marking reagent kit for rapidly detecting HYG (Hygromycin)-resisting protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CX01 | Expiry of patent term |
Granted publication date: 20120919 |
|
| CX01 | Expiry of patent term |