CN203849263U - Gold immnnochromatography rapid combined measuring kit of global regulation and control factors IrrE, Csp (cold shock protein) and CarD proteins - Google Patents
Gold immnnochromatography rapid combined measuring kit of global regulation and control factors IrrE, Csp (cold shock protein) and CarD proteins Download PDFInfo
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- CN203849263U CN203849263U CN201320731951.7U CN201320731951U CN203849263U CN 203849263 U CN203849263 U CN 203849263U CN 201320731951 U CN201320731951 U CN 201320731951U CN 203849263 U CN203849263 U CN 203849263U
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The utility model relates to a gold immnnochromatography rapid combined measuring kit of global regulation and control factors IrrE, Csp (cold shock protein) and CarD proteins. The kit comprises a bottom plate and an upper cover, and is characterized in that: the kit is in the shape of a triangular box body, the bottom plate of the box body is provided with 3 test strips, and the upper cover is provided with 3 observation windows and 3 sample adding holes. The 3 test strips are respectively positioned on three edges of the surface of the bottom plate of the triangular box body, and the 3 sample adding holes are respectively positioned on three edges of the surface of the upper cover of the triangular box body. The kit can simultaneously detect three different kinds of genetically modified ingredients of IrrE, Csp (cold shock protein) and CarD in one step, the operation is simple, the detection result is accurate, fast and visual.
Description
Technical field:
The utility model relates to the gold mark Quick joint inspection kit of the factor IrrE of global regulation, Csp and CarD albumen.
Background technology:
IrrE, Csp and CarD albumen are in the at present research work at genetically modified organism, or transgenic product being screened or when safety evaluatio, three kinds of allogenic genes that often need to simultaneously detect.If detected respectively, not only operation is very loaded down with trivial details, and testing cost is high.
IrrE albumen is the global regulation's factor in radioresistant cocci, and irrE gene is imported in Escherichia coli and can significantly strengthen its radioresistance, oxidation resistance and salt resistance ability.And by after this gene transfered plant, obviously improve salt tolerant and the drought-resistant ability of plant.
CSP albumen is a kind of cold stress protein of coercing, and as a kind of molecular chaperones and single-chain nucleic acid non-specific binding, has the effect of stable strand nucleotide secondary structure, makes cell under extreme environment survival.Cold shock protein gene (csp) is proceeded to after plant, given plant stronger salt tolerant and drought resistance.At present, cold shock protein CSP encoding gene has been applied to the research of multiple genetically modified plants.
CarD albumen is a class global regulation transcription factor, in the bioprocess such as DNA replication dna, restructuring, reparation and genetic transcription, plays an important role.Under UV radiation stress conditions, full genome transcription group analysis finds that CarD albumen is induced after UV radiation, may participate in the regulation and control of DNA damage repair process.Can Natural Transformation and be easy to, in the type strain D.radiodurans of genetic manipulation, knock out corresponding CarD encoding gene, find compared with wild-type strain under UV irradiation, hydrogen peroxide, ethanol and hunger are coerced, its phenotype there is notable difference.
Therefore, in practice, be sought after one and can have detected IrrE in genetically modified plants, Csp and CarD albumen simultaneously, and easy and simple to handle, result accurately and reliably, device fast.
Utility model content:
The purpose of this utility model is to set up a kind of easy and simple to handle, result accurately and reliably, fast, and can detect the detection kit of 3 kinds of different transgene components simultaneously.
The technical solution of the utility model is such: the gold mark Quick joint inspection kit of the factor IrrE of global regulation, Csp and CarD albumen, is made up of base plate, upper cover; It is characterized by, described kit be shaped as triangle box body, on the base plate of box body, be provided with 3 calibration tapes; On be stamped 3 observation windows and 3 wells.
Described 3 calibration tapes lay respectively at three limits on described triangle box plate surface;
Described 3 wells lay respectively at three limits of cap surface on described triangle box body.
Described base plate or upper cover can have draw-in groove, to ensure base plate and upper cover fastening.
Preferably rectangle of described observation window.
Kit be preferably shaped as flat triangle box.
The purpose of this utility model is achieved in that and enters after upper cover when base plate cover, the corresponding calibration tape of each observation window, can be carried out application of sample and be observed testing result by the single well on lid and 3 observation windows, each observation window can check respectively a kind of gene.
In a preferred example of the present utility model, taking the detection of CarD albumen that turns plant as example:
Kit is the flat box of triangle, and box plate is respectively provided with a groove along three sides of a triangle, can place 3 calibration tapes; Upper cover and calibration tape corresponding positions are equipped with 3 observation windows, and 3 wells are arranged on three drift angle places of upper cover (seeing Fig. 1, Fig. 2).
Article 3, calibration tape distributes in this way, and the one, application of sample is convenient, and application of sample to be to check 3 kinds of genes simultaneously, and the 2nd, testing result is unaffected mutually.
Above-mentioned 3 calibration tapes, one for detection of CarD albumen, and one for detection of IrrE albumen, and another is for detection of Csp albumen.Described calibration tape is a kind of test material with detection system body membrane reaction system function, is made up of multilayer materials such as macromolecular fibre film, plastics.The calibration tape of 3 kinds of albumen all can be divided into Quality Control district and detection zone, and Quality Control district can be coated with sheep anti-mouse igg or rabbit anti-mouse igg polyclonal antibody; The coated exceptional function CarD in sample detection district of CarD albumen calibration tape, the coated IrrE in sample detection district of IrrE albumen calibration tape, monoclonal antibody or the polyclonal antibody of the coated exceptional function Csp in sample detection district of Csp albumen calibration tape.
Each observation window is divided into test section (T) and Quality Control district (C) two parts, can indicate with letter C, T upper covering;
When detection, the extract of censorship article is dripped respectively in the well of kit, after 5-10 minute, just can observe directly testing result accurately by the observation window of upper cover by naked eyes.
For each observation window, judge with the following methods testing result:
1, negative (-):
Reaction condition: an aubergine band appears in observation window Quality Control district (C).
Testing result: in sample to be checked containing gene to be checked or its content at it below threshold value.
2, positive (+):
Reaction condition: an aubergine band appears in observation window Quality Control district (C), occurs aubergine band in test section (T) simultaneously.Testing result: gene content to be checked is more than threshold value.
3, invalid:
Reaction condition: aubergine band does not appear in Quality Control district (C),
Testing result: show incorrect operating process or kit rotten damage.
When foreign gene proceeds to plant, and express in plant, will produce destination protein (CarD, IrrE and Csp albumen), therefore determine by whether containing above-mentioned 3 kinds of albumen in detection sample whether this gene exists.If foreign gene proceeds to plant, producer silence, produces without destination protein, can not accomplish the end in view.
The advantage of kit described in the utility model is:
Application of sample is simple, once completes;
Economical and efficient: a detection box can detect 3 kinds of different transgenosis compositions simultaneously, greatly reduces use cost;
Easy to be quick: 3 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, within 5-10 minute, can go out result;
Testing result is directly perceived: not by any instrument, with the naked eye can be observed result;
Be widely used: blade, seed and the converted products that can detect the multiple kinds of crops such as cotton, wheat, paddy rice, corn, rape, tobacco, soybean.
The joint inspection kit providing due to the utility model can carry out multiple transgenic analysis to biological sample simultaneously, and the departments such as laboratory, field, frontier defense, customs's plant inspection, inspection and quarantine bureau, food safety detection and seed operation sale that are specially adapted to are to suspicious sample scene, rapid screening.
Brief description of the drawings:
Fig. 1 and Fig. 2 are the structural representations that the utility model relates to the gold mark Quick joint inspection kit of the factor IrrE of global regulation, Csp and CarD albumen, and wherein Fig. 1 is the vertical view of base plate, and Fig. 2 is the vertical view of kit upper cover;
In Fig. 1,3 is calibration tapes, and in Fig. 2,1 is well, the 2nd, and observation window.
Embodiment:
The preparation of the calibration tape in the utility model kit brings and illustrates with the test of preparation detection CarD:
Embodiment 1 prepares calibration tape
The preparation of 1.CarD odd contradictive hydroperitoneum
Mouse peritoneal injecting fluid paraffin 0.5mL.After 1 week, CarD monoclonal antibody hybridoma is injected in to above BALB/C mice abdominal cavity of anticipating, every injected in mice hybridoma 1～3 × 10
6.After 10 days, gather in the crops ascites, above process all completes under aseptic condition.
The purifying of 2.CarD monoclonal antibody
1) with DEAE ion-exchange chromatography and Sephacryl S300 molecular sieve, monoclonal antibody is carried out to purifying; SDS-PAGE disk electrophoresis detects the purity of purified monoclonal antibody; ELISA method is measured monoclonal antibody activity;
2) purge process: get mouse odd contradictive hydroperitoneum → 3, the centrifugal 15min → supernatant of 000 × g adds 50% (NH
4)
2sO
4precipitation, it is centrifugal that the centrifugal 20min → precipitation of spend the night → 3,000 × g is dissolved in 0.01M phosphate buffer (pH7.2) → S-300 gel column → DEAE Blue chromatographic column → 0-800mM NaCl gradient elution → ultrafiltration, concentrated monoclonal antibody;
3) monoclonal antibody purity testing: conventional SDS-PAGE electrophoresis, the purity of the purified monoclonal antibody of said method is all more than 95%;
4) determination of protein concentration: ultraviolet spectrophotometer is measured the OD value at 279nm place, calculates as follows protein content: OD
279/1.4=mg/mL (purified monoclonal antibody).Be about 1mg/mL by monoclonal antibody concentration adjustment;
5) monoclonal antibody determination of activity: select the antigen coated elisa plate of CarD (1 μ g/ hole), and sheep anti-mouse igg-HRP compound, measure each monoclonal antibody and tire (being greater than 1:5000).
3. goat-anti DOA
tthe high-titer sero-fast preparation of immunity and purifying
1) above-mentioned monoclonal antibody+Freund's complete adjuvant → immune goat → booster immunization that purifying is good 2 times, every minor tick 1 month → strengthen for the second time getting for latter about 10 days blood → centrifuging and taking serum → (NH
4)
2sO
4precipitation → DEAE ion-exchange chromatography purifying;
2) titration: ELISA sandwich method, 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 the preparation of golden labeling antibody
1) preparation of collaurum: the collaurum of preparing 40-60nm with citrate reducing process.By 0.01%HauCl
4be heated to boil, add a certain amount of 1% trisodium citrate (Na
3c
6h
5o
72H
2o), continue heating and boil 5min, treat that colloidal gold solution color is by blue → purple → red, after stablizing, cooling.Colloidal gold solution should be limpid transparent, as need are preserved for a long time, can add 0.02%NaN
3.
2) collaurum liquid 500mL is adjusted to pH8.4 with 0.1M NaOH, under magnetic agitation, slowly add monoclonal antibody 2mL, stir 20min.The centrifugal 30min of 5,500 × g, removes unconjugated protein in supernatant.Centrifugal rear absorption colloidal gold labeled monoclonal antibody precipitation, precipitation is dissolved in 10mL and preserves in liquid, with 0.45 μ m filtering with microporous membrane.Sampling is examined and determine, and all the other put 4 DEG C of preservations.
5. the preparation of film reaction system M1
1) sheep anti-mouse igg of CarD antibody and purifying is diluted with 0.1M phosphate buffer, final concentration is about 0.2-2mg/mL.
2) sheep anti-mouse igg of purifying is dissolved in 0.1M phosphate buffer, and concentration is 2.0mg/mL;
3) nitrocellulose filter size: 10cm × 27cm, detection and control that every film can spray long 25cm are with each 4;
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.With monoclonal antibody CarDAD2(2mg/mL) coated reaction detection line on NC film, with the coated reaction of 1.0mg/mL rabbit anti-mouse igg control line, the distance of control line and detection line is 4-4.5mm;
6) complete coated after, put 37 DEG C of drying box 24h, process 0.5h with the sealing of BB confining liquid, take out and wash 1 time by WB washing lotion;
7) 37 DEG C of drying box drying for standby;
8) nitrocellulose filter that cutting is got ready: 1.8cm × 27cm/ bar, put into thin aluminum bag hermetically drying and preserve.Put room temperature preservation for subsequent use.
6. the preparation of film reaction system M2a, M2b and M3
Water-absorption fiber film is soaked in respectively in M2a, M2b, M3 solution, and after soaking into, taking-up is dried, and packs in polybag room temperature preservation into.
1) M2a preparation: the glass fibre making is cut to 27cm × 1.2cm/ bar;
2) M2b preparation: the glass fibre making is cut to 27cm × 1.8cm/ bar;
3) M3 preparation: the glass fibre making is cut to 27cm × 1.2cm/ bar.
7. the preparation of film reaction system M4
1) get collaurum-monoclonal antibody compound and add dilution, mix and be 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 DEG C of oven dry 12h, put into thin aluminum bag, add drying agent, heat sealing, room temperature preservation.
8. reaction body assembling
1) on M6 plastic base, paste 4 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 M6 upper limb and hand over 1mm with M1 upper limb;
4) on M7, paste M2a (12mm), join with 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 approximately 2/3;
7) on M7, paste M2b(18mm), upper limb is pushed down M4 approximately 2/3, 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 completely, and pushes down the about 1.5mm of M1;
9) on M5, paste colour-coded adhesive tape M9, hand over 1mm with M1 upper limb, the other end climbs over M6 upper limb, is affixed on M6 quilt cover;
10) the reaction body of assembled formation is cut into 3.5mm specification with full-automatic strip cutter.
Embodiment 2 kit assemblings
Kit flat box triangular in shape, three limits of box plate are respectively provided with a groove, place 3 calibration tapes; Upper cover and calibration tape corresponding positions are equipped with 3 observation windows, and 3 wells are arranged on three drift angle places of upper cover (seeing Fig. 1, Fig. 2).
Three kinds of calibration tapes of the detection CarD, the IrrE that prepare and Csp albumen are embedded respectively in 3 grooves of base plate, then by upper cover and base plate fastening, kit.
By after kit, dropper and operation instructions dress packaging plastic bags.
Embodiment 3 kit use procedures
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 optimum detection effect, variant sample should, according to the dilution proportion in following table, completing after grinding and dilution, mix sample, leaves standstill, and gets supernatant as detecting sample.
2. detect and result
Detection box is kept flat, draw sample liquid with joined plastic suction pipe, drip 15-20 and drip in detecting the well 1 of box, absorbent material slowly moves sample to be checked, and film reaction system is activated.No matter whether testing gene is present in plant sample, and an aubergine band all can appear at (C) in Quality Control district.The aubergine band that in Quality Control district, (C) manifests is to determine whether enough plant sample liquid, and whether chromatography process normal standard, simultaneously also as the inner quality standard of reagent.
3. result is judged:
Whether each observation window detects a kind of gene to be checked and exists.Below to detect CarD as example explanation:
1) as do not contained specific CarD albumen in sample, or its concentration is lower than detection sensitivity, colloidal gold antibody can not be fixed on the monoclonal antibody immunity in detection band on film in chromatography process, thereby in test section, (T) there will not be an aubergine to detect band, show negative (-) result, only in Quality Control district (C), occur an aubergine band.
2) if when in plant sample, CarD protein concentration is higher than its detection threshold, antigen immunity gold is combined with another monoclonal antibody that detection is brought, in test section, (T) will also occur that another aubergine detects band, show positive (+) result, i.e. an aubergine band of each appearance in Quality Control district (C) and detection zone.Attention: the aubergine band in test section (T) can show the phenomenon of shade.But, within the observing time of regulation, no matter this colour band shade all should be judged to be positive findings.
3) as aubergine band does not appear in Quality Control district (C), testing result is invalid, shows incorrect operating process or kit rotten damage.
Claims (1)
1. the gold mark Quick joint inspection kit of global regulation's factor IrrE, Csp and CarD albumen, is made up of base plate, upper cover; It is characterized by, described kit be shaped as triangle box body, on the base plate of box body, be provided with 3 calibration tapes; On be stamped 3 observation windows and 3 wells; Described 3 calibration tapes lay respectively at three limits on described triangle box plate surface; The upper surface of described base plate is provided with 3 can Embedded test band groove.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201320731951.7U CN203849263U (en) | 2013-11-19 | 2013-11-19 | Gold immnnochromatography rapid combined measuring kit of global regulation and control factors IrrE, Csp (cold shock protein) and CarD proteins |
Applications Claiming Priority (1)
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| CN201320731951.7U CN203849263U (en) | 2013-11-19 | 2013-11-19 | Gold immnnochromatography rapid combined measuring kit of global regulation and control factors IrrE, Csp (cold shock protein) and CarD proteins |
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| CN203849263U true CN203849263U (en) | 2014-09-24 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108396012A (en) * | 2018-02-06 | 2018-08-14 | 中国农业科学院生物技术研究所 | Applications of the monoclonal antibody 1DB4 in detecting IrrE transgenic crops |
| CN108396013A (en) * | 2018-02-06 | 2018-08-14 | 中国农业科学院生物技术研究所 | A kind of gold label test strip detecting global regulation's factor IrrE albumen and its transgenic crop |
-
2013
- 2013-11-19 CN CN201320731951.7U patent/CN203849263U/en not_active Expired - Lifetime
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108396012A (en) * | 2018-02-06 | 2018-08-14 | 中国农业科学院生物技术研究所 | Applications of the monoclonal antibody 1DB4 in detecting IrrE transgenic crops |
| CN108396013A (en) * | 2018-02-06 | 2018-08-14 | 中国农业科学院生物技术研究所 | A kind of gold label test strip detecting global regulation's factor IrrE albumen and its transgenic crop |
| CN108396012B (en) * | 2018-02-06 | 2020-10-23 | 中国农业科学院生物技术研究所 | Application of monoclonal antibody 1DB4 in detection of IrrE transgenic crops |
| CN108396013B (en) * | 2018-02-06 | 2020-11-27 | 中国农业科学院生物技术研究所 | Gold-labeled test strip for detecting global regulatory factor IrrE protein and transgenic crops thereof |
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