CN201600374U - Quick joint inspection kit - Google Patents
Quick joint inspection kit Download PDFInfo
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- CN201600374U CN201600374U CN2010201165723U CN201020116572U CN201600374U CN 201600374 U CN201600374 U CN 201600374U CN 2010201165723 U CN2010201165723 U CN 2010201165723U CN 201020116572 U CN201020116572 U CN 201020116572U CN 201600374 U CN201600374 U CN 201600374U
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Abstract
The utility model relates to a quick joint inspection kit, which comprises a baseplate and an upper cover, wherein a test tape is arranged on the inner surface of the baseplate; an observation window is arranged on the upper cover, and the upper cover is covered on the baseplate to form the kit. The quick joint inspection kit is characterized in that after the upper cover is covered on the baseplate, one end of the test tape is exposed out of one side of the kit. The kit has the advantage of simple and convenient operation without needing sampling instruments; when a sample is detected, one end of the kit is directly immersed into extracting solution of a submitted sample and is taken out in 5 seconds and placed flatly, and test results can be directly observed through naked eyes via the observation window after 5 to 10 minutes.
Description
Technical field:
The utility model relates to a kind of fast joint inspection kit.
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.Sometimes also need simultaneously several allogenic genes to be detected.For example need to carry out simultaneously G2-EPSPS (5-enol acetone Mang oxalic acid-3-phosphate synthase) protein coding gene (aroA) and bacillus thuringiensis (Bacillus thuringiensis, Bt) detection of two kinds of genes.
The device of methods such as exempting from is exempted from, put to existing pick-up unit as enzyme, 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 it is higher to detect cost, is difficult to apply.
Chinese patent 200520114588.X provides the kit of a kind of combined testing EPSPS and BT albumen, the extract of censorship article is used to drip in two wells of kit respectively can obtain the result, though easy and simple to handle, quick compared with said apparatus.But also need application of sample apparatuses such as sample injector or dropper in the packing of kit.
The utility model content:
The purpose of this utility model is that to set up a kind of operation easier, quick, does not use sample injector can fast detect the joint inspection kit of transgene component.
The technical solution of the utility model is such:
A kind of fast joint inspection kit comprises base plate, loam cake, and plate inner surface has calibration tape, on be stamped observation window, base plate cover is formed kit after going into loam cake, it is characterized in that: after base plate cover was gone into loam cake, an end of calibration tape exposed kit one side.
When needs detect two or more transgene components simultaneously, in base plate, can insert many different calibration tapes, and loam cake is provided with the observation window of respective numbers.
The thickness of base plate is not limit, and the upper surface of base plate can be provided with two or many can embed the calibration tape groove, and each groove can be placed a calibration tape.
Described base plate or loam cake can have draw-in groove, to guarantee base plate and loam cake fastening.
Described observation window is rectangle preferably.
The shape of kit is flat rectangle box.
During use, the calibration tape that directly will expose kit one end immerses testing sample liquid, and the observation window by the kit loam cake can observe directly testing result, and each observation window can be checked a kind of gene respectively.
The major advantage of kit described in the utility model is more convenient and quicker, need not the application of sample apparatus,
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 all kinds of crops such as corn, cotton, paddy rice, wheat, tobacco, soybean.
Because the joint inspection kit that the utility model provides detects fast, 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.
Below in conjunction with drawings and Examples, further illustrate technology contents of the present utility model.
Description of drawings:
Fig. 1 and Fig. 2 are the structural representations of a kind of joint inspection kit of the utility model, and wherein Fig. 1 is the vertical view of base plate, and base plate has two calibration tapes;
Fig. 2 is the vertical view of kit loam cake, on be stamped two observation windows;
Among the figure, the 1st, calibration tape, the 2nd, observation window, T are the test sections, C is the Quality Control district.
Embodiment:
The kit of 1 one kinds of joint inspection examinations of embodiment BT-Cry1Ah
This kit plate inner surface is inserted two different calibration tapes (1), and loam cake is provided with two observation windows (2).Each observation window is divided into test section (T) and Quality Control district (C) two parts, can indicate last covering with letter C, T;
Two calibration tapes in the described base plate, one is used to detect G2-EPSPS, and another is used to detect BT 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.The calibration tape of two kinds of albumen all can be 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 G2-EPSPS, and wrap by the monoclonal antibody of BT-Cry 1Ah or polyclonal antibody in the sample detection district of BT albumen calibration tape.
When foreign gene changes plant over to, and in plant, express, will produce destination protein (G2-EPSPS and BT-Cry1Ah), therefore determine by whether containing above-mentioned two kinds of albumen in the test sample whether this gene exists.If foreign gene changes plant over to, the producer silence, no destination protein produces, and (antiweed or desinsection) can not accomplish the end in view.
During detection, only need kit pectination device is immersed in the extract of censorship article, take out kit in 5 seconds and keep flat, after 5-10 minute, naked eyes just can observe directly testing result accurately by observation window.
The detailed preparation method of kit is as follows:
(1) preparation calibration tape
1.BT-Cry1Ah the preparation of odd contradictive hydroperitoneum
Mouse peritoneal injecting fluid paraffin 0.5mL.After 1 week, BT-Cry1Ah 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.
2.BT-Cry1Ah 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 O.D. 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 BT-Cry1Ah (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 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.
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), 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 0.1M NaOH, 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.
5. the preparation of film reaction system M1
1) sheep anti-mouse igg with BT-Cry1Ah 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 BT-Cry1AhAD2 (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.
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 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.
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 12 hours, put into thin aluminum bag, add drying agent, heat sealing, room temperature preservation.
8. 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 quilt cover;
10) the full-automatic cutting cutter of reaction body and function with assembled formation is cut into the 3.5mm specification.
(2) assembling kit
The detection G2-EPSPS for preparing and two kinds of calibration tapes that detect BT-Cry1Ah albumen are embedded respectively in two grooves of base plate,, calibration tape is exposed outside the box from kit one end then with loam cake and base plate fastening.Kit.
(3) kit use
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
Kit pectination device is immersed in the extract of censorship plant sample, take out kit in 5 seconds and keep flat, absorbent material slowly moves sample to be checked, and the film reaction system is activated.After 5-10 minute, naked eyes just can observe directly testing result accurately by observation window.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:
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.
Whether each observation window detects a kind of gene to be checked and exists.Below be the example explanation to detect BT-Cry1Ah:
1) as not containing particular B T-Cry1Ah 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, show negative (-) result, an aubergine band promptly only in Quality Control district (C), occurs.
2) if when treating that the BT-Cry1Ah 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 as Quality Control district (C), then testing result is invalid, shows the rotten damage of incorrect operating process or kit.
Claims (5)
1. the fast joint inspection kit comprises base plate, loam cake, and plate inner surface has calibration tape, on be stamped observation window, base plate cover is formed kit after going into loam cake, it is characterized in that: after base plate cover was gone into loam cake, an end of calibration tape exposed kit one side.
2. fast joint inspection kit according to claim 1, described calibration tape quantity is two or more; Described observation window quantity is two or more.
3. fast joint inspection kit according to claim 1 and 2 is characterized in that the upper surface of described base plate is provided with two or more groove, and each groove can embed a calibration tape.
4. fast joint inspection kit according to claim 1 and 2 is characterized in that described base plate or loam cake have draw-in groove.
5. fast joint inspection kit according to claim 1 and 2 is characterized in that described observation window is a rectangle.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010201165723U CN201600374U (en) | 2010-02-11 | 2010-02-11 | Quick joint inspection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010201165723U CN201600374U (en) | 2010-02-11 | 2010-02-11 | Quick joint inspection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN201600374U true CN201600374U (en) | 2010-10-06 |
Family
ID=42811438
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010201165723U Expired - Lifetime CN201600374U (en) | 2010-02-11 | 2010-02-11 | Quick joint inspection kit |
Country Status (1)
| Country | Link |
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| CN (1) | CN201600374U (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045468A (en) * | 2011-10-14 | 2013-04-17 | 深圳太太基因工程有限公司 | Kit used for detecting helicobacter pylori infection |
| CN117143832A (en) * | 2023-11-01 | 2023-12-01 | 中国农业科学院生物技术研究所 | Insect-resistant protein Cry1Ah hybridoma cell strain, antibody produced by same and application thereof |
| CN117143831A (en) * | 2023-10-31 | 2023-12-01 | 中国农业科学院生物技术研究所 | Insect-resistant protein hybridoma cell strain, antibody produced by same 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 |
-
2010
- 2010-02-11 CN CN2010201165723U patent/CN201600374U/en not_active Expired - Lifetime
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045468A (en) * | 2011-10-14 | 2013-04-17 | 深圳太太基因工程有限公司 | Kit used for detecting helicobacter pylori infection |
| CN117143831A (en) * | 2023-10-31 | 2023-12-01 | 中国农业科学院生物技术研究所 | Insect-resistant protein hybridoma cell strain, antibody produced by same and application thereof |
| CN117143831B (en) * | 2023-10-31 | 2024-01-26 | 中国农业科学院生物技术研究所 | Insect-resistant protein hybridoma cell strain, antibody produced by same and application thereof |
| CN117143832A (en) * | 2023-11-01 | 2023-12-01 | 中国农业科学院生物技术研究所 | Insect-resistant protein Cry1Ah hybridoma cell strain, antibody produced by same 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 |
| CN117143832B (en) * | 2023-11-01 | 2024-02-13 | 中国农业科学院生物技术研究所 | Insect-resistant protein Cry1Ah hybridoma cell strain, antibody produced by same and application 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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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CX01 | Expiry of patent term |
Granted publication date: 20101006 |
|
| CX01 | Expiry of patent term |