CN201796037U - Cold shock protein (CSP) one-step process colloidal gold rapid detection kit - Google Patents

Abstract

A cold shock protein (CSP) one-step process colloidal gold rapid detection kit comprises a kit body, a test tape and an upper cover. The kit body is covered by the upper cover to form the kit. The kit is characterized in that the test tape is placed on the inner surface of the kit body, and the upper cover is provided with an observation window and a sampling hole. The kit can provide direct evidence for rapidly detecting whether plant seeds, laminas and processed products contain cold shock protein (CSP) gene contents or not, does not need any other additional apparatuses, and is simple in operation and vivid, accurate and fast in detection results. People only need to drip extracting solution of a detected object into the sampling hole, and can directly observe accurate detection results from the observation window without any instruments after three to five minutes.

Description

Cold shock protein CSP single stage method gold labeled quick detection reagent box

Technical field:

The utility model relates to cold shock protein CSP single stage method gold labeled quick detection reagent box.

Background technology:

Microbial cell under extreme conditions reacts as producing cold shock in the environment such as cold, arid, and forms a kind of special albumen---cold shock protein (CSP).Cold shock protein transcribe with translation process in play important regulatory role.As a kind of molecular chaperones and single-chain nucleic acid non-specific binding, have the effect of stable strand nucleotide secondary structure, make under cell survives under extreme environment.Change the cold shock protein gene (csp) in the microorganism over to plant, can obtain to have the genetically modified plants of degeneration-resistant proterties fast.The cold shock protein csp gene clone of CSP encoding histone is from the extremely strong extreme microorganism Deinococcus gobiensis I-0 of a strain tolerance to environmental stress, and at present, cold shock protein CSP encoding gene has been applied to the research of multiple genetically modified plants.

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.

Existing pick-up unit, exempt from, put the device of methods such as exempting from 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 (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 cold shock protein CSP in a kind of fast detecting genetically modified plants in the practice, and easy and simple to handle, result accurately and reliably, device fast.

The utility model content:

The purpose of this utility model be set up a kind of easy and simple to handle, result accurately and reliably, the immunity detection reagent of fast detecting transgene component (CSP albumen).

The technical scheme that its technical matters that solves the utility model adopts is:

A kind of cold shock protein CSP single stage method gold labeled quick detection reagent box, 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, described observation window is a rectangle, and the shape of kit is a rectangle.

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 cold shock protein CSP.

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 cold shock protein CSP calibration tape is by exceptional function CSP monoclonal antibody or polyclonal antibody.

But determine by whether containing this albumen in the immune response test sample whether this gene exists.When foreign gene changes plant over to, and after expressing, will produce destination protein (CSP) in plant, its sample detection is positive, if foreign gene changes plant over to, and the producer silence, no destination protein generation, (cold shock protein CSP) 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.

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, the extract of inspecting article by ready samples is dripped in the well of kit, after 3-5 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 3-5 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, tobacco, rape.

Because can carrying out CSP to plant sample, the kit that the utility model provides 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 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:

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 a detection zone.

Embodiment:

The preparation explanation of the calibration tape in the utility model kit:

Embodiment 1 preparation calibration tape

1. the preparation of cold shock protein CSP odd contradictive hydroperitoneum

Mouse peritoneal injecting fluid paraffin 0.5mL.After 1 week, cold shock protein CSP 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. cold shock protein CSP 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 → 0-800mMNaCl 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 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 cold shock protein CSP (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 cold shock protein CSP 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 CPSAD2 (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 back side;

10) the full-automatic cutting cutter of reaction body and function with assembled formation is cut into the 3.5mm specification.

The assembling of embodiment 2 kits

The calibration tape 3 of the detection cold shock protein CSP 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 3 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 cold shock protein CSP 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.

2) if when treating that cold shock protein CSP 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 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. cold shock protein CSP single stage method gold labeled quick detection reagent box, 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; Described observation window is a rectangle, kit be shaped as rectangle.

2. cold shock protein CSP single stage method gold labeled quick detection reagent box according to claim 1 is characterized in that described cartridge inner surface is provided with the groove that can embed described calibration tape.

3. cold shock protein CSP single stage method gold labeled quick detection reagent box according to claim 1 and 2 is characterized in that described box body or loam cake have draw-in groove.

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