CN108761075A - A kind of deoxynivalenol quantifies rapid detection card and its detection method - Google Patents
A kind of deoxynivalenol quantifies rapid detection card and its detection method Download PDFInfo
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Abstract
The invention discloses a kind of deoxynivalenols to quantify rapid detection card, including sample pad, nitrocellulose filter and the water absorption pad being set in turn on bottom plate, have on the nitrocellulose filter and be coated with the detection line of DON hapten-carrier albumen coupling agent and be coated with the nature controlling line of sheep anti-mouse antibody, sample solution to be checked there are the latex beads of DON antibody to be mixed before being added drop-wise to the sample pad with coupling, incubation is handled；Its detection method includes preheating, liquid feeding, incubation, reaction and reads result；Detection card of the present invention is not necessarily to the release pad containing gold labeling antibody, coupling for being mixed with test sample solution has the latex beads of DON antibody, bright-colored easy differentiation, and coupled antibody to be coupled for chemical bond, better than colloidal gold physical absorption stability, accuracy in detection and precision are high；Otherness is small at low cost between batch；In addition, the detection method operation of detection card of the present invention is simple, it is suitable for mass production and application.
Description
Technical field
The present invention is to be related to a kind of quantitatively rapid detection card and its detection method, is to be related to a kind of deoxidation snow corruption specifically
Sickle-like bacteria enol quantifies rapid detection card and its detection method, belongs to technical field of biological, especially suitable for grain and oil and paddy
The quantitative quickly detection of the deoxynivalenol of species.
Background technology
Vomitoxin (vomitoxin), also known as deoxynivalenol (deoxynivalenol, DON), chemical name
For the trihydroxy grass Fusariumsp -9- alkene -8- ketone of 3 α, 7 α, 15 1, belong to trichothecene.Since it can cause pig
It vomits and gains the name, it is three-level carcinogenic substance also to have certain damaging effect, European Union's criteria for classification to human body.Vomitoxin is to people and moves
Object has very strong toxicity, can cause humans and animals vomiting, diarrhea, skin irritatin, food refusal, neurological disorders, miscarriage, stillborn foetus etc.,
Pig is the animal most sensitive to vomitoxin, and poultry takes second place, and for ruminant due to the effect of rumen microorganism, tolerance is most strong.
Vomitoxin belongs to one kind of mycotoxin, and the mycotoxin test strips of grain and oil and cereal field prevalence are mainly colloid at present
The qualitative product of gold, colloidal gold basis weight products are difficult to account for city always since accuracy, precision and stability are extremely difficult to require
Field is leading, and client can only lean on enzyme linked immunological kit and instrumental method such as efficient liquid phase etc., detect the concrete content of sample, enzyme-linked
Immune reagent kit and instrumental method are more demanding to personnel and environment, and detection time is longer.China standard GB/T 2761-
1000 μ g/kg of mycotoxin country limitation vomitoxin limitation in 2017 national food safety standard food.
Existing deoxynivalenol test strips mainly use colloidal gold mark technology, by the antigen or antibody of specificity
It is fixed on film with ribbon, colloid gold label reagent (antibody or monoclonal antibody) is adsorbed on bonding pad, when sample to be checked
It after being added in the sample pad of test strips one end, moves forward through capillary action, dissolves the colloid gold label reagent on bonding pad
After react to each other, then when being moved to the region of fixed antigen or antibody, the conjugate of object and gold marked reagent to be checked is sent out therewith again
Life specifically binds and is trapped, and being gathered in detection takes, and can observe by the naked eye colour developing result；As Li Peizhen et al. is adopted
With the vomitoxin in colloidal gold test strip method quantitative determination grain, (colloidal gold test strip method is to Gibberella zeae in grain
The application study of ketenes quantitative determination, Wang Guifang, Li Peizhen, Cao Yang, Guo Jian, Liu Meichen,《Food technology and economy》
2 months the 1st phases of volume 39 in 2014), to verify the applicability of the method vomitoxin in detecting grain.
There are the following problems for the right above method：
(1) for colloidal gold sheet as dark violet red, color is shallower, is not easy to differentiate the yin and yang attribute of testing result；
(2) colloidal gold a batch gold amount most about 10L or so, grain size deviation is more difficult to be precisely controlled, and mass production is quantitatively produced
Product, difference batch be easy to cause big difference between batch, and then influences the precision and result stability of testing result；
(3) colloidal gold-labeled method is physical absorption, is coupled compared with chemical bond, and product stability is poor；
(4) existing market colloidal gold strip is mainly qualitative product, cannot meet people to operating quick, simple, people
Member requires the application demands of low, at low cost, macroscopic basis weight products, and existing qualitative screening, false negative and false positive compared with
It is high；
(5) burning gold, labelled antibody workload are larger, and technique is cumbersome, and output is few, wasting manpower and material resources.
Invention content
In view of the above-mentioned problems existing in the prior art and demand, the object of the present invention is to provide a kind of deoxynivalenol bacterium
Enol quantifies rapid detection card and its detection method, and sample measuring liquid to be checked is mixed to prepare the antibody containing DON with latex beads in advance
It is added drop-wise on detection card and is detected again after reaction solution, improve accuracy and the stability of testing result, no longer need to utilize glue
Body gold marks DON antibody on detection card, and operation and making are simple, of low cost, are suitable for high-volume output and application.
To achieve the above object, the technical solution adopted by the present invention is as follows：
A kind of deoxynivalenol quantifies rapid detection card, it is characterised in that：Including being set in turn on bottom plate
Sample pad, nitrocellulose filter and water absorption pad, have on the nitrocellulose filter and be coated with deoxynivalenol
The detection line of hapten-carrier albumen coupling agent and the nature controlling line for being coated with sheep anti-mouse antibody, sample solution to be checked are being added drop-wise to
There are the latex beads of DON antibody to be mixed with coupling before the sample pad, incubation is handled.
Preferably, the package amount of the detection line and the nature controlling line is respectively 0.1mg/mL and 0.4mg/mL.
As further preferred scheme, the nitrocellulose filter is in the coating for completing the detection line and the nature controlling line
Afterwards, it first carries out drying overnight processing in 37 DEG C of conditions.
Preferably, the sample pad, the nitrocellulose filter and the water absorption pad are pasted and fixed on institute successively
It states on bottom plate, the sample pad and the water absorption pad are overlapped with the nitrocellulose filter respectively.
As further preferred scheme, the sample pad, the nitrocellulose filter, the water absorption pad, the detection line
Length with the nature controlling line along the floor length direction is followed successively by 25mm, 21mm, 20mm, 3-4mm and 3-4mm, described
Sample pad and the water absorption pad are respectively 1-2mm with the lap width of the nitrocellulose filter.
Preferably, the deoxynivalenol hapten-carrier albumen coupling agent passes through following sides
Method prepares：First deoxynivalenol haptens is dissolved in DMF, adds EDC, it then will be obtained molten
It after liquid is stirred at room temperature 24 hours, is slowly dropped to dropwise in the CB solution dissolved with OVA, and stirring 24 is small at room temperature
When, finally obtained solution PBS is dialysed 3 days.
As further preferred scheme, under the deoxynivalenol hapten-carrier albumen coupling agent passes through
The method of stating prepares：It first takes 8mg deoxynivalenol haptens to be dissolved in the DMF of 0.8mL, then is used being added
After the EDC for the 20mg that the water of 0.2mL fully dissolves, it is stirred at room temperature 24 hours, you can obtain reaction solution A, weigh 36mg's
OVA simultaneously be substantially dissolved in 3mL a concentration of 0.1mol/L, pH be 9.6 CB in, you can obtain protein solution A, by reaction solution A by
Drop is slowly dropped in protein solution A, and is stirred 24 hours at room temperature, finally, with the PBS of a concentration of 0.01mol/L 4
It dialyses 3 days under the conditions of DEG C and replaces 3 dialyzates daily to get to the deoxynivalenol hapten-carrier
Albumen coupling agent.
Preferably, the coupling has the latex beads of DON antibody to be prepared by following methods：
S1：The MES buffer solutions of 25mM, pH6.0 are added in latex beads, are carried out using MES buffer solutions after mixing
Centrifuge washing；
S2：The ratio that the EDC of the NHS and 40 μ L of 40 μ L are corresponded to 1L microballoons is separately added into NHS and the EDC reaction of 25mM
Liquid carries out after mixing being incubated 10min processing；
S3：Centrifuge washing is carried out using MES buffer solutions, supernatant is gone to handle；
S4：Mixing after addition DON antibody to its final concentration of 0.6mg/mL；React 2h；
S5：Centrifugation removal supernatant processing is carried out, and the BSA of addition 2% is closed, again mixing；It is laggard in reaction 1h
Row centrifugation removal supernatant processing, later, be added 2% BSA cleaned, mixing；
S6：Centrifugation removal supernatant processing is carried out, microballoon is suspended in storage 10mMpH7.0PBS buffer solutions, and mixing is simultaneously protected
It is spare to get to coupled antibody in 2-8 DEG C of refrigerator to be stored in storage temperature；
S7：After taking coupled antibody freeze-drying diluted obtained above, frozen dried is carried out, coupled antibody is lyophilized
Reaction micropore is formed in the micropore of latex beads, that is, completes freeze-drying and the coating of the coupled antibody, and obtaining coupling has DON
The latex beads of antibody.
As further preferred scheme, the preparation method of the MES buffer solutions of described 25mM, pH6.0 is：By 0.53g
MES be dissolved in 90mL pure water, adjust pH be 6.0, be used in combination pure water constant volume in 100mL.
As further preferred scheme, the DON antibody is prepared by following methods：
(1) making of immunogene
It first takes 8mg deoxynivalenol haptens to be dissolved in the DMF of 0.8mL, adds and filled with the water of 0.2mL
The EDC for dividing the 20mg of dissolving, is then stirred at room temperature 24 hours, you can obtain reaction solution A；Weigh the OVA of 36mg and abundant
It is dissolved in the CB that 3mL a concentration of 0.1mol/L, pH are 9.6, you can obtain protein solution A；Reaction solution A is slowly dripped dropwise
It is added in protein solution A, and stirs 24 hours at room temperature, 3 are dialysed under the conditions of 4 DEG C with the PBS of a concentration of 0.01mol/L
It and replace 3 dialyzates daily to get to immunogene；
(2) it is immunized
With the immunogen immune Balb/c mouse obtained in step (1), metering is immunized as 100 μ g/, its is made to generate anti-blood
Clearly；
(3) cell fusion and cloning
The immune Balb/C mouse boosting cells in step (2) are taken, are 8 by quantitative proportion:1 ratio and SP2/0 myeloma
Cell fusion measures cell supernatant using indirect competitive ELISA method, screens positive hole；Using limiting dilution assay to the positive
Hole carries out cloning, until obtaining the hybridoma cell strain of stably excreting monoclonal antibody；
(4) cell cryopreservation and recovery
The hybridoma obtained in step (3) is made to the cell suspension of 1 × 106/mL of frozen stock solution, it is long in liquid nitrogen
Phase preserves；The cryopreservation tube preserved for a long time in liquid nitrogen is taken out when recovery, 37 DEG C of water-bath middling speeds is immediately placed in and melts, and centrifugation removal freezes
After liquid, culture bottle culture is moved into；
(5) the preparation and purification antibody of ascites
Using in vivo method, cell is beaten in mouse peritoneal, mouse ascites are taken out after 7 days, purifies to obtain with saturated ammonium sulfate method
Antibody.
The present invention also provides the detection method that a kind of deoxynivalenol quantifies rapid detection card, features
It is：Include the following steps：
(1) it preheats：Couveuse is opened to switch and be warming up to 45 ± 1 DEG C before detection；
(2) liquid feeding：Sample solution 120ul to be checked is drawn with pipettor, is vertically added dropwise to processed by freeze-drying and packet
Coupling has in the latex beads of DON antibody, inhales repeatedly and makes a call to 10 times with up to uniformly mixed；
(3) it is incubated：The solution that detection card and step (2) obtain is placed on couveuse, is incubated under conditions of 45 ± 1 DEG C
Educate 2min；
(4) it reacts：The solution after being incubated is taken out, inhales and makes a call to 4 times or more repeatedly, the liquid 120ul after careful absorption reaction ±
10ul is added dropwise in the sample pad of detection card (well at S in Fig. 2) according to sampfe order, reacts 5min；
(5) result is read：Testing result is directly obtained according to the display of detection card, alternatively, will take out from couveuse
The detection is stuck in 1min interpolations and enters the quantitative detection that readout instrument carries out batch, reads or/and print testing result.
Preferably, further comprising the steps of after step (1)：Mark sample：According to sample size to be checked, take out
Respective numbers have the latex beads of DON antibody and detection to block by freeze-drying with processed coupling is wrapped, and in detection card subscript
Note is to distinguish sample solution to be checked.
As further preferred scheme, it is 125 μ g/kg-2500 μ g/ that the sample solution to be checked, which is suitable for detection range,
Kg, preparation method include the following steps：
(1) it crushes：Cereals class sample is crushed；
(2) it weighs：Cereals class sample that 5g is crushed is weighed in 50ml centrifuge tubes；
(3) mixing：25mL deionized waters are added in centrifuge tube, after sealing high speed acutely shakes mixing 3min, in room temperature
Lower standing 2min；
(4) it centrifuges：It stands or 4000rpm centrifuges 5min；
(5) it dilutes：100 μ L supernatant liquids are taken, 900 μ L sample dilutions are added, mixing is spare.
Compared with prior art, the present invention has the advantages that：
Deoxynivalenol of the present invention quantifies rapid detection card, is not necessarily to the release pad containing gold labeling antibody,
It detecting blocking and makees simple, the coupling for being mixed with test sample solution has a latex beads of DON antibody, bright and full color,
Easily differentiate, and latex beads coupled antibody is coupled for chemical bond, it is better than colloidal gold physical absorption stability, accuracy in detection,
Precision and stability are high；Every batch of latex beads yield at least 50L-100L, with being 5-10 times of colloidal gold in batches, between batch
Otherness it is small and of low cost；Latex beads coupling process is easy to operate, and experimental article is not necessarily to steep acid every time, by environment, people
Member and reagent influence are smaller, greatly save manpower and materials；In addition, the detection method operation of detection card of the present invention it is simple, at
Power is high, and readout instrument also can be used and carry out the reading of batch, printing testing result, have compared with the existing technology conspicuousness into
Step and advantageous effect outstanding.
Description of the drawings
The structure that Fig. 1 quantifies rapid detection card for a kind of deoxynivalenol that the embodiment of the present invention 1 provides is shown
It is intended to；
Fig. 2 is the detection side that the deoxynivalenol that the embodiment of the present invention 3 provides quantifies rapid detection card
The flow diagram of method.
Figure label is schematically as follows：1, bottom plate；2, sample pad；3, nitrocellulose filter；4, water absorption pad；5, detection line；6,
Nature controlling line.
Specific embodiment
Technical scheme of the present invention is described in further detail below in conjunction with drawings and examples.
Embodiment 1
In conjunction with shown in Fig. 1, a kind of deoxynivalenol provided in this embodiment quantifies rapid detection card, including according to
The secondary sample pad 2 being set on bottom plate 1, nitrocellulose filter 3 and water absorption pad 4, and do not include the antibody containing colloid gold label
Or the release pad of monoclonal antibody, have on the nitrocellulose filter 3 and is coated with deoxynivalenol haptens-load
The detection line (T lines) 5 of body protein coupling agent (DON-BSA) and the nature controlling line (C lines) 6 for being coated with sheep anti-mouse antibody, sample to be checked
Solution has the latex beads of DON antibody to be mixed before being added drop-wise to the sample pad with coupling, incubation is handled.
In the present embodiment, the package amount of the detection line 5 and the nature controlling line 6 is respectively 0.1mg/mL and 0.4mg/
ML, the nitrocellulose filter 3 first carry out after completing the coating of the detection line 5 and the nature controlling line 6 in 37 DEG C of conditions
Drying processing overnight.
In the present embodiment, the sample pad 2, the nitrocellulose filter 3 and the water absorption pad are pasted and fixed on successively
On the bottom plate 1, the sample pad 2 and the water absorption pad 4 are overlapped with the nitrocellulose filter 3 respectively；The sample pad 2,
The nitrocellulose filter 3, the water absorption pad 4, the detection line 5 and the nature controlling line 6 are along 1 length direction of the bottom plate
Length be followed successively by 25mm, 21mm, 20mm, 3-4mm and 3-4mm, the sample pad 2 and the water absorption pad 4 respectively with the nitre
The lap width of acid cellulose film 3 is 1-2mm.
The deoxynivalenol hapten-carrier albumen coupling agent is prepared by following methods：Institute
Deoxynivalenol hapten-carrier albumen coupling agent is stated to prepare by following methods：8mg deoxidations are first taken to avenge
Rotten sickle-like bacteria enol haptens is dissolved in the DMF of 0.8mL, then the EDC that the 20mg fully dissolved with the water of 0.2mL will be added
Afterwards, it is stirred at room temperature 24 hours, you can obtain reaction solution A, weigh the OVA of 36mg and to be substantially dissolved in 3mL a concentration of
In the CB that 0.1mol/L, pH are 9.6, you can protein solution A is obtained, reaction solution A is slowly dropped to dropwise in protein solution A,
And stir 24 hours at room temperature, finally, dialyse 3 days under the conditions of 4 DEG C with the PBS of a concentration of 0.01mol/L and it is daily more
3 dialyzates are changed to get to the deoxynivalenol hapten-carrier albumen coupling agent.
The coupling has the latex beads of DON antibody to be prepared by following methods：
S1：The MES buffer solutions of 25mM, pH6.0 are added in latex beads, are carried out using MES buffer solutions after mixing
Centrifuge washing；
S2：The ratio that the EDC of the NHS and 40 μ L of 40 μ L are corresponded to 1L latex beads is separately added into the NHS and EDC of 25mM
Reaction solution carries out after mixing being incubated 10min processing；
S3：Centrifuge washing is carried out using MES buffer solutions, supernatant is gone to handle；
S4：Mixing after addition DON antibody to its final concentration of 0.6mg/mL；React 2h；
S5：Centrifugation removal supernatant processing is carried out, and the BSA of addition 2% is closed, again mixing；It is laggard in reaction 1h
Row centrifugation removal supernatant processing, later, be added 2% BSA cleaned, mixing；
S6：Centrifugation removal supernatant processing is carried out, latex beads are suspended in storage 10mMpH7.0PBS buffer solutions, mixing
And it is spare to get to coupled antibody in 2-8 DEG C of refrigerator to be stored in storage temperature；
S7：After taking coupled antibody freeze-drying diluted obtained above, frozen dried is carried out, coupled antibody is lyophilized
Reaction micropore is formed in the micropore of latex beads, that is, completes freeze-drying and the coating of the coupled antibody, and obtaining coupling has DON
The latex beads of antibody.
Preferably, the preparation method of the MES buffer solutions of described 25mM, pH6.0 is：0.53gMES is dissolved in 90mL
In pure water, it is 6.0 to adjust pH, is used in combination pure water constant volume in 100mL.
Embodiment 2
Deoxynivalenol described in the present embodiment quantifies rapid detection card with embodiment 1, and difference only exists
In the DON antibody is monoclonal antibody, and the monoclonal antibody is prepared by following methods：
(1) making of immunogene
It first takes 8mg deoxynivalenol haptens to be dissolved in the DMF of 0.8mL, adds and filled with the water of 0.2mL
The EDC for dividing the 20mg of dissolving, is then stirred at room temperature 24 hours, you can obtain reaction solution A；Weigh the OVA of 36mg and abundant
It is dissolved in the CB that 3mL a concentration of 0.1mol/L, pH are 9.6, you can obtain protein solution A；Reaction solution A is slowly dripped dropwise
It is added in protein solution A, and stirs 24 hours at room temperature, 3 are dialysed under the conditions of 4 DEG C with the PBS of a concentration of 0.01mol/L
It and replace 3 dialyzates daily to get to immunogene；
(2) it is immunized
With the immunogen immune Balb/c mouse obtained in step (1), metering is immunized as 100 μ g/, its is made to generate anti-blood
Clearly；
(3) cell fusion and cloning
The immune Balb/C mouse boosting cells in step (2) are taken, are 8 by quantitative proportion:1 ratio and SP2/0 myeloma
Cell fusion measures cell supernatant using indirect competitive ELISA method, screens positive hole；Using limiting dilution assay to the positive
Hole carries out cloning, until obtaining the hybridoma cell strain of stably excreting monoclonal antibody；
(4) cell cryopreservation and recovery
The hybridoma obtained in step (3) is made to the cell suspension of 1 × 106/mL of frozen stock solution, it is long in liquid nitrogen
Phase preserves；The cryopreservation tube preserved for a long time in liquid nitrogen is taken out when recovery, 37 DEG C of water-bath middling speeds is immediately placed in and melts, and centrifugation removal freezes
After liquid, culture bottle culture is moved into；
(5) the preparation and purification antibody of ascites
Using in vivo method, cell is beaten in mouse peritoneal, mouse ascites are taken out after 7 days, purifies to obtain with saturated ammonium sulfate method
Antibody.
Embodiment 3
In conjunction with shown in Fig. 2, the present embodiment provides a kind of quantitative quickly inspections of deoxynivalenol described in embodiment 1
The detection method for surveying card, includes the following steps：
(1) it preheats：Couveuse is opened to switch and be warming up to 45 ± 1 DEG C before detection；
(2) sample is marked：According to sample size to be checked, that takes out respective numbers has by freeze-drying with processed coupling is wrapped
The latex beads of DON antibody and detection block, and block label detecting to distinguish sample solution to be checked
(3) liquid feeding：Sample solution 120ul to be checked is drawn with pipettor, is vertically added dropwise to processed by freeze-drying and packet
Coupling has in the latex beads of DON antibody, inhales repeatedly and makes a call to 10 times with up to uniformly mixed；
(4) it is incubated：The solution that detection card and step (2) obtain is placed on couveuse, is incubated under conditions of 45 ± 1 DEG C
Educate 2min；
(5) it reacts：The solution after being incubated is taken out, inhales and makes a call to 4 times or more repeatedly, the liquid 120ul after careful absorption reaction ±
10ul is added dropwise in the sample pad 2 of detection card (in such as Fig. 2 at S) according to sampfe order, reacts 5min；
(6) result is read：Testing result is directly obtained according to the display of detection card, alternatively, will take out from couveuse
The detection is stuck in 1min interpolations and enters the quantitative detection that readout instrument carries out batch, reads or/and print testing result.
The present embodiment also provides the preparation method of the sample solution to be checked, and it is 125 μ g/kg- to be suitable for detection range
2500 μ g/kg, specifically comprise the following steps：
(1) it crushes：Cereals class sample is crushed；
(2) it weighs：Cereals class sample that 5g is crushed is weighed in 50ml centrifuge tubes；
(3) mixing：25mL deionized waters are added in centrifuge tube, after sealing high speed acutely shakes mixing 3min, in room temperature
Lower standing 2min；
(4) it centrifuges：It stands or 4000rpm centrifuges 5min；
(5) it dilutes：100 μ L supernatant liquids are taken, 900 μ L sample dilutions are added, mixing is spare.
It is of the present invention detection card detection reaction principle be：
Treated for dropwise addition on detection card, if containing equal to or higher than detection limit deoxynivalenol bacterium in sample
Enol, deoxynivalenol are combined to form compound with the antibody of latex beads coupling first, this compound is together with breast
Glue microballoon spring up forward detection line position, coupled antibody surface site due to being occupied by antigen, and prevent its from detection line
5 deoxynivalenol hapten-carrier albumen coupling agent combines, this detection line 5 is due to that cannot intercept latex beads
It is weak without developing the color or developing the color, this testing result is considered as the positive, and when containing in sample deoxynivalenol bacterium is limited less than detection
Nivalenol hapten-carrier albumen coupling agent will be deoxidized when enol, when latex beads spring up detection line 5 to catch
It obtains, latex beads, which are gathered in detection line 5, shows macroscopic red stripes, this testing result is considered as feminine gender, works as detection
When line 5 is colourless with nature controlling line 6 or nature controlling line does not develop the color, show incorrect operating process or the deterioration failure of detection card.
Pattern detection 1
Cereal sample corn, 705 μ g/kg of final concentration, 1000 μ g/kg, 1410 μ g/kg is taken to be made respectively through the above method
It is detected using above-mentioned detection card after sample solution to be checked, 21 Duplicate Samples of each Concentration Testing point are evaluated in 3 appraisers
Detection, the results are shown in Table 1 for reading.
Table 1.DON detection card evaluation results (corn)
Pattern detection 2
Take cereal sample wheat, 500 μ g/kg of final concentration, 1000 μ g/kg, 1870 μ g/kg.It is made respectively through the above method
It is detected using above-mentioned detection card after sample solution to be checked, 21 Duplicate Samples of each Concentration Testing point are evaluated in 3 appraisers
Detection, the results are shown in Table 2 for reading.
Table 2.DON detection card evaluation results (wheat)
By table 1, table 2 as it can be seen that measuring the deoxidation in corn, wheat respectively using detection of the present invention card and detection method
Nivalenol, respectively between 102%-106% between 99%-106%, RSD's rate of recovery exists respectively
Between 2.3%-6.1% between 2.9%-5.7%, illustrate the rate of recovery of detection card of the present invention and detection method it is good,
The coefficient of variation is smaller, accuracy is high.
Detection fixture provided by the invention has the advantage that：
1, red latex microballoon bright and full color, the fabulous differentiation of color；
2, every batch of latex beads yield at least 50L-100L, same is 5-10 times of colloidal gold in batches；
3, latex beads coupled antibody is coupled for chemical bond, accuracy and precision better than colloidal gold physical absorption stability
Degree is high；
4, latex beads coupling process is easy to operate, and experimental article is not necessarily to steep acid every time, is influenced by environment, personnel and reagent
It is smaller, greatly save manpower and materials；Layman operates to specifications can also complete to detect.
It is last it is necessarily pointed out that：The foregoing is merely the preferable specific embodiment of the present invention, but the present invention
Protection domain be not limited thereto, any one skilled in the art in the technical scope disclosed by the present invention,
The change or replacement that can be readily occurred in, should be covered by the protection scope of the present invention.
Claims (10)
1. a kind of deoxynivalenol quantifies rapid detection card, it is characterised in that：Including being set in turn on bottom plate
Sample pad, nitrocellulose filter and water absorption pad have on the nitrocellulose filter and are coated with deoxynivalenol half
The detection line of antigen-carrier albumen coupling agent and the nature controlling line for being coated with sheep anti-mouse antibody, sample solution to be checked is being added drop-wise to
There are the latex beads of DON antibody to be mixed with coupling before stating sample pad, incubation is handled.
2. detection card according to claim 1, it is characterised in that：The package amount of the detection line and the nature controlling line is distinguished
For 0.1mg/mL and 0.4mg/mL.
3. detection card according to claim 1, it is characterised in that：The sample pad, the nitrocellulose filter and described
Water absorption pad is pasted and fixed on successively on the bottom plate, and the sample pad and the water absorption pad are taken with the nitrocellulose filter respectively
It connects.
4. detection card according to claim 1, it is characterised in that：The deoxynivalenol hapten-carrier
Albumen coupling agent is prepared by following methods：First deoxynivalenol haptens is dissolved in DMF, then is added
Enter EDC, after solution obtained is then stirred at room temperature 24 hours, be slowly dropped to dropwise in the CB solution dissolved with OVA,
And stir at room temperature 24 hours, finally obtained solution PBS is dialysed 3 days.
5. detection card according to claim 4, it is characterised in that：The deoxynivalenol hapten-carrier
Albumen coupling agent is prepared by following methods：8mg deoxynivalenol haptens is first taken to be dissolved in 0.8mL's
In DMF, then after the EDC of the 20mg fully dissolved with the water of 0.2mL being added, it is stirred at room temperature 24 hours, you can obtain anti-
Liquid A is answered, the OVA of 36mg is weighed and is substantially dissolved in the CB that 3mL a concentration of 0.1mol/L, pH are 9.6, you can it is molten to obtain albumen
Reaction solution A is slowly dropped in protein solution A by liquid A dropwise, and is stirred 24 hours at room temperature, and finally, use is a concentration of
The PBS of 0.01mol/L dialyses 3 days under the conditions of 4 DEG C and replaces 3 dialyzates daily to get to the deoxynivalenol
Bacterium enol hapten-carrier albumen coupling agent.
6. detection card according to claim 1, it is characterised in that：Under the coupling has the latex beads of DON antibody to pass through
The method of stating prepares：
S1：The MES buffer solutions of 25mM, pH6.0 are added in latex beads, are centrifuged using MES buffer solutions after mixing
Washing；
S2：The ratio that the EDC of the NHS and 40 μ L of 40 μ L are corresponded to 1L microballoons is separately added into NHS the and EDC reaction solutions of 25mM, mixes
It carries out being incubated 10min processing after even；
S3：Centrifuge washing is carried out using MES buffer solutions, supernatant is gone to handle；
S4：Mixing after addition DON antibody to its final concentration of 0.6mg/mL, reacts 2h；
S5：Carry out centrifugation removal supernatant processing, and 2% BSA be added and is closed, mixing again, carried out after reacting 1h from
The heart remove supernatant processing, later, be added 2% BSA cleaned, mixing；
S6：Centrifugation removal supernatant processing is carried out, microballoon is suspended in storage 10mMpH7.0PBS buffer solutions, and mixing is simultaneously stored in
Storage temperature is spare to get to coupled antibody in 2-8 DEG C of refrigerator；
S7：After taking coupled antibody freeze-drying diluted obtained above, frozen dried is carried out, coupled antibody is lyophilized in breast
Reaction micropore is formed in the micropore of glue microballoon, that is, completes freeze-drying and the coating of the coupled antibody, and obtaining coupling has DON antibody
Latex beads.
7. detection card according to claim 6, it is characterised in that：The system of the MES buffer solutions of described 25mM, pH6.0
Preparation Method is：The MES of 0.53g is dissolved in 90mL pure water, it is 6.0 to adjust pH, is used in combination pure water constant volume in 100mL.
8. detection card according to claim 6, it is characterised in that：The DON antibody is prepared by following methods：
(1) making of immunogene
It first takes 8mg deoxynivalenol haptens to be dissolved in the DMF of 0.8mL, adds fully molten with the water of 0.2mL
Then the EDC of the 20mg of solution is stirred at room temperature 24 hours, you can obtain reaction solution A；It weighs the OVA of 36mg and fully dissolves
In the CB that 3mL a concentration of 0.1mol/L, pH are 9.6, you can obtain protein solution A；Reaction solution A is slowly dropped to egg dropwise
It in white solution A, and stirs 24 hours, dialyse 3 days under the conditions of 4 DEG C with the PBS of a concentration of 0.01mol/L and often at room temperature
It replaces 3 dialyzates to get to immunogene；
(2) it is immunized
With the immunogen immune Balb/c mouse obtained in step (1), metering is immunized as 100 μ g/, its is made to generate antiserum；
(3) cell fusion and cloning
The immune Balb/C mouse boosting cells in step (2) are taken, are 8 by quantitative proportion:1 ratio and SP2/0 myeloma cell
Fusion measures cell supernatant using indirect competitive ELISA method, screens positive hole；Using limiting dilution assay to positive hole into
Row cloning, until obtaining the hybridoma cell strain of stably excreting monoclonal antibody；
(4) cell cryopreservation and recovery
The hybridoma obtained in step (3) is made to the cell suspension of 1 × 106/mL of frozen stock solution, is protected for a long time in liquid nitrogen
It deposits；The cryopreservation tube preserved for a long time in liquid nitrogen is taken out when recovery, is immediately placed in 37 DEG C of water-bath middling speeds and is melted, after centrifugation removal frozen stock solution,
Move into culture bottle culture；
(5) the preparation and purification antibody of ascites
Using in vivo method, cell is beaten in mouse peritoneal, mouse ascites are taken out after 7 days, purifies to obtain antibody with saturated ammonium sulfate method.
9. a kind of deoxynivalenol described in claim 1 quantifies the detection method of rapid detection card, feature exists
In：Include the following steps：
(1) it preheats：Couveuse is opened to switch and be warming up to 45 ± 1 DEG C before detection；
(2) liquid feeding：Sample solution 120ul to be checked is drawn with pipettor, is vertically added dropwise to by freeze-drying and the processed coupling of packet
In the latex beads for having DON antibody, inhales repeatedly and make a call to 10 times with up to uniformly mixed；
(3) it is incubated：The solution that detection card and step (2) obtain is placed on couveuse, is incubated under conditions of 45 ± 1 DEG C
2min；
(4) it reacts：The solution after being incubated is taken out, inhales and makes a call to 4 times or more repeatedly, the careful liquid 120ul ± 10ul drawn after reaction,
It is added dropwise according to sampfe order in the sample pad of detection card, reacts 5min；
(5) result is read：Testing result is directly obtained according to the display of detection card, alternatively, described in being taken out from couveuse
Detection is stuck in 1min interpolations and enters the quantitative detection that readout instrument carries out batch, reads or/and print testing result.
10. detection method according to claim 9, it is characterised in that：The sample solution to be checked is suitable for detection range
For 125 μ g/kg-2500 μ g/kg, preparation method includes the following steps：
(1) it crushes：Cereals class sample is crushed；
(2) it weighs：Cereals class sample that 5g is crushed is weighed in 50ml centrifuge tubes；
(3) mixing：25mL deionized waters are added in centrifuge tube, it is quiet at room temperature after sealing high speed acutely shakes mixing 3min
Set 2min；
(4) it centrifuges：It stands or 4000rpm centrifuges 5min；
(5) it dilutes：100 μ L supernatant liquids are taken, 900 μ L sample dilutions are added, mixing is spare.
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| CN110687294A (en) * | 2019-09-04 | 2020-01-14 | 武玉香 | Detection card for rapidly and quantitatively detecting mycotoxin and application thereof |
| CN112881697A (en) * | 2021-01-13 | 2021-06-01 | 北京中检葆泰生物技术有限公司 | Method for stably detecting aflatoxin content |
| CN113759118A (en) * | 2021-11-05 | 2021-12-07 | 山东畜牧兽医职业学院 | Avian influenza virus infection and vaccine immunity differential diagnosis detection card and preparation method thereof |
| CN114957359A (en) * | 2022-04-28 | 2022-08-30 | 华南农业大学 | Hapten, artificial antigen and ELISA method for detecting DON marker DON-15-GlcA |
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