CN107976541A - Test card, preparation and the detection method of synchronous detection zearalenone and aflatoxin B1 - Google Patents
Test card, preparation and the detection method of synchronous detection zearalenone and aflatoxin B1 Download PDFInfo
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Abstract
The present invention relates to synchronous detection zearalenone and test card, preparation and the detection method of aflatoxin B1, belong to field of immunological detection, test card includes getting stuck and test strips, and test strips include bottom plate and overlap joint is pasted onto water absorption pad, detecting pad, bonding pad and sample pad on bottom plate successively；Wherein, the detecting pad is the NC films equipped with nature controlling line C, detection line T1 and detection line T2, and nature controlling line C coating sheep anti mouse secondary antibodies, detection line T1 coating ZEN BSA, detection line T2 are coated with AFB1 BSA；Bonding pad is the anti-zearalenone monoclonal antibody of embedding time-resolved fluorescence microballoon mark and the glass fibre element film of aspergillus flavus resisting toxin B1 monoclonal antibodies；The sample pad is dry glass fibre element film after sample pad treatment fluid immersion treatment.The present invention is systematization, facilitation, the quantitative synchronous detection zearalenone in the ground that standardizes and aflatoxin provide a kind of new method, and stability is good, susceptibility is high.
Description
Technical field
The invention belongs to field of immunological detection, and in particular, to synchronous detection zearalenone and aspergillus flavus
Test card, preparation and the detection method of toxin.
Background technology
Zearalenone and aflatoxin B1 are two kinds of common mycotoxins, both toxin concentrations in feed
Height has close relationship with animal epidemics prevalence.Zearalenone (F-2) is the secondary metabolite that gibberella produces,
Initially isolated from the corn for have gibberella, it has estrogen action, can cause animal acute and chronic poisoning, so as to cause
Reproduction causes death extremely, and huge economic loss is caused to animal farm.Aflatoxin B1 (AFB1) is as
Know one kind that carcinogenicity is most strong in chemical substance, toxicity is 30 times stronger than vomitoxin (Don), 25 times stronger than F-2；It can cause birds
Feed intake, body weight gains and the feed conversion rate of domestic animals reduce, pulmonary edema, infertile or miscarriage, the increase of epidemic disease neurological susceptibility.Both poison
Plain serious threat the health of animals and humans, and establishes aflatoxin in a kind of fast and accurately detection grain and feed
The method of B1 and zearalenone has great importance.
The current detection method in relation to aflatoxin B1 and zearalenone has membrane chromatographic and liquid chromatogram etc., this
Although alanysis method is accurate, stablizes, because of the shortcomings of of high cost, cumbersome, detection cycle is long, testing agency is only applicable to
And actual production can not be applied to and lived.It is that in the market is most widely used with the simple and efficient immunoassay technology for advantage,
Wherein, it is the current research hotspot in this area using fluorescent microsphere as the immunolabelling technique of tracer.Fluorescent microsphere refers to glimmering
Stimulative substance is introduced into organic or inorganic nano-particle by modes such as embedding, covalent key connections, and the parcel of nano material makes its tool
There is good stability.Compared with the Enzyme-linked Immunosorbent Assay (ELISA) to grow up at the same time, fluorescent micro-ball immune chromatography technology behaviour
Make simplicity, more fast.Compared to the colloidal gold immunochromatographimethod technology of same type, embed a large amount of fluorescence molecules and make it have higher
Sensitivity；Fluorescence signal can reach the effect of fast quantification by fluorescence analyzer detection at the same time.
The content of the invention
In view of the above-mentioned problems, it is an object of the invention to provide a kind of synchronous detection zearalenone and aflatoxin
The test card of B1, and its prepare and detection method, be systematization, facilitation, standardization ground quantitative detection of zearalenone and
Aflatoxin provides a kind of new method.
The test card of synchronous detection zearalenone and aflatoxin B1, including get stuck and be located at the interior test paper that gets stuck
Bar；Get stuck including the Ka Gai and deck mutually fastened；Test strips include bottom plate and overlap joint is pasted onto the water suction on bottom plate successively
Pad, detecting pad, bonding pad and sample pad；The region that card is covered corresponding to detecting pad is equipped with form, blocks and covers corresponding to sample pad
Region be equipped with well, the card slot of fixed test strips is equipped with deck；The bottom plate is the PVC board not absorbed water, and water absorption pad is
Absorbent filter；It is characterized in that：The detecting pad is the nitrocellulose filter equipped with nature controlling line C, detection line T1 and detection line T2,
Nature controlling line C is coated with goat-anti mouse monoclonal antibody, detection line T1 coating zearalenone haptens-bovine serum albumin(BSA) coupling
Thing, detection line T2 coating aflatoxin B1s haptens-bovine serum albumin(BSA) conjugate；The bonding pad is embedding time resolution
The zearalenone monoclonal antibody of fluorescent microsphere mark and the glass fibre element film of aflatoxin B1 monoclonal antibody；Institute
It is dry glass fibre element film after sample pad treatment fluid immersion treatment to state sample pad.
Further, the nature controlling line C, detection line T1 or detection line T2 are that coating buffer is drawn film to be made in detecting pad, its
In, the concentration of goat-anti mouse monoclonal antibody coating buffer is 0.5mg/mL, and zearalenone haptens-bovine serum albumin(BSA) is coupled
The concentration of thing coating buffer is 0.3mg/mL, the concentration of aflatoxin B1 haptens-bovine serum albumin(BSA) conjugate coating buffer
0.3mg/mL。
Further, the carboxyl polyphenyl containing highlighted rare-earth dye that the time-resolved fluorescence microballoon is diameter 200nm
Ethene microballoon.
Further, the bonding pad is that fluorescent microsphere-antibody complex solution is sprayed onto on glass fibre element film to be made,
In the fluorescent microsphere-antibody complex solution, the concentration of zearalenone monoclonal antibody is 8 μ g/mL, aflatoxin
The concentration of B1 monoclonal antibodies is 6 μ g/mL.
Further, the sample pad treatment fluid is the PBS buffer of pH 8.0, wherein the component and its concentration that contain point
It is not：Mass fraction is 0.5% sucrose, and mass fraction is 0.5% PVP-K30, and mass fraction is 0.5% PEG-
20000, mass fraction is 0.1% PEG-6000, and mass fraction is 0.3% casein, and volume fraction is 0.5%
Tween 20, mass fraction are 0.1% S9, the S21 that the S17 and mass fraction that mass fraction is 0.1% are 0.1%.
The present invention also protection prepares the method for the test card of above-mentioned synchronous detection zearalenone and aflatoxin, bag
Include following steps：
The preparation of the detecting pad：According to the coating thing on nature controlling line C, detection line T1 and detection line T2, prepare respectively containing phase
Three kinds of coating buffers of thing should be coated with, coating buffer concentration is 0.3-0.5mg/mL, by coating buffer each independent suck-back to Film-cutting machine
In pipeline, mark, complete on nature controlling line C, detection line T1 and detection line T2 according to 0.8 μ L/cm on nitrocellulose filter
Coating, is subsequently placed in 37 DEG C of air dry ovens dry 3-4h, detecting pad is made；
The preparation of the bonding pad：Fluorescent microsphere dispersion liquid is placed in centrifuge tube；MES buffer solutions are added, under ultrasound condition
Mix, centrifugation abandons supernatant and collects microballoon precipitation, repeats this step to clean microballoon；Microballoon is precipitated and dissolved in MES buffer solutions,
Mix, add EDC solution, 22-25 DEG C of lucifuge activation, supernatant is abandoned in centrifugation, adds MES buffer solutions, and ultrasound breaks up microballoon, and centrifugation is abandoned
Clearly；Borate buffer is added, ultrasound is broken up, and is then added zearalenone monoclonal antibody and is mixed, and adds aspergillus flavus poison
Plain B1 monoclonal antibodies, lucifuge reaction；Glycine solution is added, lucifuge reaction 30min, adds BSA solution, lucifuge reaction
30min, completes closing；Centrifugation is redissolved, and forms fluorescent microsphere-antibody complex solution；By fluorescent microsphere-antibody complex solution
It is sprayed onto on glass fibre element film, it is dry, bonding pad is made；
The preparation of the sample pad：Glass fibre element film is put into the sample pad treatment fluid configured, 0.5h is soaked, is put into 37
In DEG C air dry oven, dry 8-10h, is made sample pad；
The assembling of test card：Using PVC board as bottom plate, sample pad, bonding pad, detecting pad and water absorption pad are overlapped successively and are pasted onto bottom
On plate, there is when each adjacent overlap joint that 1-2mm is overlapping, and make detection line T1 on detecting pad and detection line T2 close to sample pad one
The PVC board posted is cut into strip with cutting machine, combines and be sealed with getting stuck by side, nature controlling line C close to water absorption pad side.
In addition the present invention goes back the above-mentioned test card of Sustainable use and quantitatively detects zearalenone and aflatoxin in feed
The detection method of B1：Weigh the Feed Sample through homogenizer homogeneous to be placed in centrifuge tube, add 70% methanol aqueous solution, vibrate
Device acutely shakes at least 5min, and centrifuging and taking supernatant, is diluted with sample diluting liquid, obtains sample detection liquid；Sample detection liquid is added
In well, 10min is reacted, by the card slot of test card insertion fluorescence immunity analyzer, T/C values are read, according to concentration standard
Curve, is calculated the concentration that toxin is examined in counter sample.
Synchronously the principle of detection zearalenone and aflatoxin B1 is test card of the present invention：Need when that will contain
The sample drop for surveying toxin is added in sample application zone, the fluorescent nanometer microsphere mark in the small molecule toxins and bonding pad in sample to be tested
Antibody complex is combined and chromatographed through capillary action to water absorption pad side, after the detection line of fixed antigen is reached, detection
The binding site on toxin competition antibody in antigen and sample on line, and unnecessary Fluorescent microsphere marker continues to front layer
Analysis, is combined with the sheep anti mouse monoclonal antibody for being fixed on nature controlling line.After reaction, detecting pad is scanned with ultraviolet source, detection line T1,
The fluorescent microsphere of T2 and nature controlling line C send high intensity fluorescence.In addition the fluorescence decay time of fluorescent microsphere rare earth elements is compared
Common fluorescent regimental commander, is its 103~106 times.By delaying time of measuring, the short life fluorescence to shine naturally in sample substrate is treated
All after decay, then the specificity fluorescent of rare earth element is measured, reach the effect that background interference is completely eliminated, realize high noise
Than.
Beneficial effect：
1st, test card of the present invention uses fluorescent microsphere technology, can detect zearalenone at the same time and aflatoxin B1 is residual
Stay, stability is good, compared to the electrostatic attraction combination of colloid gold particle in colloidal gold strip and protein, with c-terminus
Fluorescent microsphere be connected with antibody with covalent bond, more stablize；High sensitivity, compared to colloidal gold, common fluorescent immunochromatography skill
2~3 orders of magnitude of art high sensitivity；The quantitative detection of batch is realized in short time, the ELISA test strip time of the present invention only needs
10min, shortens compared to the ELISA kit time, while detects fluorescence signal by fluorescence analyser, bent according to built-in standard
Line calculates the content of tie element automatically, is zearalenone compared to the qualitative detection of colloidal gold immuno-chromatography test paper strip
Detection with aflatoxin provides more comprehensive detection means.
2nd, the present invention except using fluorescent microsphere as in addition to immune labeled, the preparation process in relation to test card also it is particularly important that,
It is directly related to the sensitivity and stability of test card detection, wherein, bonding pad is prepared using the preparation method of original creation, through clear
Wash, activate, marking, closing and etc., the monoclonal antibody that fluorescent microsphere marks is embedded in be formed on glass fibre element film and is tied
Close pad, during EDC activation microsphere surface carboxylic group, for mark prepare, add sealer with close microsphere surface not with
The free carboxy that antibody protein combines, improves the accuracy of quantitative result, is sequentially added glycine and BSA carries out closed for second time,
It can avoid interference of the nonspecific reaction to testing result.Sample pad prepare in soaked first through sample treatment, by S9,
S17, S21 are added in sample treatment, are acted synergistically between component, the good immune effect of more general processing method, is more suitable for raising
Expect the analysis detection of sample.
Brief description of the drawings
Fig. 1 is test strips of the present invention and stuck structure schematic diagram；Wherein：1st, sample pad；2nd, bonding pad；3rd, detecting pad；4、
Water absorption pad；
Fig. 2 is zearalenone canonical plotting；
Fig. 3 is aflatoxin B1 canonical plotting.
Embodiment
Below by specific embodiment the present invention will be further explained explanation.
It is prepared by embodiment 1, test card
1st, the preparation of fluorescent microsphere-antibody complex
(1) clean：50 μ L fluorescent microspheres are taken into 1.5mL centrifuge tubes, are added in 1mL 0.01M MES buffer solutions, concussion mixes,
15000r/min centrifuges 15min, abandons supernatant, adds 1mL 0.01M MES buffer solutions, and ultrasound breaks up microballoon；Repeat this step 3
It is secondary, achieve the purpose that to clean microballoon, finally obtained the microballoon liquid after cleaning；
(2) activate：Into the 1mL microballoon liquid after cleaning, 250 μ L EDC solutions, lucifuge activation 2h, 15000r/min centrifugation are added
15min, abandons supernatant, adds 1mL 0.01M MES buffer solutions, and ultrasound breaks up microballoon, and 15000r/min centrifugation 15min, abandon supernatant；
(3) mark：1mL 0.05M borate buffer solutions are added, ultrasound is broken up, and adds 8 μ g zearalenone monoclonal antibodies
Mix, add 6 μ g aflatoxin B1 monoclonal antibodies, lucifuge reaction 2h；
(4) 1 is closed：Add 100 μ L 1M glycine solutions, lucifuge reaction 30min；
(5) 2 are closed：Add 100 μ L 10%BSA solution, lucifuge reaction 30min；
(6) centrifugation is redissolved：15000r/min centrifuges 15min, abandons supernatant, adds 100 μ L dilutions, and ultrasound breaks up microballoon；
(7) spray：The compound that will have been marked, is sprayed onto on glass fibre element film according to 3 μ L/cm；
(8) it is dry：The glass fibre element film for being sprayed with label is put into 37 DEG C of air dry ovens, dry 3-4h；After dry
Bonding pad be put into drier.
The carboxyl polystyrene containing highlighted rare-earth dye that fluorescent microsphere described in above step is diameter 200nm is micro-
Ball；Its absorbing wavelength is 365nm, and Detection wavelength is 610nm.
The EDC is (1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides), is a kind of water-soluble carbon
Diimine.
2nd, the preparation of bonding pad
Glass fibre element film by 37 DEG C of drying after fluorescent microsphere-antibody complex solution immersion 0.5h is the bonding pad.
3rd, prepared by sample pad
The glass fibre element film of 37 DEG C of drying is the sample pad after sample pad treatment fluid soaks 0.5h, and sample pad is handled
Liquid component is：0.5% sucrose, 0.5%PVP-K30,0.5%PEG-20000,0.1%PEG-6000,0.3% casein,
0.5%Tween 20,0.1%S9,0.1%S17,0.1%S21, pH 8.0PBS.Wherein, S9 is Tetronic 1307, is one
Kind amphoteric surfactant；S17 is Ohodasurf ON-870, is a kind of emulsifying agent；S21 is BRIJ 35, is a kind of nonionic
Surfactant.After S9, S17 and S21 are added to the sample pad treatment fluid, the good immune effect of more general processing method, more
Analysis suitable for Feed Sample detects.Sample pad play the role of control sample flow rate, and be capable of Buffer samples pH value and
Influence of the zwitterion intensity to immunization.
4th, the preparation of detecting pad
The detecting pad refers to the nitric acid for being coated with a horizontal nature controlling line (C lines) and two horizontal detection lines (T1 lines, T2 lines)
Cellulose membrane, C lines are 5-6mm with T1 line spacing, and T1 lines and T2 line spacing are 3-5mm.From sample pad to water absorption pad direction successively
It is C lines from detection line to water absorption pad direction for T1 lines and T2 lines.Coated C lines are goat-anti mouse monoclonal antibodies, specific implementation step
Suddenly it is that dense sheep anti mouse monoclonal antibody is diluted to 0.5mg/mL with coating working solution, suck-back is into Film-cutting machine pipeline, according to 0.8 μ L/cm
Mark, be put into 37 DEG C of air dry ovens, dry 3-4h；T1 lines coating zearalenone haptens-bovine serum albumin(BSA) is even
Join thing (ZEN-BSA), specific implementation step is that ZEN-BSA is diluted to 0.3mg/mL, suck-back to Film-cutting machine with film working solution is drawn
In pipeline, mark, be put into 37 DEG C of air dry ovens according to 0.8 μ L/cm, dry 3-4h；T2 lines coating aflatoxin B1 half
Antigen-bovine serum albumin(BSA) conjugate (AFB1-BSA), specific implementation step are to be diluted to AFB1-BSA with film working solution is drawn
0.3mg/mL, suck-back are marked according to 0.8 μ L/cm into Film-cutting machine pipeline, are put into 37 DEG C of air dry ovens, dry 3-4h；Put
Enter stand-by in drier.The detecting pad is that effective ingredient is specifically bound with the active material being fixed on film in sample
Region.
5th, the assembling of test strips
Using the PVC adhesive sheets not absorbed water as bottom plate, by sample pad, sample bonding pad, detecting pad and water absorption pad from top to bottom successively
Overlap joint is pasted onto on bottom plate, and each component has that 1-2mm is overlapping in adjacent, and the PVC board posted is cut into the examination of 3mm wide with cutting machine
Paper slip, is combined by the card slot in deck with getting stuck.Wherein, the T1 lines on the detecting pad and T2 lines close to sample pad side,
Corresponding card has covered " T1 " and " T2 " mark；Nature controlling line C line has covered " C " mark close to water absorption pad side, corresponding card, such as
Shown in Fig. 1.The water absorption pad is absorbent filter, and absorbent filter cuts growth 30cm, wide 1.9-2.1cm.The water absorption pad has
Siphon effect.
The specification of the Ka Gai is 70mm*20mm*5mm, and there are the form of a 16mm*3mm sizes, Ka Gai and sample in Ka Gai centers
There is the well for being identified as " S " in product pad corresponding region.
The drafting of embodiment 2, standard curve
1st, the configuration of standard items：
Zearalenone gradient standard items are prepared：With the phosphate buffer of 0.1M pH7.4 by zearalenone standard items
It is diluted to 0.2ppb, 0.4ppb, 0.8ppb, 1.6ppb, 3.2ppb；
Aflatoxin B1 gradient standard items are prepared：With the phosphate buffer of 0.1M pH7.4 by aflatoxin B1 standard items
It is diluted to 0.2ppb, 0.4ppb, 0.8ppb, 1.6ppb, 3.2ppb；
2nd, reading：
By above-mentioned standard product point card, after reacting 10min, correspond to card slot in Portable fluorescence immunity analysis instrument and carry out reading, reading
The result is shown in shown in table 1 below：
3rd, the verification of computational methods：
(1) four parametric method, after zearalenone curve relevant parameter is inputted Portable fluorescence immunity analysis instrument, in gradient mark
Select two point 0.4ppb and 1.6ppb in product at random, point card, reacts 10min, read two concentration be respectively 0.339ppb and
0.788ppb
(2) piecewise linearity, after zearalenone curve relevant parameter is inputted Portable fluorescence immunity analysis instrument, in gradient mark
Select two point 0.4ppb and 1.6ppb in product at random, point card, reacts 10min, read two concentration be respectively 0.392ppb and
1.588ppb
By two groups of data comparisons, can be concluded that：The computational methods of sample concentration preferably select Piecewise.
4th, the verification and drafting of standard curve：
Reading value in step 2 is inputted into recurrence/the Fitting Calculation program, draws the standard of zearalenone and aflatoxin B1
Curve, as shown in Figure 2 and Figure 3.
Test example：Comparability test
To detect in corn flour exemplified by the content of zearalenone and aflatoxin B1, test strips more of the present invention with it is common
The detection result of colloidal gold strip.
1st, with homogenizer homogeneous Feed Sample
The sample of two parts of 5.0 ± 0.05g grindings is weighed into two 50mL polystyrene centrifuge tubes, its middle pipe 2 adds 5ng corns
Zeranol and 5ng aflatoxin B1s, are separately added into 70% methanol solutions of 25mL, acutely vibrate 5min with oscillator；
2nd, by the sample after vibration, centrifuge, 6000r/min centrifugation 5min are put into, or filtered with quantitative analysis filter paper；
3rd, 1 is pressed with sample diluting liquid:80 dilution proportion (examples：+ 790 μ L samples dilution of 10 μ L samples extracting solution)；The sample
Dilution is the PBS of pH7.4,0.05mol/L；
4th, 50 μ L of liquid after being diluted after being diluted in pipe 1 in 50 μ L of liquid and pipe 2 are taken respectively to add in card 1 and card 2 respectively, beginning timing
10min；
5th, card 1 is inserted into reading in Portable fluorescence immunity analysis instrument respective card slot, display density value is 0；It is portable that 2 insertions will be blocked
Reading in formula fluorescence immunity analyzer card slot, display density value are 0.985ng/mL；
6th, compared with the testing result of colloidal gold strip：The dilution handled well in pipe 1 is separately added into zearalenone
In colloidal-gold detecting-card 1 and aflatoxin B1 colloidal-gold detecting-card 1, the dilution of processing number in pipe 2 is separately added into corn
In zeranol colloidal-gold detecting-card 2 and aflatoxin B1 colloidal-gold detecting-card 2,10min is reacted, as a result：Gibberella zeae alkene
1 outlet two lines of ketone card, are presented negative findings, and two lines occurs in aflatoxin B1 card 1, and negative findings is presented；Gibberella zeae
2 outlet two lines of ketenes card, are presented negative findings, and two lines occurs in aflatoxin B1 card 2, and negative findings is presented.Test table
Bright, fluorescent microsphere test strips sensitiveness is compared with colloidal gold strip height
Gibberella zeae is marked to colloid gold label zearalenone monoclonal antibody complex and fluorescent microsphere further below
Ketenes monoclonal antibody complex stability compares.
1st, by the bonding pad and fluorescent microsphere-jade of the colloidal gold prepared-zearalenone monoclonal antibody complex
The bonding pad of Zearlenone monoclonal antibody complex is placed in 37 DEG C of air dry ovens.
2nd, on day 3, detected respectively at 5 days, 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 20 weeks, 36 weeks, 48 weeks negative and positive
Sample, observes its stability.
3rd, testing result is as shown in table 2 below：
By upper table it could be assumed that：The stability of fluorescent microsphere labelled antibody compound is compared with colloidal gold labeled monoclonal antibody compound
By force.
Claims (7)
1. the test card of synchronous detection zearalenone and aflatoxin B1, including get stuck and be located at the interior test paper that gets stuck
Bar；Get stuck including the Ka Gai and deck mutually fastened；Test strips include bottom plate and overlap joint is pasted onto the water suction on bottom plate successively
Pad, detecting pad, bonding pad and sample pad；The region that card is covered corresponding to detecting pad is equipped with form, blocks and covers corresponding to sample pad
Region be equipped with well, the card slot of fixed test strips is equipped with deck；The bottom plate is the PVC board not absorbed water, and water absorption pad is
Absorbent filter；It is characterized in that：The detecting pad is the nitrocellulose filter equipped with nature controlling line C, detection line T1 and detection line T2,
Nature controlling line C is coated with goat-anti mouse monoclonal antibody, detection line T1 coating zearalenone haptens-bovine serum albumin(BSA) coupling
Thing, detection line T2 coating aflatoxin B1s haptens-bovine serum albumin(BSA) conjugate；The bonding pad is embedding time resolution
The zearalenone monoclonal antibody of fluorescent microsphere mark and the glass fibre element film of aflatoxin B1 monoclonal antibody；Institute
It is dry glass fibre element film after sample pad treatment fluid immersion treatment to state sample pad.
2. synchronous detection zearalenone and the test card of aflatoxin B1 as claimed in claim 1, it is characterised in that：Institute
It is that coating buffer is drawn film to be made in detecting pad to state nature controlling line C, detection line T1 or detection line T2, wherein, goat-anti mouse monoclonal antibody
The concentration of coating buffer is 0.5mg/mL, and the concentration of zearalenone haptens-bovine serum albumin(BSA) conjugate coating buffer is 0.3
Mg/mL, 0.3 mg/mL of concentration of aflatoxin B1 haptens-bovine serum albumin(BSA) conjugate coating buffer.
3. synchronous detection zearalenone and the test card of aflatoxin B1 as claimed in claim 1, it is characterised in that：Institute
State the carboxyl polystyrene microsphere containing highlighted rare-earth dye that time-resolved fluorescence microballoon is diameter 200nm.
4. synchronous detection zearalenone and the test card of aflatoxin B1 as claimed in claim 1, it is characterised in that：
The bonding pad is that fluorescent microsphere-antibody complex solution is sprayed onto on glass fibre element film to be made, the fluorescent microsphere-antibody
In complex solution, the concentration of zearalenone monoclonal antibody is 8 μ g/mL, aflatoxin B1 monoclonal antibody it is dense
Spend for 6 μ g/mL.
5. synchronous detection zearalenone and the test card of aflatoxin B1 as claimed in claim 1, it is characterised in that：
The sample pad treatment fluid is the PBS buffer of pH 8.0, wherein the component and its concentration that contain are respectively：Mass fraction is
0.5% sucrose, mass fraction be 0.5% PVP-K30, mass fraction be 0.5% PEG-20000, mass fraction 0.1%
PEG-6000, mass fraction is 0.3% casein, and volume fraction is 0.5% Tween 20, and mass fraction is 0.1%
S9, the S21 that the S17 and mass fraction that mass fraction is 0.1% are 0.1%.
6. prepare the side of the test card such as any one synchronization detection zearalenone of claim 1-5 and aflatoxin B1
Method, it is characterised in that：
The preparation of the detecting pad：According to the coating thing on nature controlling line C, detection line T1 and detection line T2, prepare respectively containing phase
Three kinds of coating buffers of thing should be coated with, coating buffer concentration is 0.3-0.5 mg/mL, by coating buffer each independent suck-back to Film-cutting machine
In pipeline, mark, complete on nature controlling line C, detection line T1 and detection line T2 according to 0.8 μ L/cm on nitrocellulose filter
Coating, is subsequently placed in 37 DEG C of air dry ovens dry 3-4 h, detecting pad is made；
The preparation of the bonding pad：Fluorescent microsphere dispersion liquid is placed in centrifuge tube；MES buffer solutions are added, under ultrasound condition
Mix, centrifugation abandons supernatant and collects microballoon precipitation, repeats this step to clean microballoon；Microballoon is precipitated and dissolved in MES buffer solutions,
Mix, add EDC solution, 22-25 DEG C of lucifuge activation, supernatant is abandoned in centrifugation, adds MES buffer solutions, and ultrasound breaks up microballoon, and centrifugation is abandoned
Clearly；Borate buffer is added, ultrasound is broken up, and is then added zearalenone monoclonal antibody and is mixed, and adds aspergillus flavus poison
Plain B1 monoclonal antibodies, lucifuge reaction；Glycine solution is added, lucifuge reaction 30min, adds BSA solution, lucifuge reaction
30min, completes closing；Centrifugation is redissolved, and forms fluorescent microsphere-antibody complex solution；By fluorescent microsphere-antibody complex solution
It is sprayed onto on glass fibre element film, it is dry, bonding pad is made；
The preparation of the sample pad：Glass fibre element film is put into the sample pad treatment fluid configured, 0.5 h is soaked, is put into
In 37 DEG C of air dry ovens, dry 8-10 h, are made sample pad；
The assembling of test card：Using PVC board as bottom plate, sample pad, bonding pad, detecting pad and water absorption pad are overlapped successively and are pasted onto bottom
On plate, there is when each adjacent overlap joint that 1-2mm is overlapping, and make detection line T1 on detecting pad and detection line T2 close to sample pad one
The PVC board posted is cut into strip with cutting machine, combines and be sealed with getting stuck by side, nature controlling line C close to water absorption pad side.
7. any one described test card quantitatively detects zearalenone and aflatoxin in feed using claim 1-5
The detection method of B1, it is characterised in that：Weigh the Feed Sample through homogenizer homogeneous to be placed in centrifuge tube, add 70% methanol
Aqueous solution, oscillator acutely shake at least 5min, and centrifuging and taking supernatant, is diluted with sample diluting liquid, obtains sample detection liquid；By sample
Detect liquid to add in well, react 10min, by the card slot of test card insertion fluorescence immunity analyzer, read T/C values, root
According to concentration standard curve, the concentration that toxin is examined in counter sample is calculated.
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