CN207263761U - The effluent piece and kit of mycotoxin in a kind of detection food - Google Patents

Abstract

The utility model discloses a kind of effluent piece for detecting mycotoxin, the effluent piece includes a detection and blocks, and the detection card includes backing, and sample pad, gold-labelled pad, nitrocellulose filter, detection zone, quality control region and absorption pad on backing.The effluent piece of the utility model can detect fumonisin, ochratoxin, vomitoxin at the same time, suitable for the quick detection of mycotoxin.

Description

The effluent piece and kit of mycotoxin in a kind of detection food

Technical field

Detection inspection technology field is the utility model is related to, by antigen-antibody immune response in immunochemistry by means of layer Analysis technology reaches testing goal, more particularly to a kind of effluent piece for detecting mycotoxin in food.

Background technology

Fungi, is that one kind has cell membrane, and without chlorophyll, unrooted base of leaf, is survived with saprophytic or parasitic method, can be had Property or the eukaryotic microorganisms of vegetative propagation.All kinds of Mushrooms are most commonly that in fungi, in addition also mould and yeast.Although fungi It is widely used in food industry, such as makes wine, the manufacture of sauce processed, bread, benefit, but some fungies are brought to mankind's production and living Also can be endangered by producing mycotoxin contaminated food products this mode to human body health care belt.

Mycotoxin is mycetogenetic secondary metabolite, mainly including aflatoxin, fusarium toxin etc..Earliest The description that mycotoxin causes poisoning is just related in 11 Century European icon paintings, but until more than 100,000 fire of nineteen sixty Britain After the chicken event dead because of the feed of feeding aflatoxin contamination occurs, mycotoxin is just re-recognized by people.By complete The influence of the factors such as ball climate warming, humidity, edible and feeding agricultural product are on the rise by mycotoxin pollution, and long-term consumption is true Verticillium toxin contaminated food products, not only causes three causes of being poisoned but also cause, carcinogenic, cause strange, mutagenesis, all over the world for mycotoxin The report of pollution is increased.

China realizes that behave is not in place, and mycotoxin pollution is even more serious plus people due to geographical climate environment feature, Counted according to processing of farm products research institute of the Chinese Academy of Agricultural Sciences, 2001 to 2011 years are during the decade, influenced by mycotoxin pollution, I State exports European Union's food relevant contravention up to 2559, and wherein mycotoxin is exceeded accounts for 28.6%, higher than heavy metal known to the public, The factors such as food additives, agricultural residual, the ratio highest in single incident.Academy of agricultural sciences processes institute researcher Liu Yang and thinks, very The exceeded maximum obstruction for having become agricultural products in China outlet European Union of verticillium toxin, causes huge to the processing of China's grain and oil and export enterprise Big economic loss.

The common method of mycotoxin detection includes：Thin-layered chromatography (TLC), high performance liquid chromatography (HPLC), ELISA method etc..Above-mentioned detection method needs to coordinate detecting instrument to use, and detection time is longer, complicated, is not easy to existing Field detecting.Therefore, the reagent and method of detection mycotoxin easy to use, reliable results are badly in need of in this area.

Utility model content

The purpose of this utility model is to provide a kind of effluent piece of detection mycotoxin easy to use, reliable results.

In the first aspect of the utility model, there is provided a kind of effluent piece for detecting mycotoxin, the effluent piece include One detection card, the detection card include backing (0), and on the backing proximally arrive distal end with lower structure： Sample pad (1), gold-labelled pad (2), nitrocellulose filter (8), detection zone, quality control region (6) and absorption pad (7)；

And the gold-labelled pad (2) includes：First antibody load region, secondary antibody load region and the load of the 3rd antibody Area, wherein, the first antibody is directed to the antibody of fumonisin for the specificity of colloid gold label, and the secondary antibody is The specificity of colloid gold label is directed to the antibody of ochratoxin, and the 3rd antibody is directed to for the specificity of colloid gold label The antibody of vomitoxin；

And the detection zone includes：First detection zone (3), the second detection zone (4) and the 3rd detection zone (5), wherein, institute The first detection zone (3) stated is coated with the comlete antigen of fumonisin, and second detection zone (4) is coated with ochratoxin Comlete antigen, the 3rd detection zone (5) is coated with the comlete antigen of vomitoxin.

In another preference, the comlete antigen of the fumonisin is the conjugate of fumonisin and carrier protein.

In another preference, the comlete antigen of the ochratoxin is the coupling of ochratoxin and carrier protein Thing.

In another preference, the comlete antigen of the vomitoxin is the conjugate of vomitoxin and carrier protein.

In another preference, the carrier protein is selected from the group：BSA, OVA or its combination.

In another preference, the backing (0) is PVC board.

In another preference, the sample pad (1) is glass fibre.

In another preference, the absorption pad (7) is blotting paper.

In another preference, the sample pad (1), gold-labelled pad (2), nitrocellulose filter (8) and absorption pad (7) It is pasted on the backing.

In another preference, the sample pad (1) and gold-labelled pad (2) partly overlap.

In another preference, the detection zone is positioned close to the side of gold-labelled pad (2), and the quality control region (6) it is positioned close to the side of absorption pad (7).

In another preference, the effluent piece further includes cartridge (9), and the well in the cartridge (10) and observation window (11).

In another preference, the detection card is completely set up in cartridge (9), and the well (10) corresponds to institute The sample pad (1) on detection card is stated, and the observation window (11) corresponds to the detection zone and quality control region on the detection card.

In another preference, the length of the cartridge is 80-90.0mm, width 10-20mm, and height is 4-5mm.

In another preference, the length of the effluent piece is 60-70mm, and width is 3-4mm.

In another preference, the quality control region (6) includes the 4th antibody, and the 4th antibody specificity is incorporated into described First antibody, secondary antibody and the 3rd antibody.

In another preference, first detection zone (3), the second detection zone (4), the 3rd detection zone (5) and Quality Control Area (6) is located on nitrocellulose filter (8).

In another preference, the effluent piece is used to detect the mycotoxin in food or feed.

In another preference, the food includes edible oil, feed, cereal and beans etc..

In the second aspect of the utility model, there is provided a kind of kit for being used to detect mycotoxin, the kit Including：

(i) container；And

(ii) the effluent piece of the detection mycotoxin as described in the utility model first aspect.

In another preference, the kit further includes specification.

It is to be understood that in the scope of the utility model, above-mentioned each technical characteristic of the utility model and below (such as implementation Example) in specifically describe each technical characteristic between can be combined with each other, so as to form new or preferable technical solution.It is limited to Length, not repeated them here.

Brief description of the drawings

Fig. 1 is a kind of structure sectional view of the detection card of preference of the utility model.Wherein, 1 is sample pad, and 2 be gold mark Pad, 3 be the first detection zone, and 4 be the second detection zone, and 5 be the 3rd detection zone, and 6 be quality control region, and 7 be absorption pad, and 8 be cellulose nitrate Plain film, 0 is backing.

Fig. 2 is a kind of top view of the effluent piece of the detection mycotoxin of preference of the utility model.Wherein, 9 be card Box, 10 be well, and 11 be observation window.

Embodiment

The present inventor by depth studying extensively, it has unexpectedly been found that a kind of effluent for detecting mycotoxin in food Piece.Develop the color the difference of position when specifically, according to detection, which can quickly detect a variety of mycotoxins at the same time, including but Fumonisin, ochratoxin and vomitoxin are not limited to, changes the previous reagent of mesh and only detects a kind of present situation of component.Herein On the basis of, complete the utility model.

Mycotoxin

Mycotoxin is metabolite caused by fungi grows in food or feed, all harmful to human and animal.

As used herein, " fumonisin ", " fumonisins " are used interchangeably, and are by fusarium moniliforme The water soluble metabolites that (Fusarium moniliforme Sheld) is produced is one kind by different more hydrogen alcohol and the third tricarboxylic The similar diester compound of structure of acid composition.Main pollution grain and its product, and some domestic animals are produced acute toxicity and Potential carcinogenicity.Up to the present, the fumonisins of discovery have FA1, FA2, FB1, FB2, FB3, FB4, FC1, FC2, FC3, Totally 11 kinds of FC4 and FP1, wherein FB1 is its key component.FB1 to the situation of food pollution worldwide generally existing, Main pollution corn and corn product.FB1 is water-soluble mycotoxin, to thermostabilization, is not easy to be cooked destruction, so same AFT (aflatoxin) equally, mould contamination of the control crops during growth, harvest and storage is still most important.

Ochratoxin is the one group of important, pollution produced by 7 kinds of aspergillus of aspergillus and 6 kinds of Penicillium notatums of Penicillium The mycotoxin of food, including the compound that 7 kinds of structures are similar, general structure：R1=Cl or H；R2=H, CH3 or C2H5, its Poisoning is maximum, distribution is most wide, production poison amount highest, the pollution to agricultural product are most heavy, with human health it is most close be reddish brown Aspertoxin A.

Vomitoxin (vomitoxin), also known as deoxynivalenol (DON), chemistry entitled 3 α, 7 α, 15 13 Hydroxyl grass Fusariumsp -9- alkene -8- ketone, belongs to trichothecene.Gain the name since it can cause the vomiting of pig, to people Body has certain damaging effect, and European Union's criteria for classification is three-level carcinogenic substance.

As used herein, " mycotoxin to be measured " refers to the mycotoxin of the lateral flow strip detection of the utility model, Specifically include fumonisin, ochratoxin and vomitoxin.

Detection method and operation principle

For the ease of understanding the utility model, the testing principle of the utility model effluent piece is provided.It is to be understood that this practicality is new The protection domain of type and from the influence or limitation of the principle.

The utility model use competition law immunochromatography principle, by the comlete antigen (the first compound) of fumonisin, The comlete antigen (the second compound) of ochratoxin and the comlete antigen (triplex thing) of vomitoxin are individually fixed in The first detection zone, the second detection zone and the 3rd detection zone (solid phase antigen) on nitrocellulose membrane, by the special of colloid gold label Property for the specificity of the antibody (first antibody) of fumonisin, colloid gold label, for the antibody of ochratoxin, (second is anti- Body) and the specificity of colloid gold label be fixed on gold-labelled pad for the antibody (the 3rd antibody) of vomitoxin, and formed by Fig. 1 In detecting strip and being loaded.After sample to be checked is added in the sample pad of detection card one end by well, through capillary action Move forward, dissolving combines first antibody, secondary antibody and the 3rd antibody of the colloid gold label in gold-labelled pad, then moves successively To the first detection zone, the second detection zone, the 3rd detection zone, quality control region.If mycotoxin to be measured, colloidal gold are not contained in sample First antibody, secondary antibody and the 3rd antibody of mark can be fixed on the first compound on nitrocellulose filter, respectively Two compounds and the capture of triplex thing, so as to form red stripes in the first detection zone, the second detection zone, the 3rd detection zone. If containing a certain concentration certain mycotoxin to be measured in sample, such as contain fumonisin, the of colloid gold label in gold-labelled pad One antibody can be combined first with fumonisin in sample after being dissolved, so as to suppress first antibody and the on nitrocellulose filter The combination of one compound, the band color of the first detection zone will weaken or disappear；Secondary antibody and the knot of the second compound The combination of conjunction, the 3rd antibody and triplex thing is unaffected, and the second detection zone and the 3rd detection zone still form red bar Band.As contained two or more mycotoxins to be measured in sample at the same time, such as contain fumonisin, ochratoxin and vomiting at the same time Toxin, first antibody, secondary antibody and the 3rd antibody and the first compound on nitrocellulose filter, second of colloid gold label The combination of compound and triplex thing can be suppressed, the first detection zone, the second detection zone, the band face of the 3rd detection zone Color can weaken or disappear.In other words, when containing certain mycotoxin in sample to be checked, corresponding antibody and detection The combination in region is affected, and the band color of corresponding detection zone can weaken or disappear.

The utility model is provided with the 4th antibody in the quality control region of nitrocellulose filter, and the 4th antibody specificity combines In the first antibody, secondary antibody and the 3rd antibody, no matter whether contain mycotoxin to be measured, colloidal gold mark in measuring samples First antibody, secondary antibody and the 3rd antibody of note can form red stripes, the band with the 4th antibody binding of quality control region It is to judge whether chromatography process normal and whether detection plate fails standard, while also serves as control difference between batch in quantitative analysis Parameter.

Sensitivity

The effluent piece sensitivity of the utility model is as follows.

Fumonisin detects thresholding：200ug/l

Ochratoxin detects thresholding：5ug/l

Vomitoxin detects thresholding：100ug/l

The manufacture of effluent piece

In the utility model, the existing material system in this area can be selected in each constituent element (or component) of the effluent piece Into.

Backing 0 can be made of any stabilization, non-porous material, its intensity should be enough supporting material and adhere thereto Each constituent element.Because many measure are by the use of water as dispersive medium, therefore backing 0 is preferably substantially water-impervious.One In a preference, backing 0 is made of polymer film, is used made of polychloroethylene film (such as PVC offset plates).

Sample pad 1 can be made of any absorbent material.Workable example of material includes：Cellulose, nitrocellulose, Cellulose acetate, glass fibre, nylon, polyelectrolyte ion exchange membrane, propylene copolymer/nylon and polyether sulfone.

Absorption pad 7 can be made of any material that can be absorbed as sample and the liquid of buffer solution.The suction of absorption pad 7 Receipts ability is sufficiently large, to absorb the liquid for being added to test-strips.Include cellulose suitable for the example of the material of absorption pad 7 And absorbent filter.

Antibody

First antibody, secondary antibody and the 3rd antibody are the antibody of colloid gold label, can respectively with fumonisin, reddish brown Aspertoxin, vomitoxin combine.

4th antibody is sheep anti-mouse igg in combination with first antibody, secondary antibody and the 3rd antibody, preferable 4th antibody It is more anti-.

Antibody used in the utility model is commercially available.

Colloid gold label

Colloidal gold is to be acted on by gold chloride (HAuCl4) in reducing agent such as white phosphorus, ascorbic acid, sodium citrate, tannic acid etc. Under, a certain size gold grain is can be grouped to, and since electrostatic interaction becomes a kind of colloidal state of stabilization, formed electronegative Hydrophobic sol solution, becomes stable colloidal state due to electrostatic interaction, therefore claims colloidal gold.

Colloidal gold is negatively charged under mild alkaline conditions, can form firm combination with the positive charge group of protein molecule, Since this combination is electrostatical binding, so not influencing the biological nature of protein.Colloidal gold in addition to being combined with protein, It can also be combined with many other large biological molecules, such as SPA, PHA, ConA.According to some physical behaviors of colloidal gold, such as High electron density, granular size, shape and color reaction, plus the immune and biological characteristics of conjugate, thus make colloidal gold It is widely used in the fields such as immunology, histology, pathology and cell biology.

Colloid gold label, is substantially the coating process that the macromolecules such as protein are adsorbed to colloid gold particle surface.Inhale Random reason is probably colloid gold particle surface negative charge, and firm knot is formed because of Electrostatic Absorption with the positive charge group of protein Close.The colloid gold particle of various different-grain diameters, namely different colours can be easily prepared from gold chloride with reduction method.It is this Spherical particle has protein very strong adsorption function, can be with staphylococcal protein A, immunoglobulin, toxin, sugared egg In vain, the Non-covalent binding such as enzyme, antibiotic, hormone, bovine serum albumin(BSA), polypeptide, thus in basic research and clinical trial As highly useful instrument.

Immuno-gold labeling technology (Immunogold labelling technique) mainly make use of gold grain to have height The characteristic of electron density, is marked at protein binding, under the microscope visible dark brown coloured particles in gold, when these labels are corresponding When largely assembling at ligand, naked eyes red color visible or pink spot, thus detected for qualitative or sxemiquantitative tachysynthesis In method, this reaction can also be exaggerated by the deposition of Argent grain, be referred to as immunogold silver staining.

In the preparation of the utility model lateral flow strip, it is preferable that 40nm colloidal golds are taken, with 0.1M K₂CO₃PH is adjusted, with every milli Rising 5 μ g antibody of colloidal gold to be marked, must be precipitated after marking fluid centrifugation, precipitation 10mM pH7.2PBS dissolve, and in 650nm Lower measure light absorption value OD, is applied on paving polyester material and is dried with 1-3OD, is the gold-labelled pad in the utility model detection card.

The preparation of lateral flow strip

Each constituent element (or component) of the utility model effluent piece is using technical method manufacture generally in the art and group Dress.

Preferably, the first compound, the second compound, triplex thing and Quality Control antibody are diluted with coating buffer solution, will Four kinds of capture liquid after dilution are equably coated on the nitrocellulose membrane as detection part respectively, and four kinds of capture liquid penetrate into The first detection zone, the second detection zone, the 3rd detection zone and quality control region are formed after nitrocellulose filter respectively.

Sample pad, gold-labelled pad, nitrocellulose filter, absorption pad are pasted on backing by adhesive respectively.With slitting Machine-cut is placed in cartridge, well alignment detection card sample pad location into the detection card of 4mm, detection reagent is assembled into after compression Box, adds drier foil sealing packaging and preserves.

The advantages of the utility model：

It is described in the utility model detection mycotoxin effluent piece overcome need to detect one by one in existing method it is not of the same race The drawbacks of class mycotoxin, by same lateral flow strip set three detection zones, quickly, efficiently and accurately specific detection it is more Kind mycotoxin.

Below in conjunction with specific embodiment, the utility model is further illustrated.It should be understood that following description is only this practicality New most preferred embodiment, and it is not construed as the limitation for scope of protection of the utility model.Fully understanding On the basis of the utility model, usually according to normal condition, or according to the condition proposed by manufacturer, those skilled in the art Nonessential change can be made to the technical solution of the utility model, such change should be considered as being included in this practicality newly Among the protection domain of type.

Embodiment 1

The structure of effluent piece

The effluent piece (or test-strips) of the detection mycotoxin of the utility model includes cartridge and the detection in cartridge Card.

A kind of structure of preferable detection card is as shown in Figure 1, it includes backing 0, and on backing：Sample pad 1, Gold-labelled pad 2, nitrocellulose filter 8, detection zone (or detection line), including the first detection zone 3 and the detection of the second detection zone the 4, the 3rd Area 5, quality control region 6 (or check plot, control line, nature controlling line) and absorption pad 7.

A kind of structure of preferable cartridge 9 as shown in Fig. 2, include well 10 and observation window 11 thereon.It is also, described Detection card is completely set up in cartridge 9, and the well 10 corresponds to the sample pad 1 on the detection card, and the observation window 11 correspond to the first detection zone 3, the second detection zone 4, the 3rd detection zone 5 and the quality control region 6 on the detection card

Specifically, the effluent piece of the utility model has the characteristics that：

The length of backing 0 is identical with effluent piece.

Sample pad 1 is located at one end of test-strips, and absorption pad 7 is located at the other end of test-strips.

Three kinds of antibody are loaded with gold-labelled pad 2, first antibody is directed to the anti-of fumonisin for the specificity of colloid gold label Body, secondary antibody are directed to the antibody of ochratoxin for the specificity of colloid gold label, and the 3rd antibody is the spy of colloid gold label The opposite sex is directed to the antibody of vomitoxin, and above-mentioned antibody is commercially available.

Detection zone (also referred to as detection line) is between sample pad 1 and absorption pad 7, including the first detection zone 3, the second detection zone 4 and the 3rd detection zone 5, it is preferable that the first detection zone 3 is located at close to one end of sample pad 1, and usual detection zone is arranged on nitric acid fibre On the plain film 8 of dimension, pass through the gold-labelled pad 2 and water absorption pad 7 of the nitrocellulose filter linkage flag.Preferably, the cellulose nitrate Quality control region 6 (also referred to as nature controlling line) is additionally provided with plain film 8, quality control region 6 is between detection zone and absorption pad 7.

Wherein, first detection zone 3 is coated with the comlete antigen of fumonisin；Second detection zone 4 is coated with There is the comlete antigen of ochratoxin, the 3rd detection zone 5 is coated with the comlete antigen of vomitoxin.Quality control region 6 is coated with There is the 4th antibody, the 4th antibody specificity is incorporated into first antibody, secondary antibody and the 3rd antibody.

Embodiment 2

The use of effluent piece

1. taking the representational sample comminutions of more than 5g (crossing 20 mesh sieves), the present embodiment is accurately weighed using wheat as sample 1g uniformly crushes sample and is added in centrifuge tube, then 4mL dilutions are accurately added into centrifuge tube, and bottle stopper tightening seal is used Forced oscillation 5 minutes, 4000rpm centrifuge 1 minute to obtain supernatant.This supernatant is sample detection liquid.

2. detection card is kept flat, sample solution to be checked is drawn with dropper, vertical 3 drops that are added dropwise are opened in well after sample-adding Beginning timing.

3. read testing result in 5 minutes.

As a result judge：

A：First detection zone, the second detection zone, the 3rd detection zone and quality control region change colour (red) at the same time, show in sample not It is less than minimum detected value containing mycotoxin to be measured or its concentration, testing result is feminine gender.

B:Quality control region changes colour (red), and the first detection zone, the second detection zone and the 3rd detection zone are non-discolouring, show sample In contain fumonisin, ochratoxin and vomitoxin at the same time, testing result is the positive.

C：Quality control region and some or two detection zone discolorations (red), and other detection zones are non-discolouring, show that sample contains It is the positive to have with the corresponding mycotoxin of non-discolouring detection zone, testing result.

D：Quality control region is non-discolouring, and no matter whether detection zone changes colour, and shows that this detection is invalid.

It is incorporated herein by reference in all documents that the utility model refers to, just as each document quilt It is individually recited as reference.In addition, it should also be understood that, after the above-mentioned instruction content of the utility model has been read, this area skill Art personnel can make various changes or modifications the utility model, and such equivalent forms equally fall within the application appended claims Book limited range.

Claims (10)

1. a kind of effluent piece for detecting mycotoxin, it is characterised in that the effluent piece includes a detection and blocks, the detection card Including backing (0), and on the backing proximally arrive distal end with lower structure：Sample pad (1), gold-labelled pad (2), nitre Acid cellulose film (8), detection zone, quality control region (6) and absorption pad (7)；

And the gold-labelled pad (2) includes：First antibody load region, secondary antibody load region and the 3rd antibody load region, its In, the first antibody is directed to the antibody of fumonisin for the specificity of colloid gold label, and the secondary antibody is colloid The specificity of gold mark is directed to the antibody of ochratoxin, and the 3rd antibody is directed to vomiting for the specificity of colloid gold label The antibody of toxin；

And the detection zone includes：First detection zone (3), the second detection zone (4) and the 3rd detection zone (5), wherein, it is described First detection zone (3) is coated with the comlete antigen of fumonisin, and second detection zone (4) is coated with the complete of ochratoxin Holoantigen, the 3rd detection zone (5) are coated with the comlete antigen of vomitoxin.

2. effluent piece as claimed in claim 1, it is characterised in that the detection zone is positioned close to the one of gold-labelled pad (2) Side, and the quality control region (6) is positioned close to the side of absorption pad (7).

3. effluent piece as claimed in claim 1, it is characterised in that the effluent piece further includes cartridge (9), and positioned at described Well (10) and observation window (11) in cartridge.

4. effluent piece as claimed in claim 3, it is characterised in that the detection card is completely set up in cartridge (9), described Well (10) corresponds to the sample pad (1) on the detection card, and the observation window (11) corresponds on the detection card Detection zone and quality control region (6).

5. effluent piece as claimed in claim 4, it is characterised in that the length of the cartridge is 80-90.0mm, width 10- 20mm, and height is 4-5mm.

6. effluent piece as claimed in claim 1, it is characterised in that the length of the effluent piece is 60-70mm, and width is 3- 4mm。

7. effluent piece as claimed in claim 1, it is characterised in that the quality control region (6) includes the 4th antibody, and the described 4th is anti- Body specifically binds to the first antibody, secondary antibody and the 3rd antibody.

8. effluent piece as claimed in claim 1, it is characterised in that first detection zone (3), the second detection zone (4), Three detection zones (5) and quality control region (6) are located on nitrocellulose filter (8).

9. a kind of kit for being used to detect mycotoxin, it is characterised in that the kit includes：

(i) container；And

(ii) the effluent piece of the detection mycotoxin as described in claim 1-8 is any.

10. kit as claimed in claim 9, it is characterised in that the kit further includes specification.