CN105785014A - Colloidal-gold-based quantitative mycotoxin detection device and preparation method - Google Patents
Colloidal-gold-based quantitative mycotoxin detection device and preparation method Download PDFInfo
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Abstract
The invention belongs to the field of biological detection, and particularly relates to a colloidal-gold-based quantitative mycotoxin detection device and a preparation method. The detection device comprises a colloidal-gold-based quantitative detection kit and a handheld card reader, and by the detection device, an accurate quantitative detection result is provided for a toxin product to be detected. The detection kit comprises a cartridge and a reagent strip, wherein the reagent strip consists of a substrate, a sample pad, a colloidal gold pad, a reaction film and an absorbent pad; a colloidal gold labeled mycotoxin and bovine serum albumin are adsorbed to the colloidal gold pad; the reaction film is coated with a detection antigen and a quality control antibody, the detection antigen is displayed as a detection line T, and is a mycotoxin antigen, and the quality control antibody is displayed as a quality control line C, and is a goat anti-mouse IgG antibody. The detection device can be operated conveniently and rapidly, is applicable to various kinds of field detection, and is matched with a colloidal-gold-based quantitative analyzer and a specific curve establishment method to quantitatively detect the content of a sample by virtue of colloidal gold and integrate the dual advantages of the colloidal gold and ELISA to make the two technologies complementary.
Description
Technical field
The invention belongs to field of biological detection, particularly relate to a kind of mycotoxin class quantitative testing device and preparation method.
Background technology
Mycete is a kind of many cells microorganism, is widely present in nature, belongs to fungus on microbiology.It is multiplied by the form of spore.Mycotoxin is by mycete or mycetogenetic poisonous and harmful substance.In soil, on plant, all can find mycotoxin including corn, forage grass and ensilage.Hot and humid environmental condition typically favors the growth of mycete in feedstuff and the generation of mycotoxin.
Separated up to now and qualification mycotoxin out has kind more than 300.Generally speaking, mycotoxin is mainly produced by 4 kinds of mould Pseudomonas: Eurotium (Major Secretory aflatoxin, ochratoxin etc.), Penicillium (Major Secretory notalin etc.), Claviceps (Major Secretory ergotoxin), Fusarium (Major Secretory 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone, vomitoxin, T-2 toxin, fumonism), be also several mycotoxins seen most.
The harm of animal is global problem by mycotoxin, mycete belongs to middle warm type microorganism mostly, optimum growth temperature is generally 20～30 DEG C, it is 25～30 DEG C that the optimum temperature of poison is produced in mycete breeding, wherein Eurotium optimum growth temperature is 30 DEG C, and Penicillium is about 28 DEG C, and Fusarium spp. is generally about 20 DEG C, the general mycotic spore endangering feedstuff can germinate when 7 DEG C, and when temperature is higher than 49 DEG C, mycete can be killed or enter Spore Stages.The harm major embodiment of mycotoxin both ways, the first aviculture: plant fowl take in go mouldy feedstuff easily occur egg drop reduction, rate of fertilization decline, incubation rate decline；Offspring Growth is bad, bowel syndrome occurs and is difficult to control to.After the feedstuff that goes mouldy taken in by broiler, easily there are the symptoms such as ascites, renal swelling, undergrowth in body, also results in vaccine immunity failure, it is easy to bringing out the viral infectious such as newcastle, biography, influenza, clinic is difficult to control to.Second aspect human health: health can be produced impact by mycotoxin residual in livestock products.Current mycotoxin has become the maximum hidden danger of food safety, has influence on human health.After animal foods is contaminated, people is edible can be occurred acute and chronic poisoning, or teratogenesis, carcinogenic, mutagenesis occurs.
China is large agricultural country, but China seed grow industry proportion bigger be that peasant household raises scattered or the cultivation of small-sized plant, scale is disperseed, and plant mycotoxin class epidemic prevention and control level and production management ability fall behind, when pestilence occurs, it is difficult to effectively control.There is crop field supervision to as kind is many, quantity big, distribution is wide, the not first-class problem of scale, crop link especially has the feature of extremely strong disguise and timeliness, if check by eye face fundamentally can not a settle practice is hard to reform its problem existing and hidden danger in on-site law-enforcing inspection.Traditional experiment detection there is also the technical problem of program complexity, detection time length, and testing result out rear product has been sold or shifted, it is impossible to process in time, it is impossible to meet Site Detection needs needs timely, efficient, quick.And, Fast Detection Technique ripe at present is carry out for single epidemic disease or drug residue mostly, it is also difficult to reach the purpose to the detection detection that sample type is various, detection project is various.
Along with the development of biotechnology, start, by biotechnology, vegetable plague, plant product quality are carried out safety detection.Such as PCR detection method, euzymelinked immunosorbent assay (ELISA) (ELISA) and the fairly simple animal epidemic immunochromatography detection method of operation, PCR detection method is usually used in the detection of nucleic acid aspect, sensitivity is good and accuracy is of a relatively high, euzymelinked immunosorbent assay (ELISA) is usually used in detection residue of veterinary drug and animal epidemic, there is specificity and the higher advantage of remolding sensitivity, good application prospect is had for on-the-spot primary dcreening operation, the operation but the instrument and equipment of both of which needs specialty and operator test, experimental condition is required higher, implementing inconvenience, cost is high.
The method that the detection of current mycotoxin is commonly used is specific as follows several:
Thin layer chromatography:
TLC method is for different samples, being extracted from sample by mycotoxin with suitable Extraction solvent, through column chromatography purification, then chromatography launches, separates on lamellae, utilize the fluorescence of mycotoxin, measure its minimum content according to the power of fluorescence speckle and standard comparing.TLC method sample pre-treatments is loaded down with trivial details, and extracts not ideal enough with clean-up effect, and in extracting solution, impurity is more, affects the fluorescence intensity of speckle upon deployment.
Chromatography:
Chromatography, including thin layer chromatography, gas chromatogram, liquid chromatograph etc., is always up the chemical analysis method of most important mycotoxin.The analysis method of now commonplace mycotoxin or liquid chromatography, including LC-MS-MS.This method is quickly and accurately, but need expensive instrument and equipment, it is only limitted to that specialty testing agency obtains scientific research and investigation and analysis, monitoring uses, fails to be widely popularized use in enterprise and basic unit, and the hysteresis effect of its result greatly reduces and actual instructs effect to producing.
Immunochemistry detection method: (colloidal gold immunity chromatography, enzyme linked immunosorbent assay)
A kind of biochemical analysis that immunological detection method is based on the selective reaction between antibody and antigen or hapten and sets up.It is generally of high selectivity and very low detection limit, be widely used in various antigen, half anti-or the mensuration of antibody, generally can be divided into non-radioactive immunization and the radioimmunologies such as fluorescent immune method, electrochemiluminescent immunoassay method, immunization and electro-chemistry immunity method, in mycotoxins in feed detects, wherein apply wider mainly enzyme linked immunosorbent assay (ELISA) and colloidal gold immunity chromatography.
Immunological detection method owing to it is quick, sensitive, accurate, can quantitative, easy and simple to handle, without valuable instrument and equipment, and to characteristics such as sample purity are less demanding, be particularly well-suited to feed factory, grain and oil/enterprise such as food processing factory, plant carries out detection and the industrial and commercial quality supervision department Site Detection of raw material or finished product.Such method has following characteristics: 1) highly sensitive
2) disturb little: 3) simple and efficient to handle:
4) safety is high, pollutes few, with low cost:
The field fast detection method of current Application comparison maturation also has immune colloid gold test paper detection method, colloidal gold technique has sensitivity convenient and swift, special, stability is strong, do not need special installation and reagent, result judge the advantages such as directly perceived, is suitable for mass detection and large area generaI investigation etc..
There is efficient, quick, result and judge that the method for mycotoxin class in directly perceived and stable detection plant and relevant product are needed for existing market.
Summary of the invention
In order to solve above-mentioned technical problem, the present invention provides a kind of mycotoxin class gold colloidal quantitative testing device and preparation method, fully apply gold colloidal easy and simple to handle, quick, be practically applicable to the technological merit of various Site Detection, supporting gold colloidal quantitative analysis instrument and distinctive curve method for building up, reach gold colloidal energy detection by quantitative and go out the content of sample, combine the two-fold advantage of gold colloidal and ELISA, form the complementation of two kinds of technology.
Solve a kind of mycotoxin class gold colloidal quantitative testing device in the present invention of above technical problem, including gold colloidal detection by quantitative box and hand-held Card Reader instrument, it is characterized in that: toxin product to be detected can be provided detection by quantitative result accurately by described detecting device, and described mycotoxin is Aspergillus flavus B1, M1,6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone, vomitoxin, T2 toxin, ochratoxin or fumonisins.
Described detection by quantitative result is that in the colored intensity Ratio control of the detection colored intensity of line T line and the C line measured according to hand-held chart scanner and mycotoxin sample to be measured, the content of mycotoxin quantitatively contains number relation in relevant, this culvert number is the functional relation of curtain or logarithm, it is set to the internal standard curve of instrument, this hand-held Card Reader instrument measures the intensity of reflected light of C/T line by imaging system, and by random chip, testing result is transferred to by infinite network center system or cloud server；This hand-held Card Reader instrument includes smart mobile phone, mould of plastics, eyeglass, glass plate, 9V battery, LED, drawer type tray.
Described mycotoxin sample is corn and soybean, feedstuff, Semen Tritici aestivi, distiller grains, bean cake；Wherein sample weighing grams is at 2-5g, mycotoxin extraction is carried out by the methanol buffer of the 60-90% of 1:5 volume, the sample treatment liquid taking 50-150ul adds in detection card, after reaction 10-20min, detection card is put into the T/C value having read variable concentrations in hand-held chart scanner and carries out Function Fitting and set up curve.
The internal standard curve of the product of described each batch can adjust accordingly, and which reduces the difference of quantitative analysis relation between different batches product, it is ensured that the different batches product concordance to same determinand detection by quantitative relation.
Described functional relation is: y=aln (x)-b or y=ax-b, wherein y represents T/C value；, x represents concentration；A represents slope；B represents displacement.
In the present invention, the variable concentrations according to standard substance sets up interior mark curve with corresponding T/C value, goes to detect the natural sample containing variable concentrations with interior mark curve, calculates the actual extracting rate of each concentration nature sample；After original standard concentration is respectively divided by extraction ratio, then calculates standard concentration T/C value of variable concentrations after conversion and set up final curves, upload to software and make Quick Response Code and facilitate guest operation to use.
Described mycotoxin class gold colloidal detection by quantitative box, including cartridge and reagent strip, reagent strip is made up of substrate, sample pad, colloidal gold pad, reaction film and adsorptive pads, colloidal gold pad is adsorbed with mycotoxin class monoclonal antibody and the bovine serum albumin of colloid gold label, reaction film is coated with detection antigen and quality control antibody, detection antigen is shown as detection line T, for mycotoxin class antigen；Quality control antibody is shown as nature controlling line C, for sheep anti-mouse igg antibody.
The detection by quantitative principle of this product: mycotoxin glue immue quantitative detection reagent box, based on colloidal gold immunochromatographimethod technology, the nitrocellulose membrane of detection card has been coated AFB1 antigen (T line) and sheep anti-mouse igg polyclonal antibody (C line) in advance.Wherein T line is detection line, and C line is nature controlling line.The gold specific binding AFB1 of labeling antibody, the T line colour developing degree of depth linearly changes with AFB1 concentration, gets final product quantitative assay by the analysis of hand-held detector and goes out the content of sample AFB1.
The distance of described detection line T1 and detection line T2 is 3mm, so can better control the chromatography time difference of gold colloidal；Spacing is too big it is possible that the difference of result, and spacing between nature controlling line can be caused again too big；Detection line T and nature controlling line C general control at 5-10mm, spacing is oversize can the growth detection time, and detect the form got stuck more and require just longer.
Mycotoxin class antigenic quality concentration 0.5-3mg/ml in described T line, optimizes concentration 0.7-2.5mg/ml.
Described colloid gold particle size controls within the scope of 20-80nm, and especially at 30-60nm, gold colloidal concentration is the concentration of 0.8-1.2%.
T line concentration is too low, and color developing effect below is undesirable, is too high the sensitivity that can reduce detector bar, so the concentration of T line is to need to select a suitable concn in experiment research and development, reaches prescription.
The detection sample of gold colloidal detection by quantitative box is mainly: corn and soybean, feedstuff, Semen Tritici aestivi, distiller grains etc..
Described reagent strip preparation method comprises the following steps, and raw materials used consumption number is weight portion:
(1) preparation of gold colloidal:
After 500 parts of distilled water heating to boiling, add gold chloride 1-10 part and trisodium citrate 1-10 part, stir and boil 10min, being cooled to room temperature, detecting its peak value and half-peak breadth with visible spectrophotometer；The peak value 229-231, half-peak breadth 580-585 of gold colloidal.
Raw material, before use through purification, detects its purity by instrument and reaches more than 97%, and high specificity, without non-specific binding.Gold colloidal liquid quality standard: limpid transparent Chinese red or aubergine, without particle, liquid surface without waft gold situation, with ultraviolet detection crest should more precipitous half-peak breadth more narrow, the quality of gold colloidal is more good.
(2) labelling of gold colloidal and metal spraying:
Take 100 parts of colloidal gold solutions, add borate buffer 8-12 part of 1.38%, the mycotoxin class monoclonal antibody 50 parts adding 2.2-2.8mg/ml in stirring continues stirring 8-12min, the bovine serum albumin 18-22 part adding 15-18% carries out coupling, 10000r/min is centrifuged 4-7min, remove supernatant, stay clearing heat in the lung particle solution to carry out metal spraying use；
With metal spraying Film-cutting machine metal spraying on glass fibre element film, consumption 3-10ul/cm, concentration is 0.5-10ug/cm dry 10-14h at temperature 35-38 DEG C again, and sealing saves backup；
(3) the C/T setting-out of nitrocellulose filter:
With BC buffer, sheep anti-mouse igg is diluted to the sheep anti-mouse igg solution of 0.5-3mg/ml, PVC base plate posts nitrocellulose filter, middle with sheep anti-mouse igg picture C line, consumption 1-3ul/cm；
With BT buffer, mycotoxin class antigen diluent is become 0.5-3mg/ml, PVC base plate posts nitrocellulose filter C line and down draws T line, consumption 0.5-9ul/cm with the mycotoxin class antigen after dilution；
To finish PVC base plate dry 12-24h at temperature 35-38 DEG C of line, sealing saves backup；
(4) making of the big plate of gold colloidal:
PVC base plate in step (3) sticks the glass fibre mold of the good gold of spray, sticks sample pad and absorbent paper.
Described colloid gold particle is sized to 15-60nm, and optimization granular size is 20-40nm, and gold colloidal concentration is 0.8-1.2%, and optimizing concentration is 1%.
Described BC buffer formulation is: 0.08gNaCl+0.05gKCl+0.144gNa2HPO4+0.067gNaH2PO4, with distilled water constant volume to 100ml, adds sucrose 5g.
Described BT buffer formulation is: 0.08gNaCl+0.15gKCl+0.288gNa2HPO4+0.1347gNaH2PO4, with distilled water constant volume to 100ml, adds the trehalose of 5% volume.
Described sample pad processes before using:
Process step: the Alhstorm8964 of import is cut into 2cm*30cm, carries out uniform application with quantitative DY buffer, it is ensured that the homogeneity of sample pad；
Wherein, the BY buffer formulation of 100ml is: 10%tw20+ surfactant 5-10%+5%PEG20000+15% trehalose+PVPK405g, with distilled water constant volume to 100ml.
Surfactant is TritonX-100, Tween-20 or Tween-80.
Described colloidal gold solution pH value is 6.2-8.0, and the isoelectric point, IP ph value of raw material adds 0.5.
The spray film amount of described gold colloidal mark pad controls: 0.5ug-10ug/cm；
Test kit in the present invention, based on gold colloidal double antibodies sandwich technology, the nitrocellulose membrane of detection card has been coated mycotoxin antigen (T line) and sheep anti-mouse igg antibody (C line) in advance.Wherein T line is detection line, and C line is nature controlling line.Gold mark antigenic specificity is in conjunction with mycotoxin antibody, and the T line colour developing degree of depth linearly changes with mycotoxin antibody concentration, can measure mycotoxin antibody titer height in sample by the analysis of hand-held detector.
The test kit detection time in the present invention is fast, convenient, stable and accurate, is suitable for scope wide.Not i.e. only colour developing, it is also possible to quantitative assay, can determine concrete numerical value, and testing result is accurate, easily holds, and error is few, reduces and artificially assesses the deviation brought.
Accompanying drawing explanation
Fig. 1 is that in the present invention, Aspergillus flavus B1 detects Semen Maydis curve chart
Fig. 2 is 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone detection Semen sojae atricolor curve chart in the present invention
Fig. 3 is vomitoxin detection feed yeast line chart in the present invention
Detailed description of the invention
Hereinafter with specific embodiment, the present invention is further illustrated:
In following example, detection by quantitative result is that in the colored intensity Ratio control of the detection colored intensity of line T line and the C line measured according to hand-held chart scanner and mycotoxin sample to be measured, the content of mycotoxin quantitatively contains number relation in relevant, this culvert number is the functional relation of curtain or logarithm, it is set to the internal standard curve of instrument, this hand-held Card Reader instrument measures the intensity of reflected light of C/T line by imaging system, and by random chip, testing result is transferred to by infinite network center system or cloud server；This hand-held Card Reader instrument includes smart mobile phone, mould of plastics, eyeglass, glass plate, 9V battery, LED, drawer type tray.
Mycotoxin sample is corn and soybean, feedstuff, Semen Tritici aestivi, distiller grains, bean cake；Wherein sample weighing grams is at 2-5g, mycotoxin extraction is carried out by the methanol buffer of the 60-90% of 1:5 volume, the sample treatment liquid taking 50-150ul adds in detection card, after reaction 10-20min, detection card is put into the T/C value having read variable concentrations in hand-held chart scanner and carries out Function Fitting and set up curve.
The internal standard curve of the product of each batch can adjust accordingly, and which reduces the difference of quantitative analysis relation between different batches product, it is ensured that the different batches product concordance to same determinand detection by quantitative relation.Functional relation is: y=aln (x)-b or y=ax-b, wherein y represents T/C value；, x represents concentration；A represents slope；B represents displacement.
Variable concentrations according to standard substance and corresponding T/C value mark curve in setting up, and remove the detection natural sample containing variable concentrations with interior mark curve, calculate the actual extracting rate of each concentration nature sample；After original standard concentration is respectively divided by extraction ratio, then calculates standard concentration T/C value of variable concentrations after conversion and set up final curves, upload to software and make Quick Response Code and facilitate guest operation to use.
Embodiment 1
AFB1 gold colloidal quantitative testing device, including gold colloidal detection by quantitative box and hand-held Card Reader instrument, toxin product to be detected can be provided detection by quantitative result accurately by detecting device.
Detection by quantitative result is that in the colored intensity Ratio control of the detection colored intensity of line T line and the C line measured according to hand-held chart scanner and testing sample, the content of mycotoxin quantitatively contains number relation in relevant, this culvert number is the functional relation of curtain or logarithm, is set to the internal standard curve of instrument.
AFB1 gold colloidal quantitatively tries to survey box, including cartridge and reagent strip, reagent strip is made up of substrate, sample pad, colloidal gold pad, reaction film and adsorptive pads, colloidal gold pad is adsorbed with AFB1 monoclonal antibody and the bovine serum albumin of colloid gold label, reaction film is coated with detection antigen and quality control antibody, detection antigen is shown as detection line T, for AFB1 antigen；Quality control antibody is shown as nature controlling line C, for sheep anti-mouse igg antibody.
Detection line T and nature controlling line C control at 5mm, spacing is oversize can the growth detection time, and detect the form got stuck more and require just longer.
In T line, mycotoxin antigenic quality concentration is 0.5mg/ml, and AFB1 monoclonal antibody mass concentration is 2.2mg/ml, and protein amount concentration is 15%.
Reagent strip preparation method comprises the following steps, and raw materials used consumption number is weight portion:
(1) preparation of gold colloidal:
After 500 parts of distilled water heating to boiling, add gold chloride 1 part and trisodium citrate 1 part, stir and boil 10min, being cooled to room temperature, detecting its peak value and half-peak breadth with visible spectrophotometer；The peak value 229-231, half-peak breadth 580-585 of gold colloidal.
Raw material, before use through purification, detects its purity by instrument and reaches more than 97%, and high specificity, without non-specific binding.Gold colloidal liquid quality standard: limpid transparent redness or aubergine, without particle, liquid surface without waft gold situation, with ultraviolet detection crest should more precipitous half-peak breadth more narrow, the quality of gold colloidal is more good.
Colloidal gold solution pH value is 6.2, and the isoelectric point, IP ph value of raw material adds 0.5.Colloid gold particle is sized to 15-60nm, and gold colloidal concentration is 0.8%.
(2) 100 parts of colloidal gold solutions are taken, add borate buffer 8-12 part of 1.38%, the mycotoxin monoclonal antibody 50 parts adding 2.2mg/ml in stirring continues stirring 8-12min, the bovine serum albumin 18 parts adding 15% carries out coupling, 10000r/min is centrifuged 7min, remove supernatant, stay clearing heat in the lung particle solution to carry out metal spraying use；
With metal spraying Film-cutting machine metal spraying on glass fibre element film, consumption 3ul/cm, then at temperature 35 DEG C dry 14h, sealing saves backup；
(3) the C/T setting-out of nitrocellulose filter:
With BC buffer, sheep anti-mouse igg is diluted to the sheep anti-mouse igg solution of 0.5mg/ml, PVC base plate posts nitrocellulose filter, middle with sheep anti-mouse igg picture C line, consumption 1ul/cm；
BC buffer formulation is: 0.08gNaCl+0.05gKCl+0.144gNa2HPO4+0.067gNaH2PO4, with distilled water constant volume to 100ml, adds sucrose 5g.
With BT buffer, AFB1 antigen diluent is become 0.5mg/ml, PVC base plate posts nitrocellulose filter C line and down draws T line, consumption 0.5ul/cm with the AFB1 antigen after dilution；
BT buffer formulation is: 0.08gNaCl+0.15gKCl+0.288gNa2HPO4+0.1347gNaH2PO4, with distilled water constant volume to 100ml, adds the trehalose of 5% volume.
To finish PVC base plate dry 24h at temperature 35 DEG C of line, sealing saves backup；
(4) making of the big plate of gold colloidal:
Sticking the glass fibre mold of the good gold of spray on PVC base plate in step (3), glass fibre element film wants superposition nitrocellulose filter 1-2mm in the process of patch；Patch sample pad is alignd with PVC bottom edge according to sample pad lower end, superposes 1-2mm with nitrocellulose filter upper end during patch absorbent paper.
Must noting in taping process not again leaving a trace on nitrocellulose filter, pasting place must be firm, and overlapping portion must contact with each other, it is impossible to is disengaged from.
Sample pad processes before using: the Alhstorm8964 of import is cut into 2cm*30cm, carries out uniform application with quantitative DY buffer, it is ensured that the homogeneity of sample pad.
Wherein, the BY buffer formulation of 100ml is: 10%tw20+ surface activity Tween-805%+5%PEG20000+15% trehalose+PVPK405g, with distilled water constant volume to 100ml.
The preparation of AFB1 standard curve:
The foundation of standard curve: (buy from national standard material net purchase according to known, the standard substance that external sigma company buys) standard substance of 1mg concentration of concentration, it is diluted to 2ppb, 5ppb, 10ppb, 15ppb, 20ppb, 25ppb, 30ppb, 35ppb, nine Concentraton gradient of 40ppb, the mensuration of 5 Duplicate Samples is carried out by also ready-made finished product AFB1 test strip, according to its concentration change difference, its C value is measured with chart scanner, T value, T/C value, according to the principle that the more high colour developing of its principle concentration is more shallow, (abscissa also standard concentration is unit to set up its standard curve, vertical coordinate also T/C value carries out the foundation of standard curve for unit), filter out R2 value and reach the meansigma methods of more than 0.99 point, concrete such as Fig. 1；
The checking (being tested with the positive sample of occurrence according to mass spectrograph) of positive sample: take the standard curve that checking is also set up by the positive of high, medium and low three kinds of content respectively, see its deviation range.It is standard results that the result finally measured reaches within the scope of the 90-110% of legitimate reading.
Colourity change according to T and C line calculates numerical value by the detection of instrument and carries out the calculating of formula, y=ax-b, concrete numerical value is shown in Fig. 1.
Different samples are abscissa according to concentration, and T/C value is vertical coordinate, is calculated according to power relational expression matching；Ratio finally according to unknown sample can calculate concrete concentration value according to curve.
Interpretation of result: Quality Control C line develops the color, it was shown that detection effectively, namely can use detector reading.Quality Control C line does not develop the color, then show that operating process is incorrect or test strip lost efficacy.
T/C values different measured by the detection line of the different colourity of detections according to quantitative chart scanner, can the actual content of detection by quantitative embodiment sample in conjunction with the standard curve made；Current Aspergillus flavus B1 detects Semen Maydis and requires that content is less than or equal to 10ppb, is positive more than 10ppb in the industry, and Aspergillus flavus B1 content overproof is described；
Test example
AFB1 sample (corn and soybean, Semen Tritici aestivi, feedstuff, distiller grains) pre-treatment:
Sample extraction processes:
A, take 2g sample (sample its granularity size-reduced is less than 2mm) in extraction flask；
B, addition 4mL extractant, jolting 5 minutes, stand 3 minutes；
C, use syringe and filtering with microporous membrane, collect 1～1.5mL sample liquid standby in 1.5mL plastic tube；
AFB1 sample detection
Taking 100 μ L sample liquids and add sample buffer 400 μ L mixing, accurately draw 120 μ L sample liquids to be checked with pipettor, instill in well, panel is reacted 15 minutes, puts into hand-held chart scanner and reads result.
AFB1 sample detection result and HPLC contrast
(grains bureau of Sichuan Province) mass spectrometry instrument testing result correction data table of test kit and authorized by state
Detect the data after sample and gold colloidal quantitative reagent bar comparing result according to mass spectrometry, the testing result of gold colloidal than actual sample testing result very close to, the accuracy of sample detection result and legitimate reading all controls between 90%-120%；The average deviation CV% of duplicate detection result controls within 15%, illustrates that the accuracy of the testing result of colloid gold reagent bar is higher, can be suitably used for product quality that the market demand reaches and meets on-the-spot a large amount of sample and screen the quantitative colloid gold reagent bar that can select.
Embodiment 2
6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone gold colloidal quantitative testing device, including gold colloidal detection by quantitative box and hand-held Card Reader instrument, toxin product to be detected can be provided detection by quantitative result accurately by detecting device.
Detection by quantitative result is that in the colored intensity Ratio control of the detection colored intensity of line T line and the C line measured according to hand-held chart scanner and testing sample, the content of mycotoxin quantitatively contains number relation in relevant, this culvert number is the functional relation of curtain or logarithm, is set to the internal standard curve of instrument.
6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone gold colloidal detection by quantitative box, including cartridge and reagent strip, reagent strip is made up of substrate, sample pad, colloidal gold pad, reaction film and adsorptive pads, colloidal gold pad is adsorbed with 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone monoclonal antibody and the bovine serum albumin of colloid gold label, reaction film is coated with detection antigen and quality control antibody, detection antigen is shown as detection line T, for 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone antigen；Quality control antibody is shown as nature controlling line C, for sheep anti-mouse igg antibody.
Detection line T and nature controlling line C general control at 10mm, spacing is oversize can the growth detection time, and detect the form got stuck more and require just longer.
In T line, 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone antigenic quality concentration is 3mg/ml, and 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone monoclonal antibody mass concentration is 2.8ug/ml, and protein amount concentration is 18%.
Reagent strip preparation method comprises the following steps, and raw materials used consumption number is weight portion:
(1) preparation of gold colloidal:
After 500 parts of distilled water heating to boiling, add gold chloride 10 parts and trisodium citrate 10 parts, stir and boil 10min, being cooled to room temperature, detecting its peak value and half-peak breadth with visible spectrophotometer；
Colloidal gold solution pH value is 7.3, and the isoelectric point, IP ph value of raw material adds 0.5.Colloid gold particle is sized to 20-40nm, and gold colloidal concentration is 1.2%.
(2) 100 parts of colloidal gold solutions are taken, add borate buffer 8-12 part of 1.38%, the fumonisins monoclonal antibody 50 parts adding 2.5mg/ml in stirring continues stirring 12min, the bovine serum albumin 22 parts adding 15% carries out coupling, 10000r/min is centrifuged 4min, remove supernatant, stay clearing heat in the lung particle solution to carry out metal spraying use；
With metal spraying Film-cutting machine metal spraying on glass fibre element film, consumption 8ul/cm, then at temperature 38 DEG C dry 10h, sealing saves backup；
(3) the C/T setting-out of nitrocellulose filter:
With BC buffer, sheep anti-mouse igg is diluted to the sheep anti-mouse igg solution of 3mg/ml, PVC base plate posts nitrocellulose filter, middle with sheep anti-mouse igg picture C line, consumption 3ul/cm；
BC buffer formulation is: 0.08gNaCl+0.05gKCl+0.144gNa2HPO4+0.067gNaH2PO4, with distilled water constant volume to 100ml, adds sucrose 5g.
With BT buffer, 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone antigen diluent is become 3mg/ml, PVC base plate posts nitrocellulose filter C line and down draws T line, consumption 9ul/cm with the 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone antigen after dilution；
BT buffer formulation is: 0.08gNaCl+0.15gKCl+0.288gNa2HPO4+0.1347gNaH2PO4, with distilled water constant volume to 100ml, adds the trehalose of 5% volume.
To finish PVC base plate dry 12h at temperature 38 DEG C of line, sealing saves backup；
(4) making of the big plate of gold colloidal:
Sticking the glass fibre mold of the good gold of spray on PVC base plate in step (3), glass fibre element film wants superposition nitrocellulose filter 1-2mm in the process of patch；Patch sample pad is alignd with PVC bottom according to sample pad lower end, superposes 1-2mm with nitrocellulose filter upper end during patch absorbent paper.
Wherein, the BY buffer formulation of 100ml is: 10%tw20+ surfactant TritonX-10010%+5%PEG20000+15% trehalose+PVPK405g, with distilled water constant volume to 100ml.
nullThe foundation of 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone standard curve: (buy from national standard material net purchase according to known、The standard substance that external sigma company buys) standard substance of 1mg concentration of concentration，It is diluted to 20ppb、50ppb、100ppb、300ppb、500ppb、700ppb、900ppb、1100ppb、Nine Concentraton gradient of 1400ppb，The mensuration of 5 Duplicate Samples is carried out by also ready-made finished product 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone test strip，According to its concentration change difference，Its C value is measured with chart scanner、T value、T/C value，According to the principle that the more high colour developing of its principle concentration is more shallow，(abscissa also standard concentration is unit to set up its standard curve、Vertical coordinate also T/C value carries out the foundation of standard curve for unit)，Filter out R2 value and reach the meansigma methods of more than 0.99 point，Concrete such as Fig. 2；
The checking (being tested with the positive sample of occurrence according to mass spectrograph) of positive sample: take the standard curve that checking is also set up by the positive of high, medium and low three kinds of content respectively, see its deviation range.It is standard results that the result finally measured reaches within the scope of the 90-110% of legitimate reading.
Colourity change according to T and C line calculates numerical value by the detection of instrument and carries out the calculating of formula, y=aln (x)+b, and concrete numerical value is shown in Fig. 2.
Different samples are abscissa according to concentration, and T/C value is vertical coordinate, is calculated according to logarithmic relationship formula matching；Ratio finally according to unknown sample can calculate concrete concentration value according to curve.
Interpretation of result: Quality Control C line develops the color, it was shown that detection effectively, namely can use detector reading.Quality Control C line does not develop the color, then show that operating process is incorrect or test strip lost efficacy.
T/C values different measured by the detection line of the different colourity of detections according to quantitative chart scanner, can the actual content of detection by quantitative embodiment sample in conjunction with the standard curve made；Current 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone detection Semen Maydis requires that content is less than or equal to 500ppb, is positive more than 500ppb in the industry, and 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone content overproof is described；
Test example
6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone sample (corn and soybean, Semen Tritici aestivi, feedstuff, distiller grains) pre-treatment:
Sample extraction processes:
A, take 2g sample (sample its granularity size-reduced is less than 2mm) in extraction flask；
B, addition 4mL extractant, jolting 5 minutes, stand 3 minutes；
C, use syringe and filtering with microporous membrane, collect 1～1.5mL sample liquid standby in 1.5mL plastic tube；
AFB1 sample detection
Taking 100 μ L sample liquids and add sample buffer 400 μ L mixing, accurately draw 120 μ L sample liquids to be checked with pipettor, instill in well, panel is reacted 15 minutes, puts into hand-held chart scanner and reads result.
6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone sample detection result and HPLC contrast
(grains bureau of Sichuan Province) mass spectrometry instrument testing result correction data table of test kit and authorized by state
Detect the data after sample and gold colloidal quantitative reagent bar comparing result according to mass spectrometry, the testing result of gold colloidal is slightly more higher than the testing result of actual sample, and the accuracy of sample detection result and legitimate reading all controls between 90%-120%；The average deviation CV% of duplicate detection result controls within 15%, illustrates that the accuracy of the testing result of colloid gold reagent bar is higher, can be suitably used for product quality that the market demand reaches and meets on-the-spot a large amount of sample and screen the quantitative colloid gold reagent bar that can select.
Embodiment 3
The quantitative gold colloidal detecting device of vomitoxin, including gold colloidal detection by quantitative box and hand-held Card Reader instrument, toxin product to be detected can be provided detection by quantitative result accurately by detecting device.
Detection by quantitative result is that in the colored intensity Ratio control of the detection colored intensity of line T line and the C line measured according to hand-held chart scanner and testing sample, the content of mycotoxin quantitatively contains number relation in relevant, this culvert number is the functional relation of curtain or logarithm, is set to the internal standard curve of instrument.
The quantitative gold-immunochromatographyreagent reagent for assay box of vomitoxin, including cartridge and reagent strip, reagent strip is made up of substrate, sample pad, colloidal gold pad, reaction film and adsorptive pads, colloidal gold pad is adsorbed with vomitoxin monoclonal antibody and the bovine serum albumin of colloid gold label, reaction film is coated with detection antigen and quality control antibody, detection antigen is shown as detection line T, for vomitoxin antigen；Quality control antibody is shown as nature controlling line C, for sheep anti-mouse igg antibody.
Detection line T and nature controlling line C general control at 8mm, spacing is oversize can the growth detection time, and detect the form got stuck more and require just longer.
In T line, vomitoxin antigenic quality concentration is 3mg/ml, and vomitoxin monoclonal antibody mass concentration is 2.8ug/ml, and protein amount concentration is 15%.
Reagent strip preparation method comprises the following steps, and raw materials used consumption number is weight portion:
(1) preparation of gold colloidal:
After 500 parts of distilled water heating to boiling, add gold chloride 5 parts and trisodium citrate 5 parts, stir and boil 10min, being cooled to room temperature, detecting its peak value and half-peak breadth with visible spectrophotometer；
Raw material, before use through purification, detects its purity by instrument and reaches more than 97%, and high specificity, without non-specific binding.Gold colloidal liquid quality standard: limpid transparent Chinese red or aubergine, without particle, liquid surface without waft gold situation, with ultraviolet detection crest should more precipitous half-peak breadth more narrow, the quality of gold colloidal is more good.
Colloidal gold solution pH value is 7, and the isoelectric point, IP ph value of raw material adds 0.5.Colloid gold particle is sized to 20-40nm, and gold colloidal concentration is 1%.
(2) 100 parts of colloidal gold solutions are taken, add the borate buffer 9 parts of 1.38%, the ochracin monoclonal antibody 50 parts adding 2.5mg/ml in stirring continues stirring 11min, the bovine serum albumin 21 parts adding 15% carries out coupling, 10000r/min is centrifuged 6min, remove supernatant, stay clearing heat in the lung particle solution to carry out metal spraying use；
With metal spraying Film-cutting machine metal spraying on glass fibre element film, consumption 6ul/cm, then at temperature 35-38 DEG C dry 12h, sealing saves backup；
(3) the C/T setting-out of nitrocellulose filter:
With BC buffer, sheep anti-mouse igg is diluted to the sheep anti-mouse igg solution of 2 or 2.5mg/ml, PVC base plate posts nitrocellulose filter, middle with sheep anti-mouse igg picture C line, consumption 2ul/cm；
BC buffer formulation is: 0.08gNaCl+0.05gKCl+0.144gNa2HPO4+0.067gNaH2PO4, with distilled water constant volume to 100ml, adds sucrose 5g.
With BT buffer, vomiting mycin antigen diluent is become 2 or 2.5mg/ml, PVC base plate posts nitrocellulose filter C line and down draws T line, consumption 5ul/cm with the vomiting mycin antigen after dilution；
BT buffer formulation is: 0.08gNaCl+0.15gKCl+0.288gNa2HPO4+0.1347gNaH2PO4, with distilled water constant volume to 100ml, adds the trehalose of 5% volume.
To finish PVC base plate dry 18h at temperature 36 DEG C of line, sealing saves backup；
(4) making of the big plate of gold colloidal:
Sticking the glass fibre mold of the good gold of spray on PVC base plate in step (3), glass fibre element film wants superposition nitrocellulose filter 1-2mm in the process of patch；Patch sample pad is alignd with PVC bottom according to sample pad lower end, superposes 1-2mm with nitrocellulose filter upper end during patch absorbent paper.
Wherein, the BY buffer formulation of 100ml is: 10%tw20+ surfactant Tween-208%+5%PEG20000+15% trehalose+PVPK405g, with distilled water constant volume to 100ml.
nullThe foundation of vomiting mycin standard curve: (buy from national standard material net purchase according to known、The standard substance that external sigma company buys) standard substance of 1mg concentration of concentration，It is diluted to 200ppb、500ppb、1000ppb、1500ppb、2000ppb、2500ppb、3000ppb、4000ppb、Nine Concentraton gradient of 5000ppb，The mensuration of 5 Duplicate Samples is carried out by also ready-made finished product 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone test strip，According to its concentration change difference，Its C value is measured with chart scanner、T value、T/C value，According to the principle that the more high colour developing of its principle concentration is more shallow，(abscissa also standard concentration is unit to set up its standard curve、Vertical coordinate also T/C value carries out the foundation of standard curve for unit)，Filter out R2 value and reach the meansigma methods of more than 0.99 point，Concrete such as Fig. 1；
The checking (being tested with the positive sample of occurrence according to mass spectrograph) of positive sample: take the standard curve that checking is also set up by the positive of high, medium and low three kinds of content respectively, see its deviation range.It is standard results that the result finally measured reaches within the scope of the 90-110% of legitimate reading.
Colourity change according to T and C line calculates numerical value by the detection of instrument and carries out the calculating of formula, y=aln (x)+b, and concrete numerical value is shown in Fig. 1.
Different samples are abscissa according to concentration, and T/C value is vertical coordinate, is calculated according to logarithmic relationship formula matching；Ratio finally according to unknown sample can calculate concrete concentration value according to curve.
Interpretation of result: Quality Control C line develops the color, it was shown that detection effectively, namely can use detector reading.Quality Control C line does not develop the color, then show that operating process is incorrect or test strip lost efficacy.
T/C values different measured by the detection line of the different colourity of detections according to quantitative chart scanner, can the actual content of detection by quantitative embodiment sample in conjunction with the standard curve made；Current 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone detection Semen Maydis requires that content is less than or equal to 2000ppb, is positive more than 2000ppb in the industry, and vomiting mycin content overproof is described；
Test example
Vomiting mycin sample (corn and soybean, Semen Tritici aestivi, feedstuff, distiller grains) pre-treatment:
Sample extraction processes:
A, take 2g sample (sample its granularity size-reduced is less than 2mm) in extraction flask；
B, addition 4mL extractant, jolting 5 minutes, stand 3 minutes；
C, use syringe and filtering with microporous membrane, collect 1～1.5mL sample liquid standby in 1.5mL plastic tube；
Vomiting mycin sample detection
Taking 100 μ L sample liquids and add sample buffer 400 μ L mixing, accurately draw 120 μ L sample liquids to be checked with pipettor, instill in well, panel is reacted 15 minutes, puts into hand-held chart scanner and reads result.
Vomiting mycin sample detection result and HPLC contrast
(grains bureau of Sichuan Province) mass spectrometry instrument testing result correction data table of test kit and authorized by state
Detect the data after sample and gold colloidal quantitative reagent bar comparing result according to mass spectrometry, the testing result of gold colloidal is slightly more higher than the testing result of actual sample, and the accuracy of sample detection result and legitimate reading all controls between 90%-120%；The average deviation CV% of duplicate detection result controls within 15%, illustrates that the accuracy of the testing result of colloid gold reagent bar is higher, can be suitably used for product quality that the market demand reaches and meets on-the-spot a large amount of sample and screen the quantitative colloid gold reagent bar that can select.
Embodiment 4
Content in other content such as embodiment 1, is changed to ochratoxin by vomitoxin, and preparation process is as follows:
(1) preparation of gold colloidal:
The preparation of gold colloidal, the heating of 500ml distilled water, to boiling, rapidly joins gold chloride 5mg, at the trisodium citrate reducing agent adding 8mg, continuously stirred and boil 10 minutes, it is cooled to room temperature after stopping heating, is detected its peak value and half-peak breadth by visible spectrophotometer.
(2) labelling of gold colloidal and metal spraying:
Take 100ml colloidal gold solution, add 10mg borate buffer and (weigh 0.6183g boric acid, constant volume is to 100ml) buffer, the ochratoxin monoclonal antibody 3ml adding 2.4mg/ml in stirring continues stirring 10min, bovine serum albumin (weigh 16gBSA and the add distilled water 100ml) 20ml adding 16% carries out coupling, 10000r/min is centrifuged 5min, removes supernatant；Clearing heat in the lung particle solution is stayed to carry out metal spraying use.On glass fibre element film, carry out uniform metal spraying according to the amount of 4ul/cm by metal spraying Film-cutting machine, in 37 DEG C of thermostatic drying chambers, carry out 12h dry；Carry out sealing with valve bag and preserve.
(3) setting-out of the c/t of nitrocellulose filter:
First sheep anti-mouse igg BC buffer (0.08gNaCl+0.05gKCl+0.144gNa2HPO4+0.067gNaH2PO4 distilled water constant volume to 100ml again with sucrose 5g) is diluted to 1mg/ml or 1.5mg/ml, PVC base plate posts and in the middle of nitrocellulose filter, carries out picture C line with sheep anti-mouse igg according to 3ul/cm；(0.08gNaCl+0.15gKCl+0.288gNa2HPO4+0.134gNaH2PO4 distilled water constant volume is to 100ml with BT buffer for T line, trehalose adding 5% volume) ochratoxin antigen diluent is become 1mg/ml or 1.5mg/ml, PVC base plate posts nitrocellulose filter C line and down carries out picture T line with the mycotoxin B1 antigen after dilution according to 4ul/cm；At the dry 20h of the thermostatic drying chambers of 37 DEG C, carry out sealing preservation with valve bag.
(4) making of the big plate of gold colloidal:
PVC base plate sticks the glass fibre mold of the good gold of spray, sticks sample pad and absorbent paper.
The process of sample pad: (the BY buffer formulation of 100ml: 10%tw20+ surfactant 6%+5%PEG20000+15% trehalose+PVPK405g)
Embodiment 5
Other content such as embodiment 4, are changed to fumonisins by ochratoxin, wherein with metal spraying Film-cutting machine metal spraying on glass fibre element film, and consumption 10ul/cm, then at temperature 35 DEG C, dry 14h, sealing saves backup.
Carrying out mycotoxin class detection in the present invention, step is as follows:
(1) zooming to inside casing by the pull bar of detection case, casing faces up and lies against table top, opens two safety fasteners in front；
(2) operation instructions inside detection case, technical parameter table are read in detail；
(3) sample is uniformly smashed by taking-up disintegrating machine, serum plate, weighing balance, weighs a certain amount of sample in 15ml centrifuge tube with weighing balance；
(4) take out extracting solution bottle, add extracting solution and carry out the extraction of sample；
(5) centrifuge is taken out, 4000r/min, centrifugal 5 minutes, obtain supernatant；
(6) take out detectable bar, timer, open outer package and lie against table top, with the supernatant of pipettor measured amounts in the well of detector bar, start timing 20min；
(7) take out hand-held detector, turn on the power switch, until 20 minutes time, detector bar is put into detector and carries out quantitative readout.
In the present invention, test kit sample consumption is minimum, sample size can be low to moderate 2～5g, do not need the expensive instruments such as γ-enumerator, fluorescence microscope, enzyme mark detector, it is more suitable for on-the-spot application, such as the harmful substance such as radiosiotope, o-phenylenediamine does not participate in, technical merit for testing staff requires relatively low, it is not necessary to special technical operation personnel；The detection time is greatly shortened, and improves detection speed.In gold mark process, formed without covalent bond, be the physical absorption under certain ion concentration.Therefore gold colloidal immue quantitative detection reagent box can be used widely in mycotoxin detection industry, from all coincidence detection requirements repeatability, accuracy, sensitivity three aspect result；For a large amount of sample selective mechanisms in scene, for client provide simple to operate, instrumentation degree is low, the professional advantage such as low, quick, shorten the detection time of client greatly, improve work efficiency.
Claims (10)
1. a mycotoxin class gold colloidal quantitative testing device, it is characterized in that: include gold colloidal detection by quantitative box and hand-held Card Reader instrument, mycotoxin sample to be detected is provided detection by quantitative result accurately by described detecting device, and this mycotoxin includes Aspergillus flavus B1, M1,6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone, vomitoxin, T2 toxin, ochratoxin and fumonisins.
2. a kind of mycotoxin class gold colloidal quantitative testing device according to claim 1, it is characterized in that: described detection by quantitative result is that in the colored intensity Ratio control of the detection colored intensity of line T line and the C line measured according to hand-held chart scanner and mycotoxin sample to be measured, the content of mycotoxin quantitatively contains number relation in relevant, it is set to the internal standard curve of instrument, this hand-held Card Reader instrument measures the intensity of reflected light of C/T line by imaging system, and by random chip, testing result is transferred to by infinite network center system or cloud server；This hand-held Card Reader instrument includes smart mobile phone, mould of plastics, eyeglass, glass plate, 9V battery, LED, drawer type tray.
3. a kind of mycotoxin gold colloidal quantitative testing device according to claim 1 and 2, it is characterised in that: described mycotoxin sample is corn and soybean, feedstuff, Semen Tritici aestivi, distiller grains, bean cake；Wherein sample weighing grams is at 2-5g, mycotoxin extraction is carried out by the methanol buffer of the 60-90% of 1:5 volume, the sample treatment liquid taking 50-150ul adds in detection card, after reaction 10-20min, detection card is put into the T/C value having read variable concentrations in hand-held chart scanner and carries out Function Fitting and set up internal standard curve.
4. a kind of mycotoxin gold colloidal quantitative testing device according to claim 3, it is characterized in that: in described internal standard curve, the internal standard curve of the product of each batch adjusts accordingly, which reduce the difference of quantitative analysis relation between different batches product, it is ensured that the different batches product concordance to same determinand detection by quantitative relation.
5. a kind of mycotoxin gold colloidal quantitative testing device according to claim 2, it is characterised in that: described functional relation is: y=aln (x)-b or y=ax-b, wherein y represents T/C value；, x represents concentration；A represents slope；B represents displacement；
6. a kind of mycotoxin gold colloidal quantitative testing device according to claim 3, it is characterized in that: mark curve in setting up according to the variable concentrations of standard substance and corresponding T/C value, remove the detection natural sample containing variable concentrations with interior mark curve, calculate the actual extracting rate of each concentration nature sample；After original standard concentration is respectively divided by extraction ratio, then calculates standard concentration T/C value of variable concentrations after conversion and set up final curves, upload to software and make Quick Response Code and facilitate guest operation to use.
7. a kind of mycotoxin class gold colloidal quantitative testing device according to any one of claim 1-3, it is characterized in that: described mycotoxin class gold colloidal detection by quantitative box, including cartridge and reagent strip, reagent strip is made up of substrate, sample pad, colloidal gold pad, reaction film and adsorptive pads, colloidal gold pad is adsorbed with mycotoxin class monoclonal antibody and the bovine serum albumin of colloid gold label, reaction film is coated with detection antigen and quality control antibody, detection antigen is shown as detection line T, for mycotoxin class antigen；Quality control antibody is shown as nature controlling line C, for sheep anti-mouse igg antibody.
8. the preparation method of a kind of mycotoxin class gold colloidal quantitative testing device according to claim 1 or 7, it is characterised in that: the reagent strip preparation method in described detection box comprises the following steps, and raw materials used consumption number is weight portion:
(1) preparation of gold colloidal:
After 500 parts of distilled water heating to boiling, add gold chloride 1-10 part and trisodium citrate 1-10 part, stir and boil 10min, being cooled to room temperature；
(2) labelling of gold colloidal and metal spraying:
Take 100 parts of colloidal gold solutions, add borate buffer 8-12 part of 1.38%, the mycotoxin class monoclonal antibody 45-53 part adding 2.2-2.8mg/ml in stirring continues stirring 8-12min, the bovine serum albumin 18-22 part adding 15-18% carries out coupling, 10000r/min is centrifuged 4-7min, remove supernatant, stay clearing heat in the lung particle solution to carry out metal spraying use；
With metal spraying Film-cutting machine metal spraying, consumption 3-10ul/cm on glass fibre element film, concentration is 0.5ug-10ug/cm, then dries 10-14h at temperature 35-38 DEG C, and sealing saves backup；
(3) the C/T setting-out of nitrocellulose filter:
With BC buffer, sheep anti-mouse igg is diluted to the sheep anti-mouse igg solution of 0.5-3mg/ml, PVC base plate posts nitrocellulose filter centre sheep anti-mouse igg and draws C line according to 0.5-3ul/cm；
With BT buffer, mycotoxin class antigen diluent is become 0.5-3mg/ml, PVC base plate posts nitrocellulose filter C line and down draws T line with the mycotoxin antigen after dilution according to 0.5-9ul/cm；
To finish PVC base plate dry 12-24h at temperature 35-38 DEG C of line, sealing saves backup；
(4) making of the big plate of gold colloidal:
PVC base plate in step (3) sticks the glass fibre mold of the good gold of spray, sticks sample pad and absorbent paper.
9. the preparation method of a kind of mycotoxin class gold colloidal detection by quantitative box according to claim 8, it is characterised in that: described colloid gold particle is sized to 20-80nm, and gold colloidal mass concentration is 0.8-1.2%.
10. the preparation method of a kind of mycotoxin class gold colloidal quantitative testing device according to claim 8, it is characterised in that: described sample pad processes before using:
Wherein, the BY buffer formulation of 100ml is: 10%tw20+ surfactant 5-10%+5%PEG20000+15% trehalose+PVPK405g, with distilled water constant volume to 100ml.
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