CN101482566A - Production and use of test paper for fast detecting deoxynivalenol in cereal - Google Patents
Production and use of test paper for fast detecting deoxynivalenol in cereal Download PDFInfo
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Abstract
The invention provides a preparation method of test paper scrip capable of quickly detecting deoxynivalenol test paper in grain and use thereof, comprising (1) DON monoclonal antibody-colloidal gold label; (2) coating of conjugate releasing mat; (3) coating of cellulose nitrate film; (4) production of test paper scrip. The preparation method has features of specificity, high sensitivity; visual detection result; simple operation and high speed, no need of professional operation, obtaining the detection result in 10min; easy large-scale popularization and application, widely application on foodstuff purchasing scene detection, food safety detection, customs quarantine control or the like, with wide market prospect, bigger economic, social benefits.
Description
Technical field
The present invention relates to the fast detection method of mycotoxin, be specifically related to the preparation and the application thereof of the quick detection test paper bar of deoxynivalenol in the cereal grain (DON).
Background technology
(deoxynivalenol DON), claims vomitoxin (vomi toxin) again to deoxynivalenol, mainly is a kind of by in some chemical constitution that plant to produce of sickle-like bacteria toxic metabolite product-trichothecene similar with biologically active.DON has stronger toxicity, mainly pollutes cereal crops such as wheat, barley, corn.Humans and animals can produce poisonous effect widely after eating by the food of this endotoxin contamination or feed, often cause vomiting, have a headache, generate heat and feel sick.In recent years, discover that DON may be relevant with human esophagus cancer, IgA nephropathy, the health of the mankind and animal is constituted a threat to.At present, various countries have formulated strict limit standard at the content of DON in historical relics and the feed, and the limit standard of DON is 1.0mg/kg in China's cereal.
The detection method of at present relevant DON mainly contains: thin layer chromatography, vapor-phase chromatography (adopting electron capture detector or Mass Spectrometer Method), liquid phase chromatography (adopting ultraviolet, fluorescence, mass spectrum or mass spectrum-Mass Spectrometer Method) etc.The generally only suitable laboratory of said method is detected, and is consuming time, and the expense costliness needs complicated instrument and complicated sample pre-treatment, and needs the professional to operate, and is difficult to popularize and promote in basic unit, more is not suitable for on-the-spot and open-air large-scale fast detecting.
Enzyme linked immunosorbent assay (ELISA) is though at present domestic have relevant kit to sell, can detect a plurality of samples, but can qualitative also detection by quantitative, but ELISA also needs the professional to finish, its operating process more complicated, detection time is long, and also necessary supporting microplate reader reading, also can't implement on-the-spot the detection.
Based on the colloid gold immune fast detection method of antigen on the NC film and antibody response have simple to operate, be fit to on-the-spotly detect, fast, do not need complex instrument and only need eyes observation, stability better and detect characteristics such as cost is low.This method successfully has been used for the fast detecting in fields such as medical science and food security, and for example very early pregnancy detects test paper, hepatitis B surface antigen detects test paper, clenbuterol hydrochloride detection test paper etc.Now having utilizes immune colloid gold to make the preparation and the using method of the report of DON test strip: publication CN101158685A vomitoxin semiquantitative test paper bar.But this patent of invention adopts the antibody of specific recognition vomitoxin to prepare a kind of vomitoxin sxemiquantitative speed test paper slip.The detection antigen of this patent is DON-BSA, and BSA wherein is not cationization BSA (MBSA), and preparation process needs to seal processing to the NC film, and the production technology more complicated has increased production cost.People such as Deng Shun continent, this laboratory have delivered and have adopted MBSA coupling DON as detecting antigen and being applied to research paper (Food Science, 2007, Vol.28, No.02, the P149-152 that ELISA detects; Food science and technology, 2006, No.8 P222-224), but does not see that as yet the detection antigen of MBSA coupling is used for the report of the fast detecting trial-production bar of DON.
Summary of the invention
The present invention's purpose provides the method for the preparation and the application thereof of deoxynivalenol test strips in the fast detecting cereal, to realize the fast detecting of DON.
The test strips of deoxynivalenol (DON) comprises that nitrocellulose filter, sample pad, bond discharge pad, adsorptive pads and base plate in the fast detecting grain provided by the invention.Wherein have on the nitrocellulose filter bag by the test section of DON-MBSA conjugate and bag by the Quality Control district of sheep anti-mouse igg; Described bond discharges the pad bag by DON monoclonal antibody-colloid gold label thing.
1, preparation method of the present invention comprises the steps:
(1) DON monoclonal antibody-colloid gold label thing
A. add K to colloidal gold solution
2CO
3Solution, the pH of regulator solution are 5.5-7.5.
B. add 2-10 μ g DON antibody, stirring reaction 15-60min fast to every mL collaurum.
C. add BSA, final concentration is 0.5-1.0%, reaction 10-30min.
D. add PEG-20000, final concentration is 0.1%, reaction 10-30min.
E.6000-10000rpm centrifugal 25-45min.
F. supernatant discarded precipitates and adopts the PBS solution that contains 2%-10% sucrose, 0.1%-1%BSA, 0.1%-0.5%PEG-20000,0.5%-3% trehalose to redissolve.
(2) bond discharges the bag quilt of pad
Bond is discharged pad, and to put into the concentration that contains 0.05%-0.5%BSA, 2%-10% sucrose, 0.01%-0.5%PEG-20000 be that the PBS solution of 0.01-0.1M soaks 5-30min, 37 ℃ of vacuum drying.Above-mentioned DON monoclonal antibody-colloid gold label thing is sprayed onto bond uniformly discharges on the pad every 1cm
2Pad spray 3-15 μ L DON monoclonal antibody-colloid gold label thing, 37 ℃ of vacuum drying.
(3) the bag quilt of nitrocellulose filter
The detection antigen of nitrocellulose filter bag quilt is DON, N, and N-carbonyl dimidazoles (CDI) and MBSA conjugate, this detection antigen do not find to be used for the quick detection test paper bar of DON at present as yet.With concentration is that the detection antigen of 0.1-1.5mg/mL MBSA coupling DON is sprayed on the nitrocellulose filter, as detection line (T line), discharge rate is 0.4-1.0 μ L/cm, is that 5mm place spray concentration is the 1-2mg/mL sheep anti-mouse igg in spacing, as nature controlling line (C line), discharge rate is 0.4-1.0 μ L/cm.37 ℃ of vacuum drying, sealing.
(4) making of test strips
The nitrocellulose filter (NC film) that the above-mentioned DON of being sprayed with is detected antigen (T line) and sheep anti-mouse igg (C line) sticks on the PVP base plate; End at base plate is a sample pad, and the other end is an absorption layer, and NC film two ends discharge pad with sample absorption layer and bond respectively and overlap mutually and is connected (coupling part can in the 1-2mm scope), and bond is pressed with sample pad on discharging and filling up.With the width is the test strips that is fast detecting DON behind the 3-4mm/ bar slitting.
2, the application of the test strips of the prepared fast detecting DON of the present invention.
(1) preparation of sample
Detect the DON in the cereal (wheat, corn and paddy): the sample that takes by weighing pulverizing adds 5-20 distilled water doubly as extract in conical flask, fully shake up 3-10min, filters with qualitative filter paper, collects filtrate.Get filtrate, the PBS solution that adds the 0.05-0.1M that is equivalent to the 1/10-1/5 filtrate volume contains anionic surfactant and the 0.5%-5% Tween-20 of 0.01-0.5% as sample solution in this sample solution;
(2) detection of sample
Get above-mentioned sample solution and drip on test strips, promptly can be observed testing result behind the 10min.
Anionic surfactant of the present invention can be the anionic surfactant of sodium n-alkylbenzenesulfonate, lauryl sodium sulfate, alpha-alkene sulfonate, alkyl sulfonate, alpha-sulfo monocarboxylic acid and derivant thereof, fatty acid sulfoalkyl ester and fatty acid sulfoalkyl acid amides, oily acyloxy ethyl sulfonic acid sodium, dioctyl sodium sulfosuccinate, sodium glycocholate, sulfuric acid, phosphate ester salt etc. and similar structures thereof.
Percentage concentration of the present invention (%) all is meant g/mL except that particularly pointing out.
Deoxynivalenol (DON) test strips has following characteristics in the fast detecting grain of the present invention:
(1) specificity, highly sensitive.DON monoclonal antibody with high-affinity of the present invention is come the mark gold grain at pH5.5-7.5, has higher mark rate.The present invention adopts MBSA coupling DON as detecting antigen, can improve the coupling ratio of BSA and DON.The present invention compares with existing report, adds anion surface active in damping fluid, need not the NC film is sealed processing, has simplified production technology.The DON quick detection test paper bar product of the present invention's preparation can detect 1 μ g/mL.And with no cross reactions such as aflatoxin, ZEN, OTA.
(2) testing result image, directly perceived.Test strips shows that the content that does not contain DON or DON in two red line (T line and C line) expression sample is lower than 1 μ g/mL; The content that shows a red line (C line) expression DON is higher than 1 μ g/mL positive; Article two, line does not all develop the color and represents that test strips is invalid.
(3) easy and simple to handle, quick.Do not need the professional to operate, can go out testing result in the 10min.
(4) be easy to apply on a large scale.The quick detection test paper bar is simple to operate, do not need other instrument and equipments, only need by specification can finish detection, be easy to popularize, be widely used in demands such as the on-the-spot detection of grain purchases, food safety detection, customs quarantine control, have vast market prospect and bigger economical, societal benefits.
Description of drawings
Fig. 1 analyzes synoptic diagram for test strip testing result of the present invention.(a) represent that negative, (b) represents positive, (c) and (d) expression test strips inefficacy.
Fig. 2 is result's (two red line is negative, and a red line is positive) of the detection wheat samples of test strip of the present invention.
Fig. 3 is the test result of the detection sensitivity of test strip of the present invention.
Embodiment
Embodiment 1.The preparation of DON fast detecting trial-production bar
1, the preparation of DON monoclonal antibody-colloid gold label thing
(1) preparation of collaurum
With ultrapure water 1% gold chloride is diluted to 0.01%, puts oil bath band magnetic agitation heated and boiled, the gold chloride of every 500mL0.01% adds the trisodium citrate of 10mL1%, continue to boil, taking on a red color to liquid is to stop heating, supplies moisture evaporated after being cooled to room temperature, obtains colloidal gold solution.
(2) preparation of DON monoclonal antibody-colloid gold label thing
Under magnetic agitation, add K to colloidal gold solution
2CO
3The pH of regulator solution is 7.5.20 μ g/mL antibody colloidal golds are added antibody DON monoclonal antibody, continue stirring reaction 15min.Add 10%BSA to final concentration 1.0%, continue to stir 15min.Add 5%PEG-20000 to final concentration 0.1%, continue to stir 15min.6000rmp, 4 ℃ of centrifugal 30min.Supernatant discarded, precipitation adopt and contain 3% sucrose solution, 0.1%BSA, 0.3%PEG-20000 and the redissolution of 0.5% trehalose PBS redissolution liquid, obtain DON monoclonal antibody-colloid gold label thing.
2, bond discharges the bag quilt of pad
Bond release pad is put into the PBS solution that contains 0.1%BSA and 3% sucrose soak 10min, 37 ℃ of oven dry.With Biodot point film instrument DON monoclonal antibody-colloid gold label thing is injected to bond uniformly and discharges on the pad every 1cm
2Pad sprays 5 μ L DON monoclonal antibody-colloid gold label things, vacuum drying, put 4 ℃ standby.
3, the bag quilt of nitrocellulose filter
Adopting Biodot point film instrument is that the detection antigen of 1.2mg/mL MBSA coupling DON is sprayed on the nitrocellulose filter with concentration, as detection line (T line), discharge rate is 0.74 μ L/cm, in spacing is that 5mm place spray concentration is the 1.5mg/mL sheep anti-mouse igg, as nature controlling line (C line), discharge rate is 0.74 μ L/cm.Vacuum drying, sealing.
4, the assembling of colloidal gold strip
The assembling of test strips comprises: base plate one end termination is an adsorptive pads, and other end termination is a sample pad, and the nitrocellulose filter two ends discharge mutual overlapping of pad with adsorptive pads and bond respectively and are connected, and discharges on the pad at bond to be pressed with sample pad.
Embodiment 2.The detection of DON in the sample
The detection of DON in paddy, wheat and the corn
(1) sample pre-treatments
The sample to be checked (as wheat, corn and paddy) that takes by weighing the 5g pulverizing adds 50mL distilled water and extracts in triangular flask, and concussion 5min gets qualitative filter paper and filters fast, after getting 160 μ L filtrates and 40 μ L damping fluids mixing, detects.
(2) interpretation of result
Get 50 μ L and slowly be added drop-wise on the sample pad of test strips, observe the colour developing situation of test strips behind the 10min, judge the content of DON in the sample according to the detection principle of test strips.If detection line (T line) and nature controlling line (C line) all show red expression sample negative (less than 1 μ g/mL); If have only the T line to show red expression sample positive (greater than 1 μ g/mL); Represent that test strips lost efficacy if having only colour developing of T line or T line and C line all not to develop the color.
Embodiment 3.(Application Example)
1, specificity experiment
Experimentize by embodiment 2 described methods, aspergillus flavus B1, zearalenone, ochratoxin, the dilution of T-2 toxin isoconcentration are 50ng/mL, detect with this patent test strips, two red lines appear, the result is negative, is no intercrossing reaction such as DON test strip and aspergillus flavus B1, zearalenone, ochratoxin, T-2 toxin.
2, sensitivity experiment
Adopt to implement 2 described methods, detect 0,6.25,12.5,25,50,100,200 and the DON standard items of 400ng/mL respectively, repeats 10 times with test strips, wherein 0,6.25,12.5 and two red lines of 25ng/mL appearance, promptly negative; 50,100,200 and 400ng/mL a red line only appears, promptly positive.Therefore the sensitivity of this test strips is 50ng/mL.
3, homogeneity experiment
Adopt the 2 described methods of enforcement, to 0ng/mL, 50ng/mL DON carries out 10 parallel detections respectively, observations behind the reaction 10min, and two red lines appear in 0ng/mL, and a red line then only appears in 100ng/mL, detects colour developing degree of depth homogeneous, the reaction result unanimity.
4, stability experiment
The test strips that vacuum packet is installed is placed in 37 ℃ of baking ovens and is carried out destructive test, time is 10 days, and every index all meets above 1-3 item index, promptly detects other toxin and does not have intercrossing reaction, sensitivity reaches 50ng/mL, detect colour developing degree of depth homogeneous, reflection is unanimity as a result.
Claims (2)
1, deoxynivalenol test strips preparation method in a kind of fast detecting cereal is characterized in that may further comprise the steps:
(1) DON monoclonal antibody-colloid gold label thing
A. add K to colloidal gold solution
2CO
3Solution, the pH of regulator solution are 5.5-7.5;
B. add 2-10 μ g DON antibody, stirring reaction 15-60min fast to every mL collaurum;
C. add BSA, final concentration is 0.5-1.0%, reaction 10-30min;
D. add PEG-20000, final concentration is 0.1%, reaction 10-30min;
E.6000-10000rpm centrifugal 25-45min;
F. supernatant discarded precipitates and adopts the PBS solution that contains 2%-10% sucrose, 0.1%-1%BSA, 0.1%-0.5%PEG-20000,0.5%-3% trehalose to redissolve;
(2) bond discharges the bag quilt of pad
Bond is discharged pad, and to put into the concentration that contains 0.05%-0.5%BSA, 2%-10% sucrose, 0.01%-0.5%PEG-20000 be that the PBS solution of 0.01-0.1M soaks 5-30min, 37 ℃ of vacuum drying; Above-mentioned DON monoclonal antibody-colloid gold label thing is injected to bond uniformly discharges on the pad every 1cm
2Pad spray 3-15 μ L DON monoclonal antibody-colloid gold label thing, 37 ℃ of vacuum drying;
(3) the bag quilt of nitrocellulose filter
With concentration is that the detection antigen of 0.1-1.5mg/mL MBSA coupling DON is sprayed on the nitrocellulose filter, as detection line, discharge rate is 0.4-1.0 μ L/cm, in spacing is that 5mm place spray concentration is 1-2mg/mL sheep anti mouse Ig G, as nature controlling line, discharge rate is 0.4-1.0 μ L/cm, 37 ℃ of vacuum drying, sealing;
(4) making of test strips
The nitrocellulose filter that the above-mentioned DON of being sprayed with is detected antigen and sheep anti-mouse igg sticks on the PVP base plate; End at base plate is a sample pad, and the other end is an adsorptive pads, and NC film two ends discharge mutual overlapping of pad with sample absorption layer and bond respectively and are connected, and bond discharges on the pad and is pressed with sample pad; With the width is the test strips that is fast detecting DON behind the 3-4mm/ bar slitting.
2, the application of the described test strips of claim 1 is characterized in that:
(1) sample that takes by weighing pulverizing adds 5-20 distilled water doubly as extract in conical flask, fully shakes up 3-10min, filters with qualitative filter paper, collects filtrate, and is to be detected;
(2) get above-mentioned filtrate, the PBS solution that adds the 0.05-0.1M that is equivalent to the 1/10-1/5 filtrate volume contains anionic surfactant and the 0.5%-5% Tween-20 of 0.01-0.5% as sample solution in this sample solution;
(3) get above-mentioned sample solution and drip on test strips to be detected, promptly can be observed testing result behind the 10min.
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| CN102071169A (en) * | 2010-09-09 | 2011-05-25 | 中国疾病预防控制中心营养与食品安全所 | Hybridoma of DON (Deoxynivalenol) monoclonal antibody as well as preparation method and application thereof |
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| CN101851677A (en) * | 2010-04-30 | 2010-10-06 | 中国科学院广州生物医药与健康研究院 | Nucleic acid nano-gold biosensor used for detecting Hg2<+> |
| CN102071169A (en) * | 2010-09-09 | 2011-05-25 | 中国疾病预防控制中心营养与食品安全所 | Hybridoma of DON (Deoxynivalenol) monoclonal antibody as well as preparation method and application thereof |
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