CN102768275A - Staphylococcal enterotoxin assay kit, method for preparing same and application - Google Patents
Staphylococcal enterotoxin assay kit, method for preparing same and application Download PDFInfo
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Abstract
The invention discloses a staphylococcal enterotoxin assay kit, a method for preparing the same and an application, which belong to the field of immunology. Three types of staphylococcal enterotoxin A, B and C in the food can be rapidly tested at the same time. The assay kit mainly comprises a staphylococcal enterotoxin test card, wherein the staphylococcal enterotoxin test card consists of a criterion connection card and a test strip; and the test strip sequentially comprises a sample pad, a criterion antibody fixation pad, a test line and a control line which are positioned on a nitrocellulose membrane, and an absorbent pad. The rapid assay kit is simple to operate and convenient to carry, has an efficient test method, is accurate, quickly determines the result, and only takes 15 minutes for the whole test process, thus being applied in the on-site supervision of food poisoning samples, clinical specimens, dairy products and other samples and the determination screening of a large number of samples, and playing an important role in food safety testing.
Description
Technical field:
The invention belongs to field of immunology, relate to staphylococcus aureus toxin A in the fast detecting food, B, residual kit and the method for C three types.
Background technology:
The food poisoning that staphylococcus aureus causes mainly is because the enterotoxin that such bacterium is secreted into outside the born of the same parents in the growth and breeding process causes that the power of pathogenicity depends on also how much toxigenic it is.It is the food poisoning of cardinal symptom that Staphylococcus aureus enterotoxin can cause with vomiting, diarrhoea; According to statistics; The food poisoning that is caused by golden staphylococcal enterotoxin in the U.S. accounts for 33% of whole bacterium food poisoning, and Canada accounts for 45%, and etesian this type of poisoning of China is also very many.Therefore, the food poisoning that is caused by Staphylococcus aureus enterotoxin has become a worldwide hygienic issues.On the other hand, enterotoxin also plays an important role in the pyogenic infection that is caused by staphylococcus aureus, can cause multiple organ dysfunction when serious, jeopardizes the human life.This shows that pathogenic microorganisms in the food and toxin thereof have brought serious harm for human health and life security.
Known staphylococcal enterotoxin has 7 serotypes, and called after SEA, SEB, SEC1-3, SED and SEE are wherein common with SEA, account for 75%.For processed food, thermal treatment can destroy staphylococcus, but the toxin thermotolerance is strong, and 100 ℃ of lasting 30min still have activity, under general cook temperature, exists if any enterotoxin in the food, still can cause food poisoning; Mechanical damage in the process also can impact the microorganism in the food, but not influence of contratoxin.Therefore, set up the method for quick of Staphylococcus aureus enterotoxin in the food, development directly detects the quick product of enterotoxin in the food, all has great realistic meaning to ensuring food safety, ensure people's life health.
The enterotoxin detection method of stipulating among China standard GB/T4789.10-2003 is loaded down with trivial details, detects consuming timely, and application limitation is big in real work.At present, the biological Mei Liai VIDAS instrument of the many employings of detection of China's gold Portugal enterotoxin, testing cost is expensive, make detection regulator all face huge detected pressures, and enterprise can't bear especially.Cause the basic reason of this present situation to be the commercialized supply of domestic shortage enterotoxin detectable product.Therefore, the research task of this project is urgent.
The immune chromatograph testing technology is a new technology that grows up the early 1990s; Because of its easy (without any need for equipment), quick, directly perceived, sensitivity, special; Be used widely at numerous areas such as clinical diagnosises, be used for the antigenic component of fast detecting serum or urine sample; Also can be used for pathogenic bacteria and other objectionable constituent etc. in the test sample.Highly sensitive and the high specificity of this method, reaction fast, and is easy and simple to handle, do not need any special instruments and equipment, is applicable to that rapid screening detects with on-the-spot.This project is through development staphylococcus aureus toxin A, B, C type colloidal gold immunochromatographiassay assay reagent box; Can really realize rapid screening and on-the-spot the detection to staphylococcus aureus toxin A, B, C type; Can satisfy the detection demand of inspection and quarantine one line, for improving China in the monitoring level in this field and ensure food safety strong technical support is provided.
Summary of the invention:
The technical matters that the present invention solves provides Staphylococcus aureus enterotoxin detection kit in the food; To be to overcome traditional detection method loaded down with trivial details for the invention fundamental purpose, detect consuming time, defective such as application limitation is big in real work, and a kind of quicker, simple and direct, safe detection kit and detection method thereof are provided.
Staphylococcus aureus enterotoxin detection kit in the food comprises plastics cartridge and test strips, and said test strips is overlapped in order to paste on end liner and formed by sample pad, golden labeling antibody pad, nitrocellulose filter, adsorptive pads.Encapsulate staphylococcus aureus A, B or the C three type polyclonal antibodies of colloid gold particle mark on the gold labeling antibody pad, have on the nitrocellulose filter to encapsulate to form effectively sandwich monoclonal antibody detection line and the control line that encapsulates the goat-anti rabbit polyclonal antibody with golden labeling antibody.
Said test strips outer setting is the plastics cartridge, and the plastics cartridge is three yoke plates, from left to right be followed successively by SEA, SEB, SEC and in the yoke plate upper end correspondence position do sign, set gradually shrinkage pool and observation port on each plate;
The sample pad of said test strips is positioned over the shrinkage pool corresponding position, and control line that is provided with on the nitrocellulose filter and detection line are in the observation port corresponding position;
End liner is PVC;
Encapsulating the colloid gold particle particle diameter on the pad is 25nm; 1mL colloid gold particle mark staphylococcus aureus toxin A, B, C three type polyclonal antibodies; Labelled amount is respectively 10 μ g/mL, 8 μ g/mL, 15 μ g/mL; Collaurum-the antibody complex that forms encapsulates on the binding site pad after concentrated through corresponding, and package amount is 2mL/30cm.
Detection line encapsulates A, B, the corresponding polyclonal antibody of C three types on the said nitrocellulose filter, and concentration is respectively 1mg/mL, 0.95mg/mL, 1.1mg/mL, and package amount is 1 μ L/cm;
Encapsulating goat anti-rabbit antibody concentration on the control line is 1mg/mL, and package amount is 1 μ L/cm.A kind of method for preparing the staphylococcus aureus quick detection kit is:
1, the preparation of golden labeling antibody pad
A. the preparation of blank colloidal gold solution:
Adopt the citrate reducing process to prepare colloid gold particle; Adding 97.5mL distilled water and 1mL1% gold chloride are heated to boiling in Erlenmeyer flask; Add concentration and be 1% citric acid three sodium solution 1.5mL, continue to boil to liquid and present salmon pink, keep behind the fluidized state 10min flask being placed room temperature; After fully cooling off, stored refrigerated;
B. the preparation of colloid gold label antibody:
Get blank colloidal gold solution 100mL; Add 0.1M sal tartari 300～400uL, under magnetic agitation, dropwise add the good antibody of dilution, be respectively 10 μ g/mL, 8 μ g/mL, 15 μ g/mL adding A, B, C three type polyclonal antibodies by labelled amount; Continue to stir 30min; Centrifugal, absorb supernatant, with the resuspended liquid of golden labeling antibody that the precipitation of gold labeling antibody is fully resuspended;
The preparation of c. golden labeling antibody pad:
Resuspended good golden labeling antibody is encapsulated on the plain film of the spun glass that is of a size of 20cm * 30cm, and package amount is 36mL, 37 ℃ of dried overnight, and the dry place of sealing lucifuge preserves, and cuts into the size of 1cm * 30cm during use.
2, the encapsulating of T, C line on the nitrocellulose filter
The good two-dimentional specking platform of debugging; Be coated on the nitrocellulose filter as detection line through the mode of line diluting SEA, SEB, the SEC polyclonal antibody of getting well; That draws system simultaneously also has the good goat-anti rabbit polyclonal antibody of dilution as control line; 37 ℃ of dry 2h, the dry place of sealing lucifuge preserves;
3, the preparation of sample pad
A. the preparation of sample pad damping fluid:
In concentration is to add surfactant and protein in the borate buffer system of 0.1M, and surfactant is 1% PVP-10 and 8% Tetronic1307, and protein is 0.5% casein, through fully dissolve mix after, 4 ℃ of placements are subsequent use;
B. the preparation of sample pad:
The sample pad treating fluid that in a clean pallet, adds 250mL took out 50 undercuts and cuts the spun glass that is of a size of 2cm * 30cm and soak in pallet, fully soaked excessive solution in back taking-up and the removal sample pad through 5 minutes.Sample pad is lain on layer frame item by item, put into 37 ℃ baking oven baking to the layer frame that is placed with sample pad to more than 16 hours at last.
Sample pad after the oven dry should have been put into the drying agent valve bag and not make moist with assurance, should be labelled on the valve bag, should indicate specification, date and the quantity of having handled sample pad well on the label, and put into lucifuge dryer place at last and preserve.
Detection and golden labeling antibody that the sample pad of handling through this method can be used for multiple different samples are not prone to the phenomenon of separating with water, can improve the detection sensitivity of kit.
4, the selection of nitrocellulose filter (NC) model
The NC film of choosing model and be HF09002 is used for the preparation of immuno-chromatographic test paper strip.Simultaneously, select that 4000g is centrifugal, 40 ℃ of preheatings, 2 times of these three kinds of sample treatments of dilution carry out subsequent experimental, to confirm the detection sensitivity under the different sample treatments.
Model is the NC film of HF09002, reaches 4000g at centrifugal force and begins to have the chromatography reaction when above; Adopt the method for thermal pretreatment, only model is the NC film of HF09002, when preheat temperature reaches more than 40 ℃, begins to have the chromatography reaction; Adopting the directly method of dilution, is the NC film of HF09002 for model, and only diluting 2 times just has the chromatography reaction.
5, the assembling of test strips
The sample pad of final cut lengths, golden labeling antibody pad, nitrocellulose filter and adsorptive pads overlapped according to the order of sequence paste on the PVC base plate.The base plate that posts is cut into wide test strips for 4mm also be positioned in order in the draw-in groove of townhouse test card on cutting cutter, at last test card is positioned in the aluminium foil bag that has drying agent.It is 24 ℃ ± 5 ℃ that the whole assembling process of test strips must keep indoor temperature, and humidity is 30 ± 5%, must the operation of omnidistance band gloves for avoiding polluting assembler.
6, the assembling of kit
In the packing box of blank, put into the aluminium foil bag that includes test card, sample diluting liquid and the operation instruction postscript packing box that closes is stained with labeling outside the kit at the packing box outside surface.Kit is placed in room temperature, drying, lucifuge place, and the maintenance phase is 1 year.
Said test strips outer setting is the plastics cartridge; The plastics cartridge is three yoke plates; From left to right be followed successively by SEA, SEB, SEC and in the yoke plate upper end correspondence position do sign; Set gradually shrinkage pool and observation port on each plate, the sample pad of said test strips is positioned over the shrinkage pool corresponding position, and control line that is provided with on the nitrocellulose filter and detection line are in the observation port corresponding position;
Using kit of the present invention, to detect in the food Staphylococcus aureus enterotoxin residual, may further comprise the steps:
1 food samples can increase bacterium with reference to increasing in the standards such as standard GB/T4789.10-2008, inspection and quarantine industry standard SN0172-92, FDA BAM, ISO and cultivate before the bacterium method is carried out; Get and increase the centrifugal 10min of bacterial context soup 8000-10000rpm before a certain amount of, it is subsequent use to get supernatant;
2 food poisoning samples, clinical samples etc. are preferably collected fresh sample, and can add an amount of SPSS mixes, the centrifugal 10min of 8000-10000rpm, and it is subsequent use to get supernatant;
3 milk samples detect:
3.1 milk to be checked is placed 4 ℃ of precoolings;
3.2 degreasing: with 4 ℃ in milk, the centrifugal 10min of 2000g;
3.3 remove the fat deposit part;
3.4 the milk 200uL after the absorption degreasing is used for detecting.
4 results judge
Every part of yoke plate test card has three sense channels, detects A type enterotoxin (SEA), Type B enterotoxin (SEB), C type enterotoxin (SEC) from left to right respectively, and the judgment mode as a result of each sense channel is all identical, is described below:
Positive (+): two red stripes occur, one is detection line (T), and another is nature controlling line (C).
Positive findings shows: the enterotoxin that contains the corresponding model of this sense channel in the sample.
Negative (-): only a red stripes appears in nature controlling line (C), occurs at detection line (T) redfree band.
Negative findings shows: the enterotoxin that does not detect the corresponding model of this sense channel in the sample.
Invalid: red stripes does not appear in nature controlling line (C), shows that incorrect operating process or test card lost efficacy.
According to the test strips colour developing, directly read testing result.
1) quality control standard
A nature controlling line appears in each test sample book at least, has or do not have detection line.
2) result describes and judges
Only a red line occurs at nature controlling line C, Staphylococcus aureus enterotoxin residual is lower than the kit LDL in the expression sample;
When nature controlling line C and detection line T occurred simultaneously, residual being of Staphylococcus aureus enterotoxin was higher than the kit LDL in the expression sample;
No C line occurs on test card, and no matter the T line has or not, and all is judged to be null result.
Resuspended formula of liquid of 5 gold medal labeling antibodies such as following table:
The know-why of kit of the present invention is following:
According to big molecular antigen antibody response principle, carry out the mark of antibody colloidal gold, on nitrocellulose filter, to draw system simultaneously and can form effectively sandwich polyclonal antibody as detection line with golden labeling antibody, anti-mouse antibody is as control line.Gold mark compound combines on object in the chromatography process in the positive sample and the gold-marking binding pad; When chromatography arrives detection line; Form " sandwich " with polyclonal antibody on the detection line thus the seized survey line of sandwich structure compound is caught colour developing; Unnecessary gold mark compound continues chromatography, combine with the sheep anti-mouse antibody that encapsulates when reaching control line, thereby the Be Controlled line is caught and developed the color.Negative sample is then effectively sandwich because there is not the existence of object not form, and does not develop the color thereby therefore can not seized survey line in the chromatography process catch.
Beneficial effect:
This kit is that modal three kinds of toxin in the golden yellow grape ball enterotoxin are realized the plain residual fast detecting of sitotoxismus is made toxin detect and need not through coagulating after pcr amplification and the amplification through immunochromatography technique
The gel electrophoresis experiment can realize the fast detecting to Staphylococcus aureus enterotoxin in 10-20min, compare with classic method, and this detection kit has following characteristics:
1. simple to operate: as only to need pre-treatment operation just can carry out the detection of three kinds of enterotoxins simultaneously;
2. weak point consuming time: in 10-20min, can realize detection time has been shortened in the detection of SEA, SEB, three kinds of enterotoxins of SEC;
3. cost is low: detecting does not need complicated instrument, does not need expensive special reagent yet, and it is low to detect cost;
4. the experimenter requires low: owing in whole testing process, only need a desk centrifuge, operating process is simple, and the testing result naked eyes just can carry out interpretation, so the experimenter is required low, only the simple training of need can be competent at.
5. safe: no any poisonous and harmful substance in the kit all is safe and harmless to operator and experimental enviroment.
The Staphylococcus aureus enterotoxin detection kit that the present invention studied detects when having realized golden Portugal enterotoxin A, B, C three types, has shortened detection time, has reduced the detection cost; Also realized operating personnel's nonhazardous, environmentally safe simultaneously.Therefore; It meets the demand for development of country to detection technique in food security and the inspection and quarantining for import/export; Help improving the efficient and the accuracy rate of food security and inspection and quarantining for import/export work, help guaranteeing food security and foreign trade, thereby play a significant role at public safety field.After the present invention researchs and develops basic completion, just reach the sale association agreement with many enterprises.
Description of drawings
Fig. 1: the kit result judges
Fig. 2: test strips structural representation:
Fig. 3: kit side analysis synoptic diagram
Wherein: 1-well, 2-detection line, 3-control line, 4-plastics cartridge, 5-adsorptive pads, 6-nitrocellulose filter, 7-pad, 8-sample pad, 9-PVC base plate.
Embodiment
Below the present invention is done further practical implementation explanation:
The sensitivity experiment of embodiment 1 kit
100 parts of positive former milk sample detection sensitivity experimental results after three kinds of different sample treatments are handled of adding are seen table 1.The result shows, when adopting the centrifugal disposal route of 4000g, the detection sensitivity of SEA, SEB, SEC is respectively 5ng/mL,, 2ng/mL and 3ng/mL; When adopting the disposal route of 40 ℃ of preheatings, the three detection sensitivity be respectively 6ng/mL, 2ng/mL, and 3ng/mL; And when adopting the disposal route of 2 times of dilutions, the detection sensitivity of SEA is 10ng/mL, and the detection sensitivity of SEB is 4ng/mL, and the detection sensitivity of SEC is 6ng/mL.
Table 1 sample adds the sensitivity test result
Can know that from above-mentioned experimental result centrifugal sensitivity with the heat treated correspondence is higher,, and need higher sensitivity, then can select perhaps centrifugal disposal route of heating according to instrument condition if therefore detect in the laboratory.Though the method for dilution process can reduce the sensitivity of test strips, because it, can realize on-the-spot the detection without any need for instrument and equipment; Operate also more simple and convenient; Therefore carry out the scene if desired and detect, when perhaps lacking instrument condition, then can select the method for dilution process.From realizing that the on-the-spot angle that detects considers, this experimental selection the disposal route of 2 times of dilutions carry out subsequent experimental.
Add former milk sample for 100 parts of former milk samples of the unknown and 50 parts of standard items, use immuno-chromatographic test paper strip and 3M respectively
TMTecra
TMTwo kinds of detection methods of kit detect, and the result sees table 2.In the interpretation of result of immuno-chromatographic test paper strip, as long as have a kind of result to be shown as the positive in SEA, SEB and three kinds of test strips of SEC, this sample all is judged to the positive; When having only three kinds of test strips all negative, this sample just is judged to feminine gender.3M
TMTecra
TMKit carries out the result to specifications and judges.The result shows, two kinds of detection methods to 150 test result of samples through Chi-square Test analysis, P 0.05, the there was no significant difference as a result of two kinds of detection methods is described.
Table 2150 duplicate samples control experiment result
Annotate: X
2=0.00 P>0.05.
Claims (6)
1. the Staphylococcus aureus enterotoxin detection kit comprises plastics cartridge and test strips, and said test strips is overlapped in order to paste on end liner and formed by sample pad, golden labeling antibody pad, nitrocellulose filter, adsorptive pads; Encapsulate staphylococcus aureus A, B or the C three type polyclonal antibodies of colloid gold particle mark on the gold labeling antibody pad, the control line of Sheet clonal antibody detection line and goat-anti rabbit polyclonal antibody on the nitrocellulose filter.
2. according to the said Staphylococcus aureus enterotoxin detection kit of claim 1; It is characterized in that; The colloid gold particle particle diameter that encapsulates on the said golden labeling antibody pad is 25nm; 1mL colloid gold particle mark staphylococcus aureus toxin A, B, C three type polyclonal antibodies, labelled amount is respectively 10 μ g/mL, 8 μ g/mL, 15 μ g/mL.
3. according to the said Staphylococcus aureus enterotoxin detection kit of claim 1; It is characterized in that; Detection line coated with staphylococal A, B, the corresponding polyclonal antibody of C three types on the said nitrocellulose filter; Concentration is respectively 1mg/mL, 0.95mg/mL, 1.1mg/mL, and package amount is 1 μ L/cm.
4. according to the said Staphylococcus aureus enterotoxin detection kit of claim 1, it is characterized in that goat anti-rabbit antibody concentration is 1mg/mL on the said control line, package amount is 1 μ L/cm.
5. according to the preparation method of the said Staphylococcus aureus enterotoxin detection kit of claim 1-4, comprise the steps:
(1) preparation of golden labeling antibody pad
A. the preparation of blank colloidal gold solution:
Adopt the citrate reducing process to prepare colloid gold particle; Adding 97.5mL distilled water and 1mL1% gold chloride are heated to boiling in Erlenmeyer flask; Add concentration and be 1% citric acid three sodium solution 1.5mL, continue to boil to liquid and present salmon pink, keep behind the fluidized state 10min flask being placed room temperature; After fully cooling off, stored refrigerated;
B. the preparation of colloid gold label antibody:
Get blank colloidal gold solution 100mL; Add 0.1M sal tartari 300～400ul, under magnetic agitation, dropwise add the good antibody of dilution, be respectively 10 μ g/mL, 8 μ g/mL, 15 μ g/mL adding A, B, C three type polyclonal antibodies by labelled amount; Continue to stir 30min; Centrifugal, absorb supernatant, with the resuspended liquid of golden labeling antibody that the precipitation of gold labeling antibody is fully resuspended;
The preparation of c. golden labeling antibody pad:
Resuspended good golden labeling antibody is encapsulated on the plain film of the spun glass that is of a size of 20cm * 30cm, and package amount is 36mL, 37 ℃ of dried overnight, and the dry place of sealing lucifuge preserves, and cuts into the size of 1cm * 30cm during use;
(2) the encapsulating of T, C line on the nitrocellulose filter
The good two-dimentional specking platform of debugging; Be coated on the nitrocellulose filter as detection line through the mode of line diluting SEA, SEB, the SEC polyclonal antibody of getting well; That draws system simultaneously also has the good goat-anti rabbit polyclonal antibody of dilution as control line; 37 ℃ of dry 2h, the dry place of sealing lucifuge preserves;
(3) preparation of sample pad
A. the preparation of sample pad damping fluid:
In concentration is to add surfactant and protein in the borate buffer system of 0.1M, and surfactant is 1% PVP-10 and 8%Tetronic1307, and protein is 0.5% casein, through fully dissolve mix after, 4 ℃ of placements are subsequent use;
B. the preparation of sample pad:
The sample pad treating fluid that in a clean pallet, adds 250mL took out 50 undercuts and cuts the spun glass that is of a size of 2cm * 30cm and soak in pallet, fully soaked excessive solution in back taking-up and the removal sample pad through 5 minutes;
Sample pad is lain on layer frame, and layer frame put into 37 ℃ baking oven baking to more than 16 hours;
Sample pad after the oven dry has been put into the drying agent valve bag;
(4) assembling of test strips
The sample pad of cut lengths, golden labeling antibody pad, nitrocellulose filter and adsorptive pads overlapped according to the order of sequence paste on the PVC base plate; The base plate that posts is cut into wide test strips for 4mm also be positioned in order in the draw-in groove of townhouse test card on cutting cutter, at last test card is positioned in the aluminium foil bag that has drying agent; It is 24 ℃ ± 5 ℃ that assembling process keeps indoor temperature, and humidity is 30 ± 5%;
(5) assembling of kit
In the packing box of blank, put into the aluminium foil bag that includes test card, sample diluting liquid and operation instructions.
6. according to the application of the said Staphylococcus aureus enterotoxin detection kit of claim 1-4 in Staphylococcus aureus enterotoxin detects.
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| CN103134931B (en) * | 2013-01-05 | 2014-10-22 | 江南大学 | Double antibody sandwich method of detecting staphylococcus aureus enterotoxin A of food |
| CN103760354A (en) * | 2014-02-14 | 2014-04-30 | 江南大学 | Immune colloidal gold quick detection test strip of staphylococcal enterotoxin D, and preparation method of test strip |
| CN103777004A (en) * | 2014-02-14 | 2014-05-07 | 江南大学 | Immune colloidal gold rapid detection test strip for staphylococcus aureus enterotoxin E and preparation method thereof |
| CN103777004B (en) * | 2014-02-14 | 2015-12-02 | 江南大学 | Immune colloid gold Rapid detection test strip of a kind of staphylococcus aureus enterotoxin E and preparation method thereof |
| CN105181977A (en) * | 2015-11-05 | 2015-12-23 | 四川沃文特生物技术有限公司 | Kit for detecting calprotectin in excrement |
| CN111983217A (en) * | 2020-09-03 | 2020-11-24 | 菲鹏生物股份有限公司 | Sample treatment fluid and application thereof |
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