CN205246668U - Quick detect reagent strip of vibrio alginolyticus - Google Patents
Quick detect reagent strip of vibrio alginolyticus Download PDFInfo
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- CN205246668U CN205246668U CN201520962182.0U CN201520962182U CN205246668U CN 205246668 U CN205246668 U CN 205246668U CN 201520962182 U CN201520962182 U CN 201520962182U CN 205246668 U CN205246668 U CN 205246668U
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- collaurum
- vibrio parahaemolytious
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- 241000607594 Vibrio alginolyticus Species 0.000 title abstract 5
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- 238000001514 detection method Methods 0.000 claims abstract description 34
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Abstract
The utility model provides a quick detect reagent strip of vibrio alginolyticus, includes plastic substrate, absorbs pad, nitrocellulose membranes, colloidal gold pad and each lamellar structure of sample pad, absorbing to fill up and fixing in plastic substrate's upper end, nitrocellulose membranes fixes and is absorbing the lower part of filling up, plastic substrate's mid portion, the sample pad is including sample pad M2, the two -layer structure of M3, the colloidal gold pad updraft type is with the vibrio alginolyticus antibody of colloidal gold granule mark, nitrocellulose membranes divide into matter accuse district and detection zone, forms matter accuse C line at matter accuse district peridium goat -anti mouse igG antibody, forms at detection zone peridium vibrio alginolyticus antibody and detects the T line, the utility model provides a quick detect reagent strip of vibrio alginolyticus sample pad is two -layer, has improved reagent strips's chromatography speed, the shortening reaction time, and easy operation, the specificity is strong, and sensitivity is high.
Description
Technical field
The utility model relates to a kind of in-vitro diagnosis class medicine equipment, relates in particular to a kind of vibrio parahaemolytious fast detecting examinationAgent bar.
Background technology
Vibrio parahaemolytious (Vibrioparahaemolyticus), is called again enteritis vibrios, belongs to vibrionaceae, vibriosBelong to Gram-negative bacteria. Vibrio parahaemolytious ocean and the estuarine environment existence of being everlasting, is present in the sea-farmings such as fish, shrimp, shellfish movingIn the body surface or enteron aisle of thing, be the conditioned pathogen of aquatic/marine animals, mainly cause that the marine products such as fish septicemia, red appendages disease of prawn are movingThing disease. Except infecting aquatic/marine animals, vibrio parahaemolytious is also a kind of common pathogen that causes food origin disease. FeedThe food that contains this bacterium, as the marine products such as fish, shrimp, crab, shellfish and marine alga can cause food poisoning. In recent years, the whole world all byIn the diarrhoea enterogastritis case of making a definite diagnosis, the infected who has 50%-70% is because the edible marine product not boiling causes, and facesOn bed taking Acute onset, stomachache, vomiting, diarrhoea and watery stool as cardinal symptom. At present, the whole world is more next to Safety of Aquatic ProductsMore pay close attention to, timely vibrio parahaemolytious is identified, all had by this microbial disease realizing production prevention and treatmentSignificance.
The detection method of vibrio parahaemolytious is commonly used following two kinds:
1, traditional micro-biological process, adopts microbiology separation, cultivation and biochemical test etc. to identify, length consuming time,Be unfavorable for clinical quick diagnosis; Operating process is numerous and diverse, is prone to cross pollution, causes the inaccurate of result;
2, molecular biology method, the more traditional micro-biological process specificity of this method is higher, sensitivity is strong, but needs specialtyOperating personnel and equipment, cost is higher, consuming time longer.
Summary of the invention
In order to overcome the detection method complex operation of existing vibrio parahaemolytious, professional technique requires high, and cost is high, consuming timeLong deficiency, the invention provides a kind of novel vibrio parahaemolytious quick detection reagent bar, and this reagent strip is simple to operate, ordinary peopleMember gets final product complete operation after reading description, does not need to mate professional instrument and equipment; Consuming time short, can complete inspection in 20 minutesSurvey; High specificity, only specificity is for vibrio parahaemolytious, to no cross reactions such as Escherichia coli, Vibrio vulnificus, comma bacillus;Highly sensitive, detectable least concentration is 1ng/ml; Its detection specificity and sensitivity are all higher than traditional microorganism discriminating sideMethod, result is accurate.
For addressing the aforementioned drawbacks, the technical scheme that the utility model provides is:
A kind of vibrio parahaemolytious quick detection reagent bar, comprises plastic base, absorption pad, nitrocellulose filter, collaurumPad and the each layer structure of sample pad, described absorption pad is fixed on the upper end of plastic base, and nitrocellulose filter is fixed on absorptionThe bottom of pad, the mid portion of plastic base, is characterized in that: described sample pad comprises sample pad M2, M3 double-layer structure; ColloidGold pad is fixed on bottom, the nitrocellulose filter bottom of plastic base, with nitrocellulose filter near but not overlapping, collaurumOn pad, absorption has the vibrio parahaemolytious monoclonal antibody of colloid gold particle mark; Sample pad M2 is positioned at the outer middle and lower part of collaurum pad, under itEnd is longer than collaurum pad; Sample pad M3 is positioned at the skin of sample pad M2, and transparency protected adhesive tape is positioned at the outer upper end of sample pad M3,And attach to the lower end of nitrocellulose filter; Described nitrocellulose filter is coated with anti-specificity vibrio parahaemolytious antibody, nitric acid fibreTie up plain film and be divided into Quality Control district and detection zone, in Quality Control district, coated sheep anti-mouse igg forms Quality Control C line, at the coated secondary haemolysis of detection zoneVibrios monoclonal antibody or polyclonal antibody form and detect T line.
The vibrio parahaemolytious quick detection reagent bar sample pad that the utility model provides is two-layer, has improved the layer of reagent stripAnalyse speed, Reaction time shorten.
Further, the vibrio parahaemolytious quick detection reagent bar that the utility model provides, is characterized in that: sample pad M2Upper end and collaurum are leveled up together, and lower end is longer than collaurum pad but is slightly shorter than plastic base lower end; Sample pad M3 upper end is apart from colloidGold pad upper end 0.5-1cm, sample pad M3 lower end is concordant with plastic base lower end.
Further, for the performance of fortifying fibre element film, the vibrio parahaemolytious fast detecting examination that the utility model providesAgent bar, is characterized in that: described nitrocellulose filter is the high molecular cellulose film of high protein compatibility.
Further, in order to strengthen the adsorption capacity of collaurum pad, the vibrio parahaemolytious that the utility model provides is examined fastTest agent bar, is characterized in that: described collaurum pad is nonwoven.
Further, in order to strengthen the water absorbing capacity of sample pad, the vibrio parahaemolytious fast detecting that the utility model providesReagent strip, is characterized in that: described sample pad is nonwoven or glass fibre.
Further, remain the ability of sample in order to strengthen the absorption of absorption pad, the secondary haemolysis arc that the utility model providesBacterium quick detection reagent bar, is characterized in that: described absorption pad is high-hydroscopicity blotting paper.
Further, for by each layer structure fixation of the application's reagent strip, the pair that the utility model provides is moltenBlood vibrios quick detection reagent bar, is characterized in that: described plastic base, absorption pad, nitrocellulose filter, collaurum pad and sampleBetween the each layer structure of this pad M2, M3, fix by two-sided tape.
The film reaction system of the application vibrio parahaemolytious quick detection reagent bar is made up of following part, by highly completeKind film reaction system, provide the idiosyncrasy realizing liquid sample parallel and complete immunoaffinity chromatography must variousCondition:
Nitrocellulose filter: the high molecular cellulose film of high protein compatibility, coated anti-specificity pair is molten after pretreatmentBlood vibrios antibody, is prepared as reaction film and is used as the carrier of system response and shows reaction result.
M2, M3: sample pad, select glass fibre, the nonwoven of stronger water absorbing capacity, after specially treated, be reactionSystem provides applicable reaction condition, and helps to draw sample.
Collaurum pad: the nonwoven that absorption affinity is stronger is the solid phase of the anti-specificity vibrio parahaemolytious of colloid gold label antibodyCarrier.
Absorption pad: the sample after absorption detecting.
Plastic base: as the supporter of reaction system.
Two-sided and the one side adhesive tape of different size: for fixing each reaction film and absorbent material in plastic base
Transparency protected adhesive tape: fixing and protection sample pad and collaurum pad
Core component of the present invention is nitrocellulose filter and collaurum pad, is the main reflection material of immune response generationMaterial.
Nitrocellulose filter: use macromolecular fibre film as solid phase carrier. This macromolecular fibre film has very strong eggWhite adsorption function, vibrio parahaemolytious antibody is coated in film drying process after as solid phase, can with liquid phase in corresponding anti-Former-quick combination of colloidal gold labeled monoclonal antibody compound. Reaction film nitrocellulose filter is divided into Quality Control district and detection zone by the present invention.In Quality Control district, coated sheep anti-mouse igg forms Quality Control C line, forms inspection at the coated vibrio parahaemolytious monoclonal antibody of detection zone or polyclonal antibodySurvey T line.
Collaurum pad: use nonwoven as solid phase carrier, by the vibrio parahaemolytious monoclonal antibody solution of colloid gold particle markBe sprayed on nonwoven surface. Colloid gold label is the process that the large molecule of protein (antibody) is adsorbed to colloid gold particle surface.Adsorption mechanism is colloid gold particle surface negative charge, forms strong bonded with the positive charge group of protein because of Electrostatic Absorption,Belong to physical absorption. Colloidal gold labeled monoclonal antibody is by the antigen-antibody reaction (double antibody sandwich method: antibody-anti-of high specialAntigen-antibody), realize the specific detection to transgene product. Under moisture state, the antigen-colloidal gold labeled monoclonal antibody in liquid phaseCompound can be done slewing swimming and the specific antibody on immobilon-p is combined because of " wick drainage phenomenon ", forms special goldLabelled antibody-Ag-Ab cement line.
The utility model, by antibody is sprayed on collaurum pad 6, is coated on nitrocellulose filter 8, by increasing sampleThis bed course number, is that sample pad M2, M3 are two-layer by 1 layer of traditional thickening, has improved creep speed and the reaction time of sample.
The beneficial effects of the utility model are the vibrio parahaemolytious detection reaction time can be controlled in 20min and finish, behaviourDo simply, ordinary person gets final product complete operation after reading description; Do not need to mate professional instrument and equipment; High specificity, rightThe no cross reactions such as secondary hemolytic bacteria, Escherichia coli, Vibrio vulnificus, comma bacillus; Highly sensitive, detectable least concentration is 1Ng/ml; Its detection specificity and sensitivity are all higher than traditional microorganism discrimination method, and result is accurate.
Brief description of the drawings
Below in conjunction with drawings and Examples, the utility model is further illustrated:
Fig. 1 is the utility model vibrio parahaemolytious quick detection reagent bar schematic diagram
In figure, be labeled as: 1. Quality Control C line, 2. detects T line, 3. plastic base, 4. sample pad M2,5. sample pad M3,6. glueBody gold pad, 7. transparency protected adhesive tape, 8. nitrocellulose filter, 9. absorption pad.
Detailed description of the invention
As shown in Figure 1, the utility model comprises plastic base 3, absorption pad 9, nitrocellulose filter 8, collaurum pad 6 and sampleThe layer structure of this pad 4,5, each structure is from top to bottom, as described below by left-to-right concrete assembling:
Described absorption pad 9, length 22mm, by two-sided tape be fixed on plastic base 3 upper end, with plastic base 3Upper end alignment; Nitrocellulose filter 8, length 18mm, is fixed on bottom, the plastic base 3 of absorption pad 9 by two-sided tapeMid portion, nitrocellulose filter 8 upper ends are apart from the upper end 22mm of plastic base 3.
Sample pad comprises sample pad M24, sample pad M35 double-layer structure, can improve creep speed and the reaction of sampleTime.
Collaurum pad 6 is fixed on bottom, nitrocellulose filter 8 bottoms of plastic base 3, collaurum by two-sided tapePad 6 bottoms near nitrocellulose filter but not overlapping.
Sample pad M24 is positioned at the outer middle and lower part of collaurum pad 6.
Preferably, sample pad M24 upper end is concordant with collaurum pad 6, and sample pad M24 is longer than lower end collaurum pad but is slightly shortIn plastic base lower end.
Sample pad M35 is positioned at the skin of sample pad M24, and sample pad M35 upper end is apart from collaurum pad upper end 0.5-1cm,Sample pad M35 is longer than lower end collaurum pad 6 and concordant with plastic base lower end.
The bottom of transparency protected adhesive tape 7 attach to sample pad M35 upper end, top attach to nitrocellulose filter 8 underEnd, transparency protected adhesive tape 7 is for fixing nitrocellulose filter 8 and sample pad M35, collaurum pad 6.
Outer with colour-coded adhesive tape at absorption pad 9, its lower end and the outer crossing 1mm in nitrocellulose filter upper end, anotherOne end climbs over plastic base 3 upper limbs, is affixed on plastic base quilt cover, as the signature identification of this inspection test-strips.
The reagent strip of assembled formation is cut into 3.5mm specification with full-automatic strip cutter.
On nitrocellulose filter 8, be provided with Quality Control C line 1, detect T line 2, be convenient to observe judgement testing result.
The application can realize by following steps:
1. the preparation of vibrio parahaemolytious monoclonal antibody
Mouse peritoneal injecting fluid paraffin 0.5ml, after 1 week, is injected in above by vibrio parahaemolytious monoclonal antibody hybridomaThe BALB/C mice abdominal cavity of anticipating, every injected in mice hybridoma 1～3 × 106. After 10 days, gather in the crops ascites, withUpper process all completes under aseptic condition. Ascites through DEAE ion-exchange chromatography and SephacrylS300 molecular sieve to monoclonal antibodyCarry out purifying; SDS-PAGE disk electrophoresis detects the purity of purified monoclonal antibody; ELISA method is measured monoclonal antibody activity; Measure each monoclonal antibody effectValency (requirement is greater than 1:5000).
2. goat-anti DOATThe high-titer sero-fast preparation of immunity and purifying
1) the good above-mentioned monoclonal antibody+Freund's complete adjuvant immune goat booster immunization secondary of purifying, every minor tick January secondBlood centrifuging and taking serum ammonium sulfate precipitation DEAE ion-exchange chromatography purifying is got in inferior reinforcement for latter about 10 days;
2) titration: ELISA sandwich method, antibody titer dilution factor should be greater than 1:256;
3) determination of protein concentration: ultraviolet spectrometry is measured OD279, calculates protein concentration.
3. collaurum and the preparation of golden labeling antibody
1) preparation of collaurum: the collaurum of preparing 40-60nm with citrate reducing process. 0.01%HauCl4 is addedHeat, to boiling, adds a certain amount of 1% trisodium citrate (Na3C6H5O72H2O), continues heating and boils 5 minutes, treats collaurumSolution colour is purplish red by orchid, after stablizing, cooling. Colloidal gold solution should be limpid transparent, as need are preserved for a long time, can add0.02%NaN3。
2) collaurum liquid 500ml is adjusted to pH7-8 with 0.1MNaOH, under magnetic agitation, slowly add monoclonal antibody 2ml, stir20 minutes. The centrifugal 30Min of 5500rpm, removes unconjugated protein in supernatant. Centrifugal rear absorption colloidal gold labeled monoclonal antibody is heavyForm sediment, precipitation is dissolved in 10ml and preserves in liquid, with 0.45 μ m filtering with microporous membrane. Sampling is examined and determine, and all the other put 4 DEG C of preservations.
4. the preparation of film reaction system nitrocellulose filter
1) sheep anti-mouse igg of vibrio parahaemolytious antibody mab and purifying is diluted to final concentration with 0.1M phosphate bufferFor about 0.2-2mg/ml.
2) sheep anti-mouse igg of purifying is dissolved in 0.1M phosphate buffer, and concentration is 2.0mg/ml;
3) nitrocellulose filter size: 10cm × 27cm, detection and control that every film can spray long 25cm are with each 4;
4) above two kinds of solution are added respectively in two shower nozzle storage bottle of BiodotXYZ3000 flush coater;
5) spray speed being set is 2 μ l/cm, and the speed of dividing a word with a hyphen at the end of a line of NC film is 50mm/s. Exist with vibrio parahaemolytious monoclonal antibody (2mg/ml)Coated reaction detection line (Fig. 1) on NC film, with the coated reaction of 1.0mg/ml rabbit anti-mouse igg control line, control line and detection lineDistance is 4-4.5mm;
6) complete coated after, put 37C drying box 24 hours, process half an hour with the sealing of BB confining liquid, take out and wash by WB washing lotionOnce;
7) 37C drying box drying for standby;
8) nitrocellulose filter that cutting is got ready: 1.8cm × 27cm/ bar, put into thin aluminum bag hermetically drying and preserve. Put chamberTemperature saves backup.
5. the preparation of film reaction system collaurum pad
1) get collaurum-monoclonal antibody compound and add dilution, mix and be made into working concentration;
2) solution is added in the Airjet shower nozzle storage bottle of flush coater Biodot;
3) setting pressure is 15PSI, and the translational speed of glass fibre membrane is 50mm/s;
4) spray specification is 0.5 ~ 1.5cm × 25cm/ bar, and every sprays 2 times;
5) in 37 DEG C of oven dry 12 hours, put into thin aluminum bag, add drier, heat sealing, room temperature preservation.
When use, the fixed card slot of reagent strip being put into corresponding plastic clip is fixing, the sample pad region pair of reagent stripAnswer well position, reagent strip Quality Control C line 1, corresponding watch window positions, detection T line 2 regions, splash into sample in well,Under capillarity, upwards chromatography, through sample pad M24, the two-layer sample pad of sample pad M35.
Preferably, sample pad adopts nonwoven or glass fiber material, and chromatography speed promotes, highly sensitive.
Gold grain-specific antibody the compound of specific detection object in sample in collaurum pad 6 is combined, shapeBecome gold grain-antibody-specific detection object compound.
In order to strengthen the adsorption capacity of collaurum pad 6, collaurum pad 6 materials preferably adopt the nonwoven that absorption affinity is stronger.
Sample continues upwards chromatography, by transparency protected adhesive tape 7, with the anti-pair being coated in advance on nitrocellulose filter 8The combination of hemolysis vibrion specific antibody, forms gold grain-antibody-vibrio parahaemolytious antigen-antibody complex, and gold grain is gathered inNitrocellulose filter 8 film detection zones, form and detect T line 2, continue upwards chromatography, and are coated in advance on nitrocellulose filter 8Multispecific antibody combination, forms Quality Control C line 1, passes through to observe Quality Control C line 1, detects T in the watch window of the plastic clip being used in conjunction withLine 2 develops the color, and judges testing result.
Residue sample continues upwards chromatography, is absorbed pad 9 and absorbs.
For the performance of fortifying fibre element film, nitrocellulose filter is the high molecular cellulose film of high protein compatibility, warpCoated anti-specificity vibrio parahaemolytious antibody after pretreatment, is prepared as reaction film and is used as the carrier of system response and shows anti-Should result.
The ability that remains sample in order to strengthen the absorption of absorption pad, absorption pad 9 materials preferably adopt high-hydroscopicity blotting paperMaterial.
The above embodiment, has carried out further in detail the purpose of this utility model, technical scheme and beneficial effectDescribe in detail brightly, institute it should be understood that and the foregoing is only specific embodiment of the utility model, is not limited to this practicality newlyType, all within spirit of the present utility model and principle, to amendment of the present utility model or replacement, all should be of the present utility modelWithin protection domain.
Claims (7)
1. a vibrio parahaemolytious quick detection reagent bar, comprise plastic base (3), absorption pad (9), nitrocellulose filter (8),Collaurum pad (6) and the each layer structure of sample pad, described absorption pad (9) is fixed on the upper end of plastic base (3), cellulose nitrateElement film (8) is fixed on the bottom of absorption pad (9), the mid portion of plastic base (3), it is characterized in that: described sample pad comprisesSample pad M2(4), sample pad M3(5) double-layer structure; Collaurum pad (6) is fixed on bottom, the cellulose nitrate of plastic base (3)Element film (8) bottom, near but not overlapping, collaurum pad (6) upper absorption has colloid gold particle mark with nitrocellulose filter (8)Vibrio parahaemolytious antibody; Sample pad M2(4) be positioned at the outer middle and lower part of collaurum pad (6), collaurum pad (6) is longer than in bottom; SampleThis pad M3(5) be positioned at sample pad M2(4) skin, transparency protected glue (7) band is positioned at sample pad M3(5) outer upper end, and glutinousInvest the lower end of nitrocellulose filter (8); Described nitrocellulose filter (8) is divided into Quality Control district and detection zone, coated in Quality Control districtSheep anti-mouse igg antibody forms Quality Control C line (1), forms and detects T line (2) at the coated vibrio parahaemolytious antibody of detection zone.
2. vibrio parahaemolytious quick detection reagent bar according to claim 1, is characterized in that: sample pad M2(4) upper endConcordant with collaurum pad (6), lower end is longer than collaurum pad (6) but is slightly shorter than plastic base (3) lower end; Sample pad M3(5) upper endApart from collaurum pad (6) upper end 0.5-1cm, sample pad M3(5) lower end is concordant with plastic base (3) lower end.
3. vibrio parahaemolytious quick detection reagent bar according to claim 1, is characterized in that: described nitrocellulose filter(8) be the high molecular cellulose film of high protein compatibility.
4. vibrio parahaemolytious quick detection reagent bar according to claim 1, is characterized in that: described collaurum pad (6)For nonwoven.
5. vibrio parahaemolytious quick detection reagent bar according to claim 1, is characterized in that: described sample pad is nonwovenCloth or glass fibre.
6. vibrio parahaemolytious quick detection reagent bar according to claim 1, is characterized in that: described absorption pad (9) isHigh-hydroscopicity blotting paper.
7. according to the wherein vibrio parahaemolytious quick detection reagent bar described in any one claim of claim 1-6, its featureBe: described plastic base (3), absorption pad (9), nitrocellulose filter (8), collaurum pad (6) and sample pad M2(4), samplePad M3(5) fix by two-sided tape between each layer structure.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201520962182.0U CN205246668U (en) | 2015-11-26 | 2015-11-26 | Quick detect reagent strip of vibrio alginolyticus |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201520962182.0U CN205246668U (en) | 2015-11-26 | 2015-11-26 | Quick detect reagent strip of vibrio alginolyticus |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106336458A (en) * | 2016-07-29 | 2017-01-18 | 浙江省海洋水产养殖研究所 | Vibrio parahaemolyticus colloidal gold-yolk antibody rapid detection test paper and preparation method thereof |
| CN107356749A (en) * | 2017-08-06 | 2017-11-17 | 潘金文 | Noxious pollutant immunochromatography quick detection kit in a kind of aquatic products |
| CN110836975A (en) * | 2019-11-29 | 2020-02-25 | 扬州大学 | Colloidal gold plastic-sealed detection card for detecting vibrio parahaemolyticus TD toxin and preparation method and application thereof |
-
2015
- 2015-11-26 CN CN201520962182.0U patent/CN205246668U/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106336458A (en) * | 2016-07-29 | 2017-01-18 | 浙江省海洋水产养殖研究所 | Vibrio parahaemolyticus colloidal gold-yolk antibody rapid detection test paper and preparation method thereof |
| CN107356749A (en) * | 2017-08-06 | 2017-11-17 | 潘金文 | Noxious pollutant immunochromatography quick detection kit in a kind of aquatic products |
| CN110836975A (en) * | 2019-11-29 | 2020-02-25 | 扬州大学 | Colloidal gold plastic-sealed detection card for detecting vibrio parahaemolyticus TD toxin and preparation method and application thereof |
| CN110836975B (en) * | 2019-11-29 | 2023-05-02 | 扬州大学 | Colloidal gold plastic package detection card for detecting vibrio parahaemolyticus TD toxin and preparation method and application thereof |
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Effective date of registration: 20231026 Address after: Building 1-2, Building 24, No. 28 Yuefu Avenue, Beibei District, Chongqing, 400700 Patentee after: Chongqing Aochuang Biotechnology Co.,Ltd. Address before: 101, Diya Production Building, No. 2766 Yingxiu Road, High tech Zone, Jinan City, Shandong Province, 250000 Patentee before: JINAN KANGBO BIOTECHNOLOGY Co.,Ltd. |
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