CN102393455A - Preparation method of immunogold rapid test paper for vibrio parahaemolyticus - Google Patents
Preparation method of immunogold rapid test paper for vibrio parahaemolyticus Download PDFInfo
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Abstract
The invention discloses a preparation method of immunogold rapid test paper for vibrio parahaemolyticus. The method comprises the following steps of: (a) preparing a vibrio parahaemolyticus antigen; (b) preparing a vibrio parahaemolyticus inactivated vaccine; (c) preparing a polyvalent antiserum with a vaccine immune experimental rabbit; (d) purifying the antiserum; (e) marking with colloidal gold; and (f) preparing an immunogold test strip. A detection technology in which the immunogold rapid test paper for vibrio parahaemolyticus is adopted has the advantages of easiness, sensitivity, rapidness, specificity and the like; the customs clearance speed of port imported and exported goods can be increased greatly; and meanwhile, the test paper has the advantages of low price, no need of instrument, easiness and rapidness for operationing and easiness in judging results.
Description
Technical field
The present invention relates to the detection technique field, be specifically related to the preparation method of the golden quick detection test paper of a kind of vibrio parahaemolytious immunity.
Background technology
Vibrio parahemolyticus all belongs to vibrionaceae vibrio bacterial, is through food, water-borne pathogen.Vibrio parahemolyticus has similar biological and same pathogenic, all can cause diarrhoea and food poisoning.
Vibrio parahaemolytious equals nineteen fifty-three by Fujino the earliest and obtains first the separation from food poisoning patient of Japan; Claim Halophiles again; This bacterium be because of growing in the nutrient culture media that contains salt denseer (more than 3% ~ 4%), and irreproducible and gain the name in salt-free nutrient culture media.Vibrio parahaemolytious is distributed in all over the world, is present in usually in bay and zone, shore line and the marine product, and people Duo Yin is edible to pollute this bacterium again without the marine product of well processed such as crab class, oyster! Shrimp etc. and cause food poisoning.The food poisoning that vibrio parahaemolytious causes, the ratio that in food posioning, accounts for is very high, and its harm is only second to salmonella, Escherichia coli, staphylococcus and clostridium botulinum.China's Coastal Areas all has the report that causes food poisoning because of vibrio parahemolyticus every year.Cardinal symptom after the people poisons comprises: diarrhea (being watery stool or blood sample just), abdominal cramps, feel sick, vomiting and headache, the report that fever was also once arranged and feel cold, but less.
At present, the U.S. and European Union to the import aquatic products all Compulsory Feature carry out the detection of vibrio parahaemolytious, assay is negative can be open to the custom, otherwise the aquatic products of outlet will all be detained automatically or destroy on the spot.This is provided with technology barriers for the outlet of China's aquatic products.Therefore, the outlet aquatic product manufactory is conscientiously implemented HACCP plan, strengthens the detection to vibrio parahaemolytious, and quarantine departments is strengthened factory implemented the supervision of HACCP plan situation and the check of outlet aquatic products just seemed being even more important.
At present, domestic detection for vibrio parahemolyticus has had corresponding national standards and industry standard, and method roughly is divided into following process: (1) preceding to increase bacterium; (2) selective enrichment; (3) selectivity is cultivated; (4) biochemical identification; (5) Kahagawa phenomenon and serology experiment.Checkout procedure needs 5d-6d could report positive findings.The method of inspection is loaded down with trivial details, wastes time and energy, and not only influences the work efficiency of inspection body, and makes the Product transport and storage time lengthening of production division, and production cost strengthens.
Collaurum fast diagnose test paper method is a novel vitro diagnostic techniques that on monoclonal antibody technique, colloidal gold immunochromatographimethod technology and new material technology basis, has grown up since 20th century the nineties.Simple to operate, only must sample drop to be checked be added in the sample cell and get final product, spent for 10 a few minutes and just can reach the purpose of detection according to the color judgment result.This method does not need special instrument, and those skilled in the art can accomplish, and it is low to detect cost, can significantly reduce workload, is adapted to the needs of port fast detecting.Therefore, set up as early as possible a cover fast detecting vibrio parahemolyticus accurately, collaurum fast diagnose test paper method reliable, science is imperative.This all has crucial meaning for the quick test quarantine of strengthening importing and exporting vibrios in the aquatic products, investigation and control, nuisanceless aquatic products detection and the aspects such as production and aquatic products hygienic quality supervision and inspection of aquiculture disease.
In view of this, the present invention provides the preparation method of the immune golden quick detection test paper of a kind of vibrio parahaemolytious,, detection method fast easy to set up.
Summary of the invention
It is material that the present invention is intended to the vibrio parahemolyticus, and how anti-preparation is, utilizes advanced collaurum production technology to develop and detects the vibrio parahemolyticus mark detection test paper.To achieve these goals, solution of the present invention is following:
The preparation method of the golden quick detection test paper of vibrio parahaemolytious immunity may further comprise the steps:
A) preparation of vibrio parahaemolytious antigen: with peptone water medium with the vibrio parahaemolytious overnight incubation;
B) make the vibrio parahaemolytious inactivated vaccine;
C) prepare polyvalent antiserum with the vaccine immunity experimental rabbit: adopt the direct injecting immune rabbit of subcutaneous multi-point injection and vein to prepare the many antiantibodys of the anti-vibrio parahaemolytious of rabbit; The anti-vibrio parahaemolytious antibody of rabbit that obtains subcutaneous multi-point injection preparation respectively (abbreviates rabbit anti--1 as; Down with) directly inject the anti-vibrio parahaemolytious antibody of rabbit (it is anti--2 to abbreviate rabbit as, down together) of preparation with vein;
D) antiserum purifying;
E) colloid gold label: the rabbit anti-serum collaurum of purifying is carried out mark;
F) preparation of immune gold test paper strip: the rabbit anti--2 with purifying in the step d) is sprayed on the miillpore filter detection line earlier; Goat anti-rabbit antibody is sprayed on the control line; Rabbit anti--1 with the colloid gold label for preparing in the step e) is sprayed on the glass fibre membrane again, is assembled into immune gold test paper strip then;
Described step b) makes the vibrio parahaemolytious inactivated vaccine, is specially: earlier through 37 ℃ of deactivation 24h of formaldehyde of 0.3%, then through cultivate detect certain no viable bacteria after as antigen;
Described step c) prepares polyvalent antiserum with the vaccine immunity experimental rabbit, adopts the direct injecting immune rabbit of subcutaneous multi-point injection and vein to prepare the many antiantibodys of the anti-vibrio parahaemolytious of rabbit, is specially:
1) intravenous immune: injection in per 10 days once, 1mL first, 1.5mL for the second time, 2mL for the third time, the 4th 3mL, bloodletting when reaching 1:1280 or 1:2560 when tiring;
2) the subcutaneous multi-point injection of Freund immunity: Freund's complete adjuvant is with after bacterium liquid equal-volume mixes; The subcutaneous multi-point injection 4mL of rabbit nape portion; After 2 weeks, with incomplete Freund and the bacterium liquid equal-volume mixed liquor nape subcutaneous multi-point injection 4mL of portion, after 2 weeks; 5-7 days intravenous injection 4mL before the blood sampling, bloodletting when reaching 1:1280 or 1:2560 when tiring.
Described step f), the preparation of immune gold test paper strip is specially:
Resist-1 with the colloid gold label rabbit, set up the method for colloidal gold immunochromatographimethod technology fast detecting vibrio parahaemolytious; Use miillpore filter and make the immunoassay technology of carrier; Earlier rabbit anti--2 is sprayed on the miillpore filter detection line, goat anti-rabbit antibody is sprayed on the control line, and the rabbit of colloid gold label anti--1 is sprayed on the glass fibre membrane; Dry back adds confining liquid and mixes, and is assembled into behind the drying liquid to detect vibrio parahaemolytious; Amplify immune response system through gold grain again, reaction result is shown on the solid phase carrier miillpore filter.
Above-mentioned confining liquid prescription adopts: the concentration that contains 1% (W/V) casein and 0.05% (V/V) Tween-20 is the PBS solution of 0.01 mol/L, and the pH value of PBS solution is 7.2.
The preparation method of the golden quick detection test paper of vibrio parahaemolytious immunity; When prepared immune golden quick detection test paper is used for on-the-spot the detection, test strips is kept flat on the table, add the sample of 100uL at the application of sample end; Sample at first dissolves the gold mark rabbit anti--1 that is sprayed on the glass fibre membrane; Solution moves toward the NC film then, when the rabbit that runs into detection line (T) resists-2, judges that detection paper result's standard is following:
Contain vibrio parahaemolytious in the sample: on detection line and control line (C), all manifest a purplish red colo(u)r streak (forming sandwich reaction, i.e. anti--1-vibrio parahaemolytious of gold mark rabbit-rabbit anti--2);
Do not contain vibrio parahaemolytious in the sample: a purplish red colo(u)r streak only on control line, occurs;
Test paper is invalid: all do not occur a purplish red colo(u)r streak on detection line and the control line.
Beneficial effect of the present invention is following:
Adopt the easy and simple to handle, quick of the golden quick detection test paper of this method vibrio parahaemolytious immunity, do not need special instruments and equipment, the aubergine response line on the detection line is bright in colour, be easy to judge, and testing result can be preserved.Through using the detection of this test strips method to 150 duplicate samples, test strips method positive rate is 2% as a result, and bacterium separation detection positive rate is 2%, and both reach 100% by coincidence rate.The vibrio parahaemolytious antigen immune originality that the present invention selects for use is good, and the antibody agglutination titer that immune rabbit makes reaches 2
11-2
12, form test strip with colloid gold label and be used to detect vibrio parahaemolytious, have high specificity, sensitivity is high, and is simple to operate, weak point consuming time, the result is easy to judge, and with vibrio alginolyticus, Escherichia coli, salmonella, Kazakhstan Vickers bacterium with cross reaction does not take place.The successful development of this method, simply fast detecting vibrio parahaemolytious.
Adopt the detection technique of the golden quick detection test paper of vibrio parahaemolytious of the present invention immunity to have simply, sensitive, quick, special advantage; Can make things convenient for the speed of being open to the custom of port cargoes imported and exported greatly, simultaneously because this detections test paper is cheap, do not need instrument and equipment, quick, result easy and simple to handle to judge easily.This has important practical significance for prevention and control China aquatic products safety.Do not see at present and utilize immune colloidal gold technique to detect the report of importing and exporting vibrio parahemolyticus in aquatic animal and products thereof both at home and abroad; This technology is expected to for the detection of entry and exit vibrio parahemolyticus a kind of rapid sensitive, simple and reliable method are provided, and is particularly suitable for port quarantine department and implements to test fast to put needs.
Embodiment
1, present embodiment at first prepares the vibrio parahaemolytious inactivated bacterial liquid
The nutrient solution of formalin-inactivated vibrio parahaemolytious with 0.3%; There is not the survival bacterium through detecting; With this bacterium with 1,600,000,000 bacterium of content/every milliliter of immunizing rabbit; Adopt direct injecting immune of vein and subcutaneous multi-point injection immunity dual mode, adopt the reaction of reporting to the leadship after accomplishing a task of antigen absorption method sealing mixed antibody and other enteric bacillis, cryopreservation by document.
2, the preparation of the anti-vibrio parahaemolytious antibody of rabbit
The bacterium liquid of deactivation is adopted intravenous injection and Freund immunity, and concrete immune programme for children is following:
1) intravenous immune: injection in per 10 days once, 1mL first, 1.5mL for the second time, 2mL for the third time, the 4th 3mL, bloodletting when reaching 1:1280 or 1:2560 when tiring;
2) the subcutaneous multi-point injection of Freund immunity: Freund's complete adjuvant is with after bacterium liquid equal-volume mixes; The subcutaneous multi-point injection 4mL of rabbit nape portion; 2 all backs are with incomplete Freund and the bacterium liquid equal-volume mixed liquor nape subcutaneous multi-point injection 4mL of portion; 5-7 days intravenous injection 4mL before the back blood sampling of 2 weeks, bloodletting when reaching 1:1280 or 1:2560 when tiring;
3) agglutination titer: adopt slide aggegation experiment to detect the anti-vibrio parahaemolytious antibody titer of rabbit, the concentration of adjuvant immunity is 28.72 mg/mL behind the anti-vibrio parahaemolytious antibody purification of rabbit, and slide agglutination test is tired and is 1:2
11, the concentration of intravenous immune is 28.72 mg/mL, slide agglutination test is tired and is 1:2
7
3, the preparation of the anti-vibrio parahaemolytious antibody colloidal gold of rabbit
1) preparation of colloidal gold solution: prepare with trisodium citrate reduction method; Getting 1% (W/V) gold chloride, 1 mL joins in the 99 mL ultrapure waters; Gained chlorauric acid solution concentration is 0.01 (V/V) %; Be heated to boiling with water-bath magnetic agitation pot, under magnetic stirring apparatus constantly stirs with certain rotating speed, add 1% trisodium citrate aqueous solution of 2 mL rapidly, continue about 15 min of agitating heating and stablize constant to solution colour.The collaurum that this method makes is vinicolor, surveys its absorption peak at 520 nm places through ultraviolet spectrophotometer, and the colloid gold particle diameter that obtains is about about 20 nm, and the rearmounted 4 ℃ of refrigerators of cooling keep in Dark Place;
2) anti--1 mark of rabbit: get the colloidal gold solution that needs preparation amount, with 0.1 mol/L K
2CO
3Transfer its pH to 8.5 with the HCl of 0.1 mol/L.The rabbit anti--1 that under magnetic agitation, dropwise adds purifying then is (with the rabbit antibody standard gold of adjuvant immunity; Every mL colloidal gold solution adds anti--1 16.7ug of rabbit); Continue to add 5% (W/V) PEG (MW 6000) to final concentration 0.1% (W/V) behind magnetic agitation 30 min; Continue again to stir 15 min, place 4 ℃ of refrigerators at last, hold over night.The anti-centrifugal 10min of-1 collaurum 4000 r/min of rabbit that next day is good with mark gets centrifugal 30 min of supernatant 15000 r/min, and careful the suction abandoned supernatant.Deposition returns to 1/20th of original volume with resuspended damping fluid (containing 1% casein, 0.05% (W/V) PEG6000,0.05% (V/V) Tween-20, the PBS damping fluid of 0.01 mol/L pH7.2), and it is subsequent use to put 4 ℃ of refrigerators;
4, the making of reaction test strips
It is the test strips about 5.0 cm-6.0cm * 0.4cm-0.6 cm that the reaction test paper is processed size, and specifically consist of: the bottom is the PVC offset plate, and the upper end of test strips is a thieving paper; The lower end is glass fibre membrane and the sample pad that has combined collaurum, and the centre is nitrocellulose filter (a NC film), has wherein sprayed quality control reagent by the upper end; Like goat anti-rabbit antibody; It is anti--2 to have sprayed rabbit by the lower end, the test strips surface coverage thin film, shield.
When prepared immune golden quick detection test paper is used for on-the-spot the detection, test strips is kept flat on the table, add the sample of 100uL at the application of sample end; Sample at first dissolves the gold mark rabbit that is sprayed on the glass fibre membrane and resists-1, and solution moves toward the NC film then, when the rabbit that runs into detection line (T) resists-2; If contain vibrio parahaemolytious in the sample; Then form sandwich reaction, promptly anti--1-vibrio parahaemolytious of gold mark rabbit-rabbit resists-2, on detection line and control line (C), all manifests a purplish red colo(u)r streak; If do not contain vibrio parahaemolytious in the sample; A purplish red colo(u)r streak then only on control line, occurs, if a purplish red colo(u)r streak does not all appear in detection line and control line, this test strips is invalid test strips so.
Embodiment 1
The anti-vibrio parahaemolytious antibody labeling of rabbit is got the colloidal gold solution that needs preparation amount, with 0.1 mol/L K
2CO
3Transfer its pH to 8.5 with the HCl of 0.1 mol/L.The anti-vibrio parahaemolytious antibody of rabbit that under magnetic agitation, dropwise adds purifying then is (with the rabbit antibody standard gold of adjuvant immunity; Every mL colloidal gold solution adds the anti-vibrio parahaemolytious antibody of rabbit 16.7ug); Continue to add 5%PEG (MW 6000) to final concentration 0.1% behind magnetic agitation 30 min; Continue again to stir 15 min, place 4 ℃ of refrigerators at last, hold over night.The centrifugal 10min of anti-vibrio parahaemolytious antibody colloidal gold 4000 r/min of rabbit that next day is good with mark gets centrifugal 30 min of supernatant 15000 r/min, and careful the suction abandoned supernatant.Deposition returns to 1/20th of original volume with resuspended damping fluid (containing 1% casein, 0.05% PEG6000,0.05% Tween-20, the PBS damping fluid of 0.01 mol/L pH7.2), and it is subsequent use to put 4 ℃ of refrigerators.
Embodiment 2
The reaction test strips consists of: size is about 5.0 cm ~ 6.0cm * 0.4cm ~ 0.6 cm, specifically consists of: the bottom is the PVC offset plate, and the upper end of test strips is a thieving paper; The lower end is glass fibre membrane and the sample pad that has combined collaurum, and the centre is nitrocellulose filter (a NC film), has wherein sprayed quality control reagent by the upper end; Like goat anti-rabbit igg or SPA; It is anti--2 to have sprayed rabbit by the lower end, the test strips surface coverage thin film, shield.
Embodiment 3
The formulation selection experiment of confining liquid:
The NC film that is coated with T, C line seals with following 8 kinds of confining liquids respectively:
(1) PBS of the 0.01mol/L pH7.2 of 1%BSA, 0.5%Tween-20
(2) PBS of the 0.01 mol/L pH7.2 of 0.5%BSA, 0.5%Tween-20
(3) PBS of the 0.01 mol/L pH7.2 of 1%BSA, 0.05%Tween-20
(4) PBS of the 0.01 mol/L pH7.2 of 0.5%BSA, 0.05%Tween-20;
(5) PBS of the 0.01 mol/L pH7.2 of 1%casein, 0.5%Tween-20;
The PBS of the 0.01 mol/L pH7.2 of (6) 0.5% casein, 0.5%Tween-20;
The PBS of the 0.01 mol/L pH7.2 of (7) 1% casein, 0.05% Tween-20;
The PBS of the 0.01 mol/L pH7.2 of (8) 0.5% casein, 0.05%Tween-20.
Through experiment test repeatedly, obtain the result and be: adopt with the PBS sealing effect of the 0.01 mol/L pH7.2 that contains 1% casein, 0.05%Tween-20 preferably, the spot colour developing is clear, and background is more shallow.
Embodiment 4
The AC that encapsulates rabbit anti--2 through detection line is 0,24.32,48.64,72.96,97.28,6 concentration gradients of 121.6mg/ml; When the volume of collaurum rabbit anti--1 is mark 1/5,1/10,1/15 of the collaurum volume and 1/20 o'clock; Be assembled into the square formation test strips, the vibrio parahaemolytious bacteria suspension with 2 times of serial dilutions detects then.Anti--2 concentration of detection line rabbit are that the above detection line colour developing of 72.96mg/ml is clearer as a result; When the volume of collaurum rabbit anti--1 is mark 1/15 o'clock of the collaurum volume, detection line, the control line colour developing was clear and background color is more shallow.Therefore finally confirm that the concentration of the detection line rabbit anti--2 of test strips is 72.96mg/ml, anti--1 dilution of collaurum rabbit for labelled precursor amass 1/15, sensitivity can reach 2.6 * 10
3Cfu/ml.
Same; Control line encapsulates staphylococcal protein A; Concentration is 0,2,4,6,8,6 concentration gradients of 10mg/ml, and anti--1 concentration of confirming with the front again of collaurum rabbit is reacted, and drawing the least concentration that control line encapsulates staphylococcal protein A at last is 6mg/ml.
Control line encapsulates goat anti-rabbit igg, and concentration is 6 concentration gradients of 2,3,4,5,6,7 mg/ml, and anti--1 concentration of confirming with the front again of collaurum rabbit is reacted, and drawing the least concentration that control line encapsulates staphylococcal protein A at last is 4mg/ml.
Embodiment 5
The stability test experiment of test strips:
Follow-on test 6 months, test period, detecting the control line (C) of vibrio parahaemolytious bacteria suspension appearance and the color of detection line (T) does not all have ANOMALOUS VARIATIONS, and this explanation test strips is stable strong.
Through detecting, adopt the golden quick detection test paper of vibrio parahaemolytious immunity of the present invention's preparation to have excellent specificity and stability, specific as follows:
The specificity of the golden quick detection test paper of vibrio parahaemolytious immunity:
Detect 7 strain bacterial strains such as vibrio parahaemolytious, vibrio alginolyticus, comma bacillus, mermaid vibrios, Maxwell vibrios, Fu Shi citrobacter, salmonella; The control line (C) and the detection line (T) that have only vibrio parahaemolytious; Aubergine all occurs, show positive findings; And all the other 6 strain bacterial strains have only control line (C) aubergine to occur, and aubergine does not appear in detection line (T), show negative findings.
The stability of the golden quick detection test paper of vibrio parahaemolytious immunity
Follow-on test 6 months, test period, detecting the control line (C) of vibrio parahaemolytious bacteria suspension appearance and the color of detection line (T) does not all have ANOMALOUS VARIATIONS, and this explanation test strips is stable strong.
The variation that is appreciated that a lot of details is possible, but therefore this do not run counter to scope of the present invention and spirit, and the those of ordinary skill of any affiliated technical field all should be regarded as not breaking away from the category of patent of the present invention to its suitable variation of doing.
Claims (6)
1. the preparation method of the golden quick detection test paper of vibrio parahaemolytious immunity is characterized in that, may further comprise the steps:
A) preparation of vibrio parahaemolytious antigen: with peptone water medium with the vibrio parahaemolytious overnight incubation;
B) make the vibrio parahaemolytious inactivated vaccine;
C) prepare polyvalent antiserum with the vaccine immunity experimental rabbit: adopt the direct injecting immune rabbit of subcutaneous multi-point injection and vein to prepare the many antiantibodys of the anti-vibrio parahaemolytious of rabbit, obtain the anti-vibrio parahaemolytious antibody of rabbit of subcutaneous multi-point injection preparation and the anti-vibrio parahaemolytious antibody of rabbit that vein is directly injected preparation respectively;
D) antiserum purifying;
E) colloid gold label: the anti-vibrio parahaemolytious antibody of rabbit of the subcutaneous multi-point injection preparation of purifying is carried out mark with collaurum;
F) preparation of immune gold test paper strip: the anti-vibrio parahaemolytious antibody of rabbit that preparation is directly injected with the vein of purifying in the step d) by elder generation is sprayed on the miillpore filter detection line; Goat anti-rabbit antibody is sprayed on the control line; The anti-vibrio parahaemolytious antibody of rabbit with the subcutaneous multi-point injection preparation of the colloid gold label for preparing in the step e) is sprayed on the glass fibre membrane again, is assembled into immune gold test paper strip then.
2. the preparation method of the golden quick detection test paper of vibrio parahaemolytious immunity according to claim 1; It is characterized in that; Described step b); Make the vibrio parahaemolytious inactivated vaccine, be specially: earlier through 37 ℃ of deactivation 24h of formaldehyde of 0.3%, then through cultivate detect certain no viable bacteria after as antigen.
3. the preparation method of the golden quick detection test paper of vibrio parahaemolytious immunity according to claim 1; It is characterized in that; Described step c); Prepare polyvalent antiserum with the vaccine immunity experimental rabbit, adopt the direct injecting immune rabbit of subcutaneous multi-point injection and vein to prepare the many antiantibodys of the anti-vibrio parahaemolytious of rabbit, be specially:
1) intravenous immune: injection in per 10 days once, 1mL first, 1.5mL for the second time, 2mL for the third time, the 4th 3mL, bloodletting when reaching 1:1280 or 1:2560 when tiring;
2) the subcutaneous multi-point injection of Freund immunity: Freund's complete adjuvant is with after bacterium liquid equal-volume mixes; The subcutaneous multi-point injection 4mL of rabbit nape portion; After 2 weeks, with incomplete Freund and the bacterium liquid equal-volume mixed liquor nape subcutaneous multi-point injection 4mL of portion, after 2 weeks; 5-7 days intravenous injection 4mL before the blood sampling, bloodletting when reaching 1:1280 or 1:2560 when tiring.
4. the preparation method of the golden quick detection test paper of vibrio parahaemolytious immunity according to claim 1 is characterized in that, described step f), and the preparation of immune gold test paper strip is specially:
With the anti-vibrio parahaemolytious antibody of rabbit of the subcutaneous multi-point injection preparation of colloid gold label, set up the method for colloidal gold immunochromatographimethod fast detecting vibrio parahaemolytious; Use miillpore filter and make the immune detection of carrier; The anti-vibrio parahaemolytious antibody of rabbit that preparation is directly injected with vein by elder generation is sprayed on the miillpore filter detection line; Goat anti-rabbit antibody is sprayed on the control line; The anti-vibrio parahaemolytious antibody of rabbit of the subcutaneous multi-point injection preparation of colloid gold label is sprayed on the glass fibre membrane, and dry back adds confining liquid on miillpore filter, is assembled into behind the drying liquid and detects the vibrio parahaemolytious test paper; Amplify immune response system through gold grain again, reaction result is shown on the solid phase carrier miillpore filter.
5. the preparation method of the golden quick detection test paper of vibrio parahaemolytious immunity according to claim 4; It is characterized in that; Said confining liquid prescription adopts: the concentration that contains 1% (W/V) casein and 0.05% (V/V) Tween-20 is the PBS solution of 0.01 mol/L, and the pH value of PBS solution is 7.2.
6. the preparation method of the golden quick detection test paper of vibrio parahaemolytious immunity according to claim 1 is characterized in that, when prepared immune golden quick detection test paper is used for on-the-spot the detection; Test strips is kept flat on the table; Add that at the application of sample end sample of 100uL, sample at first dissolve the anti-vibrio parahaemolytious antibody of gold mark rabbit that is sprayed on the glass fibre membrane, solution moves toward miillpore filter then; When the anti-vibrio parahaemolytious antibody of the rabbit that runs into detection line, judge that detection paper result's standard is following:
Contain vibrio parahaemolytious in the sample: on detection line and control line, all manifest a purplish red colo(u)r streak;
Do not contain vibrio parahaemolytious in the sample: a purplish red colo(u)r streak only on control line, occurs;
Test paper is invalid: all do not occur a purplish red colo(u)r streak on detection line and the control line.
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| CN103134933A (en) * | 2013-02-05 | 2013-06-05 | 江西中德生物工程有限公司 | Test strip used for detecting vibrio parahemolytocus and purpose thereof |
| CN103773851A (en) * | 2013-12-10 | 2014-05-07 | 天津出入境检验检疫局动植物与食品检测中心 | Preparation and application of nucleic acid sidewise-flow test strip detection kit for detecting Bibrio parahemolyticus |
| CN104316690A (en) * | 2014-11-03 | 2015-01-28 | 上海理工大学 | Rapid vibrio parahaemolyticus immune colloidal gold test strip |
| CN105445465A (en) * | 2015-11-16 | 2016-03-30 | 珠海国际旅行卫生保健中心 | Fluorescent nano-immunochromatography kit for quickly detecting vibrio parahaemolyticus and preparation method |
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| CN106645718A (en) * | 2017-03-11 | 2017-05-10 | 中国海洋大学 | Rapid test paper for pathogenic Vibrio parahaemolyticus |
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| 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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