CN105548551A - Method for rapidly detecting vibrio parahaemolyticus - Google Patents

Method for rapidly detecting vibrio parahaemolyticus Download PDF

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CN105548551A
CN105548551A CN201610032264.4A CN201610032264A CN105548551A CN 105548551 A CN105548551 A CN 105548551A CN 201610032264 A CN201610032264 A CN 201610032264A CN 105548551 A CN105548551 A CN 105548551A
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赖卫华
王景云
刘道峰
邓省亮
山珊
熊勇华
魏华
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Nanchang University
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Abstract

The invention provides a detection method for rapidly detecting vibrio parahaemolyticus. According to the scheme, bacteria are enriched by Fe3O4/Ru(bqy) 3<2+> microspheres, a test strip is prepared, and a sample is loaded for detection. The step of eluting the vibrio parahaemolyticus from immunomagnetic beads is eliminated, so that the capturing efficiency is improved; the step of spraying the Fe3O4/Ru(bqy) 3<2+> microspheres onto a conjugate pad is eliminated, so that immunological reaction is more uniform, and a variation coefficient during quantitative detection is small; the workload and the sundry fungus contamination probability are reduced; the detection scheme is high in sensitivity and stability.

Description

A kind of method of quick detection vibrio parahemolyticus
Technical field
The present invention relates to microorganism detection field, specifically adopt Fe 3o 4/ Ru (bqy) 3 2+nano microsphere integrated immune magnetic capture technology and immunochromatography detect vibrio parahemolyticus fast.
Technical background
Vibrio parahemolyticus is one of common food-borne pathogens, and this bacterium is mainly derived from the marine products such as shrimp, fish and shellfish.According to the report of national food origin disease monitoring net, the food poisoning caused by vibrio parahemolyticus to China since 1998 is in rising trend, and exceedes Salmonella food poisoning and leap to the first, and becomes the most serious foodborne bacterial pathogens.The marine product that the mankind eat the infection of this bacterium can cause the gastrointestinal disease such as enterogastritis and septicemia, is the primary pathogen in the food poisoning case of China's part coastland.
Find vibrio parahemolyticus so far from nineteen fifty, detect the method for this bacterium both at home and abroad still based on normal isolation culture method.This method complicated operation, sense cycle long (4-7d), can not adapt to the needs that hygienic emergency detects; ELISA method, PCR method and hexavalent chrome bio-removal are high to vibrio parahemolyticus detection sensitivity, but they need longer detection time, expensive instrument and technical professional.Therefore it is quick, the sensitive detection method of necessary foundation.
Colloidal gold immuno-chromatography test paper strip (10-15min) simple to operate with it, quick, the feature such as accurate become the important tool of basic unit's screening, but due to the optical signalling of collaurum limited, the sensitivity of colloidal gold immuno-chromatography test paper strip is not high, usual not higher than 10 to the detectability of vibrio parahemolyticus 5cFU/mL, its application in the food and detection of agricultural products of this drawbacks limit.Therefore, the detection for vibrio parahemolyticus provides simply by the sensitivity improving test strips, efficient approach.
Summary of the invention
The object of the invention is to provide a kind of quick, sensitive, easy vibrio parahemolyticus qualitative and quantitative analysis technology.
Concrete scheme of the present invention is as follows:
A method for quick detection vibrio parahemolyticus, comprises the following steps:
1) preparation of Nano microsphere:
A. 0.4-0.8mmolFeCl is added 36H 2o and 0.2-1.6mmolFeCl 24H 2in the deionized water of O to 100mL, in solution, pass into nitrogen and be heated to 80-120 DEG C, then by the NH of 3-7mL25% 3h 2o adds in mixed liquor, reaction 2h; The solid matter high purity water isolating black with permanent magnet from reaction solution cleans 3 ~ 5 times, obtains Fe 3o 4nano particle;
B. 12mgFe is got 3o 4the mixed liquor of nano particle 1-10mL deionized water and 20mL ethanol is resuspended, under the condition slowly stirred, first add the NH of 0.3-0.9mL 4oH solution, then 10-300uL ethyl orthosilicate is dissolved in 50uL ethanolic solution dropwise adds, react 12h under room temperature, at Fe 3o 4nanoparticle surface forms layer of silicon dioxide, cleans several times with deionized water solution, and in ethanolic solution, redissolution obtains the Fe of coated with silica 3o 4nano particle;
C. the ethyl orthosilicate of 10-300uL, 20mL absolute ethyl alcohol, the phenanthroline connection ruthenium (Ru (bqy) of 1-10mL deionized water and 1mL0.5-3mg/L 3 2+) mixing, mixed solution is added the Fe of 0.5mL coated with silica 3o 4nano particle, finally adds the NH of 600-900 μ L 4oH, vigorous stirring 3h, obtain Fe 3o 4/ Ru (bqy) 3 2+nano microsphere, for subsequent use by washed with de-ionized water;
D. the mercaptopropyl trimethoxysilane of 1mL is added in the ethanolic solution of 10mL, with this potpourri redissolution Fe 3o 4/ Ru (bqy) 3 2+nano microsphere, after 300rpm stirs 12h at normal temperatures, then 80 DEG C of 300rpm stir 1h, the centrifugal Fe obtaining silanization 3o 4/ Ru (bqy) 3 2+nano microsphere;
E. by the Fe of silanization 3o 4/ Ru (bqy) 3 2+nano microsphere adds to and comprises 0.06gNaHCO 3, 0.08g neopelex, 0.05mL styrene, the aqueous solution of the 50mL of 0.15mL acrylic acid and 0.5g potassium persulfate solution, water-bath 70 DEG C, stirs under 200r/min, obtains carboxylated Fe after reaction 5h 3o 4/ Ru (bqy) 3 2+nano microsphere;
2) Fe that 0.5-2mg is carboxylated is got 3o 4/ Ru (bqy) 3 2+nano microsphere adds in 1mL coupling buffer, regulate pH to 5-10, add 0.05-0.18mg1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) activated carboxyl, and the anti-vibrio parahemolyticus monoclonal antibody of 50-300 μ g, when temperature 37 DEG C, coupling 60-120min on the gyroscope being placed on 10-15rpm, Magneto separate 3-5min, abandons supernatant; Rinse 3-5 all over afterwards with cleaning buffer solution, get 1mL sealer and mix closed 0.5-1h with Nano microsphere, obtain Fe 3o 4/ Ru (bqy) 3 2+nano microsphere-monoclonal antibody;
Described coupling buffer compound method is as follows: after being mixed with the boric acid of 7mL12.37g/mL by the borax of 3mL19.07g/mL, dilutes 10 times of volumes;
Described cleaning buffer solution compound method is as follows: take 0.43g2-(N-morpholine) ethyl sulfonic acid (MES) and be dissolved in the sterile distilled water of 200mL, tune pH is 5.5-6.0;
Described sealer compound method is as follows: get 100mg bovine serum albumin(BSA) (BSA) and add 1mL phosphate (PBS) damping fluid and be made into sealer;
3) cultivating vibrio parahemolyticus, is 10 by bacterium liquid adjustment concentration 6cFU/mL, 10 5cFU/mL, 10 4cFU/mL, 10 3cFU/mL, respectively gets 1mL for subsequent use; Get testing sample solution 1mL, each concentration bacterium liquid 1mL, respectively with 100-150 μ gFe 3o 4/ Ru (bqy) 3 2+nano microsphere-monoclonal antibody, in temperature 37 DEG C, 30-60min is hatched in gyroscope rotating speed 10-15rpm mixing, after hatching rear Magneto separate 3-5min, abandons supernatant, and after PBS buffer solution for cleaning, redissolving must Fe in PBS 3o 4/ Ru (bqy) 3 2+nano microsphere-monoclonal antibody-bacterium;
4) preparation of immuno-chromatographic test paper strip: by sample pad pH8.50.1MTris-HCl damping fluid (1%BSA, 0.5%Tween-20) immersion treatment, is placed in 60 DEG C of air dry ovens, takes out for subsequent use after 2h; Vibrio parahemolyticus rabbit resisting is sprayed onto on nitrocellulose membrane as detection line (T line) and nature controlling line (C line) with donkey against murine two is anti-more, concentration is 1-2mg/mL, discharge rate is 0.75uL/cm, and 37 DEG C of dried in vacuo overnight take out that to be placed in dry cylinder for subsequent use; Filter pad, sample pad, nitrocellulose membrane, thieving paper are pasted onto successively on PVC base plate, are cut into the test strips that 4mm is wide after posting, are installed; The test strips prepared is loaded in aluminium foil bag, adds drying agent sealing, be placed in dry cylinder and save backup;
5) test strips is to the detection of sample: by the Fe collected 3o 4/ Ru (bqy) 3 2+nano microsphere-monoclonal antibody-bacterium is diluted to 50-150 μ g/mL, gets 100 μ L and is added drop-wise in test strips well, after 10-15min, by the value of fluorescence reader record T line, C line fluorescence intensity and T/C;
6) qualitative analysis: the result that detects by an unaided eye carries out qualitative analysis, T line develops the color, and has vibrio parahemolyticus in interpret sample, and T line does not develop the color, and does not have vibrio parahemolyticus or the amount containing vibrio parahemolyticus lower than 10 in interpret sample 3cFU/mL;
7) quantitative test: use fluorescence reader to measure the fluorescence intensity of T line, C line, and T/C value, with the concentration of different bacterium for horizontal ordinate, T/C value is ordinate drawing standard curve, determines the vibrio parahemolyticus quantity in common sample.
Described step 1) Fe 3o 4/ Ru (bqy) 3 2+nano microsphere particle diameter is 80-210nm;
Step 3) described immuno-chromatographic test paper strip be paste successively in adhesive base filter paper, sample pad, nitrocellulose membrane, thieving paper composition.Use vibrio parahemolyticus Fe 3o 4/ Ru (bqy) 3 2+nano microsphere immuno-chromatographic test paper strip, use the method that fluorescence reader quantitatively detects simultaneously, it is characterized in that: the vibrio parahemolyticus solution preparing known series concentration, the fluorescence intensity of its correspondence is measured by fluorescence reader, according to this series of values and corresponding concentration Criterion curve, then the test strips detecting sample is put into fluorescence reader, according to the numerical value that fluorescence reader exports, look into the content that canonical plotting can draw vibrio parahemolyticus in sample.
The present invention has following advantage:
1) the present invention has simple to operate, detection time is short advantages such as (10-15min), is suitable for carrying out Site Detection;
2) technical solution of the present invention detects good stability, and detection sensitivity is high, and detectability can reach 10 3cFU/mL.The Fe adopted 3o 4/ Ru (bqy) 3 2+nano microsphere, not only has the superparamagnetic performance of magnetic nano-particle, carries out enrichment method to sample, and has Ru (bqy) 3 2+the optical signalling that Nano microsphere is strong, Ru (bqy) 3 2+the performance of the efficient coupled antibody in Nano microsphere surface, thus improve the detection sensitivity of test strips.
3) the present invention is without the need to the step eluted from immunomagnetic beads by vibrio parahemolyticus, improves capture rate; Eliminate the step be sprayed on by immune marker on pad, immunological response is more homogeneous, and when quantitatively detecting, the coefficient of variation is little; Decrease workload and living contaminants probability.
4) can carry out qualitative to object vibrio parahemolyticus simultaneously and quantitatively detect.
5) label of the present invention is carboxylated Fe 3o 4/ Ru (bqy) 3 2+nano microsphere, this label is mainly through the mode labelled antibody of chemical coupling, compare traditional collaurum (physisorption), more multispecific antibody can be caught, thus raising detection sensitivity, in addition, the rock-steady structure of this label carboxyl modified can improve the stability of material, improving storage life is 1 year, and traditional collaurum storage life is 6 months.
6) Fe of the present invention 3o 4/ Ru (bqy) 3 2+nano microsphere is due to Fe in core 3o 4the effect of nano particle, can better prevent fluorescent dye Ru (bqy) in shell 3 2+leakage, thus improve fluorescence intensity.
Accompanying drawing explanation
Fig. 1 Fe of the present invention 3o 4/ Ru (bqy) 3 2+nano microsphere immuno-chromatographic test paper strip is to the detection of vibrio parahemolyticus
Embodiment
Fe prepared by technical solution of the present invention 3o 4/ Ru (bqy) 3 2+nano microsphere and the coupling of vibrio parahemolyticus monoclonal antibody, prepare immune Fe 3o 4/ Ru (bqy) 3 2+nano microsphere, and be applied to immuno-chromatographic test paper strip vibrio parahemolyticus is detected.This test strips is based on the pattern of double antibodies sandwich, nitrocellulose membrane sprays vibrio parahemolyticus rabbit respectively how anti-anti-as detection line and nature controlling line with donkey against murine two, if in sample containing certain density vibrio parahemolyticus time, vibrio parahemolyticus will first with immune Fe 3o 4/ Ru (bqy) 3 2+nano microsphere combines and forms Fe 3o 4/ Ru (bqy) 3 2+nano microsphere-Antibody-antigen complex, this complex logistics after testing line anti-is captured in detection line region clustering by vibrio parahemolyticus rabbit is many, gathers finite concentration and forms the signal that macroscopic band or test strips reader can detect, unnecessary immune Fe 3o 4/ Ru (bqy) 3 2+nano microsphere moves to nature controlling line and is assembled the macroscopic band of formation by anti-the catching of donkey against murine two, judges that it is positive, if not containing thing to be checked in sample, and immune Fe 3o 4/ Ru (bqy) 3 2+nano microsphere only should form macroscopic band with donkey against murine two anti-reflective on control line, and detection line does not develop the color, and judges that it is negative.If nature controlling line place does not have color or do not have clear signal, illustrate that test strips is defective in quality, test invalidation.
Embodiment is provided below in conjunction with technical scheme of the present invention.The form example that following examples operate with specific experiment the solution of the present invention, experiment condition wherein and setup parameter should not be considered as the limitation to basic technical scheme of the present invention.And protection scope of the present invention is not limited to following embodiment.
Fluorescence reader is purchased from Shanghai Hu Guo tech equipment company limited.
Coupling buffer compound method is as follows: after the boric acid be borax and the 7mL concentration of 19.07g/mL by 3mL concentration being 12.37g/mL mixes, dilute 10 times of volumes;
Cleaning buffer solution compound method is as follows: take 0.43g2-(N-morpholine) ethyl sulfonic acid (MES) and be dissolved in the sterile distilled water of 200mL, tune pH is 5.5-6.0;
Sealer compound method is as follows: get 100mg bovine serum albumin(BSA) (BSA) and add 1mL phosphate (PBS) damping fluid and be made into sealer;
Embodiment one: use Fe 3o 4/ Ru (bqy) 3 2+nano microsphere immuno-chromatographic test paper strip is to the detection of vibrio parahemolyticus in milk
1. prepare the Fe of coupling monoclonal antibody 3o 4/ Ru (bqy) 3 2+nano microsphere
1.1Fe 3o 4/ Ru (bqy) 3 2+the preparation of Nano microsphere: add 0.6mmolFeCl 36H 2o and 0.3mmolFeCl 24H 2in the deionized water of O to 100mL, in solution, pass into nitrogen and be heated to 90 DEG C, then by the NH of 4.7mL25% 3h 2o adds in mixed liquor, reaction 2h.The solid matter high purity water isolating black with permanent magnet from reaction solution cleans 3 ~ 5 times, obtains Fe 3o 4nano particle; Get 12mgFe 3o 4the mixed liquor of nano particle 3mL deionized water and 20mL ethanol is resuspended, under the condition slowly stirred, first add the NH of 0.5mL 4oH solution, then 50uL ethyl orthosilicate is dissolved in 50uL ethanolic solution dropwise adds, react 12h under room temperature, at Fe 3o 4nanoparticle surface forms layer of silicon dioxide, cleans several times with deionized water solution, and in ethanolic solution, redissolution obtains the Fe of coated with silica 3o 4nano particle; The ethyl orthosilicate of 100uL, 20mL absolute ethyl alcohol, the phenanthroline connection ruthenium (Ru (bqy) of 3mL deionized water and 1mL0.5-3mg/L 3 2+) mixing, mixed solution is added the Fe of 0.5mL coated with silica 3o 4nano particle, finally adds the NH of 750 μ L 4oH, vigorous stirring 3h, obtain Fe 3o 4/ Ru (bqy) 3 2+nano microsphere, for subsequent use by washed with de-ionized water; The mercaptopropyl trimethoxysilane of 1mL is added in the ethanolic solution of 10mL, with this potpourri redissolution Fe 3o 4/ Ru (bqy) 3 2+nano microsphere, after 300rpm stirs 12h at normal temperatures, then 80 DEG C of 300rpm stir 1h, the centrifugal Fe obtaining silanization 3o 4/ Ru (bqy) 3 2+nano microsphere; By the Fe of silanization 3o 4/ Ru (bqy) 3 2+nano microsphere adds to and comprises 0.06gNaHCO 3, 0.08g neopelex, 0.05mL styrene, the aqueous solution of the 50mL of 0.15mL acrylic acid and 0.5g potassium persulfate solution, water-bath 70 DEG C, stirs under 200r/min, obtains carboxylated Fe after reaction 5h 3o 4/ Ru (bqy) 3 2+nano microsphere;
1.2. coupling reaction: get the Fe that 1.0mg is carboxylated 3o 4/ Ru (bqy) 3 2+nano microsphere adds in 1mL coupling buffer, regulate pH to 8, add 0.05-0.18mg1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) activated carboxyl, and the anti-vibrio parahemolyticus monoclonal antibody of 150 μ g, when temperature 37 DEG C, coupling 60-120min on the gyroscope being placed on 10-15rpm, Magneto separate 3-5min, abandons supernatant; Rinse 3-5 all over afterwards with cleaning buffer solution, get 1mL sealer and mix closed 0.5-1h with magnetic bead, obtain Fe 3o 4/ Ru (bqy) 3 2+nano microsphere-monoclonal antibody.
2. use immune Fe 3o 4/ Ru (bqy) 3 2+nano microsphere catches the vibrio parahemolyticus in milk
After getting the sterilizing of 25mL, milk joins in 225mL nutrient culture media, inoculates certain density vibrio parahemolyticus, and under the condition of 36 DEG C, 8-18h is cultivated in concussion.Bacterial concentration is adjusted to 10 6cFU/mL, 10 5cFU/mL, 10 4cFU/mL, 10 3cFU/mL.
Get 1mL each concentration bacterium liquid, 1mL testing sample solution, add 120 μ gFe respectively 3o 4/ Ru (bqy) 3 2+nano microsphere-monoclonal antibody, temperature 37 DEG C, 30-60min is hatched in rotating speed 10-15rpm mixing; After hatching rear Magneto separate 3-5min, abandon supernatant, after PBS buffer solution for cleaning, redissolving must Fe in PBS 3o 4/ Ru (bqy) 3 2+nano microsphere-monoclonal antibody-bacterium.
3. make vibrio parahemolyticus immuno-chromatographic test paper strip
By sample pad pH8.50.1MTris-HCl damping fluid (1%BSA, 0.5%Tween-20) process, be placed in 60 DEG C of air dry ovens, after 2h, take out that to be placed in dry cylinder for subsequent use; Using many for vibrio parahemolyticus rabbit anti-and donkey against murine two is anti-is sprayed onto as detection line and nature controlling line on nitrocellulose membrane, concentration is 1.0mg/mL, and discharge rate is 0.75uL/cm, and 37 DEG C of dried in vacuo overnight take out that to be placed in dry cylinder for subsequent use; Filter pad, sample pad, nitrocellulose membrane, thieving paper are pasted onto successively on PVC base plate, are cut into the test strips that 4mm is wide after posting, are installed.The test strips prepared is loaded in aluminium foil bag, adds drying agent sealing, be placed in dry cylinder and save backup.
4. utilize double-antibody method estimate sample and use Instrumental results
The Fe collected will be caught 3o 4/ Ru (bqy) 3 2+nano microsphere-monoclonal antibody-bacterium is diluted to 100 μ g/mL, gets 100 μ L and is added drop-wise in test strips well, after 10-15min, by the value of fluorescence reader record T line, C line fluorescence intensity and T/C; The result that detects by an unaided eye carries out qualitative analysis, and T line develops the color, and has vibrio parahemolyticus in interpret sample, and T line does not develop the color, and does not have vibrio parahemolyticus or the amount containing vibrio parahemolyticus lower than 10 in interpret sample 3cFU/mL.
With reference to the canonical plotting done, determine the quantity of vibrio parahemolyticus in sample.The scope of quantitative testing bacteria concentration is 10 3-10 6cFU/mL.The inventive method detects stable, and detection line can be low to moderate 10 3cFU/mL, speed is fast, effective.
Embodiment two: use Fe 3o 4/ Ru (bqy) 3 2+nano microsphere immuno-chromatographic test paper strip is to the detection of vibrio parahemolyticus in beef
1. prepare the Fe of coupling monoclonal antibody 3o 4/ Ru (bqy) 3 2+nano microsphere
1.1Fe 3o 4/ Ru (bqy) 3 2+the preparation of Nano microsphere: add 0.6mmolFeCl 36H 2o and 0.3mmolFeCl 24H 2in the deionized water of O to 100mL, in solution, pass into nitrogen and be heated to 90 DEG C, then by the NH of 4.7mL25% 3h 2o adds in mixed liquor, reaction 2h.The solid matter high purity water isolating black with permanent magnet from reaction solution cleans 3 ~ 5 times, obtains Fe 3o 4nano particle; Get 12mgFe 3o 4the mixed liquor of nano particle 3mL deionized water and 20mL ethanol is resuspended, under the condition slowly stirred, first add the NH of 0.5mL 4oH solution, then 50uL ethyl orthosilicate is dissolved in 50uL ethanolic solution dropwise adds, react 12h under room temperature, at Fe 3o 4nanoparticle surface forms layer of silicon dioxide, cleans several times with deionized water solution, and in ethanolic solution, redissolution obtains the Fe of coated with silica 3o 4nano particle; The ethyl orthosilicate of 100uL, 20mL absolute ethyl alcohol, the phenanthroline connection ruthenium (Ru (bqy) of 3mL deionized water and 1mL0.5-3mg/L 3 2+) mixing, mixed solution is added the Fe of 0.5mL coated with silica 3o 4nano particle, finally adds the NH of 750 μ L 4oH, vigorous stirring 3h, obtain Fe 3o 4/ Ru (bqy) 3 2+nano microsphere, for subsequent use by washed with de-ionized water; The mercaptopropyl trimethoxysilane of 1mL is added in the ethanolic solution of 10mL, with this potpourri redissolution Fe 3o 4/ Ru (bqy) 3 2+nano microsphere, after 300rpm stirs 12h at normal temperatures, then 80 DEG C of 300rpm stir 1h, the centrifugal Fe obtaining silanization 3o 4/ Ru (bqy) 3 2+nano microsphere; By the Fe of silanization 3o 4/ Ru (bqy) 3 2+nano microsphere adds to and comprises 0.06gNaHCO 3, 0.08g neopelex, 0.05mL styrene, the aqueous solution of the 50mL of 0.15mL acrylic acid and 0.5g potassium persulfate solution, water-bath 70 DEG C, stirs under 200r/min, obtains carboxylated Fe after reaction 5h 3o 4/ Ru (bqy) 3 2+nano microsphere;
1.2. coupling reaction: get the Fe that 1.0mg is carboxylated 3o 4/ Ru (bqy) 3 2+nano microsphere adds in 1mL coupling buffer, regulate pH to 8, add 0.05-0.18mg1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) activated carboxyl, and the anti-vibrio parahemolyticus monoclonal antibody of 150 μ g, when temperature 37 DEG C, coupling 60-120min on the gyroscope being placed on 10-15rpm, Magneto separate 3-5min, abandons supernatant; Rinse 3-5 all over afterwards with cleaning buffer solution, get 1mL sealer and mix closed 0.5-1h with magnetic bead, obtain Fe 3o 4/ Ru (bqy) 3 2+nano microsphere-monoclonal antibody.
2. use the Fe of coupling monoclonal antibody 3o 4/ Ru (bqy) 3 2+nano microsphere catches the vibrio parahemolyticus in beef
The beef meat gruel getting the sterilizing of 25mg joins in 225mL nutrient culture media, inoculates certain density vibrio parahemolyticus, and under the condition of 36 DEG C, 8-18h is cultivated in concussion.Bacterial concentration is adjusted to 10 6cFU/mL, 10 5cFU/mL, 10 4cFU/mL, 10 3cFU/mL.
Get 1mL each concentration bacterium liquid, 1mL testing sample solution, add 120 μ gFe respectively 3o 4/ Ru (bqy) 3 2+nano microsphere-monoclonal antibody, temperature 37 DEG C, 30-60min is hatched in rotating speed 10-15rpm mixing; After hatching rear Magneto separate 3-5min, abandon supernatant, after PBS buffer solution for cleaning, redissolving must Fe in PBS 3o 4/ Ru (bqy) 3 2+nano microsphere-monoclonal antibody-bacterium.
3. make vibrio parahemolyticus immuno-chromatographic test paper strip
By sample pad pH8.50.1MTris-HCl damping fluid (1%BSA, 0.5%Tween-20) process, be placed in 60 DEG C of air dry ovens, after 2h, take out that to be placed in dry cylinder for subsequent use; Using many for vibrio parahemolyticus rabbit anti-and donkey against murine two is anti-is sprayed onto as detection line and nature controlling line on nitrocellulose membrane, concentration is 1.0mg/mL, and discharge rate is 0.75uL/cm, and 37 DEG C of dried in vacuo overnight take out that to be placed in dry cylinder for subsequent use; Filter pad, sample pad, nitrocellulose membrane, thieving paper are pasted onto successively on PVC base plate, are cut into the test strips that 4mm is wide after posting, are installed.The test strips prepared is loaded in aluminium foil bag, adds drying agent sealing, be placed in dry cylinder and save backup.
4. utilize double-antibody method estimate sample and use Instrumental results
The Fe collected will be caught 3o 4/ Ru (bqy) 3 2+nano microsphere-monoclonal antibody-bacterium is diluted to 100 μ g/mL, gets 100 μ L and is added drop-wise in test strips well, after 10-15min, by the value of fluorescence reader record T line, C line fluorescence intensity and T/C; The result that detects by an unaided eye carries out qualitative analysis, and T line develops the color, and has vibrio parahemolyticus in interpret sample, and T line does not develop the color, and does not have vibrio parahemolyticus or the amount containing vibrio parahemolyticus lower than 10 in interpret sample 3cFU/mL.
With reference to the canonical plotting done, determine the quantity of vibrio parahemolyticus in sample.The scope of quantitative testing bacteria concentration is 10 3-10 6cFU/mL.The inventive method detects stable, and detectability can be low to moderate 10 3cFU/mL, speed is fast, effective.

Claims (3)

1. detect a method for vibrio parahemolyticus fast, it is characterized in that comprising the following steps:
1) preparation of Nano microsphere:
A. 0.4-0.8mmolFeCl is added 36H 2o and 0.2-1.6mmolFeCl 24H 2in the deionized water of O to 100mL, in solution, pass into nitrogen and be heated to 80-120 DEG C, then by the NH of 3-7mL25% 3h 2o adds in mixed liquor, reaction 2h; The solid matter high purity water isolating black with permanent magnet from reaction solution cleans 3 ~ 5 times, obtains Fe 3o 4nano particle;
B. 12mgFe is got 3o 4the mixed liquor of nano particle 1-10mL deionized water and 20mL ethanol is resuspended, under the condition slowly stirred, first add the NH of 0.3-0.9mL 4oH solution, then 10-300uL ethyl orthosilicate is dissolved in 50uL ethanolic solution dropwise adds, react 12h under room temperature, at Fe 3o 4nanoparticle surface forms layer of silicon dioxide, cleans several times with deionized water solution, and in ethanolic solution, redissolution obtains the Fe of coated with silica 3o 4nano particle;
C. the ethyl orthosilicate of 10-300uL, 20mL absolute ethyl alcohol, the phenanthroline connection ruthenium (Ru (bqy) of 1-10mL deionized water and 1mL0.5-3mg/L 3 2+) mixing, mixed solution is added the Fe of 0.5mL coated with silica 3o 4nano particle, finally adds the NH of 600-900 μ L 4oH, vigorous stirring 3h, obtain Fe 3o 4/ Ru (bqy) 3 2+nano microsphere, for subsequent use by washed with de-ionized water;
D. the mercaptopropyl trimethoxysilane of 1mL is added in the ethanolic solution of 10mL, with this potpourri redissolution Fe 3o 4/ Ru (bqy) 3 2+nano microsphere, after 300rpm stirs 12h at normal temperatures, then 80 DEG C of 300rpm stir 1h, the centrifugal Fe obtaining silanization 3o 4/ Ru (bqy) 3 2+nano microsphere;
E. by the Fe of silanization 3o 4/ Ru (bqy) 3 2+nano microsphere adds to and comprises 0.06gNaHCO 3, 0.08g neopelex, 0.05mL styrene, the aqueous solution of the 50mL of 0.15mL acrylic acid and 0.5g potassium persulfate solution, water-bath 70 DEG C, stirs under 200r/min, obtains carboxylated Fe after reaction 5h 3o 4/ Ru (bqy) 3 2+nano microsphere;
2) Fe that 0.5-2mg is carboxylated is got 3o 4/ Ru (bqy) 3 2+nano microsphere adds in 1mL coupling buffer, regulate pH to 5-10, add 0.05-0.18mg1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) activated carboxyl, and the anti-vibrio parahemolyticus monoclonal antibody of 50-300 μ g, when temperature 37 DEG C, coupling 60-120min on the gyroscope being placed on 10-15rpm, Magneto separate 3-5min, abandons supernatant; Rinse 3-5 all over afterwards with cleaning buffer solution, get 1mL sealer and mix closed 0.5-1h with Nano microsphere, obtain Fe 3o 4/ Ru (bqy) 3 2+nano microsphere-monoclonal antibody;
Described coupling buffer compound method is as follows: after being mixed with the boric acid of 7mL12.37g/mL by the borax of 3mL19.07g/mL, dilutes 10 times of volumes;
Described cleaning buffer solution compound method is as follows: take 0.43g2-(N-morpholine) ethyl sulfonic acid (MES) and be dissolved in the sterile distilled water of 200mL, tune pH is 5.5-6.0;
Described sealer compound method is as follows: get 100mg bovine serum albumin(BSA) (BSA) and add 1mL phosphate (PBS) damping fluid and be made into sealer;
3) cultivating vibrio parahemolyticus, is 10 by bacterium liquid adjustment concentration 6cFU/mL, 10 5cFU/mL, 10 4cFU/mL, 10 3cFU/mL, respectively gets 1mL for subsequent use; Get testing sample solution 1mL, each concentration bacterium liquid 1mL, respectively with 100-150 μ gFe 3o 4/ Ru (bqy) 3 2+nano microsphere-monoclonal antibody, in temperature 37 DEG C, 30-60min is hatched in gyroscope rotating speed 10-15rpm mixing, after hatching rear Magneto separate 3-5min, abandons supernatant, and after PBS buffer solution for cleaning, redissolving must Fe in PBS 3o 4/ Ru (bqy) 3 2+nano microsphere-monoclonal antibody-bacterium;
4) preparation of immuno-chromatographic test paper strip: by sample pad pH8.50.1MTris-HCl damping fluid (1%BSA, 0.5%Tween-20) immersion treatment, is placed in 60 DEG C of air dry ovens, takes out for subsequent use after 2h; Vibrio parahemolyticus rabbit resisting is sprayed onto on nitrocellulose membrane as detection line (T line) and nature controlling line (C line) with donkey against murine two is anti-more, concentration is 1-2mg/mL, discharge rate is 0.75uL/cm, and 37 DEG C of dried in vacuo overnight take out that to be placed in dry cylinder for subsequent use; Filter pad, sample pad, nitrocellulose membrane, thieving paper are pasted onto successively on PVC base plate, are cut into the test strips that 4mm is wide after posting, are installed; The test strips prepared is loaded in aluminium foil bag, adds drying agent sealing, be placed in dry cylinder and save backup;
5) test strips is to the detection of sample: by the Fe collected 3o 4/ Ru (bqy) 3 2+nano microsphere-monoclonal antibody-bacterium is diluted to 50-150 μ g/mL, gets 100 μ L and is added drop-wise in test strips well, after 10-15min, by the value of fluorescence reader record T line, C line fluorescence intensity and T/C;
6) qualitative analysis: the result that detects by an unaided eye carries out qualitative analysis, T line develops the color, and has vibrio parahemolyticus in interpret sample, and T line does not develop the color, and does not have vibrio parahemolyticus or the amount containing vibrio parahemolyticus lower than 10 in interpret sample 3cFU/mL;
7) quantitative test: use fluorescence reader to measure the fluorescence intensity of T line, C line, and T/C value, with the concentration of different bacterium for horizontal ordinate, T/C value is ordinate drawing standard curve, determines the vibrio parahemolyticus quantity in common sample.
2. method according to claim 1, is characterized in that described step 1) Fe 3o 4/ Ru (bqy) 3 2+nano microsphere particle diameter is 80-210nm.
3. method according to claim 1, is characterized in that: step 3) described immuno-chromatographic test paper strip be paste successively in adhesive base filter paper, sample pad, nitrocellulose membrane, thieving paper composition.
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CN110470840A (en) * 2019-09-06 2019-11-19 齐鲁工业大学 A method of detection vibrio parahemolyticus
CN113252903A (en) * 2021-05-14 2021-08-13 南昌大学 Magnetic fluorescent microsphere immunochromatography kit for field detection and manufacturing method and detection method thereof
WO2021222822A1 (en) * 2020-05-01 2021-11-04 Canon Virginia, Inc. Rapid lateral flow assay for vibrio detection

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CN110470840A (en) * 2019-09-06 2019-11-19 齐鲁工业大学 A method of detection vibrio parahemolyticus
CN110470840B (en) * 2019-09-06 2022-05-27 齐鲁工业大学 Method for detecting vibrio parahaemolyticus
WO2021222822A1 (en) * 2020-05-01 2021-11-04 Canon Virginia, Inc. Rapid lateral flow assay for vibrio detection
CN113252903A (en) * 2021-05-14 2021-08-13 南昌大学 Magnetic fluorescent microsphere immunochromatography kit for field detection and manufacturing method and detection method thereof

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