CN102818894A - Colloidal gold detection card for detecting staphylococcus aureus and preparation method thereof - Google Patents
Colloidal gold detection card for detecting staphylococcus aureus and preparation method thereof Download PDFInfo
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- CN102818894A CN102818894A CN2012102363570A CN201210236357A CN102818894A CN 102818894 A CN102818894 A CN 102818894A CN 2012102363570 A CN2012102363570 A CN 2012102363570A CN 201210236357 A CN201210236357 A CN 201210236357A CN 102818894 A CN102818894 A CN 102818894A
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- staphylococcus aureus
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Abstract
The invention discloses a colloidal gold detection card for detecting staphylococcus aureus and a preparation method thereof. The method comprises the following steps of: measuring and preparing colloidal gold; preparing colloidal gold labeled antibody; centrifuging and purifying the colloidal-gold antibody; carrying out hydration and sealing treatments on a sample pad and a colloidal-gold antibody bonding pad through a treatment fluid; spotting the colloidal-gold antibody on the colloidal-gold antibody bonding pad; diluting a coating antibody staphylococcus aureus and spotting on a NC film; and successively pasting the NC film, absorbent paper, the colloidal-gold antibody bonding pad and the sample pad on a PVC rubber sheet. The detection card provided by the invention is used to greatly simplify test procedure and shorten the detection time, can provide powerful technical support for quality control in food enterprises, on-site law enforcement and daily supervision in food safety supervision departments of business, health, quality and technical supervision and the like, and responses to bacterial food poisoning public health emergency, and is of great significance for promoting sound development of our nation's food industry and guaranteeing the health of people.
Description
Technical field
The present invention relates to the staphylococcus aureus detection range, relate in particular to a kind of collaurum test card that detects staphylococcus aureus.
Background technology
Staphylococcus aureus is one of food hygiene microorganism conventional sense project.Staphylococcus aureus can produce enterotoxin owing to cause a disease, so in case bacterial contamination food, and under proper temperature environment, bacterium can breed and produce enterotoxin in a large number, thus cause that the consumer poisons by food.
Staphylococcus aureus is ubiquitous at occurring in nature, all can find in the excreta of air, water, dust and humans and animals.Thereby food receives the chance of its pollution a lot.According to the Center for Disease Control report, the infection that is caused by staphylococcus aureus accounts for second, is only second to Escherichia coli.The caused poisoning of the golden yellow grape ball public health problem that become international; By the food poisoning that Staphylococcus aureus enterotoxin causes, account for 33% of whole food posioning in the U.S., Canada is then more; Account for 45%; Etesian this type of poisoning of China is also very many, and the economic loss that causes thus is also quite heavy, so present countries in the world are all classified staphylococcus aureus as the legal test item of food hygiene.
Staphylococcus aureus at first will carry out enrichment culture per sample in the conventional method detection food; Make the quantity of staphylococcus aureus to be detected in sample, reach detectable level; Could carry out the separation of staphylococcus aureus according to the food microorganisms organon then; Morphological feature is observed and a series of physio-biochemical characteristics are identified; Need 5 days-7 days common this detection time, and complex operation, detection time are long, can not satisfy food hygiene supervision department to fast detecting, early warning of food poisoning pathogen and the needs of tackling public health emergency.Therefore, research and development staphylococcus aureus Fast Detection Technique becomes primary study direction in recent years.
Summary of the invention
For solving above-mentioned middle problem and the defective that exists, the invention provides a kind of collaurum test card that detects staphylococcus aureus, said technical scheme is following:
Sample pad, golden labeling antibody pad, NC film and thieving paper, said sample pad, golden labeling antibody pad, NC film and thieving paper are arranged on the PVC offset plate.
Be used to detect the preparation method of the collaurum test card of staphylococcus aureus, said method comprises:
A surveys the system collaurum;
B prepares colloid gold label antibody;
C handles golden labeling antibody centrifugal purification;
D carries out aquation and sealing processing with sample pad and golden labeling antibody pad through treating fluid;
E with golden labeling antibody point sample on golden labeling antibody pad;
F is with the dilution of coated antibody staphylococcus aureus, and point sample is on the NC film;
G sticks on NC film, thieving paper, golden labeling antibody pad and sample pad on the PVC offset plate successively.
The beneficial effect of technical scheme provided by the invention is:
Staphylococcus aureus immunity collaurum test card provided by the invention; Simplified check program greatly; Shortened detection time, can be the food enterprise quality control, food security supervision department on-site law-enforcings such as industry and commerce, health, quality supervision, routine monitoring and the public health emergency of reply food posioning provide strong technical support; To promoting China's food service industry to develop in a healthy way, ensure that the people ' s health aspect is significant.
Description of drawings
Fig. 1 is the preparation method's process flow diagram that is used to detect the collaurum test card of staphylococcus aureus;
Fig. 2 is the collaurum test card structural drawing that detects staphylococcus aureus.
Embodiment
For making the object of the invention, technical scheme and advantage clearer, will combine accompanying drawing that embodiment of the present invention is done to describe in detail further below:
Referring to Fig. 2, a kind of collaurum test card that detects staphylococcus aureus comprises sample pad, golden labeling antibody pad, NC film and thieving paper, and said sample pad 1, golden labeling antibody pad 3, NC film 9 and thieving paper 5 are arranged on the PVC offset plate 6.Said NC film is provided with control line (C line) 4 and detection line (T line) 8.
Above-mentioned collaurum test card in use; Sample drop is added on the sample pad, and it is as shown in fig. 1 that sample drips direction 2, through chromatography; Chromatography direction 7 is as shown in Figure 1, shows testing result through detection line control line (C line) and detection line (T line) behind the chromatography.
Present embodiment also provides a kind of preparation method who is used to detect the collaurum test card of staphylococcus aureus, and (as shown in Figure 2) this method comprises:
Above-mentioned steps can realize through following specific embodiment:
Embodiment 1
Survey the system collaurum: the chlorauric acid solution that adds 100ml ultrapure water and 1ml1% in the round-bottomed flask in condensation reflux unit; Be heated to the citric acid three sodium solution of boiling back adding 1ml1% concentration; Observe change color, continue reaction 10min when treating colour stable, cooling was left standstill one day; Utilize the particle size analyzer detection to make particle diameter and be the 40nm collaurum, its maximum absorption band is at 526nm.
Preparation colloid gold label antibody: minimum stabilize proteins amount increases about 10% again and is optimum mark albumen consumption.Collaurum is with HCl and the K of 0.1mol/L
2CO
3Transfer to pH8.2; Get optimum amount staphylococcus aureus PBP2a monoclonal antibody 16 μ g/ml, whole mass concentration and be 1% trehalose and add the 100ml colloidal gold solution simultaneously; After under magnetic stirring apparatus, mixing 10min; Add whole mass concentration and be 0.5% BSA, 4 ℃ of preservations freezing ultracentrifugation purifying in back that spends the night.
Golden labeling antibody centrifugal purification is handled: at first with the centrifugal 20min of golden labeling antibody solution normal temperature low speed (1500rpm), discard the deposition that the gold grain by cohesion forms, get red supernatant solution in 4 ℃ of centrifugal 30min of 8000 r/min again.
Solution is divided into three layers: transparent supernatant, the fine and close gold grain layer of black on flowable kermesinus deposition in the pipe end and the pipe diapire.Flowable kermesinus deposition is transferred in the another one centrifuge tube, and resuspended to commercial weight with resuspended liquid, resuspended liquid: whole mass concentration is 0.1% polysorbas20,1% trehalose, 0.5% BSA, 0.01mol/L PBS pH7.4 solution.
Repeated centrifugation 3 times, last resuspended to 1/20 of original volume, 4 ℃ of preservations of Sodium azide of adding 0.1% are subsequent use.
Sample pad and golden labeling antibody pad are carried out aquation and sealing processing through treating fluid: pretreatment liquid carries out aquation and sealing is handled through dipping after cutting out for sample pad and pad; Oven dry in 37 ℃, 2 hours behind the immersion 30min, the pad treating fluid is: containing whole mass concentration is that 0.3%% polysorbas20,2% trehalose, 10% sucrose and pH value are that 7.4 concentration are the PBS solution of 0.01mol/L.
With golden labeling antibody point sample on golden labeling antibody pad: golden labeling antibody transfers concentration to OD
526 Be 5, the some model machine is with golden labeling antibody even point sample on pad, and the point sample amount is at 30 μ l/cm
2, 37 ℃ of vacuum drying.
Detection line antibody and control line two anti-encapsulating: coated antibody staphylococcus aureus PBP2a is detected monoclonal antibody be diluted to 0.7mg/ml; Two anti-dilutabilitys are that commercialization is diluted 0.7 mg/ml doubly; Point sample is drawn T line and C line respectively on the NC film, spacing is 5mm, 37 ℃ of dryings.
The assembling of test strips: 2cm * 0.3cmNC film, 1.8cm * 0.3cm thieving paper, 1cm * 0.3cm gold mark pad, 1.5cm * 0.3cm sample pad are sticked on the PVC offset plate by Fig. 1 mode successively, the staphylococcus aureus colloidal gold strip.
With staphylococcus aureus compete standard items by 1 times the dilution 5 times the dilution 10 times the dilution 50 times of dilutions concentration drip successively on the staphylococcus aureus colloidal gold strip, with damping fluid as control group, the observation experiment result.
Embodiment 2
It is identical to survey system collaurum and above-mentioned first embodiment, is not described in detail here.
Preparation colloid gold label antibody: minimum stabilize proteins amount increases about 10% again and is optimum mark albumen consumption.Collaurum is with HCl and the K of 0.1mol/L
2CO
3Transfer to pH9.2; Get optimum amount staphylococcus aureus monoclonal antibody 16 μ g/ml, whole mass concentration and be 0.5% trehalose and add the 100ml colloidal gold solution simultaneously; After under magnetic stirring apparatus, mixing 10min; Add whole mass concentration and be 0.5% BSA, 4 ℃ of preservations freezing ultracentrifugation purifying in back that spends the night.
Gold labeling antibody centrifugal purification is handled: the centrifugal 20min of golden labeling antibody solution normal temperature low speed (1500rpm), discard the deposition that the gold grain by cohesion forms, and get red supernatant solution in 4 ℃ of centrifugal 30min of 10000 r/min again.
Solution is divided into three layers: transparent supernatant, the fine and close gold grain layer of black on negotiable kermesinus deposition in the pipe end and the pipe diapire.Flowable kermesinus deposition is transferred in the another one centrifuge tube, and resuspended to commercial weight with resuspended liquid, the whole mass concentration of resuspended liquid is that 0.2% polysorbas20,2% trehalose, 1%BSA and pH value are that 7.4 concentration are the PBS solution of 0.01mol/L.
Repeated centrifugation 3 times, last resuspended liquid are to 1/20 of original volume, and 4 ℃ of preservations of Sodium azide of adding 0.1% are subsequent use.
Sample pad and golden labeling antibody pad carry out aquation through treating fluid to be handled with sealing: pretreatment liquid carries out aquation and sealing is handled through dipping after cutting out for sample pad and pad; Oven dry in 37 ℃, 2 hours behind the immersion 30min, the pad treating fluid is: containing whole mass concentration is that 0.2% polysorbas20,1% trehalose, 8% sucrose and pH value are that 7.4 concentration are the PBS solution of 0.01mol/L.
Gold labeling antibody point sample on golden labeling antibody pad, detection line antibody and control line two anti-encapsulate and the assembling of test strips duplicates with above-mentioned first embodiment, be not described in detail here.
It is identical to survey system collaurum and above-mentioned first embodiment, is not described in detail here.
Preparation colloid gold label antibody: minimum stabilize proteins amount increases about 10% again and is optimum mark albumen consumption.Collaurum is with HCl and the K of 0.1mol/L
2CO
3Transfer to pH9.2; Get optimum amount staphylococcus aureus monoclonal antibody 16 μ g/ml, whole mass concentration and be 2% trehalose and add the 100ml colloidal gold solution simultaneously; After under magnetic stirring apparatus, mixing 10min; Add whole mass concentration and be 0.5% BSA, 4 ℃ of preservations freezing ultracentrifugation purifying in back that spends the night.
Gold labeling antibody centrifugal purification is handled: at first with the centrifugal 20min of golden labeling antibody solution normal temperature low speed (1500rpm), discard the deposition that the gold grain by cohesion forms, get red supernatant solution in 4 ℃ at the centrifugal 30min of 12000 r/min.
Solution is divided into three layers: transparent supernatant, the fine and close gold grain layer of black on flowable kermesinus deposition in the pipe end and the pipe diapire.Flowable kermesinus deposition is transferred in the another one centrifuge tube, and resuspended to commercial weight with resuspended liquid, resuspended liquid: whole mass concentration is that 0.5% polysorbas20,3% trehalose, 2% BSA and pH value are that 7.4 concentration are the PBS solution of 0.01mol/L.
Repeated centrifugation 3 times, last resuspended to 1/20 of original volume, 4 ℃ of preservations of Sodium azide of adding 0.1% are subsequent use.
Sample pad and golden labeling antibody pad carry out aquation through treating fluid to be handled with sealing: pretreatment liquid carries out aquation and sealing is handled through dipping after cutting out for sample pad and pad; Oven dry in 37 ℃, 2 hours behind the immersion 30min, the pad treating fluid is: containing whole mass concentration is that 0.5% polysorbas20,3% trehalose, 6% sucrose and pH value are that 7.4 concentration are the PBS solution of 0.01mol/L.
Gold labeling antibody point sample on golden labeling antibody pad, detection line antibody and control line two anti-encapsulate and the assembling of test strips duplicates with above-mentioned first embodiment, be not described in detail here.
Embodiment 4
It is identical to survey system collaurum and above-mentioned first embodiment, is not described in detail here.
Preparation colloid gold label antibody: minimum stabilize proteins amount increases about 10% again and is optimum mark albumen consumption.Collaurum is with HCl and the K of 0.1mol/L
2CO
3Transfer to pH9.2; Get optimum amount staphylococcus aureus monoclonal antibody 16 μ g/ml, whole mass concentration and be 1.5% trehalose and add the 100ml colloidal gold solution simultaneously; After under magnetic stirring apparatus, mixing 10min; Add whole mass concentration and be 0.5% BSA, 4 ℃ of preservations freezing ultracentrifugation purifying in back that spends the night.
Gold labeling antibody centrifugal purification is handled: the centrifugal 20min of golden labeling antibody solution normal temperature low speed (1500rpm), discard the deposition that the gold grain by cohesion forms, and get red supernatant solution in 4 ℃ of centrifugal 30min of 12000 r/min again.
Solution is divided into three layers: transparent supernatant, the fine and close gold grain layer of black on negotiable kermesinus deposition in the pipe end and the pipe diapire.Flowable kermesinus deposition is transferred in the another one centrifuge tube, and resuspended to commercial weight with resuspended liquid, the whole mass concentration of resuspended liquid is that 0.3% polysorbas20,2.5% trehalose, 1.5%BSA and pH value are that 7.4 concentration are the PBS solution of 0.01mol/L.
Repeated centrifugation 3 times, last resuspended liquid are to 1/20 of original volume, and 4 ℃ of preservations of Sodium azide of adding 0.1% are subsequent use.
Sample pad and golden labeling antibody pad carry out aquation through treating fluid to be handled with sealing: pretreatment liquid carries out aquation and sealing is handled through dipping after cutting out for sample pad and pad; Oven dry in 37 ℃, 2 hours behind the immersion 30min, the pad treating fluid is: containing whole mass concentration is that 0.3% polysorbas20,2.5% trehalose, 5% sucrose and pH value are that 7.4 concentration are the PBS solution of 0.01mol/L.
Gold labeling antibody point sample on golden labeling antibody pad, detection line antibody and control line two anti-encapsulate and the assembling of test strips duplicates with above-mentioned first embodiment, be not described in detail here.
The above is merely preferred embodiment of the present invention, and is in order to restriction the present invention, not all within spirit of the present invention and principle, any modification of being done, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. collaurum test card that detects staphylococcus aureus; It is characterized in that; Said collaurum test card comprises: sample pad, golden labeling antibody pad, NC film and thieving paper, said sample pad, golden labeling antibody pad, NC film and thieving paper are arranged on the PVC offset plate.
2. the collaurum test card of detection staphylococcus aureus according to claim 1 is characterized in that said NC film is provided with control line and detection line.
3. detect the preparation method of the collaurum test card of staphylococcus aureus, it is characterized in that said method comprises:
A surveys the system collaurum;
B prepares colloid gold label antibody;
C handles golden labeling antibody centrifugal purification;
D carries out aquation and sealing processing with sample pad and golden labeling antibody pad through treating fluid;
E with golden labeling antibody point sample on golden labeling antibody pad;
F is with the dilution of coated antibody staphylococcus aureus, and point sample is on the NC film;
G sticks on NC film, thieving paper, golden labeling antibody pad and sample pad on the PVC offset plate successively.
4. the preparation method who is used to detect the collaurum test card of staphylococcus aureus according to claim 3; It is characterized in that; Said steps A specifically comprises: pure water and chlorauric acid solution are heated to the back of boiling add citric acid three sodium solution and react and cool off, it is the 40nm collaurum that particle diameter is produced in detection.
5. the preparation method who is used to detect the collaurum test card of staphylococcus aureus according to claim 3; It is characterized in that; Said step B specifically comprises: get staphylococcus aureus monoclonal antibody 16ug/ml, whole mass concentration and be 0.5～3% trehalose and 100mL colloidal gold solution and mix; The whole mass concentration of adding is 0.5% BSA in mixed solution, obtains golden labeling antibody solution, and carries out the refrigerated centrifuge purification process.
6. the preparation method who is used to detect the collaurum test card of staphylococcus aureus according to claim 3 is characterized in that, said step C specifically comprises golden labeling antibody solution and to get supernatant and centrifugal 30min in the solution at normal temperature low-speed centrifugal 20min; Said solution comprises three layers: black gold grain layer on transparent supernatant, pipe end kermesinus precipitated liquid and the pipe diapire;
Get pipe end kermesinus precipitated liquid, and the resuspended liquid of adding is resuspended in the kermesinus precipitated liquid; Said resuspended liquid is: whole mass concentration is that 0.1%～0.5% polysorbas20,1%～3% trehalose, 0.5%～2%BSA and pH value are that 7.4 concentration are the PBS solution of 0.01mol/L;
The counterweight suspension carries out centrifugal treating, and adds 0.1% Sodium azide in the resuspended liquid after centrifugal treating, and temperature is to preserve under 4 ℃ of conditions.
7. the preparation method who is used to detect the collaurum test card of staphylococcus aureus according to claim 3; It is characterized in that, among the said step D sample pad and golden labeling antibody pad are carried out aquation and seal to handle through treating fluid comprising: is to soak under 37 ℃ of environment also to dry in 2 hours with treating fluid in temperature; Said treating fluid is: containing whole mass concentration is that 0.2%～0.5% polysorbas20,1%～3% trehalose, 5%～10% sucrose and pH value are that 7.4 concentration are the PBS solution of 0.01mol/L.
8. the preparation method who is used to detect the collaurum test card of staphylococcus aureus according to claim 3 is characterized in that said step e specifically comprises: with concentration to OD
526Be 5 the even point sample of golden labeling antibody on pad, and be 37 ℃ of following vacuum drying in temperature; Said point sample amount is 30 μ l/cm
2
9. the preparation method who is used to detect the collaurum test card of staphylococcus aureus according to claim 3 is characterized in that, the dilution of coated antibody staphylococcus aureus comprises monoclonal antibody dilution and two anti-dilutions in the said step F; Wherein, Monoclonal antibody is diluted to 0.7 mg/ml, two anti-0.7 mg/ml that are diluted to, and with the staphylococcus aureus point sample after monoclonal antibody dilution and two anti-the dilutions on the NC film; Drawing T line and C line respectively, is 37 ℃ of dryings down in temperature.
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| CN105891469A (en) * | 2014-11-14 | 2016-08-24 | 宁波大学 | Portunus trituberculatus reovirus detection test paper and preparation method thereof |
| CN106771211A (en) * | 2016-11-23 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | A kind of detection card for detecting staphylococcus aureus in milk |
| CN107727862A (en) * | 2017-11-06 | 2018-02-23 | 北京农学院 | A kind of Ribavirin test strip |
| CN112698038A (en) * | 2020-12-08 | 2021-04-23 | 黑龙江八一农垦大学 | Preparation method of test paper strip for detecting Sudan red I colloidal diamond carbon in food |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105891469A (en) * | 2014-11-14 | 2016-08-24 | 宁波大学 | Portunus trituberculatus reovirus detection test paper and preparation method thereof |
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| CN106771211A (en) * | 2016-11-23 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | A kind of detection card for detecting staphylococcus aureus in milk |
| CN107727862A (en) * | 2017-11-06 | 2018-02-23 | 北京农学院 | A kind of Ribavirin test strip |
| CN112698038A (en) * | 2020-12-08 | 2021-04-23 | 黑龙江八一农垦大学 | Preparation method of test paper strip for detecting Sudan red I colloidal diamond carbon in food |
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