CN102135540A - Detection method of staphylococcus aureus and gold-labeled rapid diagnosis kit for same and preparation method thereof - Google Patents
Detection method of staphylococcus aureus and gold-labeled rapid diagnosis kit for same and preparation method thereof Download PDFInfo
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- CN102135540A CN102135540A CN2010102728890A CN201010272889A CN102135540A CN 102135540 A CN102135540 A CN 102135540A CN 2010102728890 A CN2010102728890 A CN 2010102728890A CN 201010272889 A CN201010272889 A CN 201010272889A CN 102135540 A CN102135540 A CN 102135540A
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Abstract
The invention relates to a detection method of staphylococcus aureus, as well as a gold-labeled rapid diagnosis kit for the method and a preparation method thereof. The detection method comprises the following steps of: (1) loading colloidal gold-labeled antibodies resistant to the staphylococcus aureus on a glassfiber or nonwoven carrier, and coating a detection line composed of the antibodies resistant to the staphylococcus aureus and a control line composed of antibodies of sheep and rabbit anti-mouse IgG (Intravenous Gamma Globulin) on a detection carrier connected with the carrier of the colloidal gold-labeled antibodies; and (2) dropping sample diluent on the carrier of the colloidal gold-labeled antibodies, wherein double lines (the detection line and the control line) are formed on the detection carrier, and the result is judged to be positive if the diluent contains the staphylococcus aureus, and the result is judged to be negative if only the control line develops. The kit comprises a detection card, wherein the detection card comprises a detection strip and a plastic card box; the detection strip is arranged in a plastic card, and is provided with a water absorption layer at a sample-adding end, a detection layer and a water absorption layer at a water absorption end, on a lining board; a gold-labeled staphylococcus aureus antibody layer is arranged between the detection layer and the water absorption layer at the sample-adding end; and the detection line composed of the antibodies resistant to the staphylococcus aureus and the control line composed of the antibodies of the sheep and the rabbit anti-mouse IgG are coated on the detection layer.
Description
Technical field
The present invention relates to a kind of method that is used to detect staphylococcus aureus, and the staphylococcus aureus golden label fast diagnosis reagent box and the preparation method that are used for this method.
Background technology
Staphylococcus aureus is ubiquitous at occurring in nature, all can find staphylococcus aureus in the excreta of air, water, dust and humans and animals.Therefore, food is subjected to the chance of its pollution a lot.In recent years, the Center for Disease Control report shows that the infection that is caused by staphylococcus aureus accounts for second, is only second to Escherichia coli.The detection of Staphylococcus aureus enterotoxin is a global health difficult problem.In the U.S., by the food poisoning that Staphylococcus aureus enterotoxin causes, account for 33% of whole food posioning, Canada is then more, account for 45%, and etesian this type of poisoning of China is also very many.
The epidemiology of staphylococcus aureus generally has following characteristics: two seasons of spring and summer are more common in the infection of staphylococcus aureus; The poisoning food kind is many: as milk, meat, egg, fish and goods thereof.In addition, the staphylococcus aureus poisoning that causes because of food leftovers, fried egg, glutinous rice cakes and bean jelly etc. also has report.Infection of the upper respiratory tract patient's nasal cavity bacterial bearing rate is up to 83%, so people, animal pyogenic infection position often becomes pollution source.
Generalized case can be carried disease germs because of food processing personnel, cook or sales force by following approach contaminated food products: i through staphylococcus aureus, and be caused food pollution; Ii food itself carries disease germs before processing, or has been subjected to pollution in process, has produced enterotoxin, causes food poisoning; Iii cooked product packing department sealing, or caused pollution: iv suffers from suppurative mastitis because of milk cow or the fowl poultry is local when suppurating, to the pollution at other position of the human body in the transportation.
Staphylococcus aureus is a modal pathogen in the human suppurative infection.It can cause local suppurative infection, also can cause pneumonia, pseudomembranous enteritis, pericarditis etc., even general infection such as septicemia, pyemia.Simultaneously, the natural source of this bacterium is very extensive, and at present its mechanism of causing a disease also is not very clear.Traditional detection method mainly contains methods such as animal experiment, serological test, immunofluorescence detection test and enzyme linked immunological absorption.Though correlation detection technology has been obtained very big progress in recent years, but still there are some defectives.So the control that quick, accurate, the sensitive detection method of further studying this bacterium infects food-borne pathogens, the diagnosis of disease and epidemiology survey are very crucial.
The present invention adopts staphylococcus aureus immunity rabbit or sheep to prepare multi-epitope antibody, and immune balb/c mice prepares monoclonal antibody, develops a kind of golden label fast diagnosis reagent box applicable to execute-in-place.This kit can be fast (5-20 minute go out result) specificly detects staphylococcus aureus, can be used for the onset site quick diagnosis.
Of the present invention open
First purpose of the present invention is: the detection method that a kind of staphylococcus aureus is provided.This method is a step detection method, highly sensitive, high specificity, and reaction velocity is fast, easy and simple to handle, need not Special Equipment.This kit can be used for multiple occasions such as clinical detection, epidemiology survey and place quarantine.
Second purpose of the present invention is: a kind of staphylococcus aureus golden label fast diagnosis reagent box is provided.This kit detects at staphylococcus aureus, and a kind of easy and direct single stage method detection kit is provided.Utilize this kit to detect highly sensitive, the high specificity of staphylococcus aureus, reaction velocity is fast, easy and simple to handle, need not Special Equipment.
The 3rd purpose of the present invention is: the preparation method that a kind of staphylococcus aureus golden label fast diagnosis reagent box is provided.This method is easy and simple to handle, good stability, good reproducibility.
First purpose of the present invention can realize by following measure:
A kind of detection methods of staphylococcus aureus may further comprise the steps: (1) is with the anti-staphylococcus aureus antibody of colloid gold label, comprise many anti-or monoclonal antibody is stated from glass fibre or the nonwoven fabrics carrier, link to each other with the colloid gold label antibody carrier then, detect and wrap on the carrier by anti-staphylococcus aureus antibody (monoclonal antibody and how anti-) detection line that forms and the control line that forms by sheep, rabbit anti-mouse igg antibody; (2) sample thief dilution drop is on the carrier of colloid gold label antibody.Contain staphylococcus aureus as above-mentioned dilution, then aubergine detection line and control line two-wire occur on the detection carrier, this testing result is judged to be the positive, if only a red nature controlling line is arranged, then testing result is negative.
Second purpose of the present invention can realize by following measure:
A kind of staphylococcus aureus golden label fast diagnosis reagent box, in establish staphylococcus aureus gold mark test card and form.
Test card is made up of detector bar and plastics cartridge, and detector bar is installed in the plastic clip.Described staphylococcus aureus gold mark detector bar is to be provided with application of sample end water accepting layer, detection layers and suction side water accepting layer on liner plate, between detection layers and application of sample end water accepting layer, be provided with the anti-staphylococcus aureus antibody layer of gold mark, on detection layers, be coated with by anti-staphylococcus aureus many anti-or detection line that monoclonal antibody forms and the control lines that form by sheep, rabbit anti-mouse igg antibody.
The 3rd purpose of the present invention can realize by following measure:
A kind of preparation method of staphylococcus aureus golden label fast diagnosis reagent box; The control line and the detection line that mainly comprise golden labeling antibody layer of preparation and detection layers.And then combination staphylococcus aureus gold label test strip and check-out console on the liner plate.
The preparation method of described golden labeling antibody layer comprises the steps: the preparation of i collaurum; In concentration is that 1.5-2%, the concentration that adds its volume in the gold chloride of 0.006-0.012% is the trisodium citrate of 1-3%, boils 10-20 minute, can obtain the colloidal gold solution of 15-50 nanometer; The anti-staphylococcus aureus antibody of ii colloid gold label: in colloidal gold solution, transfer pH7.8-8.8 with the 0.2M solution of potassium carbonate, press 1-4mg/100ml and add staphylococcus aureus antibody, stirring back adding concentration in solution is the animal blood serum albumen of 0.1-0.6G/100ml, and 4 ℃ left standstill 2-4 hour; Iii through 2000 rev/mins of centrifugal 10-15 minutes, abandons sediment with above-mentioned colloidal gold solution, collects supernatant; Iv with supernatant through 10000 rev/mins of centrifugal 60-80 minutes, centrifugal sediment; V is the damping fluid of 4-10ml with the volume that sediment is dissolved in 0.02M, pH7.4Tris-HCI, gets colloidal gold solution.Containing volume ratio in this damping fluid is animal blood serum albumen and the 0.01-0.06% Sodium azide of 0.2-0.6%; Till vi immersed glass fibre or nonwoven fabrics colloidal gold solution to liquid and oozes out, 37 ℃ of dried overnight formed golden labeling antibody layer.The preparation of described detection layers is to be provided with staphylococcus aureus antibody detection line that constitutes and the control line that has sheep, rabbit anti-mouse igg antibody to form on nitrocellulose filter.Its preparation method is: in concentration is the fixing agent that adds 1-3% in the antibody-solutions of 1-2mg/ml, utilizes Membrane jetter that antibody is sprayed on the cellulose membrane again, 37 ℃ of dryings behind the spray film.The PBS that contains 10% calf serum then with 0.01M, pH7.0.Sealed 30 minutes the PBS rinsing of 0.01M, pH7.0,37 ℃ of dryings down at 37 ℃.
Described fixing agent is selected a kind of in formaldehyde, the acetone for use.
The present invention has following advantage compared to existing technology:
1, the method for the present invention's detection, diagnosis staphylococcus aureus is a single stage method.Have higher specificity and sensitivity, and easy and simple to handle, need not Special Equipment and facility.
Have higher specificity and sensitivity when 2, diagnostic kit of the present invention detects staphylococcus aureus.The testing process operation is rapid, quick and easy, and this kit is applicable to multiple occasions such as clinical examination, epidemiology survey and place quarantine.
3. easy, the good stability of the preparation method of diagnostic kit of the present invention, good reproducibility.
The drawing explanation
Fig. 1 is the surface structure synoptic diagram of invention check-out console
1-check-out console 2-well 3-inspection window
Fig. 2 is the inner structure synoptic diagram of check-out console of the present invention
4-application of sample end water accepting layer 5-gold labeling antibody layer 6-control line 7-detection line
8-detection layers 9-suction side water accepting layer 10-liner plate
Embodiments of the present invention
The present invention also will be described in further detail in conjunction with the embodiments:
Embodiment one:
A kind of detection methods of staphylococcus aureus may further comprise the steps: (1) is stated from the anti-staphylococcus aureus antibody of colloid gold label on glass fibre or the nonwoven fabrics carrier, on the detection carrier that the antibody carrier of colloid gold label is linked to each other, and with detection carrier that the colloid gold label antibody carrier links to each other on the detection line of coated with staphylococal antibody formation and the control line that forms by sheep, rabbit anti-mouse igg antibody; (2) sample thief dilution drop contains staphylococcus aureus as above-mentioned dilution on the carrier of colloid gold label antibody, is then detecting formation aubergine detection line, control line two-wire on the carrier; Show the two-wire result and be judged to be the positive, only control line colour developing result is judged to be negative.
With reference to Fig. 1, a kind of staphylococcus aureus golden label fast diagnosis reagent box, in establish the anti-mark of staphylococcus aureus test card 1.
With reference to Fig. 2, the staphylococcus aureus gold mark test card 1 of telling a story in these claims is to be provided with application of sample end water accepting layer 4, detection layers 8 and suction side water accepting layer 9 on liner plate 10.Between detection layers and application of sample end water accepting layer 9, be provided with golden standard gold staphylococcus aureus antibody layer 5, on detection layers 8, be coated with detection line 7 and control line 6.Wherein application of sample end water accepting layer 4 and suction side water accepting layer 9 are to be made by multilayer filter paper; Detection layers 8 is a cellulose membrane, and cellulose membrane can be nitrocellulose filter, cellulose acetate membrane; Gold labeling antibody layer is that glass fibre or nonwoven fabrics leaching colloid gold label antibody are made.
A kind of preparation method of staphylococcus aureus golden label fast diagnosis reagent box: mainly comprise the bag quilt of preparation golden labeling antibody layer (5) and detection layers (8), control line (6) and detection line (7), and then liner plate (10) is gone up combination staphylococcus aureus gold label test strip and test card (1).Paste application of sample end water accepting layer 4 and suction side water accepting layer 9 respectively at the two ends of plastic base plate 10; Section is pasted the cellulose rete 8 of tested survey line of bag and control line therein, at the handing-over position of application of sample end water accepting layer 4 with cellulose rete 8, folder pastes the glass fibre that contains golden labeling antibody 5, and 4/5 part of glass fibre is in the middle of application of sample end water accepting layer 4, and 1/5 part is on cellulose rete 8.Then by 4 mm wides, 8 centimeter length specification slittings, in being assembled in plastic casing.Well 2 is over against the application of sample end water accepting layer 4 of test-strips on the lid, and viewport 3 is over against the detection layers 8 of cellulose membrane.
The preparation method of described golden labeling antibody layer comprises the steps: the preparation of i collaurum: get the 100mg gold chloride and be dissolved in the 1000ml tri-distilled water, add 15ml, concentration then and be 1% trisodium citrate, boiled 15 minutes, and obtained the colloidal gold solution of 15-50 nanometer after the cooling; Ii colloid gold label staphylococcus aureus antibody; Measure the 100ml colloidal gold solution, transfer pH8.6 with the 0.2ml solution of potassium carbonate, add 4mg staphylococcus aureus antibody, stirred 20 minutes, add the 240mg bovine serum albumin(BSA) then, continue to stir 5 minutes, 4 ℃ left standstill 2-4 hour; Iii with above-mentioned colloidal gold solution through 2000 rev/mins centrifugal 10 minutes, abandon sediment, collect supernatant; Iv with supernatant through 10000 rev/mins centrifugal 60 minutes, the collecting precipitation thing; V with sediment by 4-10ml/100ml be dissolved in 0.02M, the pH7.4Tris-HCI damping fluid gets colloidal gold solution, contains 0.25% bovine serum albumin(BSA) and 0.02% Sodium azide in this damping fluid; Till vi immerses glass fibre or nonwoven fabrics colloidal gold solution and has liquid to ooze out to its surface; Form golden labeling antibody layer 5 37 ℃ of following dryings 2 hours.The described colloid gold label antibody of these claims is the anti-staphylococcus aureus antibody of sheep, rabbit.
Described detection layers 8 is to be provided with staphylococcus aureus antibody detection line 7 that constitutes and the control line 6 that has sheep or rabbit anti-mouse igg antibody to form on nitrocellulose filter.Its preparation method is to get anti-staphylococcus aureus antibody, transfers concentration to 1mg/ml, adds 2% formaldehyde, sprays detection line 7 in the cellulose membrane stage casing with Membrane jetter; Get rabbit, sheep anti-mouse igg again, transfer concentration then, add 2% formaldehyde again to 2mg/ml.With Membrane jetter in the cellulose membrane stage casing, apart from detection line 0.5cm place, spray control line 6, by ul/10cm spray film amount is set, the spray film was placed on 37 ℃ of dryings 2 hours, contained the PBS of 10% calf serum again with 0.01M, pH7.0, sealed 30 minutes down at 37 ℃, 0.01M, the PBS rinsing of pH7.0,37 ℃ of dryings.
Embodiment two:
Detection methods of staphylococcus aureus and golden label fast diagnosis reagent box thereof and example are together.
The antibody that the preparation method of this kit removes the preparation colloid gold label is that anti-staphylococcus aureus monoclonal anti is external, and other conditions and example are together.
Claims (8)
1. detection methods of staphylococcus aureus, it is characterized in that: (1) is stated from the staphylococcus aureus antibody of colloid gold label on glass fibre or the nonwoven fabrics carrier, and with detection carrier that the colloid gold label antibody carrier links to each other on bag by anti-staphylococcus aureus antibody (how anti-or monoclonal antibody), formed detection line and the control line that forms by sheep or rabbit anti-mouse igg antibody.(2) get detection sample dilution drop on the carrier of colloid gold label antibody.Be contained in if detected material contains staphylococcus aureus on the detection carrier and form detection line and control line two-wire, then testing result is judged to be the positive, and then testing result is negative if only have control line to develop the color.
2. a staphylococcus aureus golden label fast diagnosis reagent box is characterized in that establishing in this kit staphylococcus aureus gold mark test card, and test card is made up of detector bar and plastics cartridge, and detector bar is installed in the plastic clip.
3. staphylococcus aureus gold-labelled rapid diagnosis reagent card as claimed in claim 2, it is characterized in that described staphylococcus aureus antigen gold mark test card (1) is to be provided with application of sample end water accepting layer (4), detection layers (8) and suction side water accepting layer (9) on liner plate (10), between detection layers (8) and application of sample end water accepting layer (4), be provided with golden standard gold staphylococcus aureus antibody layer (5), on detection layers (8), be coated with detection line (7) and control line (6).
4. as the preparation method of the described staphylococcus aureus golden label fast diagnosis reagent box of claim (2), it is characterized in that preparing the control line (6) of golden labeling antibody layer (5) and detection layers (8), the bag quilt of detection line (7), go up combination staphylococcus aureus gold label test strip and test card (1) at liner plate (10) then.
5. the preparation method of staphylococcus aureus golden label fast diagnosis reagent box as claimed in claim 4, the preparation method who it is characterized in that described golden labeling antibody layer (5) comprises the steps: the preparation of i collaurum, concentration be add in 0.006～0.012% the gold chloride its volume 1.5～2%, concentration is 1～3% trisodium citrate, boiled 10～20 minutes, and can obtain the colloidal gold solution of 15～50 nanometers; The anti-staphylococcus aureus monoclonal antibody of ii colloid gold label, in colloidal gold solution, transfer pH7.8～8.8 with the 0.2m solution of potassium carbonate, add anti-people staphylococcus aureus monoclonal antibody by 1～4mg/100ml, after the stirring, add animal blood serum albumen by 0.1～0.6g/100ml again in solution, 4 ℃ left standstill 2-4 hour; Iii with above-mentioned colloidal gold solution through 2000 rev/mins centrifugal 10～15 minutes, abandon sediment, supernatant; Iv with supernatant through 10000 rev/mins of centrifugal 60～80 minutes centrifugal sediments; V with sediment by 4-10ml/100ml be dissolved in 0.02M, the pH7.4Tris-HCI damping fluid gets colloidal gold solution, contains animal blood serum albumen and the 0.01-0.06% Sodium azide of 0.2-0.6% in this damping fluid; Till vi immersed above-mentioned colloidal gold solution glass fibre or nonwoven fabrics to liquid and begins to ooze out, 37 ℃ of dry filters formed golden labeling antibody layer.
6. as the preparation method of claim 4 or 5 described staphylococcus aureus golden label fast diagnosis reagent boxes, it is characterized in that described colloid gold label antibody is anti-staphylococcus aureus antibody of sheep or rabbit or staphylococcus aureus monoclonal antibody.
7. the preparation method of staphylococcus aureus golden label fast diagnosis reagent box as claimed in claim 4 is characterized in that described detection layers (8) is to be provided with sheep or rabbit staphylococcus aureus antibody or the detection line (7) of staphylococcus aureus monoclonal antibody formation and the control line (6) that is formed by anti-sheep or rabbit anti-mouse igg antibody on cellulose membrane.
8. the preparation method of staphylococcus aureus golden label fast diagnosis reagent box as claimed in claim 7 is characterized in that described fixing agent is formaldehyde or acetone.
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| CN102393455A (en) * | 2011-08-26 | 2012-03-28 | 厦门出入境检验检疫局检验检疫技术中心 | Preparation method of immunogold rapid test paper for vibrio parahaemolyticus |
| CN102590506A (en) * | 2012-02-16 | 2012-07-18 | 上海师范大学 | Method for rapidly detecting and screening staphylococcus aureus |
| CN102645536A (en) * | 2012-04-19 | 2012-08-22 | 沈鹤柏 | Method for detecting staphylococcus aureus |
| CN102818894A (en) * | 2012-07-09 | 2012-12-12 | 北京六角体科技发展有限公司 | Colloidal gold detection card for detecting staphylococcus aureus and preparation method thereof |
| CN103913572A (en) * | 2014-04-02 | 2014-07-09 | 上海理工大学 | Method for detecting staphylococcus aureus and monoclonal antibodies of staphylococcus aureus |
| CN103954759A (en) * | 2014-04-02 | 2014-07-30 | 上海理工大学 | Staphylococcus aureus immunization colloidal gold rapid detection test paper strip |
| CN104614531A (en) * | 2015-02-09 | 2015-05-13 | 甘肃出入境检验检疫局检验检疫综合技术中心 | Alicyclobacillus colloidal gold label test strip and preparation method thereof |
| CN105651993A (en) * | 2016-01-19 | 2016-06-08 | 南昌大学 | Method for rapidly detecting staphylococcus aureus |
| CN105891470A (en) * | 2014-12-24 | 2016-08-24 | 瑞正浩博(天津)医疗器械销售有限公司 | Rapid detection kit |
| 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 |
| US10267792B2 (en) | 2016-09-09 | 2019-04-23 | International Business Machines Corporation | Device for detecting toxic shock syndrome toxins and method of making the same |
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| CN102393455A (en) * | 2011-08-26 | 2012-03-28 | 厦门出入境检验检疫局检验检疫技术中心 | Preparation method of immunogold rapid test paper for vibrio parahaemolyticus |
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| CN103954759A (en) * | 2014-04-02 | 2014-07-30 | 上海理工大学 | Staphylococcus aureus immunization colloidal gold rapid detection test paper strip |
| CN103913572A (en) * | 2014-04-02 | 2014-07-09 | 上海理工大学 | Method for detecting staphylococcus aureus and monoclonal antibodies of staphylococcus aureus |
| CN103954759B (en) * | 2014-04-02 | 2016-01-20 | 上海理工大学 | Staphylococcus aureus immunity colloidal gold fast detecting test paper strip |
| CN105891470A (en) * | 2014-12-24 | 2016-08-24 | 瑞正浩博(天津)医疗器械销售有限公司 | Rapid detection kit |
| CN104614531A (en) * | 2015-02-09 | 2015-05-13 | 甘肃出入境检验检疫局检验检疫综合技术中心 | Alicyclobacillus colloidal gold label test strip and preparation method thereof |
| CN105651993A (en) * | 2016-01-19 | 2016-06-08 | 南昌大学 | Method for rapidly detecting staphylococcus aureus |
| CN105651993B (en) * | 2016-01-19 | 2018-01-16 | 南昌大学 | A kind of method of fast detecting Staphylococcus aureus |
| US10267792B2 (en) | 2016-09-09 | 2019-04-23 | International Business Machines Corporation | Device for detecting toxic shock syndrome toxins and method of making the same |
| 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 |
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Application publication date: 20110727 |