CN102135538A - Detection method of escherichia coli O157:H7 and gold-labeled rapid diagnosis kit for same and preparation method thereof - Google Patents
Detection method of escherichia coli O157:H7 and gold-labeled rapid diagnosis kit for same and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a detection method of escherichia coli O157:H7, 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 escherichia coli O157:H7 on a glassfiber or nonwoven carrier, and coating a detection line composed of the antibodies resistant to the escherichia coli O157:H7 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 escherichia coli O157:H7, 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 escherichia coli O157:H7 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 escherichia coli O157:H7 and the control line composed of the antibodies of the sheep and 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 Escherichia coli O 157: H7, and the Escherichia coli O 157 that is used for this method: H7 golden label fast diagnosis reagent box and its preparation method.
Background technology
Escherichia coli are median size bacillus, and its size is 1～3um * 0.5～0.7um, amphitrichous, no brood cell, the feasible one-tenth pod membrane of the bacterial strain that has, Gram-negative, aerobic or amphimicrobian, biochemical reaction is active, be easy to breed on ordinary culture medium, and adaptability is strong.This bacterium very easily produces drug resistance to general sanitizer sensitivity to microbiotic and sulfonamides etc.
According to the antigen difference, known Escherichia coli have 70 kinds of thalline (" O ") antigen 1s, nearly 103 kinds of surface (K) antigen, and 60 kinds of flagellum (H) antigens, thereby constituted many serotypes.Pili (F) antigen is used to serological Identification recently, modal serotype K88, K99, called after F4 and F5 type respectively.In the serotype that causes people and animals' intestines problem, the branch of enteropathogenic E.Coli (being called for short EPEC), enterotoxigenic escherichia coli (being called for short ETEC) and enteroinvasive E.Coli (being called for short EIEC) etc. is arranged, most enterotoxigenic escherichia colis all have F antigen.In 170 kinds of " O " type antigen serotypes about about 1/2 fowl is had pathogenic, but maximum be O1, O2, four serotypes of O78, O35.Escherichia coli energy decomposition glucose, maltose, sweet mellow wine, wood sugar, glycerine, rhamnose, sorbierite and arabinose produce acid and aerogenesis.Most bacterial strains energy lactose fermenterses have part strain fermentation sucrose, produce indole, do not decompose dextrin, starch, inositol and urea.Do not produce sulfuretted hydrogen, liquefy gelatin, V～P do not test feminine gender, and M.R tests negative.
Enterorrhagia Bacillus coil 0157: H7 is a kind of cause of disease of emerging food transmission disease.Severe complications such as hemolytic uremic syndrome, thrombotic thrombocytopenic purpura also can take place in it except that causing diarrhoea, hemorrhagic enteritis.Since nineteen eighty-two, the U.S. found because of the microbial food poisoning of should causing a disease first, enterorrhagia Bacillus coil 0157: the H7 epidemic situation began progressively to spread and spread, and caused the outburst of diarrhea disease and popular in a plurality of countries such as Britain, Canada, Japan in succession.A province was separated to Escherichia coli O 157: H7 delicatessen food, imported food, diarrhea cases, domestic animals and fowls etc. surplus China had ten successively.
Coliform is one of important indicator of estimating food hygiene quality, is widely used in the food sanitation both at home and abroad at present.This flora is mainly derived from people and warm-blooded animal ight soil, generally is used as the fecal pollution index in the food more.Past China experience aspect colibacillary check is few, and is also abundant inadequately to the understanding of this flora.Whole nation revision food hygiene bacteriologic test method forum in 1974 and national food hygienic standard meeting suggestion in 1976 as fecal pollution index bacterium, and propose to carry out the research work of relevant coliform aspect with coliform.In the process of current revision version food sanitary testing method in 1976, coliform scientific research cooperative groups is also discussed rapid detection method for coliform group bacteria in 1983-1985 again, and measuring for the coliform in the current revision national standard food sanitary testing method microbiology part provides scientific basis.
Escherichia coli O 157: H7 infects and has the anxious characteristics of morbidity, and the natural source of this bacterium is very extensive simultaneously, and at present its mechanism of causing a disease also is not very clear.Though correlation detection technology has been obtained very big progress in recent years, but still there are some defectives.For this reason, my research and development department of company is to the method for inspection of coli-group, mainly be that method for quickly detecting and health significance thereof have carried out scientific research widely and practice, obtained certain achievement, be quick, accurate, the sensitive detection method of further this bacterium of research, and provide scientific basis the control of food-borne pathogens infection, the diagnosis and the epidemiology survey of disease.
The present invention adopts Escherichia coli O 157: H7 immunizing rabbit or sheep, and preparation multi-epitope antibody, 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 specific detection Escherichia coli O 157: H7 antigen, be swift in response (5-20 minute go out result) 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 Escherichia coli O 157: H7 is provided, this method is a step detection method, highly sensitive, high specificity, and also speed is fast, easy and simple to handle, need not Special Equipment, be applicable to multiple occasions such as clinical detection, epidemiology survey and place quarantine.
Second purpose of the present invention is: a kind of Escherichia coli O 157 is provided: the H7 golden label fast diagnosis reagent box.Be at Escherichia coli O 157: a kind of easy and direct single stage method detection kit that provides is provided H7, utilizes this kit to detect highly sensitive, the high specificity of Escherichia coli O 157: H7, and reaction velocity is fast, easy and simple to handle, need not specialized facilities.
The 3rd purpose of the present invention is: a kind of Escherichia coli O 157 is provided: the preparation method of H7 golden label fast diagnosis reagent box, this method is easy, good stability, good reproducibility.
First purpose of the present invention can realize by following measure:
A kind of Escherichia coli O 157: the H7 detection method comprises the steps: that (1) is stated from Chinese People's Anti-Japanese Military and Political College's enterobacteria O157:H7 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 the detection line that formed by the enterobacteria O157:H7 of Chinese People's Anti-Japanese Military and Political College antibody of bag and the control line that forms by sheep, rabbit anti-mouse igg antibody; (2) sample thief dilution drop contains Escherichia coli O 157 as above-mentioned dilution on the carrier of colloid gold label antibody: H7 then forms detection line, control line two-wire on the detection carrier, shows the two-wire result and is judged to be the positive, and only control line colour developing result is judged to be feminine gender.
Second purpose of the present invention can realize by following measure:
A kind of Escherichia coli O 157: establish Escherichia coli O 157: H7 gold mark test card in the H7 golden label fast diagnosis reagent box and form.
Test card is made up of detector bar and plastics cartridge, and detector bar is installed in the plastic clip.Described Escherichia coli O 157: H7 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 gold mark enterobacteria O157:H7 of Chinese People's Anti-Japanese Military and Political College antibody layer, on detection layers, be coated with detection line that forms by Chinese People's Anti-Japanese Military and Political College's enterobacteria monoclonal antibody and the control line that forms by sheep, rabbit anti-mouse igg antibody.
The 3rd purpose of the present invention can realize by following measure:
A kind of Escherichia coli O 157: the preparation of H7 golden label fast diagnosis reagent box is violated the law and is mainly prepared the golden labeling antibody layer and the control line of detection layers, the bag quilt of detection line, and then makes up Escherichia coli O 157 on the liner plate: H7 antigen gold label test strip and check-out console.
The preparation method of described golden labeling antibody layer comprises the steps: the preparation of i collaurum, in concentration is that the 1.0-1.8% concentration that adds its volume in the gold chloride of 0.004-0.010% is the trisodium citrate of 1-3%, boiled 10-20 minute, and can obtain the colloidal gold solution of 15-60 nanometer; The enterobacteria O157:H7 of ii colloid gold label Chinese People's Anti-Japanese Military and Political College antibody, in colloidal gold solution, transfer pH7.2-8.8, press 3-8mg/100ml and add Escherichia coli O 157 with the 0.2M solution of potassium carbonate: H7 antibody, after the stirring, press 0.1-0.6g/100ml again and add animal blood serum albumen in solution, 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 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 colloidal gold solution glass fibre or nonwoven fabrics to liquid and begins to ooze out, 37 ℃ of dried overnight formed golden labeling antibody layer.
The preparation of described detection layers is to be provided with Escherichia coli O 157 on cellulose membrane: detection line that H7 antibody constitutes and the control line that has sheep, rabbit anti-mouse igg antibody to form.Its preparation method be concentration be 1-2mg/ml's or antibody-solutions in just added the fixing agent of 1-3%, utilize Membrane jetter that antibody is sprayed on the cellulose membrane again, the spray film is placed on 37 ℃ of dryings, the PBS that contains 10% calf serum again with 0.01mlpH7.0, sealed 30 minutes down at 37 ℃, 0.01mlpH7.0 the PBS rinsing, 37 ℃ of dryings.
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 Escherichia coli O 157: H7 is a single stage method, has higher specificity and sensitivity, and easy and simple to handle, need not specialized facilities.
2, diagnostic kit of the present invention detects Escherichia coli O 157: have higher specificity and sensitivity during H7, the testing process operation is rapid, quick and easy, 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 a check-out console inner structure synoptic diagram 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 Escherichia coli O 157: the H7 detection method comprises the steps: that (1) is stated from Chinese People's Anti-Japanese Military and Political College's enterobacteria O157:H7 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 Escherichia coli O 157: detection line that H7 antibody forms and the control line that forms by sheep or rabbit anti-mouse igg antibody; (2) sample thief dilution drop contains Escherichia coli O 157 as above-mentioned extract on the carrier of colloid gold label antibody: H7, then on the detection carrier, form detection line, control line two-wire, and show the two-wire result and be judged to be the positive, only control line colour developing result is negative.
With reference to Fig. 1, a kind of Escherichia coli O 157: establish Escherichia coli O 157: H7 gold mark test card 1 in the H7 golden label fast diagnosis reagent box.
With reference to Fig. 2, described Escherichia coli O 157: H7 gold mark test card 1 is to be provided with application of sample end water accepting layer 4, detection layers 8 and water accepting layer 9 on liner plate 10, be provided with gold mark Escherichia coli O 157 between detection layers and application of sample end water accepting layer 4: H7 antibody layer 5 is coated with detection line 7 and control line 6 on detection layers 8.Wherein application of sample end water accepting layer 4 and suction side water accepting layer 9 are made by multilayer filter paper; Detection layers 8 is a cellulose membrane, and this 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 Escherichia coli O 157: the preparation method of H7 golden label fast diagnosis reagent box mainly comprises preparation golden labeling antibody layer (5) and the control line (6) of detection layers (8), the bag quilt of detection line (7), and then liner plate (10) is gone up and made up Escherichia coli O 157: H7 gold label test strip and test card (1).Paste application of sample end absorbent paper 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 absorbent paper layer 4 with cellulose rete 8, folder pastes the glass fibre that contains golden labeling antibody layer 5, and 4/5 part of glass fibre is in the middle of application of sample end absorbent paper layer 4, and 1/5 part is on cellulose rete 8.By 4 mm wides, 8 centimeter length specification slittings, be assembled in the plastic casing more then.Well 2 is over against the application of sample end absorbent paper 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 5 comprises the steps: the preparation of i collaurum, getting the 100mg gold chloride is dissolved in the 1000ml tri-distilled water, adding 15ml, concentration are 1% trisodium citrate, boil 15 minutes, obtain the colloidal gold solution of 15-50 nanometer after the cooling; Ii colloid gold label Escherichia coli O 157: H7 antibody, get the 100ml collaurum and be dissolved in, be dissolved in 0.2M sal tartari and transfer pH8.4, add the 6mg Escherichia coli O 157: H7 antibody, stirred 20 minutes, add the 240mg bovine serum albumin(BSA) again, continue to stir 5 minutes, 4 ℃ left standstill 2-4 hour; Iii with above-mentioned collaurum be dissolved in through 2000 rev/mins centrifugal 10 minutes, abandon sediment, collect supernatant; Iv with supernatant through 10000 rev/mins of centrifugal 60 minutes centrifugal sediments; V with sediment by 4ml/100ml be dissolved in 0.02M, the pH7.4Tris-HCI damping fluid gets collaurum and is dissolved in, and contains 0.25% bovine serum albumin(BSA) and 0.02% Sodium azide in this damping fluid; Vi with collaurum be dissolved in immerse glass fibre or nonwoven fabrics to liquid and begin to ooze out till, 37 ℃ following 2 hours dry and form golden labeling antibody layer 5.Described colloid gold label antibody is sheep, the enterobacteria O157:H7 of rabbit Chinese People's Anti-Japanese Military and Political College antibody.
Described detection layers 8 is to be provided with Escherichia coli O 157 on nitrocellulose filter: detection line 7 that H7 antibody constitutes and the nature controlling line 6 that is formed by sheep or rabbit anti-mouse igg antibody.Its preparation method is to get the enterobacteria O157:H7 of Chinese People's Anti-Japanese Military and Political College antibody, and accent concentration is 1mg/ml, adds 2% formaldehyde, sprays detection line 7 in the cellulose membrane stage casing with Membrane jetter; Getting sheep or rabbit anti-mouse igg accent concentration again is 2mg/ml, add 2% formaldehyde, in the cellulose membrane stage casing,, spray control line 6 with Membrane jetter apart from detection line 05cm place, by 20ul/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 0.01mlpH7.0, sealed 30 minutes down at 37 ℃, 0.01mlpH7.0 the PBS rinsing, 37 ℃ of dryings.
Embodiment two:
Escherichia coli O 157: H7 detection method and golden label fast diagnosis reagent box thereof and example are together.
The antibody that the preparation method of this kit removes the preparation collaurum is that Chinese People's Anti-Japanese Military and Political College's enterobacteria O157:H7 monoclonal anti is external, and other conditions and example are together.
Claims (8)
1. Escherichia coli O 157: H7 detection method, it is characterized in that: (1) is with the Escherichia coli O 157 of colloid gold label: H7 antibody is stated from 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 the enterobacteria O157:H7 of Chinese People's Anti-Japanese Military and Political College antibody (how anti-or monoclonal antibody) detection line that forms 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, contain Escherichia coli O 157 as above-mentioned dilution: H7, then detecting formation detection line and control line two-wire on the carrier, showing the two-wire result and be judged to be the positive, only control line colour developing result is judged to be feminine gender.
2. Escherichia coli O 157: the H7 golden label fast diagnosis reagent box is characterized in that: establish Escherichia coli O 157: H7 gold mark test card in this kit, test card is formed detector bar by detector bar and plastics cartridge and is installed in the plastic clip.
3. Escherichia coli O 157 as claimed in claim 2: H7 gold-labelled rapid diagnosis reagent card, it is characterized in that: described Escherichia coli O 157: H7 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), be provided with gold mark Escherichia coli O 157 between detection layers (8) and application of sample end water accepting layer (4): H7 antibody layer (5) is coated with detection line (7) and control line (6) on detection layers (8).
4. Escherichia coli O 157 as claimed in claim 2: the preparation method of H7 golden label fast diagnosis reagent box, 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 at liner plate (10) then and paste Escherichia coli O 157: H7 antigen gold label test strip and test card (1).
5. Escherichia coli O 157 as claimed in claim 4: the preparation method of H7 golden label fast diagnosis reagent box, 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 0.004～0.012% the gold chloride its volume 1.0～1.8%, concentration is 1～3% trisodium citrate, boiled 10～20 minutes, and can obtain the colloidal gold solution of 15～50 nanometers; The enterobacteria O157:H7 of ii colloid gold label Chinese People's Anti-Japanese Military and Political College monoclonal antibody, in colloidal gold solution, transfer pH7.2～8.8 with the 0.2m solution of potassium carbonate, add anti-people Escherichia coli O157:H7 monoclonal antibody by 4～12mg/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 claim 4 or 5 described Escherichia coli O 157s: the preparation method of H7 golden label fast diagnosis reagent box is characterized in that described colloid gold label antibody is sheep or rabbit Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 antibody or Escherichia coli O 157: the H7 monoclonal antibody.
7. Escherichia coli O 157 as claimed in claim 4: the preparation method of H7 golden label fast diagnosis reagent box is characterized in that described detection layers (8) is to be provided with sheep or rabbit Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 antibody and Escherichia coli O 157 on cellulose membrane: the control line (6) that detection line (7) that the H7 monoclonal antibody constitutes and anti-sheep or hairy rat IgG antibody body form.
8. Escherichia coli O 157 as claimed in claim 7: the preparation method of H7 golden label fast diagnosis reagent box is characterized in that described fixing agent is formaldehyde or acetone.
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| CN102411053A (en) * | 2011-08-11 | 2012-04-11 | 浙江省农业科学院 | Co-detection test strip of colibacillus 0157 and listeria monocytogenes |
| CN102520167A (en) * | 2011-11-15 | 2012-06-27 | 吉林出入境检验检疫局检验检疫技术中心 | Method for detecting Escherichia coli O157 by liquid-phase chip |
| CN103792362A (en) * | 2014-01-22 | 2014-05-14 | 上海理工大学 | Immune colloidal gold rapid detection test strip for enterohemorrhagic E.col O157:H7 |
| CN104316686A (en) * | 2014-10-11 | 2015-01-28 | 南昌大学 | Test paper box capable of detecting escherichia coli O157:H7 |
| 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 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102411053A (en) * | 2011-08-11 | 2012-04-11 | 浙江省农业科学院 | Co-detection test strip of colibacillus 0157 and listeria monocytogenes |
| CN102520167A (en) * | 2011-11-15 | 2012-06-27 | 吉林出入境检验检疫局检验检疫技术中心 | Method for detecting Escherichia coli O157 by liquid-phase chip |
| CN102520167B (en) * | 2011-11-15 | 2013-12-04 | 吉林出入境检验检疫局检验检疫技术中心 | Method for detecting Escherichia coli O157 by liquid-phase chip |
| CN103792362A (en) * | 2014-01-22 | 2014-05-14 | 上海理工大学 | Immune colloidal gold rapid detection test strip for enterohemorrhagic E.col O157:H7 |
| CN103792362B (en) * | 2014-01-22 | 2015-10-07 | 上海理工大学 | Enterohemorrhagic Escherichia coli O 157: H7 immune colloid gold Rapid detection test strip |
| CN104316686A (en) * | 2014-10-11 | 2015-01-28 | 南昌大学 | Test paper box capable of detecting escherichia coli O157:H7 |
| 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 |
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Application publication date: 20110727 |