CN104316686A - Test paper box capable of detecting escherichia coli O157:H7 - Google Patents
Test paper box capable of detecting escherichia coli O157:H7 Download PDFInfo
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- CN104316686A CN104316686A CN201410533005.0A CN201410533005A CN104316686A CN 104316686 A CN104316686 A CN 104316686A CN 201410533005 A CN201410533005 A CN 201410533005A CN 104316686 A CN104316686 A CN 104316686A
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- escherichia coli
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention relates to an immunochromatography test paper box based on a thallus fluorescence staining technology and used for detecting a stained escherichia coli O157:H7 thallus by using a single antibody. The immunochromatography test paper box comprises a single antibody test strip and a fluorescence dye box. The immunochromatography test paper box has the advantages that the escherichia coli O157:H7 thallus is pre-stained by using a fluorescence dye, and a specific antibody of the escherichia coli O157:H7 is sprayed on a test strip T line to capture the escherichia coli O157:H7 thallus in a mixed sample, so that the thallus in the sample is rapidly and specifically detected.
Description
Technical field
The present invention relates to bacteriology and immunological technique field.Be specifically related to immuno-chromatographic test paper strip of single antibody test bacterium and preparation method thereof.
Background technology
Immunochromatography technique (immunochromatography) is a kind of Fast Detection Technique set up on the basis of immunity percolation beginning of the nineties late 1980s, its technical characteristic is: can be coated on the ad-hoc location of the immunity-chromatography test scraps of paper in advance with band shape with the antibody (or antigen) of the antigen-antibody reaction of the antigen (or antibody) in sample to be checked, antigen (or antibody) in sample to be checked is identified by pigment in advance in the process utilizing developping solution to launch, when antigen (or antibody) in the sample to be checked identified by pigment launches ad-hoc location (namely band shape is coated with the position of antibody (or antigen)), antigen (or antibody) in the sample to be checked identified by pigment is captured by antigen-antibody reaction, therefore, form the chromogenic line of the color development by pigment on location.Because immunochromatography technique must not carry out being separated of binding label and free label, thus simple to operate, quick, be applicable to very much the use of Site Detection.Therefore, the immuno-chromatographic test paper strip development based on immunochromatography technique development is very fast, can be undertaken by immunoreactive detection by immunochromatography technique.
The immune colloidal gold chromatography method test strips extensively adopted at present has the following disadvantages:
1. collaurum is a kind of nano particle, prepares Au colloidal nanoparticles more difficult, and the more difficult control of particle diameter is even;
2. use strong reductant in colloid gold label process, the person's ion that simultaneously will contact heavy metal, have certain influence to health and safety;
3. colloid gold label process is physical adsorption process, therefore less stable during liquid phase;
4. only have when gold grain gathers a certain amount of, people's naked eyes just can observe purplish red band, and this coloured panel and background contrasts are not quite, thus limit detection sensitivity;
5. different matrix of materials effects is obvious, and background interference is very large.
Tradition test strips all adopt double antibody sandwich method (double antibody sandwich format) bacterial detection and, virus, protein, LPS etc.The method feature is known specific antibody to be coated in as detection zone on film using certain amount, can with gold mark that thing is combined two anti-as quality control band.And large antigen molecule has multiple antigen site, test strips antibody used can not select same site, needs to carry out antibody conjugates.Psma ligand is long to process consumes time, increases R&D costs.
Summary of the invention
An object of the present invention is the defect in overcoming prior art, there is provided a kind of highly sensitive, quantitative and qualitative analysis easy and simple to handle detects in mixing sample and detects Escherichia coli O 157: the paper box of H7, for the detection of China pathogenic bacteria and prevention provide a kind of easy diagnosis and detection method and instrument.
A kind of paper box detecting Escherichia coli O 157: H7, comprises monospecific antibody test strips, fluorescent dye box; Described monospecific antibody test strips, comprise sample pad, nitrocellulose filter, adsorptive pads and PVP base plate, its specific structural features is: nitrocellulose filter is pasted onto on PVP base plate; Be sample pad in one end of base plate, the other end is adsorptive pads; Be be monospecific antibody test strips after 3-4mm/ bar slitting with width; Described nitrocellulose filter is sprayed with Escherichia coli O 157: H7 antibody is as detection line T line, and anti-fluorescent dye antibody sprays nature controlling line C line;
The box-packed fluorescent dye had of described fluorescent dye is propidium iodide, fluorescein diacetate (FDA), acridine orange or SYBR Green; Described anti-fluorescent dye antibody spraying concentration is mass concentration 0.01%;
Described Escherichia coli O 157: H7 antibody spraying concentration is 1mg/mL.
The paper box detecting Escherichia coli O 157: H7 carries out the method for Bacteria Detection, and it is characterized in that comprising the steps: to get testing sample, in centrifugal rear fluorescent dye box, fluorescent dye carries out dyeing 5 min, washes 3 times and resuspended precipitation with 1 × PBS; Get 20 uL, add to the sample pad of monospecific antibody test strips with 1 × PBS after mixing, after reaction 15min, monospecific antibody test strips judges qualitative results under uviol lamp, and there is band at detection line place, illustrates in testing sample have object bacteria, and result is positive; Detection line, without bright band, illustrates driftlessness bacterium in testing sample, and result is negative;
Utilize the object bacteria of normal gradients concentration to join in different sample pad respectively, on detection line, fluorescence intensity utilizes fluorescent quantitation spectrometer to detect, and is made typical curve by the fluorescence intensity of object bacteria on detection line of gradient concentration; Read the fluorescence intensity of testing sample on detection line, reference standard curve, quantitatively detects the object bacteria concentration in testing sample.The method is preferred for the Escherichia coli O 157 in food samples: H7 detects.
The invention has the beneficial effects as follows:
1. monospecific antibody paper slip only needs monospecific antibody can complete the detection of a kind of bacterium, avoids antibody conjugates problem, uses fluorescent dye antibody to make C line;
2., owing to not needing gold chloride, do not need to consider antibody conjugates problem yet, and simple process, easy to prepare, more cost-saving than traditional colloidal gold strip;
3. a combinable dye molecule of bacterium is more than the combinative antibody of this bacterium a lot, and therefore multiple test strips is higher than the sensitivity of common test strips;
4. relatively common immunity test strip, does not need the coupling preparation process of the developers such as collaurum, does not even need glue gold pad, therefore easier.
Accompanying drawing explanation
The common colloidal gold strip structural representation of Fig. 1
The schematic diagram of Fig. 2 monospecific antibody test strips of the present invention
ELISA test strip after other dyeing thalline of Fig. 3
1. sarranine, 2. crystal violet, 3. husky yellow, 4.DiOC
18(3), 5. fluorescein isothiocynate
Fig. 4 Escherichia coli O 157: H7 colloidal gold strip testing result
Escherichia coli O 157: H7 body quantity respectively: 1.10
10, 2.10
7, 3.10
6, 4.10
5, 5.10
4, 6.10
3, 7.10
2, 8. blank
Fig. 5 Escherichia coli O 157: H7 monospecific antibody ELISA test strip result
Escherichia coli O 157: H7 body quantity respectively: 1. blank, 2.10
10, 3.10
7, 4.10
6, 5.10
5, 6.10
4, 7.10
3, 8.10
2.
Embodiment
Below in conjunction with drawings and Examples, the present invention will be further described
embodiment 1: monospecific antibody test strips structure of the present invention describes, preparation method
1. paper box of the present invention
Detect the paper box of Escherichia coli O 157: H7, comprise monospecific antibody test strips, fluorescent dye box; Described monospecific antibody test strips, comprise sample pad, nitrocellulose filter, adsorptive pads and PVP base plate, its specific structural features is: nitrocellulose filter is pasted onto on PVP base plate; Be sample pad in one end of base plate, the other end is adsorptive pads; Be be monospecific antibody test strips after 3-4mm/ bar slitting with width; Described nitrocellulose filter is sprayed with Escherichia coli O 157: H7 antibody is as detection line T line, and anti-fluorescent dye antibody sprays nature controlling line C line;
Fluorescent dye propidium iodide, fluorescein diacetate (FDA), acridine orange or SYBR Green are housed in fluorescent dye box.Anti-fluorescent dye antibody spraying concentration is mass concentration 0.01%;
Described Escherichia coli O 157: H7 antibody spraying concentration is 1mg/mL.
2. the preparation of colloidal gold strip
The preparation of 2.1 colloidal gold solutions
Employing trisodium citrate reduction method is prepared, and concrete steps are as follows: get 100 mL ultrapure waters and load in clean beaker, adding 1 mL concentration is wherein the HAuCl of 1%
4solution, to be placed on magnetic stirring apparatus after heating is boiled while stirring, to add 1% citric acid three sodium solution immediately.Finally after solution colour becomes bright redness, continue stirring 10 min and stop heating, naturally cool to after room temperature until it, 4 DEG C save backup.
The preparation of 2.2 gold medal labeling antibodies
Draw a certain amount of colloidal gold solution in the small beaker of cleaning, with 0.2 M K
2cO
3pH value is adjusted to 8.1 by solution; Under room temperature condition, dropwise adding antibody diluent to final concentration is while stirring 20 μ g/mL.After reacting 1 h, dropwise add 1% PEG 20000 of 1/10 volume, after stirring 30 min, more dropwise add the capping agent solutions of 1/10 volume; After continuing stirring 30 min, 4 DEG C, centrifugal 30 min of 5000 rpm, abandon supernatant, and the collaurum re-suspension liquid of precipitation original volume 1/10 is resuspended.
The preparation of 2.3 colloidal gold strips and assembling
Be sprayed on glue gold pad by the metal spraying amount of 10 μ L/cm, after 30 DEG C of vacuum drying 2 h, detection line and control line resist with 2.0 mg/mL anti-object bacteria antibody bodies and 1.0 mg/mL bis-respectively, close after 37 DEG C of drying 8 h with 0.5% OVA, fully dry under being then placed in 37 DEG C of conditions; Filter paper, sample pad, glue gold pad, nitrocellulose filter, thieving paper are overlapped successively and sticks on PVP base plate, loads after being cut into the wide strip of 4 mm with BIODOT cutting machine and get stuck, be placed in packaging bag, add drying agent sealing, preserve under normal temperature.Concrete as Fig. 1.
3. detect
Take out cultured bacterium in shaking table, wash 3 times with the 1*PBS containing 5% polysorbas20, period washes one time with 75% ethanol, the centrifugal 5min of 1W rpm.Thalline after washed with propidium iodide (PI) dyeing in fluorescent dye box, directly adds PI, washes 3 times and resuspended precipitation after PI concentration 25ug/ml dyeing 5min with the 1*PBS of 5% polysorbas20;
Get the mixed liquor after 20 uL thalline dyeing, add in the well of monospecific antibody test strips prepared by us and colloidal gold strip after mixing with the 1*PBS of 90 uL5% polysorbas20s, monospecific antibody test strips result of determination under uviol lamp after reaction 15min, there is band at detection line place, and result is positive.Detection line is without bright band, and result is negative, and makes comparisons with prepared colloidal gold strip;
By the schematic diagram of common colloidal gold strip and monospecific antibody test strips, Fig. 1 and Fig. 2's is more known, from with above content, monospecific antibody test strips 1, monospecific antibody test strips only need monospecific antibody can complete the detection of a kind of bacterium, avoid antibody conjugates problem, use fluorescent dye antibody to make C line.2, a combinable dye molecule of bacterium is more than the combinative antibody of this bacterium a lot, and therefore monospecific antibody test strips is higher than the sensitivity of common test strips.3, relatively common immunity test strip, does not need the coupling preparation process of the developers such as collaurum, does not even need gold pad, therefore easier.4, owing to not needing gold chloride, do not need to consider antibody conjugates problem yet, can Simplified flowsheet, cost-saving.
embodiment 2: the ELISA test strip after other dyeing thalline
With luxuriant red, the husky Huang of common dye, crystal violet and Common fluorescent dyestuff DiOC
18(3), fluorescein isothiocynate (FITC) is to Escherichia coli O 157: H7 is loaded in test strips of the present invention after carrying out dyeing 10 min.C line sprays corresponding dyestuff antibody;
As shown in Figure 3, other dyestuffs find all to develop the color in test strips result.Infer that reason may be that these dyestuffs are not suitable for being applied to test strips, while they are combined with thalline, be also combined with the nitrocellulose filter of test strips or sample pad etc., cause the thalline of dyeing can not to move in test strips, finally can not develop the color.And the propidium iodide of the present invention's screening, fluorescein diacetate (FDA), acridine orange or SYBR Green tetra-kinds of fluorescent dyes can be applicable to the use of test strips preferably, dyeing thalline can move in test strips, and develops the color on T line.
embodiment 3:paper box of the present invention detects Escherichia coli O 157: H7
Paper box prepared by Example 1;
Get thalline quantity and be respectively 10
2, 10
3, 10
4, 10
5, 10
6, 10
7, 10
10escherichia coli O 157: H7 suspension uses 0.01% fluorescent dye dye solution look 5 min.The bacterium drop that dyeing terminates is added 4 samples (about 100 μ L) in test strips well.Reaction 15min after under uviol lamp result of determination, there are two bands at detection line place, result be the positive.Detection line is without bright band, and result is negative;
With identical thalline quantity Escherichia coli O 157: H7 colloidal gold strip testing result compares, Fig. 4 Escherichia coli O 157: H7 colloidal gold strip testing result shows that its detectability of most end is in thalline quantity 10
5to 10
4between.Escherichia coli O 157: H7 monospecific antibody ELISA test strip result shows its detectability of most end in thalline quantity at least 10
3below, we can draw Escherichia coli O 157 of the present invention thus: H7 monospecific antibody test strips (see figure 5) is than Escherichia coli O 157: H7 colloidal gold strip (Fig. 4) is sensitiveer, exceeds more than 1 order of magnitude.
Claims (5)
1. detect a paper box of Escherichia coli O 157: H7, it is characterized in that comprising monospecific antibody test strips, fluorescent dye box; Described monospecific antibody test strips, comprise sample pad, nitrocellulose filter, adsorptive pads and PVP base plate, its specific structural features is: nitrocellulose filter is pasted onto on PVP base plate; Be sample pad in one end of base plate, the other end is adsorptive pads; Be be monospecific antibody test strips after 3-4mm/ bar slitting with width; Described nitrocellulose filter is sprayed with Escherichia coli O 157: H7 antibody is as detection line T line, and anti-fluorescent dye antibody sprays nature controlling line C line.
2. paper box according to claim 1, is characterized in that the box-packed fluorescent dye had of described fluorescent dye is propidium iodide, fluorescein diacetate (FDA), acridine orange or SYBR Green; Described anti-fluorescent dye antibody spraying concentration is mass concentration 0.01%.
3. paper box according to claim 1, is characterized in that described Escherichia coli O 157: H7 antibody spraying concentration is 1mg/mL.
4. a utilization detects Escherichia coli O 157 as claimed in claim 1: the paper box of H7 carries out the method for Bacteria Detection, it is characterized in that comprising the steps: to get testing sample, in centrifugal rear fluorescent dye box, fluorescent dye carries out dyeing 5 min, washes 3 times and resuspended precipitation with 1 × PBS; Get 20 uL, add to the sample pad of monospecific antibody test strips with 1 × PBS after mixing, after reaction 15min, monospecific antibody test strips judges qualitative results under uviol lamp, and there is band at detection line place, illustrates in testing sample have object bacteria, and result is positive; Detection line, without bright band, illustrates driftlessness bacterium in testing sample, and result is negative; Utilize the object bacteria of normal gradients concentration to join in different sample pad respectively, on detection line, fluorescence intensity utilizes fluorescent quantitation spectrometer to detect, and is made typical curve by the fluorescence intensity of object bacteria on detection line of gradient concentration; Read the fluorescence intensity of testing sample on detection line, reference standard curve, quantitatively detects the object bacteria concentration in testing sample.
5. method according to claim 4, is characterized in that described sample is food samples.
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| CN201410533005.0A CN104316686A (en) | 2014-10-11 | 2014-10-11 | Test paper box capable of detecting escherichia coli O157:H7 |
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| CN201410533005.0A CN104316686A (en) | 2014-10-11 | 2014-10-11 | Test paper box capable of detecting escherichia coli O157:H7 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105606806A (en) * | 2016-02-29 | 2016-05-25 | 上海理工大学 | Escherichia coli O157:H7 direct type immunofluorescence chromatography test paper |
| CN105638839A (en) * | 2016-03-16 | 2016-06-08 | 刘剑 | Meat product pressing device for detecting escherichia coli by applying immunology |
| CN105974113A (en) * | 2016-05-03 | 2016-09-28 | 上海理工大学 | Sandwiched immunochromatographic test paper used for detecting Escherichia coli O157:H7 |
| CN108680747A (en) * | 2018-06-26 | 2018-10-19 | 宁波奥丞生物科技有限公司 | A kind of placenta growth factor detection immunofluorescence technique kit |
| CN109738638A (en) * | 2019-01-03 | 2019-05-10 | 西北农林科技大学 | Direct immunization chromatographs detection method, test strips and the application for detecting Escherichia coli |
| CN111521827A (en) * | 2020-05-21 | 2020-08-11 | 上海理工大学 | Immunofluorescence test strip for rapidly determining titer of monoclonal antibody based on antigen tracing |
| CN113687072A (en) * | 2021-09-10 | 2021-11-23 | 佛山墨赛生物技术有限公司 | Test strip, kit and detection method for quantitative detection of viable bacteria of Escherichia coli O157H 7 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN108680747A (en) * | 2018-06-26 | 2018-10-19 | 宁波奥丞生物科技有限公司 | A kind of placenta growth factor detection immunofluorescence technique kit |
| CN109738638A (en) * | 2019-01-03 | 2019-05-10 | 西北农林科技大学 | Direct immunization chromatographs detection method, test strips and the application for detecting Escherichia coli |
| CN111521827A (en) * | 2020-05-21 | 2020-08-11 | 上海理工大学 | Immunofluorescence test strip for rapidly determining titer of monoclonal antibody based on antigen tracing |
| CN113687072A (en) * | 2021-09-10 | 2021-11-23 | 佛山墨赛生物技术有限公司 | Test strip, kit and detection method for quantitative detection of viable bacteria of Escherichia coli O157H 7 |
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Application publication date: 20150128 |