CN110133262A - A kind of kit for detecting bacterium - Google Patents
A kind of kit for detecting bacterium Download PDFInfo
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- CN110133262A CN110133262A CN201910337253.0A CN201910337253A CN110133262A CN 110133262 A CN110133262 A CN 110133262A CN 201910337253 A CN201910337253 A CN 201910337253A CN 110133262 A CN110133262 A CN 110133262A
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56938—Staphylococcus
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Abstract
The invention discloses double labelling gold nanorods probes, it is added dropwise in the suspension of gold nanorods by urase solution, and anti-Staphylococcus aureus antibody-solutions mixed is added dropwise in mixed；Centrifugation abandons what supernatant obtained；The dual labelled probe is converted into solution ph variation by the catalytic action of urase and the specific recognition effect of IgY, by the number signal of SA；A kind of staphylococcus aureus detection kit is provided simultaneously, it includes: double labelling gold nanorods probe, non-specific magnetic bead, saturation urea, phenolphthalein test paper；The double labelling gold nanorods probe urase and anti-Staphylococcus aureus antibody label；With the quick testing result of phenolphthalein test paper, carmetta is become from white；Advantage of the present invention: the kit is easy to operate；Testing result can carry out interpretation by naked eyes under natural light, without any instrument；And can be compared with standard color comparison card, obtain half-quantitative detection result；Detection quickly, can be completed in 20 minutes.
Description
Technical field
The invention belongs to technical field of microbial detection, and in particular to a kind of kit for detecting bacterium.
Background technique
Staphylococcus aureus (Staphylococcus aureus, SA) and it is a kind of important zoonosis pathogenic bacteria,
It is one of five big pathogen relevant to food origin disease, a variety of severe infections can be caused.Staphylococcus aureus is due to being resistant to
Widely distributed in nature by large-scale pH, temperature and humidity, there are many contaminated chance of food, cause food poisoning
One of main pathogenic bacteria.With being widely used for antibiotic, there is a large amount of resistant Staphylococcus aureus.Golden yellow grape
Disease caused by coccus has become global public health problem, seriously endangers human security and health.Studies have shown that
The general population of 30%-50% is the carrier of SA.Therefore, in the preparation and transportational process of food, product is highly susceptible to dirt
Dye.It is reported that also there are more than 240,000 people to infect SA every year even if in the safest U.S. of food supply, thus caused by payment for medical care
With up to 167,597,860 dollars/year.Meanwhile staphylococcus aureus is also the important pathogen for causing milk cow to suffer from mammitis,
Huge economic loss is caused to aquaculture.China includes resistant Staphylococcus aureus for staphylococcus aureus at present
Detection method is based on traditional microculture, biochemical identification etc., it is necessary to establish a kind of accurate, quick, sensitive detection
Method.
Traditional cultivation is SA detection method most common, that technology is most mature.However, time-consuming for its process, it is uncomfortable
In reply burst food safety affair.In order to accelerate to detect speed, researchers develop many alternatives, such as use nucleic acid
Amplification technique or advanced equipment, detection time can be reduced to several hours or even dozens of minutes from 2-4 days.But these skills
Art still limits their extensive use to the dependence of professional and precision instrument.In addition, complicated material processing is significantly
Improve total testing cost.
The appearance of nanotechnology is overcomes above-mentioned obstacle to provide possibility.In recent years, it is obtained using nanoparticle simpler
Operation and lower cost become focus concerned by people.2016, Bhaisare et al. developed a kind of non-specific magnetic
Pearl (nMB), i.e., the ferric oxide nanometer particle (Fe modified with carbon dots3O4NP).Since magnetic nanoparticle surface is attached with carbon dots,
NMB can effectively, non-specifically be enriched with various Gram-positives or negative bacteria.With traditional immune Magneto separate probe phase
Than can be used directly after nMB synthesis, without marking or modifying antibody.This means that processing cost and testing cost substantially reduce.
Unquestionably, in complicated matrix, nMB is a kind of convenience, economy, powerful Enrichment of bacteria, detection instrument.
Nano Au particle (AuNP) is the molecule of gold, and diameter has high electron density, dielectric special in 1 ~ 100nm
Property and catalytic action, can be in conjunction with a variety of large biological molecules, and do not influence its bioactivity.Because it can also easily be closed
At and functionalization, in terms of biosensor, nanogold is primarily designed as immunosensor, be using Ag-Ab it is special
Property in conjunction with and cause change in detection signal to design.In order to make nano particle that can not only identify object, and can
Catalysis substrate, a variety of double labelling AuNP probes are developed.Wherein, most of researchers select gold nanosphere (AuNS) to make
For carrier.But some researches show that under equal conditions, the protein for being adsorbed on the surface gold nanorods (AuNR), which is significantly more than, to be adsorbed
On the surface gold nanosphere (AuNS).Hinterwirth et al. and Joshi et al. have also obtained similar result.Therefore, we
It can reasonably infer that AuNR can be in combination with more antibody and enzyme, and the performance of double labelling AuNR probe is better than double labelling
AuNS probe.But as far as we know, there is presently no the detections for being applied to SA for double labelling AuNR probe.
Summary of the invention
Object of the present invention is to time-consuming, at high cost for the detection method that solves the problems, such as existing bacterium, and provide one kind
Convenient, economic kit for detecting bacterium.
Double labelling gold nanorods probe, it is to be prepared by the following method:
1) urase solution is added dropwise in the suspension of gold nanorods, mixed is incubated for；
2) incubation of anti bacterial antibody solution mixed is added dropwise；
3) it is centrifuged, abandoning supernatant obtains double labelling gold nanorods probe；
The step 1) is that the urase solution that 120 μ L concentration are 8-12mg/mL is added dropwise to mixing for 1200 μ L gold nanorods
In suspension, slow mixed 5 minutes；
The step 2) is that the anti bacterial antibody solution that 120 μ l concentration are 0.25-1mg/mL, slow mixed 25 is added dropwise
Minute；
The gold nanorods are prepared using seed mediated growth method；
The anti bacterial antibody is antibacterium Yolk antibody (IgY).The bacterium is staphylococcus aureus.
A kind of kit for detecting bacterium, it includes: double labelling gold nanorods probe, non-specific magnetic bead, saturation urea, phenol
Phthalein test paper；The double labelling gold nanorods probe urase and anti bacterial antibody label；
The double labelling gold nanorods concentration and probe concentration are as follows: 0.5-1.5 mg/mL；
The suspension containing non-specific magnetic bead that the non-specific magnetic bead is concentration 0.5-2mg/mL；
The bacterium is staphylococcus aureus；
The double labelling gold nanorods probe is prepared by the above method.
The present invention provides double labelling gold nanorods probes, it is to be prepared by the following method: 1) adding urase solution dropwise
Enter in the suspension of gold nanorods, mixed is incubated for；2) incubation of anti-Staphylococcus aureus antibody-solutions mixed is added dropwise；3) from
The heart, abandoning supernatant obtain double labelling gold nanorods probe；The dual labelled probe passes through the catalytic action of urase and the specific recognition of IgY
Effect, converts the number signal of SA to the variation of solution ph；A kind of staphylococcus aureus detection reagent is provided simultaneously
Box, it includes: double labelling gold nanorods probe, non-specific magnetic bead, saturation urea, phenolphthalein test paper；The double labelling Jenner
Rice stick probe urase and anti-Staphylococcus aureus antibody label；With phenolphthalein test paper piece quick visualization testing result,
Carmetta is become from white；Advantage of the present invention: (1) kit is easy to operate, suitable for lacking the base of professional technician
Food surveillance department uses；(2) testing result can carry out interpretation by naked eyes under natural light, without any instrument；And
It can be compared with standard color comparison card, obtain half-quantitative detection result；(3) detection quickly, can be completed in 20 minutes.
Detailed description of the invention
The transmission electron microscope picture of Fig. 1 gold nanorods；
The transmission electron microscope picture of Fig. 2 non-specificity magnetic bead；
Fig. 3 overhaul flow chart.
Specific embodiment
The preparation of 1 gold nanorods of embodiment
Gold nanorods are prepared using seed mediated growth method, the specific steps are as follows:
1) configure seed liquor: it is 0.1M that the three hydration tetra chlorauric acid aqueous solutions that 96 μ l concentration are 25 mM, which are added to 7.5mL concentration,
Cetyl trimethylammonium bromide aqueous solution in；It is water-soluble then to add the sodium borohydride that 0.5-2mL concentration is 0.02M
Liquid；After mixed solution becomes dark-brown, stirred 1-3 hours at 30 DEG C, acquired solution is seed liquor；
2) configure growth-promoting media: the cetyl trimethylammonium bromide aqueous solution for being 0.1M by 100mL concentration, 2mL concentration are
The sulfuric acid of 0.5M, the three hydration tetra chlorauric acid aqueous solutions that 1.96mL concentration is 25mM, 0.5-2mL concentration is the nitre of 0.01M
The aqueous ascorbic acid that sour silver aqueous solution and 0.5-1mL concentration are 0.1M is sufficiently mixed, and gained mixed solution is to grow
Liquid；
3) seed liquor described in 0.1-1mL is added in growth-promoting media described in 50-100mL, is sufficiently mixed；30 DEG C of standings grow 10-15
Hour；9000 rpm are centrifuged 15 minutes, are concentrated into 20 mL；Using the wet chemical that concentration is 0.2M by the pH of solution
Value is adjusted to 6.0-8.0 to get the suspension containing gold nanorods, for use；
Gold nanorods transmission electron microscope picture is as shown in Figure 1；The gold nanorods are about 40-50nm, diameter about 10-15nm.
The preparation of 2 double labelling gold nanorods probe of embodiment
The urase solution that 120 μ L concentration are 8-12mg/mL is added dropwise described in 1200 μ L embodiments 1 containing gold nanorods
In suspension, slow mixed 5 minutes；Then, the anti-Staphylococcus aureus that 120 μ l concentration are 0.25-1mg/mL is added dropwise
Yolk antibody solution, slow mixed 25 minutes；8000 rpm are centrifuged 10 minutes, abandon supernatant, sediment fraction is double labelling Jenner
Rice stick probe；
It is resuspended with 300-500 μ l ultrapure water, as the suspension containing double labelling gold nanorods probe；By using visible light-
The absorption spectrum of ultraviolet specrophotometer measurement, the more unmarked gold nanorods of absorption spectrum of double labelling gold nanorods probe occurs
Obvious red shift, it was demonstrated that double labelling gold nanorods probe is successfully prepared.
The preparation of the non-specific magnetic bead of embodiment 3
Non-specific magnetic bead is the Fe for being coated with carbon dots3O4Nanoparticle is synthesized using two-step method；
Step 1: preparation Fe3O4Nanoparticle core: 0.5-1g Iron dichloride tetrahydrate and 1-2g ferric chloride hexahydrate are sufficiently molten
Solution is in 30mL ultrapure water；35mL 28%(w/v is added dropwise) ammonium hydroxide；Under the protection of nitrogen, stirred 5 hours at 80 DEG C；With forever
Magnet isolates the solid matter of black from reaction solution, is cleaned with ultrapure water, as Fe3O4Nanoparticle core, dry to
With；
Step 2: in Fe3O4Nanoparticle core surface modification carbon dots: the addition above-mentioned Fe of 0.05-0.2g3O4Nanoparticle core
With 0.25-1g chitosan in 30mL4%(w/v) in glacial acetic acid, mix well；Gained liquid is moved in reaction kettle, 180 DEG C anti-
It answers 6-24 hours；The solid matter for isolating black from reaction solution with permanent magnet, is cleaned with ultrapure water, as non-specific
Magnetic bead dries stand-by；
Non-specific magnetic bead transmission electron microscope picture is as shown in Figure 2；The non-specificity bead diameter is about 15-20nm.
The preparation of 4 phenolphthalein test paper of embodiment
5 filter paper of Fusion is immersed in 1%(w/v) in phenolphthalein solution, after drying, it is cut into the circular paper that diameter is 5mm, to
With.
The detection of 5 staphylococcus aureus of embodiment
Overhaul flow chart is as shown in Figure 3；By 200 μ L of analyte sample fluid, 200 μ of suspension containing double labelling gold nanorods probe
L, 200 μ L of suspension (being resuspended with the ultrapure water) mixing containing non-specific magnetic bead that concentration is 0.5-2mg/mL, at room temperature
Reaction 10 minutes；Magneto separate takes 200 μ L supernatants；200 μ L saturation urea is added in taken supernatant, reacts 5 minutes；It will
Phenolphthalein test paper immerses solution after reaction, observes phenolphthalein test paper color change, interpretation result.
The method of the present invention detection is stablized, and detection limit can be down to 1000CFU/mL, and detection time is short, achievable inspection in 20 minutes
It surveys, testing result can pass through naked eyes interpretation directly under natural light.Result color is compared with standard color comparison card, can be obtained
Semi-quantitative results.To staphylococcus aureus, Escherichia coli O 157: H7, listeria monocytogenes, mouse typhus sramana
The bacterium such as Salmonella, vibrio parahemolyticus are detected, and the testing result of only staphylococcus aureus is positive, remaining is yin
Property.As it can be seen that this method high specificity, has no false positive and false negative result, as a result as shown in table 1, table 2.All experiment counterpoises
Again three times, as a result unanimously.
The detection of 6 food pollution analog sample (beef) of embodiment
5g beef is weighed, meat gruel is ground into, is laid in plane, is placed under ultraviolet lamp and irradiates 2 hours, is wherein stored naturally with removing
Long-pending pathogen；Meat gruel after taking 1g to sterilize is added 10mL sterile saline, mixes well, food samples matrix is made；It will
The S. aureus Inoculate of various concentration detects it with detection kit of the present invention in the food substrate；Detection
The results are shown in Table 3.All experiments are repeated three times, as a result unanimously.
The detection of 7 food pollution analog sample (Chinese cabbage) of embodiment
5g Chinese cabbage is weighed, dish gruel is ground into, is laid in plane, is placed under ultraviolet lamp and irradiates 2 hours, is wherein stored naturally with removing
Long-pending pathogen；Dish after taking 1g to sterilize is rotten, and 10mL sterile saline is added, mixes well, food samples matrix is made；It will
The S. aureus Inoculate of various concentration detects it with detection kit of the present invention in the food substrate；Detection
The results are shown in Table 4；All experiments are repeated three times, as a result unanimously.
。
Claims (9)
1. double labelling gold nanorods probe, it is to be prepared by the following method:
1) urase solution is added dropwise in the suspension of gold nanorods, mixed is incubated for；
2) incubation of anti bacterial antibody solution mixed is added dropwise；
3) it is centrifuged, abandoning supernatant obtains double labelling gold nanorods probe.
2. double labelling gold nanorods probe according to claim 1, it is characterised in that: the anti-micrococcus antibody is anti-
Bacterium Yolk antibody.
3. double labelling gold nanorods probe according to claim 2, it is characterised in that: the bacterium is golden yellow grape
Coccus.
4. double labelling gold nanorods probe according to claim 1,2 or 3, it is characterised in that: the step 1) is will
120 μ L concentration are that the urase solution of 8-12mg/mL is added dropwise in the suspension of 1200 μ L gold nanorods, slow mixed 5 minutes.
5. double labelling gold nanorods probe according to claim 4, it is characterised in that: the step 2) is to add dropwise
Enter the anti-Staphylococcus aureus antibody-solutions that 120 μ l concentration are 0.25-1mg/mL, slow mixed 25 minutes.
6. double labelling gold nanorods probe according to claim 5, it is characterised in that: the gold nanorods are using kind
Sub- growth method preparation.
7. a kind of kit for detecting bacterium, it includes: double labelling gold nanorods probe, non-specific magnetic bead, saturation urea, phenolphthalein
Test paper；The double labelling gold nanorods probe is for double labelling gold nanorods probe described in claim 1.
8. a kind of kit for detecting bacterium according to claim 6, it is characterised in that: the double labelling gold nanorods
Concentration and probe concentration are as follows: 0.5-1.5 mg/mL.
9. a kind of kit for detecting bacterium according to claim 7, it is characterised in that: the non-specific magnetic bead is dense
Spend the suspension containing non-specific magnetic bead of 0.5-2mg/mL.
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Cited By (5)
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| CN111323582A (en) * | 2020-03-16 | 2020-06-23 | 西南大学 | ELISA (enzyme-Linked immunosorbent assay) detection kit for staphylococcus aureus SEO and detection method thereof |
| CN111323596A (en) * | 2020-03-11 | 2020-06-23 | 赵薇 | Staphylococcus aureus detection kit and preparation method thereof |
| CN111366727A (en) * | 2020-03-19 | 2020-07-03 | 吉林大学 | Kit for detecting salmonella typhimurium and preparation method thereof |
| CN112649602A (en) * | 2020-06-17 | 2021-04-13 | 吉林大学 | Visual kit for detecting staphylococcus aureus based on immunomagnetic beads |
| CN113281507A (en) * | 2021-05-23 | 2021-08-20 | 吉林大学 | Rapid detection method and kit for staphylococcus aureus |
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| CN112649602A (en) * | 2020-06-17 | 2021-04-13 | 吉林大学 | Visual kit for detecting staphylococcus aureus based on immunomagnetic beads |
| CN113281507A (en) * | 2021-05-23 | 2021-08-20 | 吉林大学 | Rapid detection method and kit for staphylococcus aureus |
| CN113281507B (en) * | 2021-05-23 | 2022-08-16 | 吉林大学 | Rapid detection method and kit for staphylococcus aureus |
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