CN107525930B - Detect the preparation method and application of staphylococcus aureus and enterotoxin B kit - Google Patents
Detect the preparation method and application of staphylococcus aureus and enterotoxin B kit Download PDFInfo
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Abstract
The invention discloses a kind of detection staphylococcus aureus and the preparation method and applications of enterotoxin B kit.Staphylococcus aureus is adsorbed to eggshell membrane surface, the L-form staphylococcus aureus aptamers for having modified gold nanoclusters are added, carry out gold particle colour developing, it is different that eggshell membrane adsorbs staphylococcus aureus concentration, gold particle colour developing is different, realizes to staphylococcus aureus qualitative detection.Gold nanoclusters are first synthesized on eggshell membrane, the DNA aptamers with sulfydryl are accessed by golden sulfide linkage, after adding the enterotoxin B reaction of various concentration, carry out gold particle colour developing, enterotoxin B concentration is different, the amount that hydrogen peroxide is decomposed is different, so that finally different from color developing effect after trisodium citrate, polyethylene glycol, gold chloride reaction.According to the relationship of gold particle absorbance value and antigen concentration, enterotoxin B content in unknown sample is calculated.The present invention is applied to the content detection of food, cereal crops, underwater gold staphylococcus aureus or middle enterotoxin B.
Description
Technical field
The invention belongs to chemical-biological sensing and technical field of biological, and in particular to one kind is examined based on eggshell membrane
Survey the preparation method and application of staphylococcus aureus and enterotoxin B kit.
Background technique
Staphylococcus aureus (Staphylococcus aureus) be it is a kind of can lead to zoonosis it is important cause a disease
Bacterium, the bacterium is in the excreta that nature is widely present in sky gas and water, dust and humans and animals.Staphylococcus aureus can produce
Raw a variety of pathogenic substances, such as some ingredients of toxin, enzyme and thallus.
Staphylococcus aureus can generate a variety of pathogenic substances, these substances can make one once excessive enter human body
Poison.Food poisoning is a global public health problem, is either pass by or present, in flourishing industrialized country and hair
National in exhibition, food poisoning all often occurs.It is reported according to disease prevention and control center of the U.S., as caused by staphylococcus aureus
Food poisoning is only second to escherichia coli, occupies second, accounts for the 33% of food posioning, and Canadian incidence is higher,
The 45% of food posioning is accounted for, the etesian such poisoning in China is also very more, and there are report in all parts of the country, is
The disease of prevention and control of diseases department emphasis monitoring.Largely it turns out that, food-safety problem seriously affects the daily of people
Life, urgent need establish a kind of hypersensitivity rapid analysis method for staphylococcus aureus.Enterotoxin, which is one group, to be had
The bacteriotoxin of superantigen activity, the enterotoxin of human body excess intake can also cause illness, damage human organ, strong to human body
Health has great harm, thus it is also of crucial importance to establish a kind of analyzing detecting method for enterotoxin.
Protein rich in eggshell membrane (ESM), a small amount of liposome and carbohydrate, because of its unique structural property
Gold nanoclusters can be synthesized on its surface.Research shows that eggshell membrane gold nanoclusters have certain catalytic capability, in eggshell membrane
In the presence of gold nanoclusters, H2O2It is decomposed by quick catalysis.H is passed through by gold chloride2O2Reduction method prepare with can be convenient it is various not
With the nanogold particle of partial size, color is taken on a red color according to diameter to blue.Meanwhile gold nano grain is with the variation of diameter
Different colors is showed, ultraviolet-ray visible absorbing peak also can accordingly change.In the preparation of gold nano grain, dioxygen
Aqueous solution is important one of reactant, and hydrogen peroxide can be catalytically decomposed in eggshell membrane gold nanoclusters, later the concentration of hydrogen peroxide
It changes, gold nano grain will show different colors, from red, aubergine to blue.Based on this, we can be established
A kind of new method of bimodulus detection, not only carried out visualization half-quantitative detection to determinand using color change, but also can pass through
The size of absorbance carries out quantitative detection to the exact level of determinand.
Summary of the invention
One of the objects of the present invention is to provide a kind of preparation methods for detecting staphylococcus aureus kit, it includes
Following steps:
(1) staphylococcus aureus and reference bacterial Gram positive bacterium bacillus subtilis and Gram-negative bacteria is big
Intestines Escherichia is incubated for half an hour with eggshell membrane respectively, wherein bacillus subtilis and escherichia coli are as control；
(2) specificity according to staphylococcus aureus and L-form staphylococcus aureus aptamers (ATCC-aptamer)
In conjunction with adding and modified the ATCC-aptamer with the active gold nanoclusters of Mimetic Peroxidase；
(3) citric acid three sodium solution, polyglycol solution, hydrogen peroxide solution gold particle development step: are added into system
And chlorauric acid solution makes it also can be different from the amount of ATCC-aptamer connection according to the difference of staphylococcus aureus concentration,
The color that gold particle is presented is also different, so achievees the purpose that qualitative detection staphylococcus aureus.
Specifically, step (2) the ATCC-aptamer base sequence are as follows:
5’-HS-TCCCTACGGCGCTAACCTCCCAACCGCTCCACCCTGCCTCCGCCTCGCCACCGTGCTACAAC-
3’。
Preferably, the ATCC- with the active gold nanoclusters of Mimetic Peroxidase has been modified in step (2) addition
After aptamer, the hole on the BSA closing eggshell membrane surface of 1mg/mL is added.
Preferably, the ATCC- of modification described in step (2) with the active gold nanoclusters of Mimetic Peroxidase
Aptamer is that the ATCC-aptamer and gold nanoclusters for being 5:3 by volume ratio use interception for 3500 after reaction 10 hours
Bag filter dialyse 24 hours gained.
Preferably, in step (3) the gold particle development step, the citric acid three sodium solution of addition, polyglycol solution,
In hydrogen peroxide solution and chlorauric acid solution, citric acid three sodium solution concentration is 4mmol/L, and polyglycol solution concentration is pure poly-
Ethylene glycol solution dilute 50 times gained, hydrogen peroxide solution concentration be 500 μm of ol/L, chlorauric acid solution concentration be 1mmol/L, four kinds
The volume ratio of solution is 1:1:1:1.
The second object of the present invention is to provide a kind of preparation method for detecting enterotoxin B kit, it includes following step
It is rapid:
(1) eggshell membrane is reacted in alkaline environment 24 hours with gold chloride, also according to major protein on eggshell membrane
Former gold chloride and obtain the eggshell membrane gold nanoclusters with intense red fluorescence；
(2) there is the eggshell membrane of gold nanoclusters to react with glutathione solution 4 hours synthesis again, obtain with peroxidating
The eggshell membrane gold nanoclusters of object analogue enztme activity；
(3) eggshell membrane gold nanoclusters obtained in the previous step are passed through golden sulfide linkage to be adapted to the enterotoxin B DNA with sulfydryl
Body (SEB-aptamer) is connected；
(4) enterotoxin B of various concentration, reaction 20 is added in the specific binding according to SEB-aptamer and enterotoxin B
Minute；
(5) citric acid three sodium solution, polyglycol solution, hydrogen peroxide solution gold particle development step: are added into system
And chlorauric acid solution, according to the difference of enterotoxin B concentration, the area for covering eggshell membrane gold nanoclusters is different, and gold particle is in
Existing color is also different, further according to the relationship of the gold particle absorbance value at wavelength 550nm and enterotoxin B concentration, can count
Calculation obtains the content of enterotoxin B in sample to be tested.
Specifically, step (3) the SEB-aptamer base sequence are as follows:
5’-HS-GGTATTGAGGGTCGCATCCACTGGTCGTTGTTGTCTGTTGTCTGTTATGTTGTTTCGTGATG
GCTCTAACTCTCCTCT-3’。
Preferably, in step (5) the gold particle development step, the citric acid three sodium solution of addition, polyglycol solution,
In hydrogen peroxide solution and chlorauric acid solution, citric acid three sodium solution concentration is 4mmol/L, and polyglycol solution concentration is pure poly-
Ethylene glycol solution dilute 50 times gained, hydrogen peroxide solution concentration be 500 μm of ol/L, chlorauric acid solution concentration be 1mmol/L, four kinds
The volume ratio of solution is 1:1:1:1.
Specifically, being 0.04ng/mL to the detection limit value of enterotoxin B.
The third object of the present invention is to provide a kind of above-mentioned detection staphylococcus aureus or detection enterotoxin B kit
Preparation method application, it may be assumed that content detection or food applied to the staphylococcus aureus in food, cereal crops, water
The content detection of middle enterotoxin B.
Characteristic of the present invention using eggshell membrane absorption bacterium, the specific binding of foundation antigen-antibody, by itself and modification
The aptamers effect of gold nanoclusters, and then achieve the purpose that detect staphylococcus aureus；Recycle eggshell membrane gold nano
Cluster is catalyzed and decomposes the characteristic of hydrogen peroxide, and eggshell membrane gold nanoclusters and gold nano grain, both nano materials are organically tied
Altogether, replace the biological enzyme in enzyme-linked imnnoadsorption using eggshell membrane gold nanoclusters, then according to antibody and antigen
The hypersensitivity kit of detection enterotoxin B analogue enztme is successfully prepared in specific binding, and phenomenon is obvious, operation letter
Just, sensitivity superelevation effectively can accurately measure the concentration of enterotoxin B in food.
It is hypersensitivity kit that the present invention, which detects staphylococcus aureus and the kit of enterotoxin B, it can be quick
Staphylococcus aureus and enterotoxin B are accurately detected, makes eggshell membrane gold nanoclusters that traditional enzyme linked immunological successfully be replaced to inhale
Biological enzyme in reaction enclosure.Eggshell membrane is easily obtained, price, in terms of have biological enzyme macromolecular unrivaled
Advantage, it is often more important that, eggshell membrane gold nanoclusters are easy the problem of losing bioactivity there is no bio protease.Another party
Face introduces under gold nano grain different conditions and shows different colours, Visual retrieval established, in addition, the height of gold nanoparticle
Extinction coefficient provides high detection sensitivity for this detection method, compared with traditional enzyme-linked adsorption method, significantly mentions
The sensitivity and stability of high detection.
It handles by means of the present invention, the gold nano grain solution of different colours, the face of this nano particle can be obtained
Color change can be regulated and controled according to the concentration of hydrogen peroxide, reached by the variation of color or absorbance value to Staphylococcus aureus
The detection of the ultra trace of bacterium and enterotoxin B, compared with prior art, the method for the present invention is simple, at low cost, high sensitivity, Neng Goushi
It now fast and accurately detects, and all there is highly important reference valence for the detection of other large biological molecules and disease
Value has very wide application prospect.
Detailed description of the invention
Fig. 1 is that Fresh Egg shell membrane adsorbs bacterium in the embodiment of the present invention 1, then special with the DNA aptamers of having modified golden cluster
The opposite sex combines and comparative experiments color change compares picture.In Fig. 1, first row is bacillus subtilis, and second row is large intestine angstrom
Uncommon Salmonella, third row are staphylococcus aureus.
Fig. 2 is that modification gold nanoclusters detect different bacterium non-specific adsorption on eggshell membrane in the embodiment of the present invention 1
When chromogenic substrate color change compare picture.In Fig. 2, first row is staphylococcus aureus, and second row is escherichia coli,
Third row is bacillus subtilis.
Color change picture when Fig. 3 is the detection in the embodiment of the present invention 2 to enterotoxin B standard solution, parallel 3 groups.
The absorbance value variation at 550nm when Fig. 4 is the detection in the embodiment of the present invention 2 to enterotoxin B standard solution
Canonical plotting.
Specific embodiment
The present invention is described in further detail with attached drawing combined with specific embodiments below.
Embodiment 1:
Detection for staphylococcus aureus:
Equirotal Fresh Egg shell membrane is added in 21 centrifuge tubes first, every 7 centrifuge tubes are one group and are divided into three
Group, same bacterium (staphylococcus aureus, bacillus subtilis, escherichia coli) solution of every group of addition various concentration
(concentration is 0,4 × 103、4×104、4×105、4×106、2×107、4×107A/mL) reaction 30 minutes, then with sterilizing
After normal saline solution flushes three times, according to staphylococcus aureus and L-form staphylococcus aureus aptamers (ATCC-
Aptamer specific binding) adds the ATCC-aptamer (ATCC- of gold nanoclusters for having modified gold nanoclusters
Aptamer is that the ATCC-aptamer and gold nanoclusters for being 5:3 by volume ratio use interception for 3500 after reaction 10 hours
Bag filter dialyse 24 hours gained), rinsed three times with the PBS of 0.02mol/L, the BSA that 1mg/mL is then added closes egg
Eggshell membrane taking-up is placed in new centrifuge tube, it is water-soluble that 500 μ L, the dioxygen of 500 μm of ol/L is added by the hole on shell membrane surface
Liquid reacts 30 minutes, then pipettes the hydrogen peroxide solution of 200 μ L from centrifuge tube respectively and be added to 200 μ L concentration have been added and be
In the centrifuge tube that polyethylene glycol (PEG 200) solution that the trisodium citrate of 4mmol/L and 200 μ L dilute 50 times mixes, then
The chlorauric acid solution that 200 μ L concentration are 1mmol/L is added.After twenty minutes, color change comparative diagram is as shown in Figure 1.When bacterium
When concentration is 0, the color of gold nano grain is red, due to ATCC-aptamer of gold nanoclusters and staphylococcus aureus
Reaction, will not be with other bacterial actions, so while the bacillus subtilis of other concentration, escherichia coli can be adsorbed on chicken
It is aobvious red in egg shell membrane, and staphylococcus aureus becomes blue with the increase of its concentration, illustrates this method energy
Selectively detection staphylococcus aureus by other bacteriums without being interfered.
Above-mentioned ATCC-aptamer base sequence are as follows:
5’-HS-TCCCTACGGCGCTAACCTCCCAACCGCTCCACCCTGCCTCCGCCTCGCCACCGTGCTACAAC-
3’。
In order to further prove the non-specific adsorption effect of bacterium and eggshell membrane, the gold nano of catalytic will be modified with
The bacterium of the eggshell membrane absorption various concentration of cluster.Firstly, modification has the gold nanoclusters of catalytic on eggshell membrane: by chicken
After egg shell membrane reacts 24 hours with gold nanoclusters, obtained at this time with the active eggshell membrane gold nano of Mimetic Peroxidase
Cluster；Staphylococcus aureus, the escherichia coli of various concentration are separately added on the eggshell membrane that synthesis there are gold nanoclusters again
Bacterium and bacillus subtilis, reaction develop the color after twenty minutes, and certain gradient is presented in gold particle colour developing color.Fig. 2 be with not
Visual retrieval figure after congener bacterium (staphylococcus aureus, escherichia coli, bacillus subtilis) solution effects.
Its bacterial concentration is from left to right successively are as follows: 0,4 × 102、4×103、4×105、4×107A/mL.In the bacterium item of various concentration
Under part, the gold nanoclusters level of coverage on eggshell membrane surface is different, is exposed to the gold nanoclusters on eggshell membrane surface to hydrogen peroxide
The effect that solution decomposes is also different, when bacterium smaller, the exposed gold nano on surface that covers the concentration to eggshell membrane surface
Cluster is more, and the amount that hydrogen peroxide is catalytically decomposed also can be more, and gold nano grain is reunited, therefore forms blue solution；When
The concentration that bacterium covers to eggshell membrane surface is bigger, and the exposed gold nanoclusters on surface are fewer, and hydrogen peroxide is catalytically decomposed
Amount also can be fewer, gold nano grain relative distribution, therefore red is presented in solution.
Embodiment 2:
Detection for enterotoxin B content:
Equirotal eggshell membrane is added in centrifuge tube first, after gold chloride reaction being added 15 minutes, by egg shell
Film takes out, and after being flushed three times with secondary distilled water, after sodium hydroxide solution reaction being added 24 hours, rinses three with secondary distilled water
It is secondary, add glutathione solution react 4 hours, after being flushed three times again with secondary distilled water, by eggshell membrane take out as
In new centrifuge tube, 500 μ L, 1.0 × 10 are added-13The DNA aptamers (SEB-aptamer) of μm ol/L enterotoxin B, reaction 10 are small
Shi Hou is flushed three times with the PBS of 0.01mol/L, and various concentration enterotoxin B solution is added, and is reacted 20 minutes, is used secondary distilled water
After flushing three times, the hydrogen peroxide solution that 500 μ L concentration are 500 μm of ol/L is added and reacts 30 minutes, then is moved from centrifuge tube respectively
Take the hydrogen peroxide solution of 200 μ L be added to be added 200 μ L concentration be 4mmol/L trisodium citrate and 200 μ L dilute 50 times
The centrifuge tube that mixes of polyglycol solution in, add the chlorauric acid solution that 200 μ L concentration are 1mmol/L.After 30 minutes,
Its color change is as shown in Figure 3.In the quantitative detection of enterotoxin B it is parallel three times, concentration is from left to right successively are as follows: 0.04ng/
mL,0.1ng/mL,2.0ng/mL,8.0ng/mL,20.0ng/mL.Under the conditions of the enterotoxin B of various concentration, due to enterotoxin B
The specific binding of corresponding DNA aptamers, the gold nanoclusters degree for making it cover eggshell membrane surface is different, is generating gold
When nano particle, it is also different to be exposed to the effect that the gold nanoclusters on eggshell membrane surface decompose hydrogen peroxide solution, when intestines poison
Plain B concentration is smaller, and the exposed gold nanoclusters on eggshell membrane surface are more, and the amount that hydrogen peroxide is catalytically decomposed also can be more, gold
Nano particle is reunited, therefore forms blue solution, when enterotoxin B concentration is bigger, the exposed gold on eggshell membrane surface
Nano-cluster is fewer, and the amount that hydrogen peroxide is catalytically decomposed also can be fewer, gold nano grain relative distribution, therefore red is presented in solution.
When the concentration of enterotoxin B is 0.04ng/mL, the concentration of enterotoxin B very little at this time, the amount in conjunction with corresponding aptamers is
Through seldom, the detection limit to enterotoxin B detection is reached, detection is limited to 0.04ng/mL.Existed according to the gold nano grain of colour developing
Relationship between absorbance at 550nm and enterotoxin B concentration can get the good standard curve of linear relationship, as shown in figure 4,
The content of enterotoxin B in unknown sample can be calculated according to its standard curve.
Above-mentioned SEB-aptamer base sequence are as follows:
5’-HS-GGTATTGAGGGTCGCATCCACTGGTCGTTGTTGTCTGTTGTCTGTTATGTTGTTTCGTGATG
GCTCTAACTCTCCTCT-3’。
Embodiment 3:
Sample treatment and determination of recovery rates:
Corn 1.0g, rice 0.5g, the flour 0.25g bought from supermarket is weighed to be respectively placed in 5mL centrifuge tube, respectively plus
Enter 4mL 10ng/mL, 1ng/mL, 1ng/mL enterotoxin B solution, it is clear to draw upper layer for ultrasound 15 minutes, centrifugation after mixing 2 hours
Liquid, for use.
In 18 1.5mL centrifuge tubes, synthesis, which is added, to be had the gold nanoclusters of catalytic and makees to make good use of with SEB-aptamer
Eggshell membrane, then 500 μ L are separately added into through corn, the processed enterotoxin B solution of rice and flour, corn, rice and flour
Every kind is respectively one group, totally three groups, every group 6, after reacting 30 minutes, takes out the eggshell membrane in every group of 3 centrifuge tubes, use is secondary
Distilled water flushing three times is stand-by, be separately added into again in remaining every group 3 centrifuge tubes 500 μ L 1ng/mL, 20 μ L 10ng/mL,
20 μ L 10ng/mL enterotoxin B standard solution, reaction after twenty minutes, are rinsed three times with secondary distilled water, finally, this is 18 fragmented
Heart pipe is separately added into 500 μ L 0.5mmol/L H2O2, after reaction 30 minutes, then the dioxygen of 200 μ L is pipetted from centrifuge tube respectively
Aqueous solution is added to the polyglycol solution that 50 times of the trisodium citrate that 200 μ L concentration are 4mmol/L and 200 μ L dilution has been added
In the centrifuge tube mixed, the chlorauric acid solution that 200 μ L concentration are 1mmol/L is added.After 15 minutes, gold particle colour developing is basic
Stablize, measures its absorbance value at 550nm, according to standard curve, corresponding enterotoxin B concentration can be acquired.It is beautiful by calculating
Rice, rice and flour the rate of recovery be respectively: 105.3%, 98.86% and 98.76%.Above data shows that the rate of recovery is all protected
It holds in higher level, this explanation is high with the accuracy of method of the invention to detection enterotoxin B.
Claims (11)
1. a kind of preparation method for detecting staphylococcus aureus kit, it is characterised in that include the following steps:
(1) by staphylococcus aureus and with reference to bacterial Gram positive bacterium bacillus subtilis and Gram-negative bacteria large intestine angstrom
Uncommon Salmonella is incubated for half an hour with eggshell membrane respectively, wherein bacillus subtilis and escherichia coli are as control；
(2) specific binding according to staphylococcus aureus and L-form staphylococcus aureus aptamers ATCC-aptamer, then
The ATCC-aptamer with the active gold nanoclusters of Mimetic Peroxidase has been modified in addition；
(3) citric acid three sodium solution, polyglycol solution, hydrogen peroxide solution and chlorine gold particle development step: are added into system
Auric acid solution makes it also can be different from the amount of ATCC-aptamer connection according to the difference of staphylococcus aureus concentration, gold
The color that grain is presented is also different, so achievees the purpose that qualitative detection staphylococcus aureus.
2. detecting the preparation method of staphylococcus aureus kit according to claim 1, it is characterised in that: step (2)
The ATCC-aptamer base sequence are as follows:
5’-HS-TCCCTACGGCGCTAACCTCCCAACCGCTCCACCCTGCCTCCGCCTCGCCACCGTGCTACAAC-3’。
3. detecting the preparation method of staphylococcus aureus kit according to claim 1, it is characterised in that: in step
(2) addition has been modified after the ATCC-aptamer with the active gold nanoclusters of Mimetic Peroxidase, and 1mg/ is added
The hole on the BSA closing eggshell membrane surface of mL.
4. detecting the preparation method of staphylococcus aureus kit according to claim 1, it is characterised in that: step (2)
It is 5:3 that ATCC-aptamer of the modification with the active gold nanoclusters of Mimetic Peroxidase, which is by volume ratio,
ATCC-aptamer and gold nanoclusters, reaction 10 hours after, use interception be 3500 bag filter dialyse 24 hours obtained by.
5. detecting the preparation method of staphylococcus aureus kit according to claim 1, it is characterised in that: step (3)
In the gold particle development step, the citric acid three sodium solution of addition, polyglycol solution, hydrogen peroxide solution and gold chloride are molten
In liquid, citric acid three sodium solution concentration is 4mmol/L, and polyglycol solution is that pure polyglycol solution dilutes 50 times of gained, double
Oxygen concentration of aqueous solution is 500 μm of ol/L, and chlorauric acid solution concentration is 1 mmol/L, and the volume ratio of four kinds of solution is 1:1:1:1.
6. a kind of preparation method for detecting enterotoxin B kit, it is characterised in that include the following steps:
(1) eggshell membrane is reacted in alkaline environment 24 hours with gold chloride, restores chlorine according to major protein on eggshell membrane
Auric acid and obtain the eggshell membrane gold nanoclusters with intense red fluorescence；
(2) there is the eggshell membrane of gold nanoclusters to react with glutathione solution 4 hours synthesis again, obtain with peroxide mould
The eggshell membrane gold nanoclusters of quasi- enzymatic activity；
(3) eggshell membrane gold nanoclusters obtained in the previous step are passed through into golden sulfide linkage and the enterotoxin B DNA aptamers with sulfydryl
SEB-aptamer is connected；
(4) enterotoxin B of various concentration is added in the specific binding according to SEB-aptamer and enterotoxin B, reacts 20 minutes；
(5) citric acid three sodium solution, polyglycol solution, hydrogen peroxide solution and chlorine gold particle development step: are added into system
Auric acid solution, according to the difference of enterotoxin B concentration, the area for covering eggshell membrane gold nanoclusters is different, what gold particle was presented
Color is also different, further according to the relationship of the gold particle absorbance value at wavelength 550nm and enterotoxin B concentration, can calculate
The content of enterotoxin B into sample to be tested.
7. detecting the preparation method of enterotoxin B kit according to claim 6, it is characterised in that: step (3) described SEB-
Aptamer base sequence are as follows: 5 '-HS-GGTATTGAGGGTCGCATCCACTGGTCGTTGTTGTCTGTTGTCTGTTATGTT
GTTTCGTGATGGCTCTAACTCTCCTCT-3’。
8. detecting the preparation method of enterotoxin B kit according to claim 6, it is characterised in that: step (5) gold
In grain development step, in the citric acid three sodium solution of addition, polyglycol solution, hydrogen peroxide solution and chlorauric acid solution, lemon
Lemon acid trisodium solution concentration is 4mmol/L, and polyglycol solution is that pure polyglycol solution dilutes 50 times of gained, hydrogen peroxide solution
Concentration is 500 μm of ol/L, and chlorauric acid solution concentration is 1 mmol/L, and the volume ratio of four kinds of solution is 1:1:1:1.
9. detecting the preparation method of enterotoxin B kit according to claim 6, it is characterised in that: the detection to enterotoxin B
Limit value is 0.04ng/mL.
10. a kind of application of the preparation method of the staphylococcus aureus kit of detection as described in claim 1, feature exist
In: the content detection applied to the staphylococcus aureus in food, cereal crops, water.
11. a kind of application of the preparation method of the enterotoxin B kit of detection as claimed in claim 6, it is characterised in that: be applied to
The content detection of enterotoxin B in food.
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