CN105424930A - Method for rapidly detecting staphylococcus aureus - Google Patents
Method for rapidly detecting staphylococcus aureus Download PDFInfo
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- CN105424930A CN105424930A CN201510822460.7A CN201510822460A CN105424930A CN 105424930 A CN105424930 A CN 105424930A CN 201510822460 A CN201510822460 A CN 201510822460A CN 105424930 A CN105424930 A CN 105424930A
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- staphylococcus aureus
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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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
The invention provides a method for rapidly detecting staphylococcus aureus. Quantum dot fluorescence and a staphylococcus aureus antibody are coupled to rapidly gather target bacteria, and whether the target bacteria exist or not is judged through the fluorescence. The invention further discloses preparation steps. The method has the advantages that the quantum dot fluorescence is adopted as detection signals, signal gathering is conducted through combination of a sterile injector and a filter membrane, detection is conducted with the help of ultraviolet, and the method is rapid, simple, convenient and accurate. Firstly, CdTe quantum dots have very stable optical properties, and thus the stability of the final detection signals is guaranteed; secondly, the accuracy of experiments is determined by the specificity and singleness of the antibody; finally, gathering can be conducted only through the injector and the filter membrane, and the method is rapid and simple. The method can be derived to be applied to other microorganism detection, and compared with a national standard detection method, the method is rapider, simpler and more convenient, and has good application prospects.
Description
Technical field
The invention belongs to microbial rapid detection technical field, especially relate to a kind of method of fast detecting Staphylococcus aureus.
Background technology
At present, the food poisoning caused by staphylococcus aureus is increasing, and according to the Center for Disease Control report expression, the food posioning caused by staphylococcus aureus ranks first, account for 33% of whole food posioning, Canada is even up to 45%.Staphylococcus aureus is the main pathogens of food poisoning, diarrhoea with dislocation of bacillary groups, its not only in food pollution in occupation of very important position, also be simultaneously one of very important pathogen infection clinically, it and acquired immune deficiency syndrome (AIDS), hepatitis B are listed as three of the world and infect persistent ailment greatly.
Now, the detection method of staphylococcus aureus mainly contains traditional immunological detection, nucleic acid detection method and is separated identification method etc., wherein, separation identification method takes time and effort, and other two kinds of methods require conversion, the enrichment of equal microbial signals, in recent years, the method of the fast separating concentration of the fluorescent characteristic of quantum dot and function magnetic bead is utilized to be widely used, but the detection efficiency height of this method depends in part on the capture rate of magnetic bead, reduces the enrichment of detection signal.And signal enrichment is carried out in the use asepsis injector described in the present invention and filter membrane combination, owing to being the interception to all thalline, this guarantees effective enrichment of detection signal, the CdTe quantum simultaneously gone out for filtration and the compound of antibody can carry out repetitive cycling use, add the utilization factor of antibody, maximization is reduced the loss, and reduces costs.
Summary of the invention
In view of this, the invention aims to provide the method for the staphylococcus aureus that a kind of fast enriching is combined with CdTe quantum fluorescence, and with the fluorescence color of quantum dot for detection signal detects the existence of staphylococcus aureus.
For achieving the above object, the technical scheme of the invention is achieved in that
Adopt the combination of asepsis injector and 0.45um filter membrane, be that miillpore filter is connected on the injector, the potpourri of thalline and antibody and quantum dot crossed film, enrichment is carried out to quantum dot fluorescence signal.
Enrichment method of the present invention has very strong enrichment function to by the fluorescence signal of antibody in quantum dot-labeled staphylococcus aureus.
The invention also discloses the detection operation steps of enrichment method:
(1) take glutathione as stabilizing agent aqueous phase open system synthesis CdTe quantum;
(2) EDC activates the carboxyl of CdTe quantum, and with rabbit antiantibody 25 DEG C of Dual culture 1h of the peculiar gene IsdA of staphylococcus aureus, connect, ultrafiltration, arranges blank;
(3) staphylococcus aureus of 1ml logarithmic phase is got, centrifugal, PBS washs, and adds the compound of CdTe quantum that step (2) obtains and antibody, 30-33 DEG C of co-incubation 1-4h, get the staphylococcus aureus of 1ml logarithmic phase simultaneously, centrifugal, PBS washs, and does not add the compound of CdTe quantum that step (2) obtains and antibody, cultivate 1-4h, as blank for 30-33 DEG C;
(4) potpourri of Dual culture is sucked in the asepsis injector of 1ml, with the membrane filtration of 0.45um, reclaim filtered fluid, then syringe needle is changed, washing three times with PBS, PBST respectively, whether have fluorescence, if there is fluorescence, staphylococcus aureus has been described if observing under ultraviolet, otherwise then without, blank group is through washing equal unstressed configuration.0.45 μm of filter membrane is put into asepsis injector Combination application.At the potpourri of Dual culture just refers to 30-33 DEG C, the potpourri after quantum dot antibody complex, S. aureus L-forms co-incubation 1-4h
The present invention utilizes quantum dot fluorescence as signal, and Signal separator amplification is carried out in asepsis injector and filter membrane combination, and ultraviolet light detects fast.This method can be used for carrying out enrichment to chemistry of micro-organisms signal, is applicable to the enrichment of various chemistry of micro-organisms luminous signal, has potential development and application values.
Relative to prior art, the method for the CdTe quantum fluorescence that the fast enriching described in the invention is combined with staphylococcus aureus and be applied in during staphylococcus aureus detects there is following advantage:
This law adopts quantum dot as detection signal, and carries out signal enrichment with asepsis injector and filter membrane combination, and detect by ultraviolet, this method is quick, easy, accurately.First CdTe quantum has highly stable optical characteristics, this guarantees the stability of final detection signal; Secondly, the specificity of antibody and selectivity determine the accuracy of experiment, finally, only can carry out enrichment with syringe and filter membrane, fast, simply.Owing to being the interception to all thalline, this guarantees effective enrichment of detection signal, the CdTe quantum simultaneously gone out for filtration and the compound of antibody can carry out repetitive cycling use, and add the utilization factor of antibody, maximization is reduced the loss, and reduces costs.This law can derive and is applied to other microorganism detection, and relative to the method that GB detects, this law is more quick, easy, has a good application prospect.
Accompanying drawing explanation
The accompanying drawing of the part of formation the invention is used to provide the further understanding to the invention, and the schematic description and description of the invention, for explaining the invention, does not form the improper restriction to the invention.In the accompanying drawings:
Fig. 1 CdTe quantum Ultraluminescence illuminated diagram.
Fig. 2 combines for the practical operation of the asepsis injector described in the invention embodiment and filter membrane.
Fig. 3 is ultraviolet luminous for the quantum dot fluorescence of staphylococcus aureus after film enrichment described in the invention embodiment.
Embodiment
In order to make the above-mentioned feature and advantage of the present invention clearly and easy understand, below in conjunction with accompanying drawing, embodiments of the present invention are described in further detail.
CdTe quantum described in following embodiment is synthesized for oneself, and synthetic method is the NaHB weighing 80mgTe powder and 50mg
4be placed in dry flask, Homogeneous phase mixing, adds 1ml ultrapure water, and rubber stopper seals, and rubber stopper inserts a syringe needle, reacts 6 hours under room temperature.
IsdA rabbit antiantibody, staphylococcus aureus reference culture are that laboratory is existing, and other (1ml asepsis injector, 0.45um filter membrane, sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride) are commercially available, need sterilizing before using.
Embodiment 1
The invention provides the method for the CdTe quantum fluorescence that a kind of fast enriching is combined with staphylococcus aureus, concrete operation method is as follows:
(1) take the acetate dihydrate cadmium of 26.6mg, add 50mL deionized water, the glutathione of 36.84mg mixes with cadmium acetate solution, regulates pH to 10.5; Take the potassium tellurite of 5.08mg, add 50mL deionized water, by two kinds of solution mixing, 100 DEG C of backflow 1h, the CdTe quantum that synthesizing glutathion is modified, before using, uses concentrated 20 times of the super filter tube of 30kDa;
(2) drawing 100 μ lEDC (4mg/ml) adds in 200ulCdTe quantum dot, activation 30min, then with rabbit antiantibody 25 DEG C of Dual culture 1h of peculiar gene IsdA with 300ul staphylococcus aureus respectively, connect, the ultrafiltration of 100kDa super filter tube, arranges CdTe quantum blank group;
(3) get the staphylococcus aureus of 1ml logarithmic phase, centrifugal, PBS washs, and adds the compound of 200 μ lCdTe quantum dots and antibody, 33 DEG C of Dual culture 1h, arranges the rabbit antiantibody compound blank of CdTe quantum and IsdA;
(4) potpourri of Dual culture is sucked in the asepsis injector of 1ml, with the membrane filtration (practical operation combination as shown in Figure 3) of 0.45um, reclaim filtered fluid, then syringe needle is changed, washing three times with PBS, PBST respectively, whether have fluorescence, if there is fluorescence, staphylococcus aureus has been described if observing under ultraviolet, otherwise then without, blank group is through washing equal unstressed configuration.
In order to understand the effect of the method for the CdTe quantum fluorescence that fast enriching provided by the invention is combined with staphylococcus aureus better, the optical characteristics of experimental measuring point material being characterized, and UV detect has been carried out to final result.
Fig. 1 is the fluorescent characteristic of CdTe quantum.As shown in Figure 1, test CdTe quantum sends out yellow fluorescence, and optimum transmit wavelength is 555nm, and the emission peak of CdTe quantum is symmetrical and narrow, and the fluorescence intensity of CdTe quantum can reach about 28000, the signal intensity of the continuous experiment of guarantee simultaneously.
Fig. 3 is the UV detect result figure after staphylococcus aureus enrichment.Be followed successively by rabbit antiantibody compound blank group, the 300ul antibody group of quantum dot blank group, CdTe quantum and IsdA from left to right.This shows that this enrichment detecting method is feasible, in result, quantum dot fluorescence has difference, may be the impact that quantum dot concentrates.
The foregoing is only the preferred embodiment of the invention; not in order to limit the invention; within all spirit in the invention and principle, any amendment done, equivalent replacement, improvement etc., within the protection domain that all should be included in the invention.
Claims (3)
1. a method for fast detecting Staphylococcus aureus, is characterized in that: by quantum dot fluorescence and Staphylococcus aureus antibody coupling fast enriching object bacteria, and whether object bacteria exists to utilize fluorescence to differentiate.
2. the method for a kind of fast detecting Staphylococcus aureus according to claim 1, is characterized in that, comprise the steps:
(1) take glutathione as stabilizing agent open system synthesis CdTe quantum;
(2) EDC activates the carboxyl of CdTe quantum, with the rabbit antibody of the peculiar protein I sdA of staphylococcus aureus at 20-25 DEG C of co-incubation 1-4h, and ultrafiltration;
(3) get the staphylococcus aureus of 1ml logarithmic phase, centrifugal, PBS washs, add the compound of CdTe quantum that step (2) obtains and antibody, 30-33 DEG C of co-incubation 1-4h, gets the staphylococcus aureus of 1ml logarithmic phase simultaneously, centrifugal, PBS washs; Do not add the compound of CdTe quantum that step (2) obtains and antibody, cultivate 1-4h, as blank for 30-33 DEG C;
(4) the Dual culture potpourri of step (3) is sucked in the asepsis injector of 1ml, with the membrane filtration of 0.45 μm, reclaim filtered fluid, then syringe is changed, with PBS washing 3-5 time, then with PBST washing 3-5 time, the residue after washing observes whether have fluorescence under ultraviolet, if there is fluorescence, staphylococcus aureus is described, otherwise has then there is no staphylococcus aureus.
3. the method for a kind of fast detecting Staphylococcus aureus according to claim 2, is characterized in that: by 0.45 μm of filter membrane and asepsis injector Combination application in step (4).
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282305A (en) * | 2016-11-02 | 2017-01-04 | 百奥森（江苏）食品安全科技有限公司 | A kind of detection method of Staphylococcus aureus in food |
| CN106290909A (en) * | 2016-08-09 | 2017-01-04 | 中海油能源发展股份有限公司 | A kind of quantum dot-labeled method filtering enrichment quickly detection Salmonella |
| CN108204940A (en) * | 2016-12-16 | 2018-06-26 | 江苏维赛科技生物发展有限公司 | High performance liquid chromatography quickly detects staphylococcus aureus concentration in quick-frozen food |
| CN110411993A (en) * | 2019-06-27 | 2019-11-05 | 天津科技大学 | A kind of binary channels visualization quickly detects the new method of food-borne pathogens |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106290909A (en) * | 2016-08-09 | 2017-01-04 | 中海油能源发展股份有限公司 | A kind of quantum dot-labeled method filtering enrichment quickly detection Salmonella |
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| CN108204940A (en) * | 2016-12-16 | 2018-06-26 | 江苏维赛科技生物发展有限公司 | High performance liquid chromatography quickly detects staphylococcus aureus concentration in quick-frozen food |
| CN110411993A (en) * | 2019-06-27 | 2019-11-05 | 天津科技大学 | A kind of binary channels visualization quickly detects the new method of food-borne pathogens |
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Application publication date: 20160323 |