CN107228819A - A kind of flow cytometry assays of staphylococcus aureus - Google Patents
A kind of flow cytometry assays of staphylococcus aureus Download PDFInfo
- Publication number
- CN107228819A CN107228819A CN201710410954.3A CN201710410954A CN107228819A CN 107228819 A CN107228819 A CN 107228819A CN 201710410954 A CN201710410954 A CN 201710410954A CN 107228819 A CN107228819 A CN 107228819A
- Authority
- CN
- China
- Prior art keywords
- staphylococcus aureus
- flow cytometry
- staphylococcus
- culture
- flow cytometer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000191967 Staphylococcus aureus Species 0.000 title claims abstract description 50
- 238000000684 flow cytometry Methods 0.000 title claims abstract description 17
- 241000588724 Escherichia coli Species 0.000 claims abstract description 21
- 239000007853 buffer solution Substances 0.000 claims abstract description 16
- 108091023037 Aptamer Proteins 0.000 claims abstract description 13
- 239000000725 suspension Substances 0.000 claims abstract description 11
- XKRFYHLGVUSROY-UHFFFAOYSA-N argon Substances [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims abstract description 9
- 229910052786 argon Inorganic materials 0.000 claims abstract description 9
- 238000003384 imaging method Methods 0.000 claims abstract description 8
- 238000004458 analytical method Methods 0.000 claims description 10
- 238000005119 centrifugation Methods 0.000 claims description 10
- 238000011534 incubation Methods 0.000 claims description 10
- 238000001802 infusion Methods 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 claims description 9
- 241000191940 Staphylococcus Species 0.000 claims description 7
- -1 argon ion Chemical class 0.000 claims description 7
- 230000003139 buffering effect Effects 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 108010088751 Albumins Proteins 0.000 claims description 5
- 102000009027 Albumins Human genes 0.000 claims description 5
- 239000012888 bovine serum Substances 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 5
- 238000002347 injection Methods 0.000 claims description 5
- 239000007924 injection Substances 0.000 claims description 5
- 239000002184 metal Substances 0.000 claims description 5
- 229910052751 metal Inorganic materials 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 239000013589 supplement Substances 0.000 claims description 5
- 238000010257 thawing Methods 0.000 claims description 5
- 238000010792 warming Methods 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 108020004707 nucleic acids Proteins 0.000 claims description 2
- 150000007523 nucleic acids Chemical class 0.000 claims description 2
- 102000039446 nucleic acids Human genes 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 18
- 238000004043 dyeing Methods 0.000 abstract description 4
- 239000001963 growth medium Substances 0.000 abstract 1
- 238000000034 method Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 208000031729 Bacteremia Diseases 0.000 description 1
- 241001478240 Coccus Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 206010031252 Osteomyelitis Diseases 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 206010000269 abscess Diseases 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 206010014665 endocarditis Diseases 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 208000013223 septicemia Diseases 0.000 description 1
- 206010040872 skin infection Diseases 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
- G01N2015/144—Imaging characterised by its optical setup
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Engineering & Computer Science (AREA)
- Dispersion Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a kind of flow cytometry assays of staphylococcus aureus, comprise the following steps：Staphylococcus aureus strains are put into culture medium and cultivated, then staphylococcus aureus and Escherichia coli are made the suspension of suitable concn, then are combined with aptamers and sequentially enter flow cytometer using argon ion laser using different folding modes and carry out fluorescence signal detection.Compared with traditional detection method, the present invention improves the quick increase that is active and realizing staphylococcus aureus quantity of staphylococcus aureus with two groups of culture mediums successively culture staphylococcus aureus, buffer solution and laser imaging are easy to the staphylococcus aureus after dyeing to be observed and counted, the argon ion laser being electrically connected with the top of flow cytometer with calculator can improve the accuracy of detection, the present invention is simple and convenient, step operation is easy, detection cycle is short, accuracy is high, it is applied widely, be conducive to popularizing.
Description
Technical field
The invention belongs to staphylococcus aureus technical field, more specifically, more particularly to a kind of Staphylococcus aureus
The flow cytometry assays of bacterium.
Background technology
A kind of staphylococcus aureus, spherical gram-positive bacteria, is present in sky gas and water, dust, the mankind or animal
Skin, soil etc., are the most common pathogens of mankind's pyogenic infection.It from infected human or animal directly contact except being felt
Dye, can also be infected by the air spittle or mediate contact and such as beat spray Spray.The disease as caused by staphylococcus aureus can be with
From slight skin infection, disease such as pneumonia, meningitis, osteomyelitis, endocarditis, the intestines and stomach sense of abscess to threat to life
Dye, bacteremia, septicemia etc..Therefore develop highly sensitive detection methods of staphylococcus aureus to be significant.
Traditional detection methods of staphylococcus aureus is typically all based on the selectivity culture to bacterium and is enriched with form,
Such as colony counting method, dyeing observation etc..However, these detection methods need to take to day, it is fairly time consuming, laborious.And
The limitation being subject to during detection is more, poor accuracy, is not suitable for widely popularizing.
The content of the invention
The invention aims to solve shortcoming present in prior art, and a kind of staphylococcus aureus proposed
Flow cytometry assays.
To achieve the above object, the present invention provides following technical scheme：A kind of flow cytometry of staphylococcus aureus
Detection method, comprises the following steps：
S1, staphylococcus aureus strains are put into brain-heart infusion medium under 34-40 degrees Celsius of aerobic conditions overnight
Culture, then further takes out the staphylococcus aureus strains after brain-heart infusion medium culture and is put into LB culture mediums and existed with shaking table
Cultivated 3-4 hours under 150rpm/min, 34-40 degrees celsius；
S2, with the S. aureus colonies in oese picking LB culture mediums and Escherichia coli in 8000-12000rpm/
Centrifugation 3-7 minutes, are then washed 1-3 times with buffering in min centrifuge, and supernatant is abandoned in centrifugation suction；
It S3, will be preserved with four kinds of folding modes, then fitted using the nucleic acid of four kinds of folding modes after aptamer uniform stirring
Part is separately added into S. aureus colonies solution and Escherichia coli solution and supplements incubation system with buffer solution, on ice 25-
After 35 min, washing is resuspended；
S4, then four groups of staphylococcus aureuses after incubation and E. coli suspension existed with SYBRGreenI dyestuffs respectively
Dyed 10-20 minutes under 20-25 degrees Celsius；
S5, open flow cytometer and adjust the voltage mode and voltage, histogrammic voltage and threshold values in analysis face, four groups golden yellow
Color staphylococcus and E. coli suspension are sequentially placed into streaming pipe and inserted at the injection port of flow cytometer；
S6, finally use laser confocal microscope imaging technique observation analysis fluorescence signal, then be repeated several times aforesaid operations obtain
The data gone out are analyzed.
It is preferred that, four kinds of folding modes in the step S3 include respectively（1）Water-bath is warming up to 95 DEG C -100 DEG C,
Centrifuge tube is inserted on cursory, is put into water, after 3-7min, turns off water-bath, its Temperature fall is stayed overnight, finally takes out and puts
Saved backup in 2 DEG C -6 DEG C of quick secondary memory；（2）Using PCR instrument, it is 95 DEG C -100 DEG C, 2- to set PCR programs
7min, 1 DEG C -3 DEG C are dropped per 35s-45s, 35 DEG C -40 DEG C are down to, and are taken out from PCR instrument, are directly put in subzero 10 DEG C -20 DEG C
It is standby in quick secondary memory, use preceding existing thawing；（3）The 3-7min in 95 DEG C -100 DEG C of metal bath, is immediately placed on ice
8min-12min on box；（4）Without any processing, directly take out and use from 2 DEG C -6 DEG C of centrifuge tube.
It is preferred that, the buffer solution in the step S3 includes combination buffer and the BB containing 1 percent bovine serum albumins
Buffer solution.
It is preferred that, the top of the flow cytometer is provided with the argon ion laser being electrically connected with calculator.
The technique effect and advantage of the present invention：A kind of Flow cytometry for staphylococcus aureus that the present invention is provided
Method, compared with traditional detection method, the present invention is golden yellow to improve with two groups of culture mediums successively culture staphylococcus aureus
The staphylococcic activity of color and the quick increase for realizing staphylococcus aureus quantity, are easy to later stage multigroup detection to be applicable, by gold
Staphylococcus aureus are added to be incubated in the aptamer of four kinds of folding modes, effectively can be drawn in flow cytometry
Influence of the aptamers folding mode to detection, buffer solution and laser imaging are easy to the staphylococcus aureus after dyeing to be observed
And statistics, flow cytometer top and the argon ion laser of calculator electric connection can improve the accuracy of detection, be easy to
Promote the use of, the present invention is simple and convenient, easily, detection cycle is short for step operation, and accuracy is high, applied widely, is conducive to pushing away
Wide and popularization.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with specific embodiment, to this
Invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, not
For limiting the present invention.Based on the embodiment in the present invention, those of ordinary skill in the art are not before creative work is made
The every other embodiment obtained is put, the scope of protection of the invention is belonged to.
Embodiment 1
A kind of flow cytometry assays of staphylococcus aureus, comprise the following steps：
S1, staphylococcus aureus strains are put into brain-heart infusion medium under 34 degrees Celsius of aerobic conditions and trained overnight
Support, then further take out the staphylococcus aureus strains after brain-heart infusion medium culture and be put into LB culture mediums and existed with shaking table
Cultivated 3 hours under 150rpm/min, 34 degrees celsius；
S2, with the S. aureus colonies in oese picking LB culture mediums and Escherichia coli 8000rpm/min from
Centrifugation 3 minutes, are then washed 1 time with buffering in scheming, and supernatant is abandoned in centrifugation suction；
S3, it will be preserved after aptamer uniform stirring with four kinds of folding modes, four kinds of folding modes include respectively（1）By water-bath
Pot is warming up to 95 DEG C, and centrifuge tube is inserted on cursory, is put into water, after 3min, turns off water-bath, its Temperature fall is stayed overnight,
Finally take out to put to 2 DEG C of quick secondary memory and save backup；（2）Using PCR instrument, it is 95 DEG C to set PCR programs,
2min, 1 DEG C is dropped per 35s, 35 DEG C are down to, and is taken out, is directly put in standby in subzero 10 DEG C of quick secondary memory from PCR instrument
With using preceding existing thawing；（3）The 3min in 95 DEG C of metal bath, is immediately placed on 8min on ice chest；（4）Without any processing,
Directly take out and use from 2 DEG C of centrifuge tube, be then separately added into golden yellow Portugal using the aptamer of four kinds of folding modes
Grape coccus bacterium colony solution and Escherichia coli solution supplement incubation system with buffer solution, and on ice after 25min, washing is resuspended, buffer solution
BB buffer solutions including combination buffer and containing 1 percent bovine serum albumins；
S4, then four groups of staphylococcus aureuses after incubation and E. coli suspension existed with SYBRGreenI dyestuffs respectively
Dyed 10 minutes under 20 degrees Celsius；
S5, open flow cytometer and adjust the voltage mode and voltage, histogrammic voltage and threshold values in analysis face, four groups golden yellow
Color staphylococcus and E. coli suspension are sequentially placed into streaming pipe and inserted at the injection port of flow cytometer, flow cytometer
Top be provided with calculator be electrically connected with argon ion laser；
S6, finally use laser confocal microscope imaging technique observation analysis fluorescence signal, then be repeated several times aforesaid operations obtain
The data gone out are analyzed.
Embodiment 2
A kind of flow cytometry assays of staphylococcus aureus, comprise the following steps：
S1, staphylococcus aureus strains are put into brain-heart infusion medium under 37 degrees Celsius of aerobic conditions and trained overnight
Support, then further take out the staphylococcus aureus strains after brain-heart infusion medium culture and be put into LB culture mediums and existed with shaking table
Cultivated 3.5 hours under 150rpm/min, 37 degrees celsius；
S2, with the S. aureus colonies in oese picking LB culture mediums and Escherichia coli 10000rpm/min from
Centrifugation 5 minutes, are then washed 2 times with buffering in scheming, and supernatant is abandoned in centrifugation suction；
S3, it will be preserved after aptamer uniform stirring with four kinds of folding modes, four kinds of folding modes include respectively（1）By water-bath
Pot is warming up to 98 DEG C, and centrifuge tube is inserted on cursory, is put into water, after 5min, turns off water-bath, its Temperature fall is stayed overnight,
Finally take out to put to 4 DEG C of quick secondary memory and save backup；（2）Using PCR instrument, it is 97 DEG C to set PCR programs,
5min, 2 DEG C are dropped per 40s, 37 DEG C are down to, and are taken out, are directly put in standby in subzero 15 DEG C of quick secondary memory from PCR instrument
With using preceding existing thawing；（3）The 5min in 98 DEG C of metal bath, is immediately placed on 10min on ice chest；（4）Without any place
Reason, directly takes out from 4 DEG C of centrifuge tube and uses, be then separately added into golden yellow using the aptamer of four kinds of folding modes
Staphylococcus bacterium colony solution and Escherichia coli solution supplement incubation system with buffer solution, and on ice after 30 min, washing is resuspended, buffering
Liquid includes combination buffer and the BB buffer solutions containing 1 percent bovine serum albumins；
S4, then four groups of staphylococcus aureuses after incubation and E. coli suspension existed with SYBRGreenI dyestuffs respectively
Dyed 15 minutes under 23 degrees Celsius；
S5, open flow cytometer and adjust the voltage mode and voltage, histogrammic voltage and threshold values in analysis face, four groups golden yellow
Color staphylococcus and E. coli suspension are sequentially placed into streaming pipe and inserted at the injection port of flow cytometer, flow cytometer
Top be provided with calculator be electrically connected with argon ion laser；
S6, finally use laser confocal microscope imaging technique observation analysis fluorescence signal, then be repeated several times aforesaid operations obtain
The data gone out are analyzed.
Embodiment 3
A kind of flow cytometry assays of staphylococcus aureus, comprise the following steps：
S1, staphylococcus aureus strains are put into brain-heart infusion medium under 40 degrees Celsius of aerobic conditions and trained overnight
Support, then further take out the staphylococcus aureus strains after brain-heart infusion medium culture and be put into LB culture mediums and existed with shaking table
Cultivated 4 hours under 150rpm/min, 40 degrees celsius；
S2, with the S. aureus colonies in oese picking LB culture mediums and Escherichia coli 12000rpm/min from
Centrifugation 7 minutes, are then washed 3 times with buffering in scheming, and supernatant is abandoned in centrifugation suction；
S3, it will be preserved after aptamer uniform stirring with four kinds of folding modes, four kinds of folding modes include respectively（1）By water-bath
Pot is warming up to 100 DEG C, and centrifuge tube is inserted on cursory, is put into water, after 7min, turns off water-bath, makes its Temperature fall mistake
At night, finally taking-up, which is put to 6 DEG C of quick secondary memory, saves backup；（2）Using PCR instrument, it is 100 DEG C to set PCR programs,
7min, 3 DEG C are dropped per 45s, 40 DEG C are down to, and are taken out, are directly put in standby in subzero 20 DEG C of quick secondary memory from PCR instrument
With using preceding existing thawing；（3）The 7min in 100 DEG C of metal bath, is immediately placed on 12min on ice chest；（4）Without any place
Reason, directly takes out from 6 DEG C of centrifuge tube and uses, be then separately added into golden yellow using the aptamer of four kinds of folding modes
Staphylococcus bacterium colony solution and Escherichia coli solution supplement incubation system with buffer solution, and on ice after 35 min, washing is resuspended, buffering
Liquid includes combination buffer and the BB buffer solutions containing 1 percent bovine serum albumins；
S4, then four groups of staphylococcus aureuses after incubation and E. coli suspension existed with SYBRGreenI dyestuffs respectively
Dyed 20 minutes under 25 degrees Celsius；
S5, open flow cytometer and adjust the voltage mode and voltage, histogrammic voltage and threshold values in analysis face, four groups golden yellow
Color staphylococcus and E. coli suspension are sequentially placed into streaming pipe and inserted at the injection port of flow cytometer, flow cytometer
Top be provided with calculator be electrically connected with argon ion laser；
S6, finally use laser confocal microscope imaging technique observation analysis fluorescence signal, then be repeated several times aforesaid operations obtain
The data gone out are analyzed.
In summary：A kind of flow cytometry assays for staphylococcus aureus that the present invention is provided, it is and traditional
Detection method is compared, and the present invention improves the work of staphylococcus aureus with two groups of culture mediums successively culture staphylococcus aureus
Property and realize the quick increase of staphylococcus aureus quantity, be easy to later stage multigroup detection to be applicable, by staphylococcus aureus plus
It is incubated in the aptamer for entering four kinds of folding modes, can effectively draws aptamers folding mode in flow cytometry
Influence to detection, buffer solution and laser imaging are easy to the staphylococcus aureus after dyeing to be observed and counted, and streaming is thin
The argon ion laser being electrically connected with the top of born of the same parents' instrument with calculator can improve the accuracy of detection, be easy to promote the use of, this hair
Bright simple and convenient, easily, detection cycle is short for step operation, and accuracy is high, applied widely, is conducive to popularizing.
Finally it should be noted that：The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention,
Although the present invention is described in detail with reference to the foregoing embodiments, for those skilled in the art, it still may be used
To be modified to the technical scheme described in foregoing embodiments, or equivalent substitution is carried out to which part technical characteristic,
Within the spirit and principles of the invention, any modification, equivalent substitution and improvements made etc., should be included in the present invention's
Within protection domain.
Claims (4)
1. a kind of flow cytometry assays of staphylococcus aureus, it is characterised in that：Comprise the following steps：
S1, staphylococcus aureus strains are put into brain-heart infusion medium under 34-40 degrees Celsius of aerobic conditions overnight
Culture, then further takes out the staphylococcus aureus strains after brain-heart infusion medium culture and is put into LB culture mediums and existed with shaking table
Cultivated 3-4 hours under 150rpm/min, 34-40 degrees celsius；
S2, with the S. aureus colonies in oese picking LB culture mediums and Escherichia coli in 8000-12000rpm/
Centrifugation 3-7 minutes, are then washed 1-3 times with buffering in min centrifuge, and supernatant is abandoned in centrifugation suction；
It S3, will be preserved with four kinds of folding modes, then fitted using the nucleic acid of four kinds of folding modes after aptamer uniform stirring
Part is separately added into S. aureus colonies solution and Escherichia coli solution and supplements incubation system with buffer solution, on ice 25-
After 35 min, washing is resuspended；
S4, then four groups of staphylococcus aureuses after incubation and E. coli suspension existed with SYBRGreenI dyestuffs respectively
Dyed 10-20 minutes under 20-25 degrees Celsius；
S5, open flow cytometer and adjust the voltage mode and voltage, histogrammic voltage and threshold values in analysis face, four groups golden yellow
Color staphylococcus and E. coli suspension are sequentially placed into streaming pipe and inserted at the injection port of flow cytometer；
S6, finally use laser confocal microscope imaging technique observation analysis fluorescence signal, then be repeated several times aforesaid operations obtain
The data gone out are analyzed.
2. a kind of flow cytometry assays of staphylococcus aureus according to claim 1, it is characterised in that：Institute
State four in step S3 kinds of folding modes includes respectively（1）Water-bath is warming up to 95 DEG C -100 DEG C, it is cursory that centrifuge tube is inserted in
On, it is put into water, after 3-7min, turns off water-bath, stay overnight its Temperature fall, finally takes out and put quick auxiliary to 2 DEG C -6 DEG C
Help in memory and save backup；（2）Using PCR instrument, it is 95 DEG C -100 DEG C to set PCR programs, 2-7min, the drop 1 per 35s-45s
DEG C -3 DEG C, 35 DEG C -40 DEG C are down to, takes out, is directly put in standby in subzero 10 DEG C -20 DEG C of quick secondary memory from PCR instrument
With using preceding existing thawing；（3）The 3-7min in 95 DEG C -100 DEG C of metal bath, is immediately placed on 8min-12min on ice chest；（4）
Without any processing, directly take out and use from 2 DEG C -6 DEG C of centrifuge tube.
3. a kind of flow cytometry assays of staphylococcus aureus according to claim 1, it is characterised in that：Institute
The buffer solution stated in step S3 includes combination buffer and the BB buffer solutions containing 1 percent bovine serum albumins.
4. a kind of flow cytometry assays of staphylococcus aureus according to claim 1, it is characterised in that：Institute
The top for stating flow cytometer is provided with the argon ion laser being electrically connected with calculator.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710410954.3A CN107228819B (en) | 2017-06-04 | 2017-06-04 | Flow cytometry detection method for staphylococcus aureus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710410954.3A CN107228819B (en) | 2017-06-04 | 2017-06-04 | Flow cytometry detection method for staphylococcus aureus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107228819A true CN107228819A (en) | 2017-10-03 |
| CN107228819B CN107228819B (en) | 2019-12-13 |
Family
ID=59933920
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710410954.3A Expired - Fee Related CN107228819B (en) | 2017-06-04 | 2017-06-04 | Flow cytometry detection method for staphylococcus aureus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107228819B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112903995A (en) * | 2021-01-15 | 2021-06-04 | 西北农林科技大学 | Colorimetric/fluorescent probe, test strip for detecting zearalenone and application |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101603075A (en) * | 2009-06-26 | 2009-12-16 | 华南理工大学 | A kind of method that detects based on the sorting enzyme of yeast surface display |
| CN101760502A (en) * | 2010-01-28 | 2010-06-30 | 中国检验检疫科学研究院 | Method for rapidly determining drug tolerance of strain |
| CN103243166A (en) * | 2013-05-15 | 2013-08-14 | 中国农业科学院植物保护研究所 | Rapid molecular detection method and application for plasmopara viticola |
| CN104031875A (en) * | 2014-05-30 | 2014-09-10 | 浙江省农业科学院 | Engineering bacteria for producing S-equol and application |
| CN104630291A (en) * | 2015-03-13 | 2015-05-20 | 吕涛 | Method for realizing high-yield succinic acid through coupling fermenting of two bacterial strains |
| CN105506070A (en) * | 2015-08-20 | 2016-04-20 | 江苏省家禽科学研究所 | Specific probe and method thereof for rapid and sensitive quantification of staphylococcus aureus |
| CN105738342A (en) * | 2016-02-26 | 2016-07-06 | 中国人民解放军军事医学科学院军事兽医研究所 | SERS method for in-situ-synthesizing nano-silver by serving aptamer as support |
| CN105859845A (en) * | 2016-04-14 | 2016-08-17 | 石河子大学 | Mycoplasma ovipneumoniae multi-epitope fusion antigen MO-meAg5 and preparing method and application thereof |
-
2017
- 2017-06-04 CN CN201710410954.3A patent/CN107228819B/en not_active Expired - Fee Related
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101603075A (en) * | 2009-06-26 | 2009-12-16 | 华南理工大学 | A kind of method that detects based on the sorting enzyme of yeast surface display |
| CN101760502A (en) * | 2010-01-28 | 2010-06-30 | 中国检验检疫科学研究院 | Method for rapidly determining drug tolerance of strain |
| CN103243166A (en) * | 2013-05-15 | 2013-08-14 | 中国农业科学院植物保护研究所 | Rapid molecular detection method and application for plasmopara viticola |
| CN104031875A (en) * | 2014-05-30 | 2014-09-10 | 浙江省农业科学院 | Engineering bacteria for producing S-equol and application |
| CN104630291A (en) * | 2015-03-13 | 2015-05-20 | 吕涛 | Method for realizing high-yield succinic acid through coupling fermenting of two bacterial strains |
| CN105506070A (en) * | 2015-08-20 | 2016-04-20 | 江苏省家禽科学研究所 | Specific probe and method thereof for rapid and sensitive quantification of staphylococcus aureus |
| CN105738342A (en) * | 2016-02-26 | 2016-07-06 | 中国人民解放军军事医学科学院军事兽医研究所 | SERS method for in-situ-synthesizing nano-silver by serving aptamer as support |
| CN105859845A (en) * | 2016-04-14 | 2016-08-17 | 石河子大学 | Mycoplasma ovipneumoniae multi-epitope fusion antigen MO-meAg5 and preparing method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 梁华 等: "金黄色葡萄球菌超抗原样蛋白_5抑制人脐血源性内皮祖细胞黏附功能及其机制研究", 《第三军医大学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112903995A (en) * | 2021-01-15 | 2021-06-04 | 西北农林科技大学 | Colorimetric/fluorescent probe, test strip for detecting zearalenone and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107228819B (en) | 2019-12-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zheng et al. | The effect of cultivar, wilting and storage period on fermentation and the clostridial community of alfalfa silage | |
| Jarp | Classification of coagulase-negative staphylococci isolated from bovine clinical and subclinical mastitis | |
| CN101353633A (en) | Lactobacillus plantarum ZJ316, procreant antibiotic peptides, preparation and use thereof | |
| CN104593191B (en) | A kind of method of application lactic acid bacteria yellow rice wine brewage | |
| CN104336416A (en) | Lactobacillus plantarum and application thereof to alfalfa silage | |
| CN102426229A (en) | Preparation method of human IgG immunomagnetic bead for staphylococcus aureus enrichment | |
| CN107228819A (en) | A kind of flow cytometry assays of staphylococcus aureus | |
| CN113621536B (en) | Paenibacillus polymyxa SP1 and application thereof | |
| CN104483409A (en) | Staphylococcus aureus identification method based on fingerprint | |
| CN104232784A (en) | Multiplex PCR (polymerase chain reaction) method for testing three main pathogens in beef | |
| WO2022207015A2 (en) | Lactobacillus farciminis sr2 and use thereof | |
| CN104996543A (en) | Improved biological preservation agent for aquatic products, as well as preparation method and application thereof | |
| CN103224888B (en) | Long-term preservation method of blumeria graminis strains | |
| Szweda et al. | Efficient production of Staphylococcus simulans lysostaphin in a benchtop bioreactor by recombinant Escherichia coli | |
| CN100485036C (en) | Fennero penaeus chinensis antibacterial protein gene and recombinant expression and use | |
| CN102198265B (en) | Method for degrading streptococcus suis biofilm by applying phage lyase | |
| Olstorpe et al. | Growth inhibition of various Enterobacteriaceae species by the yeast Hansenula anomala during storage of moist cereal grain | |
| CN106591174B (en) | The lactobacillus curvatus of one plant of bacteriocinogeny and its application | |
| CN102706821A (en) | Method for quickly identifying food-borne pathogen bacterial biofilm formation inhibitor | |
| SAKAGUCHI et al. | Comparative study on Pediococcus halophilus, P. soyae, P. homari, P. urinae-equi and related species | |
| CN103045514B (en) | Proteus mirabilis for synthetizing acidic ethyl urethane hydrolytic enzyme and application thereof | |
| Cybulska et al. | Bacteriostatic activity of serum against staphylococci | |
| Chen et al. | Growth characteristics of a new methylomonad | |
| CN102260689A (en) | Gene engineering bacteria for food-grade heterologous secretion expression of IIa-class bacteriocin, construction method and application thereof | |
| CN101402954A (en) | Method for expressing recombinant pig lactoferrin N leaf with pichia thermophilic methanol yeast |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200407 Address after: 238000 Hefei Vocational and Technical College, Chaohu Economic Development Zone, Hefei City, Anhui Province Patentee after: HEFEI TECHNOLOGY College Address before: 238000 Anhui city of Hefei province Chaohu City Economic Development Zone South nest Laoshan Hefei Career Technical College Patentee before: Xu Zhenguo |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20201229 Address after: 224000 buildings 40-42 of national entrepreneurship Park, group 1, Anlong village, Bufeng Town, Yancheng Economic and Technological Development Zone, Jiangsu Province Patentee after: Jiangsu Yiqi integrated wood Co.,Ltd. Address before: 238000 Hefei vocational and technical college, Chaohu Economic Development Zone, Hefei City, Anhui Province Patentee before: HEFEI TECHNOLOGY College |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210507 Address after: 224000 room 2-401, building 6, No.5, South hope Avenue, Yancheng Economic and Technological Development Zone, Jiangsu Province Patentee after: Yancheng Xinhui Town Construction Development Co.,Ltd. Address before: 224000 buildings 40-42 of national entrepreneurship Park, group 1, Anlong village, Bufeng Town, Yancheng Economic and Technological Development Zone, Jiangsu Province Patentee before: Jiangsu Yiqi integrated wood Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210624 Address after: Room 604, building 4, No.5, hope Avenue South Road, Yancheng Economic and Technological Development Zone, Jiangsu Province 224000 Patentee after: Jiangsu beiyinkang Biotechnology Co.,Ltd. Address before: 224000 room 2-401, building 6, No.5, South hope Avenue, Yancheng Economic and Technological Development Zone, Jiangsu Province Patentee before: Yancheng Xinhui Town Construction Development Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230117 Address after: 224000 room 2-401, building 6, No.5, South hope Avenue, Yancheng Economic and Technological Development Zone, Jiangsu Province Patentee after: Yancheng Xinhui Town Construction Development Co.,Ltd. Address before: Room 604, building 4, No.5, hope Avenue South Road, Yancheng Economic and Technological Development Zone, Jiangsu Province 224000 Patentee before: Jiangsu beiyinkang Biotechnology Co.,Ltd. |
|
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20191213 |