CN106442987A - Staphylococcus-aureus fluorescence detection kit and application method thereof - Google Patents
Staphylococcus-aureus fluorescence detection kit and application method thereof Download PDFInfo
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- CN106442987A CN106442987A CN201610870705.8A CN201610870705A CN106442987A CN 106442987 A CN106442987 A CN 106442987A CN 201610870705 A CN201610870705 A CN 201610870705A CN 106442987 A CN106442987 A CN 106442987A
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- staphylococcus aureus
- atcc25923
- magainin
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- fluorescence
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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
-
- 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
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
Abstract
The invention discloses a staphylococcus-aureus fluorescence detection kit and an application method thereof. The staphylococcus-aureus fluorescence detection kit comprises staphylococcus-aureus specificity bacteriophage polypeptide coupling magnetic beads and fluorescein isothiocyanate coupling Magainin I. The application method includes the steps that the staphylococcus-aureus specificity bacteriophage polypeptide coupling magnetic beads are added into a to-be-test sample, and incubation and magnetic separation are carried out; then the fluorescein isothiocyanate coupling Magainin I is added, incubation and magnetic separation are carried out, fluorescence signals are detected and recorded, and the concentration of staphylococcus aureus in the to-be-test sample is obtained according to the fluorescence intensity. According to the staphylococcus-aureus fluorescence detection kit and the application method thereof, the to-be-test staphylococcus-aureus specific bacteriophage polypeptide and the broad-spectrum antibacterial peptide Magainin I serve as bacterium identification reagents, the magnetic bead rapid preconcentration technology and the high-sensitive fluorescence detection method are combined, staphylococcus-aureus whole cells are detected, and the staphylococcus-aureus fluorescence detection kit and the application method thereof have the advantages of being rapid, convenient, specific, sensitive, stable and low in cost.
Description
Technical field
The invention belongs to technical field of microbial detection, it is related to a kind of detection kit of staphylococcus aureus and its makes
Use method.
Background technology
Staphylococcus aureus is a kind of important pathogen of the mankind, is to cause the modal pathogen of pyogenic infection,
Including local pyogenic infection, systemic infection and systemic infection.Therefore, quick, convenience, sensitively golden yellow grape are set up
Coccus detection method, for health important in inhibiting.
In the existing detection method of staphylococcus aureus, the most ripe is the tradition side based on Bacteria Culture pattern
Method, most widely used method is the PCR method based on bacterial nucleic acid.Although the conventional method reliability based on Bacteria Culture pattern
Good, sensitivity is high, but time and effort consuming, it usually needs the time of a couple of days just can obtain testing result, simultaneously need to skilled specialty
Personnel and substantial amounts of experimental apparatus.Although the PCR method specificity based on bacterial nucleic acid is high, can achieve multi-analyte immunoassay, need
Will be through extracting the complicated process such as nucleic acid and synthesis specific primer.In addition, the method for detecting staphylococcus aureus
The also Southern blot hybridization technique based on DNA probe and MTT method etc., although these method high specificities,
Sensitivity but very low it is impossible to realize highly sensitive detection to staphylococcus aureus.
Content of the invention
In view of this, it is an object of the invention to provide a kind of fluorescence detection reagent kit of staphylococcus aureus and its make
With method, can quickly, convenient, special, sensitive, stable, qurer detection staphylococcus aureus.
For reaching above-mentioned purpose, the present invention provides following technical scheme：
1. the fluorescence detection reagent kit of staphylococcus aureus, including following components：Staphylococcus aureus specific bacteriophage
Polypeptide coupled bead and fluoresceinisothiocyanate Magainin I.
Magainin I is the small peptide being made up of 23 amino acid in xenopous laevis skin particles gland, and bacterium and fungi are had
Certain antibacterial activity, its act on bacterium surface and have heat endurance, has a broad antifungal spectrum, be readily synthesized, good stability etc. excellent
Point.
Preferably, described staphylococcus aureus specific phage polypeptide coupled bead is by Avidin magnetic bead and biology
After the staphylococcus aureus specific phage polypeptide reaction that element is modified, then it is obtained with gelatin closing.
Preferably, the staphylococcus aureus specific phage polypeptide of described biotin modification is in Staphylococcus aureus
After carbon teminal 3 glycine residues of connection of bacterium ATCC25923 specific bacteriophage polypeptide, modified biological is plain again.
Preferably, described fluoresceinisothiocyanate Magainin I is to modify different sulphur in the carbon teminal of Magainin I
Cyanic acid fluorescein.
In technique scheme, according to the type of staphylococcus aureus to be detected, select this type golden yellow Portugal
The specific phage polypeptide of grape coccus prepares phage polypeptide coupled bead, you can realize to this type staphylococcus aureus
Detection.
As an optimal technical scheme, described staphylococcus aureus is staphylococcus aureus ATCC25923；Described
Staphylococcus aureus specific phage polypeptide is staphylococcus aureus ATCC25923 specific bacteriophage polypeptide, its ammonia
Base acid sequence is Val-Pro-His-Asn-Pro-Gly-Leu-Ile-Ser-Leu-Gln-Gly（SEQ ID No.1）.
Further, also include following components in described kit：Staphylococcus aureus ATCC25923 bacteria suspension, reaction
Buffer solution and detection buffer solution.
Preferably, described staphylococcus aureus ATCC25923 bacteria suspension is obtained by following methods：Take Staphylococcus aureus
Bacterium ATCC25923 single bacterium colony is inoculated in LB nutrient solution, 37 DEG C of shaken cultivation, and centrifugation removes supernatant, and bacterium is cleaned with water
Afterwards, the phosphate buffer with 0.01 mol/L, pH 7.4 is resuspended and adjusts bacterial concentration；Described reaction buffer is 0.01
The phosphate buffer of mol/L, pH 7.4, described detection buffer solution is the phosphate buffer of 0.01 mol/L, pH8.0.
. the using method of the fluorescence detection reagent kit of described staphylococcus aureus, be add in testing sample golden yellow
Color aureus specific phage polypeptide coupled bead, incubation, Magnetic Isolation, obtain staphylococcus aureus-bacteriophage many
Peptide coupled bead compound, adds fluoresceinisothiocyanate Magainin I, incubation, and Magnetic Isolation obtains different sulphur cyanogen
Sour fluorescein is coupled Magainin I- staphylococcus aureus-phage polypeptide coupled bead compound, detects and records fluorescence
Signal, tries to achieve the concentration of staphylococcus aureus in testing sample according to fluorescence intensity.
As an optimal technical scheme, the user of the fluorescence detection reagent kit of staphylococcus aureus ATCC25923
Method, comprises the following steps：
A the dilution of staphylococcus aureus ATCC25923 bacteria suspension reaction buffer is made staphylococcus aureus by ()
ATCC25923 work bacterium solution；Add staphylococcus aureus in staphylococcus aureus ATCC25923 work bacterium solution
ATCC25923 specific bacteriophage polypeptide coupled bead, incubation, Magnetic Isolation, obtain staphylococcus aureus ATCC25923-
Phage polypeptide coupled bead compound, after being washed with reaction buffer, adds fluoresceinisothiocyanate Magainin
I, incubation, Magnetic Isolation, obtain isothiocyanic acid and be coupled Magainin I- staphylococcus aureus ATCC25923- phage polypeptide
Coupled bead compound, after being washed with reaction buffer, adds detection buffer solution, detects and record fluorescence signal, with fluorescence
Intensity is mapped to staphylococcus aureus ATCC25923 concentration, drawing curve；
B () adds staphylococcus aureus ATCC25923 specific bacteriophage polypeptide coupled bead in testing sample, incubation,
Magnetic Isolation, obtains staphylococcus aureus ATCC25923- phage polypeptide coupled bead compound, is washed with reaction buffer
After washing, then plus fluoresceinisothiocyanate Magainin I, incubation, Magnetic Isolation, obtain fluoresceinisothiocyanate
Magainin I- staphylococcus aureus ATCC25923- phage polypeptide coupled bead compound, is washed with reaction buffer
Afterwards, add detection buffer solution, detect and record fluorescence signal, asked according to the working curve that fluorescence intensity and step (a) are drawn
Obtain the concentration of staphylococcus aureus ATCC25923 in testing sample.
Preferably, the using method of the fluorescence detection reagent kit of staphylococcus aureus ATCC25923, walks including following
Suddenly：
A the dilution of staphylococcus aureus ATCC25923 bacteria suspension reaction buffer is made concentration 10 ~ 10 × 10 by ()5
Staphylococcus aureus ATCC25923 work bacterium solution in the range of CFU/mL；To 1.0 mL staphylococcus aureus ATCC25923
10 g staphylococcus aureus ATCC25923 specific bacteriophage polypeptide coupled bead, 37 DEG C of incubations 45 are added in work bacterium solution
Minute, Magnetic Isolation, obtain staphylococcus aureus ATCC25923- phage polypeptide coupled bead compound, with reaction buffering
After liquid washing, add 5 g/mL fluoresceinisothiocyanate Magainin I 1.0mL, 37 DEG C are incubated 45 minutes, and magnetic is divided
From, obtain fluoresceinisothiocyanate Magainin I- staphylococcus aureus ATCC25923- phage polypeptide be coupled magnetic
Pearl compound, after being washed with reaction buffer, adds 200 L detection buffer solutions, detects and record fluorescence signal, with fluorescence
Intensity is mapped to staphylococcus aureus ATCC25923 concentration, drawing curve；
B () adds 10 g staphylococcus aureus ATCC25923 specific bacteriophage polypeptides to be coupled in 1.0 mL testing samples
Magnetic bead, 37 DEG C are incubated 45 minutes, and Magnetic Isolation obtains staphylococcus aureus ATCC25923- phage polypeptide coupled bead multiple
Compound, after being washed with reaction buffer, adds 5 g/mL fluoresceinisothiocyanate Magainin I 1.0mL, incubates for 37 DEG C
Educate 45 minutes, Magnetic Isolation, obtain fluoresceinisothiocyanate Magainin I- staphylococcus aureus ATCC25923- and bite
Thalline polypeptide coupled bead compound, after being washed with reaction buffer, adds 200 L detection buffer solutions, detects and record glimmering
Optical signal, tries to achieve staphylococcus aureus in testing sample according to the working curve that fluorescence intensity and step (a) are drawn
The concentration of ATCC25923.
The beneficial effects of the present invention is：The invention provides a kind of fluorescence detection reagent kit of staphylococcus aureus,
Using staphylococcus aureus specific phage polypeptide and broad spectrum antimicrobial peptide Magainin I as bacterium identification agent, in conjunction with magnetic
Pearl fast enriching technology and high-sensitive fluorescence detection method, are detected for the full cell of staphylococcus aureus, have fast
Fast, convenient, special, sensitive, stable, low price advantage, is expected to be applied to the Site Detection of staphylococcus aureus and quick sieve
Look into, can detect that staphylococcus aureus provides strong technology to prop up for fields such as clinical diagnosis, food security and environment measurings
Fair platform.
Brief description
Fig. 1 is the use schematic flow sheet of the fluorescence detection reagent kit of staphylococcus aureus ATCC25923.
Fig. 2 is to detect staphylococcus aureus using the fluorescence detection reagent kit of staphylococcus aureus ATCC25923
The working curve of ATCC25923.
Specific embodiment
In order that the object, technical solutions and advantages of the present invention are clearer, excellent to the present invention below in conjunction with accompanying drawing
Embodiment is selected to be described in detail.
The preparation of the fluorescence detection reagent kit of embodiment 1 staphylococcus aureus ATCC25923
The fluorescence detection reagent kit of the staphylococcus aureus ATCC25923 of the present embodiment, including following components：Golden yellow grape
Coccus ATCC25923 specific bacteriophage polypeptide coupled bead, fluoresceinisothiocyanate Magainin I, golden yellow grape
Coccus ATCC25923 bacteria suspension, reaction buffer and detection buffer solution.
The preparation method of described staphylococcus aureus ATCC25923 specific bacteriophage polypeptide coupled bead is：Take parent
Mix with staphylococcus aureus ATCC25923 specific bacteriophage polypeptide 30 g of biotin modification with biscuit porcelain pearl 2mg, instead
Should, Magnetic Isolation, washed with the phosphate buffer of 0.01 mol/L, pH 7.4, add 10 g/mL aqueous gelatin solutions
1.0mL closes, and Magnetic Isolation is washed with the phosphate buffer of 0.01 mol/L, pH 7.4, obtained final product.Described golden yellow grape
The amino acid sequence of coccus ATCC25923 specific bacteriophage polypeptide is Val-Pro-His-Asn-Pro-Gly-Leu-Ile-
Ser-Leu- Gln-Gly（SEQ ID No.1）.The staphylococcus aureus ATCC25923 specificity of described biotin modification is bitten
Thalline polypeptide is after the carbon teminal of staphylococcus aureus ATCC25923 specific bacteriophage polypeptide connects 3 glycine residues
Modified biological element again.
Described fluoresceinisothiocyanate Magainin I is that isothiocyanic acid is glimmering in the carbon teminal modification of Magainin I
Light element.
The preparation method of described staphylococcus aureus ATCC25923 bacteria suspension is：Take staphylococcus aureus
ATCC25923（From Guangdong Province, Culture Collection obtains）Single bacterium colony is inoculated in LB nutrient solution, 37 DEG C of vibration trainings
Support 12 hours, centrifugation removes supernatant, with, after sterile water wash bacterium 2 times, being delayed with the phosphate of 0.01 mol/L, pH 7.4
Rush the resuspended bacterium of liquid and adjust bacterial concentration to 1.0 × 105CFU/mL.
Described reaction buffer is the phosphate buffer of 0.01 mol/L, pH 7.4.
Described detection buffer solution is the phosphate buffer of 0.01 mol/L, pH8.0.
The using method of the fluorescence detection reagent kit of embodiment 2 staphylococcus aureus ATCC25923
As shown in figure 1, the using method of the fluorescence detection reagent kit of staphylococcus aureus ATCC25923 of embodiment 1 preparation,
Comprise the following steps：
A the dilution of staphylococcus aureus ATCC25923 bacteria suspension reaction buffer is made concentration 10 ~ 10 × 10 by ()5
Staphylococcus aureus ATCC25923 work bacterium solution in the range of CFU/mL；To 1.0 mL staphylococcus aureus ATCC25923
10 g staphylococcus aureus ATCC25923 specific bacteriophage polypeptide coupled bead, 37 DEG C of incubations 45 are added in work bacterium solution
Minute, Magnetic Isolation, obtain staphylococcus aureus ATCC25923- phage polypeptide coupled bead compound, with reaction buffering
Liquid washs, and adds 5 g/mL fluoresceinisothiocyanate Magainin I 1.0mL, and 37 DEG C are incubated 45 minutes, and magnetic is divided
From, obtain fluoresceinisothiocyanate Magainin I- staphylococcus aureus ATCC25923- phage polypeptide be coupled magnetic
Pearl compound, is washed with reaction buffer, adds 200 L detection buffer solutions, detects and record fluorescence signal（Excitation light wave
Long 488nm, wavelength of transmitted light 525nm）, with fluorescence intensity, staphylococcus aureus ATCC25923 concentration is mapped, drawing
Curve；
B () adds 10 g staphylococcus aureus ATCC25923 specific bacteriophage polypeptides to be coupled in 1.0 mL testing samples
Magnetic bead, 37 DEG C are incubated 45 minutes, and Magnetic Isolation obtains staphylococcus aureus ATCC25923- phage polypeptide coupled bead multiple
Compound, is washed with reaction buffer, adds 5 g/mL fluoresceinisothiocyanate Magainin I 1.0mL, 37 DEG C of incubations
45 minutes, Magnetic Isolation, obtain fluoresceinisothiocyanate Magainin I- staphylococcus aureus ATCC25923- phagocytosis
Body polypeptide coupled bead compound, is washed with reaction buffer, adds 200 L detection buffer solutions, detects and record fluorescence letter
Number, staphylococcus aureus ATCC25923 in testing sample is tried to achieve according to the working curve that fluorescence intensity and step (a) are drawn
Concentration.
In above-mentioned steps (a), the concentration of staphylococcus aureus ATCC25923 work bacterium solution is respectively 10,1.0 × 102、
1.0×103、1.0×104With 1.0 × 105CFU/mL, is made to staphylococcus aureus ATCC25923 concentration with fluorescence intensity
Figure, gained working curve is as shown in Figure 2.With the increase of staphylococcus aureus ATCC25923 concentration, fluorescence intensity constantly increases
Greatly, the two is 10 ~ 1.0 × 105Good linear relationship, R is assumed in the range of CFU/mL2=0.9995, test limit（S/N=3）For
12 CFU/mL, illustrate can delicately detect staphylococcus aureus ATCC25923 using the detection kit of the present invention.
In above-mentioned steps (a), in staphylococcus aureus ATCC25923 work bacterium solution, it is separately added into 6 kinds of pattern bacterium（Gold
Staphylococcus aureus CCTCC AB 91093, micrococcus luteus CCTCC AB 91100, MRSE GIM1.444, verdigris
Pseudomonas CCTCC AB 93078, Escherichia coli CCTCC AB 212355 and streptococcus ATCC35668）, then press above-mentioned
Same method is detected.Result shows, in addition to MRSE GIM1.444 has weak interference, other 5 kinds of pattern bacterium
Detection to staphylococcus aureus ATCC25923 does not all constitute interference, illustrates that the detection kit using the present invention can be special
Staphylococcus aureus ATCC25923 is detected in strange land.
In above-mentioned steps (b), respectively using cider, lake water and Healthy People urine as testing sample, by its high-temperature sterilization
Afterwards, wherein cider and lake water do not dilute, and Healthy People urine deionized water dilutes ten times, is added to the gold of concentration known
Staphylococcus aureus ATCC25923 bacteria suspension, is then detected in same way as described above.As shown in table 1,3 kinds to be measured for result
The recovery of standard addition of Gold Samples staphylococcus aureus ATCC25923 is 81% ~ 105%, RSD value not higher than 5.1%, illustrates to use
The detection kit of the present invention can detect staphylococcus aureus ATCC25923 exactly.
Finally illustrate, above example only in order to technical scheme to be described and unrestricted, although by ginseng
According to the preferred embodiments of the present invention, invention has been described, it should be appreciated by those of ordinary skill in the art that can
So that in the form and details it is made with various changes, the present invention being limited without departing from appended claims
Spirit and scope.
<110>Southwest University
<120>The fluorescence detection reagent kit of staphylococcus aureus and its using method
<160> 1
<210> 1
<211> 12
<212> PRT
<213>Artificial sequence
<220>
<223>Staphylococcus aureus ATCC25923 specific bacteriophage polypeptide
<400> 1
Val Pro His Asn Pro Gly Leu Ile Ser Leu Gln Gly
1 5 10
Claims (10)
1. the fluorescence detection reagent kit of staphylococcus aureus is it is characterised in that include following components：Staphylococcus aureus is special
Different in nature phage polypeptide coupled bead and fluoresceinisothiocyanate Magainin I.
2. staphylococcus aureus as claimed in claim 1 fluorescence detection reagent kit it is characterised in that described golden yellow Portugal
Grape coccus specific bacteriophage polypeptide coupled bead is will be special with the staphylococcus aureus of biotin modification for Avidin magnetic bead
Property phage polypeptide reaction after, then with gelatin closing be obtained.
3. the fluorescence detection reagent kit of staphylococcus aureus as claimed in claim 2 is it is characterised in that described biotin is repaiied
The staphylococcus aureus specific phage polypeptide of decorations is the carbon teminal company in staphylococcus aureus specific phage polypeptide
Connect after 3 glycine residues modified biological element again.
4. the fluorescence detection reagent kit of staphylococcus aureus as claimed in claim 1 is it is characterised in that described isothiocyanic acid
It is to modify fluorescein isothiocynate in the carbon teminal of Magainin I that fluorescein is coupled Magainin I.
5. the fluorescence detection reagent kit of the staphylococcus aureus as described in any one of Claims 1-4 is it is characterised in that institute
Stating staphylococcus aureus is staphylococcus aureus ATCC25923；Described staphylococcus aureus specific phage polypeptide
For staphylococcus aureus ATCC25923 specific bacteriophage polypeptide, its amino acid sequence is Val-Pro-His-Asn-Pro-
Gly-Leu- Ile-Ser-Leu-Gln-Gly.
6. the fluorescence detection reagent kit of staphylococcus aureus as claimed in claim 5 is it is characterised in that in described kit
Also include following components：Staphylococcus aureus ATCC25923 bacteria suspension, reaction buffer and detection buffer solution.
7. staphylococcus aureus as claimed in claim 6 fluorescence detection reagent kit it is characterised in that described golden yellow Portugal
Grape coccus ATCC25923 bacteria suspension is obtained by following methods：Staphylococcus aureus ATCC25923 single bacterium colony is taken to be inoculated in LB
In nutrient solution, 37 DEG C of shaken cultivation, centrifugation removes supernatant, after bacterium is cleaned with water, with the phosphorus of 0.01 mol/L, pH 7.4
Phthalate buffer is resuspended and adjusts bacterial concentration；Described reaction buffer is the phosphate-buffered of 0.01 mol/L, pH 7.4
Liquid, described detection buffer solution is the phosphate buffer of 0.01 mol/L, pH8.0.
8. the using method of the fluorescence detection reagent kit of the staphylococcus aureus described in any one of claim 1 to 7, its feature
It is, add staphylococcus aureus specific phage polypeptide coupled bead, incubation, Magnetic Isolation in testing sample, obtain
To staphylococcus aureus-phage polypeptide coupled bead compound, add fluoresceinisothiocyanate Magainin I,
Incubation, Magnetic Isolation, obtain fluoresceinisothiocyanate Magainin I- staphylococcus aureus-phage polypeptide and be coupled
Bead complexes, detect and record fluorescence signal, try to achieve the concentration of staphylococcus aureus in testing sample according to fluorescence intensity.
9. the using method of the fluorescence detection reagent kit of staphylococcus aureus as claimed in claim 8 is it is characterised in that wrap
Include following steps：
A the dilution of staphylococcus aureus ATCC25923 bacteria suspension reaction buffer is made staphylococcus aureus ATCC by ()
25923 work bacterium solution；Add staphylococcus aureus ATCC 25923 in staphylococcus aureus ATCC25923 work bacterium solution
Specific bacteriophage polypeptide coupled bead, incubation, Magnetic Isolation, obtain staphylococcus aureus ATCC25923- phage polypeptide
Coupled bead compound, after being washed with reaction buffer, adds fluoresceinisothiocyanate Magainin I, incubation, magnetic
Property separate, obtain isothiocyanic acid be coupled Magainin I- staphylococcus aureus ATCC25923- phage polypeptide coupled bead
Compound, after being washed with reaction buffer, adds detection buffer solution, detects and record fluorescence signal, with fluorescence intensity to gold
Staphylococcus aureus ATCC25923 concentration is mapped, drawing curve；
B () adds staphylococcus aureus ATCC25923 specific bacteriophage polypeptide coupled bead in testing sample, incubation,
Magnetic Isolation, obtains staphylococcus aureus ATCC25923- phage polypeptide coupled bead compound, is washed with reaction buffer
After washing, then plus fluoresceinisothiocyanate Magainin I, incubation, Magnetic Isolation, obtain fluoresceinisothiocyanate
Magainin I- staphylococcus aureus ATCC25923- phage polypeptide coupled bead compound, is washed with reaction buffer
Afterwards, add detection buffer solution, detect and record fluorescence signal, asked according to the working curve that fluorescence intensity and step (a) are drawn
Obtain the concentration of staphylococcus aureus ATCC25923 in testing sample.
10. the fluorescence detection reagent kit of staphylococcus aureus as claimed in claim 9 using method it is characterised in that
Comprise the following steps：
A the dilution of staphylococcus aureus ATCC25923 bacteria suspension reaction buffer is made concentration 10 ~ 10 × 10 by ()5
Staphylococcus aureus ATCC25923 work bacterium solution in the range of CFU/mL；To 1.0 mL staphylococcus aureus ATCC25923
10 g staphylococcus aureus ATCC25923 specific bacteriophage polypeptide coupled bead, 37 DEG C of incubations 45 are added in work bacterium solution
Minute, Magnetic Isolation, obtain staphylococcus aureus ATCC25923- phage polypeptide coupled bead compound, with reaction buffering
After liquid washing, add 5 g/mL fluoresceinisothiocyanate Magainin I 1.0mL, 37 DEG C are incubated 45 minutes, and magnetic is divided
From, obtain fluoresceinisothiocyanate Magainin I- staphylococcus aureus ATCC25923- phage polypeptide be coupled magnetic
Pearl compound, after being washed with reaction buffer, adds 200 L detection buffer solutions, detects and record fluorescence signal, with fluorescence
Intensity is mapped to staphylococcus aureus ATCC25923 concentration, drawing curve；
B () adds 10 g staphylococcus aureus ATCC25923 specific bacteriophage polypeptides to be coupled in 1.0 mL testing samples
Magnetic bead, 37 DEG C are incubated 45 minutes, and Magnetic Isolation obtains staphylococcus aureus ATCC25923- phage polypeptide coupled bead multiple
Compound, after being washed with reaction buffer, adds 5 g/mL fluoresceinisothiocyanate Magainin I 1.0mL, incubates for 37 DEG C
Educate 45 minutes, Magnetic Isolation, obtain fluoresceinisothiocyanate Magainin I- staphylococcus aureus ATCC25923- and bite
Thalline polypeptide coupled bead compound, after being washed with reaction buffer, adds 200 L detection buffer solutions, detects and record glimmering
Optical signal, tries to achieve staphylococcus aureus in testing sample according to the working curve that fluorescence intensity and step (a) are drawn
The concentration of ATCC25923.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108872194A (en) * | 2018-08-22 | 2018-11-23 | 暨南大学 | A kind of method of sandwich structure SERS detection pathogen |
| CN114317787A (en) * | 2021-12-29 | 2022-04-12 | 成都大学 | Magnetic adsorption bead, kit and application thereof, and method for detecting staphylococcus aureus |
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| CN102590506A (en) * | 2012-02-16 | 2012-07-18 | 上海师范大学 | Method for rapidly detecting and screening staphylococcus aureus |
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| CN102590506A (en) * | 2012-02-16 | 2012-07-18 | 上海师范大学 | Method for rapidly detecting and screening staphylococcus aureus |
| CN103777013A (en) * | 2014-02-27 | 2014-05-07 | 杭州绿洁水务科技有限公司 | Micro-fluidic chip and method for detecting escherichia coli in water |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108872194A (en) * | 2018-08-22 | 2018-11-23 | 暨南大学 | A kind of method of sandwich structure SERS detection pathogen |
| CN108872194B (en) * | 2018-08-22 | 2020-09-04 | 暨南大学 | Method for detecting pathogenic bacteria by sandwich structure SERS |
| CN114317787A (en) * | 2021-12-29 | 2022-04-12 | 成都大学 | Magnetic adsorption bead, kit and application thereof, and method for detecting staphylococcus aureus |
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