CN109187826A - A kind of detection method of the outer component of candida albicans biofilm extracellular matrix protein matter - Google Patents
A kind of detection method of the outer component of candida albicans biofilm extracellular matrix protein matter Download PDFInfo
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Abstract
A kind of detection method of the outer component of candida albicans biofilm extracellular matrix protein matter, it is characterized in that, activation candida albicans is simultaneously incubated for flowable state candida albicans biofilm, biofilm extracellular matrix is extracted using ion exchange resin, identifies the ingredient in envelope extracellular matrix other than isolating protein with UPLC-TOF-MS.The pretreatment process removal protein component of sample of the invention is thorough, so that the detection effect of the outer component of protein is good.
Description
Technical field
The invention belongs to technical field of biological, and in particular to a kind of candida albicans biofilm extracellular matrix protein matter
The detection method of outer component.
Background technique
Candida albicans is a kind of metatroph, usually survives in skin, mucous membrane, alimentary canal and the other organs of human body
In, when Abwehrkraft des Koepers reduces, Candida albicans will be bred, and when reaching a certain amount of, human body will fall ill, thus, Bai Nian
Pearl bacterium is a kind of human condition pathogenic fungus.
Candida albicans can improve the drug resistance of itself by forming biofilm.The born of the same parents of candida albicans biofilm secretion
Epimatrix ingredient is extremely complex, (accounts for extracellular matrix dry weight containing protein (accounting for extracellular matrix dry weight 55%), polysaccharide on the whole
25%), the macromolecular substances such as lipid (accounting for extracellular matrix dry weight 15%) and nucleic acid (accounting for extracellular matrix dry weight 5%).
Summary of the invention
It is an object of that present invention to provide a kind of detection methods of component outside candida albicans biofilm extracellular matrix protein matter.
To achieve the above object, present invention provide the technical scheme that a kind of candida albicans biofilm extracellular matrix egg
The detection method of the outer component of white matter, including the following steps:
Step 1: candida albicans activates
Candida albicans is inoculated in SDB culture medium, shaking table is incubated for 20~28h；Fungal cell is collected after medium centrifugal,
It is cleaned, then cleaning gained fungal cell is resuspended in RPMI-1640 culture medium, then be incubated for 12- with sterile PBS buffer
18h with 2500~3500 turns/min centrifugation, collects the fungal cell of sinking, adds fungi culture medium then under the conditions of 4 DEG C
Fungal cell's concentration is adjusted to 0.8~1.2 × 106CFU/ml obtains resuspended bacterium solution；
Step 2: flowable state candida albicans biofilm is incubated for
Medical catheter piece is impregnated to keep sterile in ethanol, after sterile water wash, then is added it to described heavy
It in outstanding bacterium solution, is incubated in shaking table, candida albicans is made to be adhered to the medical catheter on piece, then to being adhered to medical catheter piece
On candida albicans carry out flowing incubation, after be incubated for the candida albicans for obtaining being formed with mature biofilm；
Step 3: the NaH that pH value is 7.8 is added in the candida albicans for being formed with mature biofilm2PO4-Na2HPO4From
In sub- buffer, be first vortexed concussion, is then then sonicated to obtain suspension, will exchange by pretreated highly acidic cation
Resin is mixed with suspension, and shaking table is incubated for 2~4h, is then 0.22 μm of filtering with microporous membrane with aperture, with 7500~
8500rpm/min is centrifuged 2~4min, obtains supernatant.
Preferred technical solution are as follows: every liter of SDB culture medium contains 10g peptone, 20g agar powder, 40g glucose and 0.1g
Chloramphenicol.
Preferred technical solution are as follows: the composition of raw materials of the RPMI-1640 culture medium includes the original of following mass percent
Material: 1% mannitol, 1% nutrient broth, 0.2% K2HPO4With 1.35% agar powder.
Preferred technical solution are as follows: every liter of fungi culture medium contain the potassium chloride of 400mg, 6000mg sodium chloride,
The glucose of 2000mg, the L-arginine of 290.00mg, the altheine of 50mg, the L-Aspartic acid of 20mg, 65.15mg
L-cysteine dihydrochloride, the Pidolidone of 20mg, the glycine of 10mg, the L-Histidine of 15mg, 20mg L- hydroxyproline,
The l-Isoleucine of 50mg, the L-Leu of 50mg, the L-lysine of 40mg, the l-methionine of 15mg, 15mg L- phenylpropyl alcohol
Propylhomoserin, the L-PROLINE of 20mg, the Serine of 30mg, the L-threonine of 20mg, the L-Trp of 5mg, 23.19mg L- junket
The Valine of propylhomoserin, 20mg, the fetal calf serum that the mass concentration of 100ml is 10%, remaining is purified water, then uses 3- (N-
Ma Lindai) propane sulfonic acid adjusts pH value to 7.0.
Preferred technical solution are as follows: the supernatant is stored in -80 DEG C of low temperature refrigerators, before testing, after thawing on ice
100 μ L samples are pipetted into EP pipe, 300 μ L methanol are added, add 10 μ L internal standard L-2- chlorophenylalanines, are vortexed after mixing,
Ultrasonic treatment ultrasound is carried out in ice-water bath；After under the conditions of -20 DEG C stand 0.5~1.5h；Then under the conditions of 4 DEG C,
10~20min is centrifuged with 10000~14000rpm speed；The supernatant for finally taking out 200 μ L, which is placed in sample injection bottle, carries out machine examination
It surveys.
Preferred technical solution are as follows: the time of ultrasonic treatment is 10min, ultrasonic frequency 25-35KHz, power density
For 0.35-0.45w/cm2。
Preferred technical solution are as follows: the upper machine testing uses high performance liquid chromatography-flight time mass spectrum method for combined use, color
Spectrum column is UPLC BEH Amide chromatographic column, having a size of 1.7 μm of 2.1 × 100mm；Gradient elution mobile phase: A:25mM ammonium acetate
And 25mM, B: acetonitrile；
| Time/min | Flow velocity/μ L/min | A% | B% |
| 0 | 500 | 5 | 95 |
| 0.5 | 500 | 5 | 95 |
| 7 | 500 | 35 | 65 |
| 8 | 500 | 60 | 40 |
| 9 | 500 | 60 | 40 |
| 9.1 | 500 | 5 | 95 |
| 12 | 500 | 5 | 95 |
Mass spectrum: bombarding energy: 30eV, ESI source parameters are provided that atomization air pressure: 60Psi, assist gas pressure:
60Psi, gas curtain air pressure: 35Psi, temperature: 650 DEG C, when positive ion mode, spray voltage 5000V, when negative ion mode, spray
Mist voltage is -4000V.
Preferred technical solution are as follows: the high performance liquid chromatography is 1290 ultra performance liquid chromatography of Agilent, and mass spectrograph is
AB 6600Triple TOF mass spectrograph.
Preferred technical solution are as follows: the storng-acid cation exchange resin is 732 storng-acid cation exchange resins.
Since above-mentioned technical proposal is used, the present invention has the advantage, that compared with prior art
The pretreatment process removal protein component of sample of the invention is thorough, so that the detection of the outer component of protein
Effect is good.
Specific embodiment
Embodiments of the present invention are illustrated by particular specific embodiment below, those skilled in the art can be by this explanation
Content disclosed by book is understood other advantages and efficacy of the present invention easily.
A kind of embodiment one: detection method of the outer component of candida albicans biofilm extracellular matrix protein matter
(1) candida albicans activates
Candida albicans SC5314 be inoculated in SDB culture medium (peptone containing 10g/L, 20g/L agar powder, 40g/L glucose,
0.1g/L chloramphenicol) in, it is incubated for for 24 hours in the shaking table of 180rpm at 37 DEG C；Then bacterium cell is collected after centrifugation through 2000rpm,
It is cleaned 2 times with sterile PBS；The bacterium cell of collection be resuspended in RPMI-1640 culture medium (containing 1% mannitol, 1% nutrient broth,
0.2%K2HPO4, 1.35% agar powder) in, it is incubated for 12-18h again at 37 DEG C.3000 turns/min is centrifuged 5min at 4 DEG C, collects
The fungal cell of sinking, addition fungi culture medium adjust bacterium cell concentration to 1 × 106CFU/ml obtains resuspended bacterium solution.
Wherein, the ingredient of fungi culture medium: every liter of fungi culture medium contains the chlorine of the potassium chloride of 400mg, 6000mg
Change sodium, the glucose of 2000mg, the L-arginine of 290.00mg, the altheine of 50mg, 20mg L-Aspartic acid,
The l-cysteine dihydrochloride of 65.15mg, the Pidolidone of 20mg, the glycine of 10mg, the L-Histidine of 15mg, 20mg L-
Hydroxyproline, the l-Isoleucine of 50mg, the L-Leu of 50mg, the L-lysine of 40mg, the l-methionine of 15mg, 15mg
L-phenylalanine, the L-PROLINE of 20mg, the Serine of 30mg, the L-threonine of 20mg, 5mg L-Trp,
The Valine of the l-tyrosine of 23.19mg, 20mg, the fetal calf serum that the mass concentration of 100ml is 10%, remaining is purifying
Then water adjusts pH value to 7.0 with 3- (N- Ma Lindai) propane sulfonic acid.
(2) flowable state candida albicans biofilm is incubated for
The groove type of every section of about 6cm medical grade PVC (PVC) conduit piece was impregnated in 75% ethyl alcohol first
Night to keep sterile, is incubated for, in shaking table (37 DEG C of 220rpm/ altogether after sterile water wash, then with the SC5314 re-suspension liquid of activation
Min it is incubated for 4 hours in), makes fungal adhesion in conduit piece.Conduit piece is taken out after adherency, is put into the centrifugation of 1640 culture medium of 40mL
Guan Zhong, flowing is incubated for 20h, flow velocity 1ml/min at 37 DEG C.
Flow the method being incubated for:
(1) fungal organism envelope flow model device constructs
Fungal organism envelope flow model device includes beaker, a digital display peristaltic pump, a use for a Sheng culture solution
Liquid separation sample injector (50ml) made of centrifuge tube, four centrifuge tubes (50ml/) and its bracket, one being incubated for for biofilm
A beaker for containing efflux.
The liquid separation sample injector the preparation method comprises the following steps: centrifugation tube wall relative position on make a call to 1 and a row 4 respectively
The hole of a 2mm, 1 hole is inlet opening in surface when placement, and 4 holes are fluid hole in underface.
The biofilm be incubated for centrifuge tube the preparation method comprises the following steps: cap and bottom end each can make a call to the hole of a 2mm, each
The medical PVC conduit piece of a 5*1mm is placed in centrifuge tube.
The timbering material is PMMA, there is four layers, and wherein first layer and third layer have a hole of 4 2cm, the second layer and the
Four layers of hole for having 4 4cm, four layers of hole is entirely concentric holes.
The fungal organism envelope flow model device, every time before use, all components need to pass through 121 DEG C of steam high temperature
Sterilizing, 4 biofilm incubation tubes should be located in same level.
The fungal organism envelope flow model device, constant environment temperature is 37 ± 1 DEG C, liquid separation sample injector when operation
Can be fixed on by cord on a metal test tubes frame, the hole of surface via emulsion tube (outer diameter of 3mm and the wall thickness of 5mm) with
Insertion fills in the beaker of aseptic culture fluid after one peristaltic pump is connected, and the hole of underface is via the emulsion tube of identical size and four
The bottom end that a biofilm is incubated for centrifuge tube is connected, and the hole above centrifuge tube pipe cap is flowed out by the emulsion tube and Sheng of identical size
The beaker of liquid is connected.
Conduit piece is placed in the centrifuge tube containing 50ml fungi culture medium, after 37 DEG C of stationary culture 2h, opens and wriggles
Pump, adjustment flow velocity to constant 0.7ml/min, 37 DEG C of flowings are incubated for 19-20h.
(3) ion-exchange-resin process extracts flowable state biofilm extracellular matrix
Sticking has the tweezers of the conduit piece sterilizing of mature biofilm gently to take out, and 6ml is added in film forming rear tube piece
NaH2PO4-Na2HPO4Ion buffer (pH7.8), be vortexed concussion 1min, mild ultrasound 20min (37 DEG C of 50W).It will pass through
Pretreated storng-acid cation exchange resin takes out the amount of twice of liquid volume in clean syringe, uses asepsis injector
Suspension in test tube is transferred in resin, shaking table is incubated for 400rpm/min 3h, the liquid asepsis injector that will be acted on resin
The filtering with microporous membrane through 0.22 μm is taken out, 3min is centrifuged under 8000rpm/min, takes supernatant to be measured；Add the NaCl of 500mM
Solution elution ionic exchanger resin, shaking table act on 2h 400rpm/min, after by eluent with asepsis injector take out through 0.22 μm
Filtering with microporous membrane, be centrifuged 3min under 8000rpm/min, take supernatant to be measured.
Wherein, 0.2M Na2HPO4: weigh 71.628g Na2HPO4-12H2O is dissolved in 1000mL distilled water；
0.2M NaH2PO4: weigh 31.228g NaH2PO4-2H2O is dissolved in 1000mL distilled water；
PH7.8NaH2PO4-Na2HPO4Buffer: the 0.2M Na of 91.5mL2HPO4With the 0.2M NaH of 8.5mL2PO4Mixing
After obtain.
(4) UPLC-TOF-MS is detected
4.1 samples: totally 6 candida albicans biofilm matrix samples are stored in -80 DEG C of low temperature refrigerators, until experiment
Detection.
4.2 instruments and reagent
| Instrument | Model | Brand |
| Ultra high efficiency liquid phase | 1290UHPLC | Agilent |
| High resolution mass spectrum | Triple TOF 6600 | AB Sciex |
| Centrifuge | Heraeus Fresco17 | Thermo Fisher Scientific |
| Balance | BSA124S-CW | Sartorius |
| Pure water meter | Bright and limpid D24UV | Merck Millipore |
| Ultrasound Instrument | PS-60AL | Electronics Co., Ltd. of Randt nation of Shenzhen |
4.3 sample pre-treatments
100 μ L samples are pipetted into EP pipe after thawing on ice, and 300 μ L methanol are added, add 10 μ L internal standard L-2- chlorobenzenes
Alanine is vortexed and mixes 30 seconds；Ultrasonic 10min (ice-water bath；Ultrasonic frequency is 25-35KHz, power density 0.35-
0.45w/cm2.)；Subzero 20 DEG C of standings 1h；By 4 DEG C of sample, 12000rpm is centrifuged 15min；Carefully take out 200 μ L supernatants in
In the sample injection bottle of included interpolation pipe, each sample respectively takes 20 μ L to be mixed into QC sample, then takes machine testing on 200 μ L.
Machine testing on 4.4
It is analyzed under the control of 1290 ultra high efficiency liquid phase of Agilent according to the mobile phase parameter in following table.Used chromatography
Column is the UPLC BEH Amide chromatographic column (1.7 μm of * 2.1*100mm) purchased from Waters.There are two types of modes for loading: cation mould
Formula (pos) and negative ion mode (neg), sampling volume are pos:4 μ L, neg:2 μ L.
Liquid chromatogram mobile phase condition:
| Time/min | Flow velocity/μ L/min | A% | B% |
| 0 | 500 | 5 | 95 |
| 0.5 | 500 | 5 | 95 |
| 7 | 500 | 35 | 65 |
| 8 | 500 | 60 | 40 |
| 9 | 500 | 60 | 40 |
| 9.1 | 500 | 5 | 95 |
| 12 | 500 | 5 | 95 |
AB 6600Triple TOF mass spectrograph can be under control software (Analyst TF 1.7, AB Sciex) control
Level-one, the acquisition of second order ms data are carried out based on IDA function.In each data acquisition cycles, it is most strong and big to filter out intensity
Molecular ion in 100 is acquired corresponding second order ms data.Bombarding energy: 30eV, 15 every 50ms of second level spectrogram.
ESI source parameters is provided that atomization air pressure (GS1): 60Psi, assist gas pressure: 60Psi, gas curtain air pressure: 35Psi, temperature:
650 DEG C, spray voltage: 5000V (positive ion mode) or -4000V (negative ion mode).
4.5 data processing
Using ProteoWizard software, by mass spectrum, original to change into mzXML various.Reuse XCMS do retention time correction,
Work, the minfrac such as peak identification, peak are extracted, peak integral, peak are aligned are set as 0.5, cutoff and are set as 0.6.It uses and writes certainly simultaneously
R program bag and self-built second order ms database carry out identification to peak.
(5) testing result of the outer component of candida albicans biofilm extracellular matrix protein matter
The pretreatment process of sample eliminates most albumen, and candida albicans biofilm extracellular matrix is through the above method
Testing result it is as follows.
Lipid, organic acid and derivative:
| Title | Scoring | Charge-mass ratio | Appearance time |
| Hydroxy-palmitic acid | 0.9124 | 271.2287 | 39.17 |
| Arachidonic acid | 0.917 | 303.2346 | 38.9335 |
| Nutmeg oleic acid | 0.9999 | 225.1869 | 47.223 |
| Oleic acid | 0.937 | 281.2498 | 38.4415 |
| Stearic acid | 1 | 283.2669 | 40.013 |
Sugar and derivative:
| Title | Scoring | Charge-mass ratio | Appearance time |
| Ribitol | 0.9791 | 151.0618 | 229.561 |
| Sucrose | 0.9364 | 341.1109 | 389.023 |
| Iditol | 0.852 | 181.0722 | 291.6155 |
| Sedoheptulose | 0.9472 | 247.0177 | 375.6955 |
| Erythrose-4-phosphate | 0.8948 | 259.0236 | 441.0255 |
Amino acid:
| Title | Scoring | Charge-mass ratio | Appearance time |
| Threonine | 0.9087 | 84.04355 | 365.614 |
| Alanine | 0.9996 | 90.054 | 338.7655 |
| Proline | 0.9998 | 116.0691 | 304.3545 |
| Glutamic acid | 0.9524 | 130.0493 | 365.519 |
| Leucine | 0.9065 | 132.1005 | 269.96 |
Nucleic acid and derivative:
A kind of embodiment two: detection method of the outer component of candida albicans biofilm extracellular matrix protein matter
A kind of detection method of the outer component of candida albicans biofilm extracellular matrix protein matter, including the following steps:
Step 1: candida albicans activates
Candida albicans is inoculated in SDB culture medium, shaking table is incubated for 25h；Fungal cell is collected after medium centrifugal, with nothing
Then cleaning gained fungal cell is resuspended in RPMI-1640 culture medium, then is incubated for 16h by the cleaning of bacterium PBS buffer solution, then
Under the conditions of 4 DEG C, with 3000 turns/min centrifugation, the fungal cell of sinking is collected, addition fungi culture medium adjustment fungal cell is dense
It spends to 1.2 × 106CFU/ml obtains resuspended bacterium solution；
Step 2: flowable state candida albicans biofilm is incubated for
Medical catheter piece is impregnated to keep sterile in ethanol, after sterile water wash, then is added it to described heavy
It in outstanding bacterium solution, is incubated in shaking table, candida albicans is made to be adhered to the medical catheter on piece, then to being adhered to medical catheter piece
On candida albicans carry out flowing incubation, after be incubated for the candida albicans for obtaining being formed with mature biofilm；
Step 3: the NaH that pH value is 7.8 is added in the candida albicans for being formed with mature biofilm2PO4-Na2HPO4From
In sub- buffer, be first vortexed concussion, is then then sonicated to obtain suspension, will exchange by pretreated highly acidic cation
Resin is mixed with suspension, and shaking table is incubated for 4h, is then 0.22 μm of filtering with microporous membrane with aperture, with 8500rpm/min from
Heart 4min, obtains supernatant.
Preferred embodiment are as follows: every liter of SDB culture medium contains 10g peptone, 20g agar powder, 40g glucose and 0.1g
Chloramphenicol.
Preferred embodiment are as follows: the composition of raw materials of the RPMI-1640 culture medium includes the original of following mass percent
Material: 1% mannitol, 1% nutrient broth, 0.2% K2HPO4With 1.35% agar powder.
Preferred embodiment are as follows: every liter of fungi culture medium contain the potassium chloride of 400mg, 6000mg sodium chloride,
The glucose of 2000mg, the L-arginine of 290.00mg, the altheine of 50mg, the L-Aspartic acid of 20mg, 65.15mg
L-cysteine dihydrochloride, the Pidolidone of 20mg, the glycine of 10mg, the L-Histidine of 15mg, 20mg L- hydroxyproline,
The l-Isoleucine of 50mg, the L-Leu of 50mg, the L-lysine of 40mg, the l-methionine of 15mg, 15mg L- phenylpropyl alcohol
Propylhomoserin, the L-PROLINE of 20mg, the Serine of 30mg, the L-threonine of 20mg, the L-Trp of 5mg, 23.19mg L- junket
The Valine of propylhomoserin, 20mg, the fetal calf serum that the mass concentration of 100ml is 10%, remaining is purified water, then uses 3- (N-
Ma Lindai) propane sulfonic acid adjusts pH value to 7.0.
Preferred embodiment are as follows: the supernatant is stored in -80 DEG C of low temperature refrigerators, before testing, after thawing on ice
100 μ L samples are pipetted into EP pipe, 300 μ L methanol are added, add 10 μ L internal standard L-2- chlorophenylalanines, are vortexed after mixing,
Ultrasonic treatment ultrasound is carried out in ice-water bath；After stand 1.5h under the conditions of -20 DEG C；Then under the conditions of 4 DEG C, with
14000rpm speed is centrifuged 10min；The supernatant for finally taking out 200 μ L, which is placed in sample injection bottle, carries out machine testing.
Preferred embodiment are as follows: the time of ultrasonic treatment is 10min, ultrasonic frequency 35KHz, and power density is
0.35w/cm2。
Preferred embodiment are as follows: the upper machine testing uses high performance liquid chromatography-flight time mass spectrum method for combined use, color
Spectrum column is UPLC BEH Amide chromatographic column, having a size of 1.7 μm of 2.1 × 100mm；Gradient elution mobile phase: A:25mM ammonium acetate
And 25mM, B: acetonitrile；
| Time/min | Flow velocity/μ L/min | A% | B% |
| 0 | 500 | 5 | 95 |
| 0.5 | 500 | 5 | 95 |
| 7 | 500 | 35 | 65 |
| 8 | 500 | 60 | 40 |
| 9 | 500 | 60 | 40 |
| 9.1 | 500 | 5 | 95 |
| 12 | 500 | 5 | 95 |
Mass spectrum: bombarding energy: 30eV, ESI source parameters are provided that atomization air pressure: 60Psi, assist gas pressure:
60Psi, gas curtain air pressure: 35Psi, temperature: 650 DEG C, when positive ion mode, spray voltage 5000V, when negative ion mode, spray
Mist voltage is -4000V.
Preferred embodiment are as follows: the high performance liquid chromatography is 1290 ultra performance liquid chromatography of Agilent, and mass spectrograph is
6600 Triple TOF mass spectrograph of AB.
Preferred embodiment are as follows: the storng-acid cation exchange resin is 732 storng-acid cation exchange resins.
A kind of embodiment three: detection method of the outer component of candida albicans biofilm extracellular matrix protein matter
A kind of detection method of the outer component of candida albicans biofilm extracellular matrix protein matter, including the following steps:
Step 1: candida albicans activates
Candida albicans is inoculated in SDB culture medium, shaking table is incubated for 20h；Fungal cell is collected after medium centrifugal, with nothing
Then cleaning gained fungal cell is resuspended in RPMI-1640 culture medium, then is incubated for 12h by the cleaning of bacterium PBS buffer solution, then
Under the conditions of 4 DEG C, with 2500 turns/min centrifugation, the fungal cell of sinking is collected, addition fungi culture medium adjustment fungal cell is dense
It spends to 0.8 × 106CFU/ml obtains resuspended bacterium solution；
Step 2: flowable state candida albicans biofilm is incubated for
Medical catheter piece is impregnated to keep sterile in ethanol, after sterile water wash, then is added it to described heavy
It in outstanding bacterium solution, is incubated in shaking table, candida albicans is made to be adhered to the medical catheter on piece, then to being adhered to medical catheter piece
On candida albicans carry out flowing incubation, after be incubated for the candida albicans for obtaining being formed with mature biofilm；
Step 3: the NaH that pH value is 7.8 is added in the candida albicans for being formed with mature biofilm2PO4-Na2HPO4From
In sub- buffer, be first vortexed concussion, is then then sonicated to obtain suspension, will exchange by pretreated highly acidic cation
Resin is mixed with suspension, and shaking table is incubated for 2h, is then 0.22 μm of filtering with microporous membrane with aperture, with 7500rpm/min from
Heart 4min, obtains supernatant.
Preferred embodiment are as follows: every liter of SDB culture medium contains 10g peptone, 20g agar powder, 40g glucose and 0.1g
Chloramphenicol.
Preferred embodiment are as follows: the composition of raw materials of the RPMI-1640 culture medium includes the original of following mass percent
Material: 1% mannitol, 1% nutrient broth, 0.2% K2HPO4With 1.35% agar powder.
Preferred embodiment are as follows: every liter of fungi culture medium contain the potassium chloride of 400mg, 6000mg sodium chloride,
The glucose of 2000mg, the L-arginine of 290.00mg, the altheine of 50mg, the L-Aspartic acid of 20mg, 65.15mg
L-cysteine dihydrochloride, the Pidolidone of 20mg, the glycine of 10mg, the L-Histidine of 15mg, 20mg L- hydroxyproline,
The l-Isoleucine of 50mg, the L-Leu of 50mg, the L-lysine of 40mg, the l-methionine of 15mg, 15mg L- phenylpropyl alcohol
Propylhomoserin, the L-PROLINE of 20mg, the Serine of 30mg, the L-threonine of 20mg, the L-Trp of 5mg, 23.19mg L- junket
The Valine of propylhomoserin, 20mg, the fetal calf serum that the mass concentration of 100ml is 10%, remaining is purified water, then uses 3- (N-
Ma Lindai) propane sulfonic acid adjusts pH value to 7.0.
Preferred embodiment are as follows: the supernatant is stored in -80 DEG C of low temperature refrigerators, before testing, after thawing on ice
100 μ L samples are pipetted into EP pipe, 300 μ L methanol are added, add 10 μ L internal standard L-2- chlorophenylalanines, are vortexed after mixing,
Ultrasonic treatment ultrasound is carried out in ice-water bath；After stand 0.5h under the conditions of -20 DEG C；Then under the conditions of 4 DEG C, with
10000rpm speed is centrifuged 20min；The supernatant for finally taking out 200 μ L, which is placed in sample injection bottle, carries out machine testing.
Preferred embodiment are as follows: the time of ultrasonic treatment is 10min, ultrasonic frequency 25KHz, and power density is
0.45w/cm2。
Preferred embodiment are as follows: the upper machine testing uses high performance liquid chromatography-flight time mass spectrum method for combined use, color
Spectrum column is UPLC BEH Amide chromatographic column, having a size of 1.7 μm of 2.1 × 100mm；Gradient elution mobile phase: A:25mM ammonium acetate
And 25mM, B: acetonitrile；
Mass spectrum: bombarding energy: 30eV, ESI source parameters are provided that atomization air pressure: 60Psi, assist gas pressure:
60Psi, gas curtain air pressure: 35Psi, temperature: 650 DEG C, when positive ion mode, spray voltage 5000V, when negative ion mode, spray
Mist voltage is -4000V.
Preferred embodiment are as follows: the high performance liquid chromatography is 1290 ultra performance liquid chromatography of Agilent, and mass spectrograph is
AB 6600Triple TOF mass spectrograph.
Preferred embodiment are as follows: the storng-acid cation exchange resin is 732 storng-acid cation exchange resins.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause
This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as
At all equivalent modifications or change, should be covered by the claims of the present invention.
Claims (8)
1. a kind of detection method of the outer component of candida albicans biofilm extracellular matrix protein matter, it is characterised in that: including following
Step:
Step 1: candida albicans activates
Candida albicans is inoculated in SDB culture medium, shaking table is incubated for 20~28h；Fungal cell is collected after medium centrifugal, with nothing
Then cleaning gained fungal cell is resuspended in RPMI-1640 culture medium, then is incubated for 12-18h by the cleaning of bacterium PBS buffer solution, so
Afterwards under the conditions of 4 DEG C, with 2500~3500 turns/min centrifugation, the fungal cell of sinking is collected, addition fungi culture medium adjustment is true
Bacterium cell concentration is to 0.8~1.2 × 106CFU/ml obtains resuspended bacterium solution；
Step 2: flowable state candida albicans biofilm is incubated for
Medical catheter piece is impregnated to keep sterile in ethanol, after sterile water wash, then adds it to the resuspension bacterium
It in liquid, is incubated in shaking table, candida albicans is made to be adhered to the medical catheter on piece, then to being adhered to medical catheter on piece
Candida albicans carries out flowing incubation, after be incubated for the candida albicans for obtaining being formed with mature biofilm；
Step 3: the NaH that pH value is 7.8 is added in the candida albicans for being formed with mature biofilm2PO4-Na2HPO4Ion buffering
In liquid, be first vortexed concussion, is then then sonicated to obtain suspension, storng-acid cation exchange resin is mixed with suspension,
Shaking table is incubated for 2~4h, is then 0.22 μm of filtering with microporous membrane with aperture, with 7500~8500rpm/min centrifugation 2~
4min obtains supernatant.
2. the detection method of the outer component of candida albicans biofilm extracellular matrix protein matter according to claim 1, special
Sign is: every liter of SDB culture medium contains 10g peptone, 20g agar powder, 40g glucose and 0.1g chloramphenicol.
3. the detection method of the outer component of candida albicans biofilm extracellular matrix protein matter according to claim 1, special
Sign is: the composition of raw materials of the RPMI-1640 culture medium includes the raw material of following mass percent: 1% mannitol, 1%
Nutrient broth, 0.2% K2HPO4With 1.35% agar powder.
4. the detection method of the outer component of candida albicans biofilm extracellular matrix protein matter according to claim 1, special
Sign is: every liter of fungi culture medium contain the potassium chloride of 400mg, the sodium chloride of 6000mg, 2000mg glucose,
The L-arginine of 290.00mg, the altheine of 50mg, the L-Aspartic acid of 20mg, 65.15mg two hydrochloric acid of l-cysteine
Salt, the Pidolidone of 20mg, the glycine of 10mg, the L-Histidine of 15mg, the L- hydroxyproline of 20mg, 50mg the different bright ammonia of L-
The L- dried meat of acid, the L-Leu of 50mg, the L-lysine of 40mg, the l-methionine of 15mg, the L-phenylalanine of 15mg, 20mg
Propylhomoserin, the Serine of 30mg, the L-threonine of 20mg, the L-Trp of 5mg, the l-tyrosine of 23.19mg, 20mg L- figured silk fabrics
Propylhomoserin, the fetal calf serum that the mass concentration of 100ml is 10%, remaining is purified water, then uses 3- (N- Ma Lindai) propane sulfonic acid tune
Whole pH value is to 7.0.
5. the detection method of the outer component of candida albicans biofilm extracellular matrix protein matter according to claim 1, special
Sign is: the supernatant is stored in -80 DEG C of low temperature refrigerators, before testing, 100 μ L samples is pipetted after thawing on ice to EP pipe
In, 300 μ L methanol are added, add 10 μ L internal standard L-2- chlorophenylalanines, are vortexed after mixing, are carried out at ultrasound in ice-water bath
Reason ultrasound；After under the conditions of -20 DEG C stand 0.5~1.5h；Then under the conditions of 4 DEG C, with 10000~14000rpm speed
It is centrifuged 10~20min；The supernatant for finally taking out 200 μ L, which is placed in sample injection bottle, carries out machine testing.
6. the detection method of the outer component of candida albicans biofilm extracellular matrix protein matter according to claim 5, special
Sign is: the time of ultrasonic treatment is 10min, ultrasonic frequency 25-35KHz, power density 0.35-0.45w/cm2。
7. the detection method of the outer component of candida albicans biofilm extracellular matrix protein matter according to claim 5, special
Sign is: the upper machine testing uses high performance liquid chromatography-flight time mass spectrum method for combined use, and chromatographic column is UPLC BEH
Amide chromatographic column, having a size of 1.7 μm of 2.1 × 100mm；Gradient elution mobile phase: A:25mM ammonium acetate and 25mM, B: acetonitrile；
Mass spectrum: bombarding energy: 30eV, ESI source parameters are provided that atomization air pressure: 60Psi, assist gas pressure: 60Psi, gas
Curtain air pressure: 35Psi, temperature: 650 DEG C, when positive ion mode, spray voltage 5000V, when negative ion mode, spray voltage is-
4000V。
8. the detection method of the outer component of candida albicans biofilm extracellular matrix protein matter according to claim 7, special
Sign is: the high performance liquid chromatography is 1290 ultra performance liquid chromatography of Agilent, and mass spectrograph is 6600 Triple TOF of AB
Mass spectrograph.
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