CN109943489A - Culture medium, cultural method and its application - Google Patents
Culture medium, cultural method and its application Download PDFInfo
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- CN109943489A CN109943489A CN201910257751.4A CN201910257751A CN109943489A CN 109943489 A CN109943489 A CN 109943489A CN 201910257751 A CN201910257751 A CN 201910257751A CN 109943489 A CN109943489 A CN 109943489A
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Abstract
The present invention provides a kind of culture medium, and the culture medium includes germfree animal tissue fluid, and the culture medium is used to prepare galactomannans.Culture medium provided by the invention at least has one of following advantage: culture medium provided by the invention can be used for preparing galactomannans, prepared galactomannans is configured to reference material, consistent with the galactomannans variation tendency in aspergillus infection person's serum.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to culture medium, cultural method and its application.
Background technique
Deep fungal infection refers to that fungi intrusion internal organ, blood, deep skin texture causes below mucous membrane or horny layer of epidermis
Infection.With the extensive application of clinically broad-spectrum antibiotic, corticosteroid, immunosuppressor, anti-tumor drug etc., AIDS
The prevalence of disease and the development of transplant operation, deep fungal infections are increasing and tend to complicate, and often result in lethal
Infection.Aspergillus is important pathogenic bacteria, is distributed widely in nature soil, plant and air, is a kind of opportunistic fungus,
With underlying lung disease, hypoimmunity, be the high-risk of deep aspergillus infection morbidity there are the patient of pulmonary shadow, infection symptoms
Crowd.Aspergillus fumigatus is main pathogenic bacteria, accounts for 70%~90% of human infection caused by aspergillus class fungi.Deep aspergillus infection without
Special clinical manifestation, it is difficult to early diagnose, treat in time, lethality is up to 60%~100%.It is counted according to GAFFI, the whole world has
More than 800 ten thousand people are influenced by chronic pulmonary aspergilosis (CPA) and allergic bronchial pulmonary aspergilosis (ABPA), and are had due to lacking
The detection method of effect, many patients cannot timely and effectively be diagnosed.
Skeleton of the galactomannans as aspergillus cell wall, while being also aspergillus Exoantigen, galactomannan is poly-
Sugar antigens are considered as the target of Aspergillosis early diagnosis.
Summary of the invention
The current existing in vitro culture for aspergillus making galactomannans antigen is usually that sabouraud culture medium is used to exist
22~30 DEG C culture Aspergillus strain 2~3 days, then with the polysaccharide in 80% ethanol precipitation sterilization fermentation liquid, and by certain
Way of purification is to extract aspergillus galactomannans glycan.Present inventor is by the discovery that studies for a long period of time, by above-mentioned
The extent of polymerization and composition of method in vitro culture galactomannans obtained are widely different, in aspergillus infection person's serum
Galactomannans variation tendency it is inconsistent, so as to cause by the obtained aspergillus galactomannans of in vitro culture as antigen
Specific antibody potency is lower in the immune serum obtained, although or the higher person's blood that can not identify aspergillus infection of specific antibody potency
Galactomannans in clear, is not inconsistent with clinic, so that the aspergillus galactomannans of in vitro culture obtains antibody and opens
The application of the reagent of sending is restricted.
In view of this, the culture medium prepares galactomannans for cultivating aspergillus the present invention provides a kind of culture medium,
Prepared aspergillus galactomannans glycan is configured to reference material, becomes with the galactomannans in aspergillus infection person's serum
Change trend is consistent.
The present invention provides a kind of culture medium, and the culture medium includes germfree animal tissue fluid, and the culture medium is used to prepare
Galactomannans.
In the specific embodiment of the present invention, the germfree animal tissue fluid is sterile cow's serum and/or sterile
Ox lung tissue grinds broken liquid.
In the specific embodiment of the present invention, the content of the sterile cow's serum is 5%~20%, for example, institute
The content for stating sterile cow's serum is 5%, 7%, 8%, 10%, 11.5%, 12%, 15%, 18% or 20% etc., it is preferable that institute
The content for stating sterile cow's serum is 10%.
In the specific embodiment of the present invention, the sterile ox lung tissue grind the content of broken liquid be 3%~
15%, for example, the content that the sterile ox lung tissue grinds broken liquid is 3%, 5%, 7%, 9%, 10%, 12.5%, 14%
Or 15% etc., it is preferable that the content that the sterile ox lung tissue grinds broken liquid is 5%.
In the specific embodiment of the present invention, according to mass ratio meter, the culture medium includes 2.5% glucose,
1% peptone, 10% sterile cow's serum and 5% sterile ox lung tissue grind broken liquid.
On the other hand the application provides a kind of cultural method comprising Aspergillus is cultivated in culture medium described above.
In the specific embodiment of the present invention, the temperature of the culture is 34~40 DEG C.
In the specific embodiment of the present invention, the cultural method is specifically included: the aspergillus hyphae of activation is connect
Kind is cultivated, when aspergillus galactomannans Glycan concentration in fermentation liquid into aforesaid liquid culture medium on 37 DEG C of constant-temperature tables
Culture is terminated when growth trend slows down.
On the other hand the application also provides a kind of galactomannans, the galactomannans utilizes training described above
It supports base and/or cultural method described above is prepared.
In the specific embodiment of the present invention, the I value of the galactomannans is more than or equal to 1.6, for example, institute
Stating I value is 1.6,1.7,1.8,1.9,2.0,2.1,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3.0,3.1 or 3.2
Deng.
Another aspect of the present invention provides the kit for detecting aspergillin infection, it includes above-mentioned galactomannans or
The antibody that above-mentioned galactomannans is prepared as antigen.
Another aspect of the present invention provides galactomannans described above and is preparing the examination for detecting aspergillin infection
Application in agent box or genetic chip.
Culture medium provided by the invention at least has one of following advantage: culture medium provided by the invention can be used for preparing half
Newborn mannosan, prepared galactomannans are configured to reference material, poly- with the galactomannan in aspergillus infection person's serum
Sugared variation tendency is consistent.
Specific embodiment
Unless otherwise defined, all technical and scientific terms used in the present invention have and technical field of the present invention
The normally understood identical meanings of those of ordinary skill.
Specifically, used herein, "comprising" and " comprising ", " containing " or " being characterized in that " it is synonymous, and be packet
It is including including or open, and it is not excluded for the other ingredient that do not state or method and step.Term "comprising" is herein
Any statement, especially when describing method of the invention, purposes or product, it is thus understood that including substantially by the component
Or element or step composition and product, method and purposes those of are formed by the component or element or step.It is exemplified here
Can suitably any one or more of element not specifically disclosed herein, one or more limits be not present in the present invention of description
It is practiced in the case where system.
The term and statement used herein is used as descriptively rather than restrictive term, and in such term and statement
Use in it is not expected exclude shown in and described feature or part thereof any equivalent, it is appreciated that various modifications are being requested
It is possible in the scope of the present invention of protection.It is therefore understood that although the present invention passed through preferred embodiment and optionally
Feature specifically discloses, but those skilled in the art can use the modification and transformation of concept disclosed herein, and such modification
It is considered as in the scope of the present invention such as defined by accessory claim with variation.
It to be illustrated more clearly that the present invention, is described in detail now in conjunction with following examples, but these embodiments are only
To exemplary description of the invention, the limitation to the application should not be construed as.
The preparation of 1 galactomannans of embodiment
The ingredient of culture medium:
Improvement sabouraud culture medium (peptone 1%, glucose 2.5%) in be added the sterile cow's serum of 5%-20% and
The sterile ox lung tissue of 3%-15% grinds broken liquid.
Except sterile cow's serum, sterile ox lung tissue are ground, broken liquid etc. is sterile to be moved the medium component provided in the present embodiment
Object tissue fluid has outside certain requirement, other compositions, for example, peptone, glucose can be according to cultivating aspergillus in the prior art
Amount needed for bacterium prepares galactomannans is adjusted.The basic ingredient of the culture mediums such as glucose, peptone is not the application
Inventive point.
Fermentation process and purification process
1) aspergillus hyphae of activation is inoculated into aforesaid liquid culture medium, on 34-40 DEG C of shaking table, 180rpm revolving speed item
It is cultivated 3~5 days under part.It is measured in fermentation liquid after starting culture 48h every 4h galactomannans glycan detection kit
Aspergillus galactomannans Glycan concentration is primary, and culture is terminated when growth trend slows down；
2) 121 DEG C of sterilizing 30min are stood to room temperature；
3) thallus is filtered with 8 layers of sterile gauze, retains filtrate, filtrate is added 3 times of volume dehydrated alcohols and stands in 2~8 DEG C
16h, alcohol precipitation product are contained with centrifugal barrel clean in advance, and 4 DEG C, 30min is centrifuged under the conditions of 8000rpm, takes out centrifugal barrel, slowly
Supernatant is poured out, it is invisible to liquid in 40 DEG C of air dry oven drying centrifugal barrels, it is redissolved with pure water, it is pure that 10~20mL is added
Water/1L fermentation liquid, repeatedly rinse centrifugal barrel inner wall 30min；3 times of volume dehydrated alcohols are added in redissolving liquid to mix, 2~8 DEG C
4h is stood, 10000rpm is centrifuged 15min, abandons supernatant, can not in 40 DEG C of air dry ovens drying 10~20min of centrifugal barrel to liquid
See, redissolved with pure water, 5~10mL pure water/1L fermentation liquid is added, and whirlpool mixes 30min, and 10000rpm is centrifuged 10min, receives
Collect supernatant；Supernatant crosses 0.45 μm of filter.
4) after lysate is diluted with 9 times of sterile waters, then with 10kD super filter tube be lower than the revolving speed ultrafiltration 10 of 5000xg~
30min, until volume of the volume concentration to before diluting, is sucked out polysaccharide solution；
5) chromatographic separation and purification product is used, the high purpose peak of aspergillus galactomannans activity is retained.
Operating procedure:
1, instrument and equipment: Dalian Yi Lite liquid chromatogram；Filler: Superdex 30increase
2, prepare mobile phase: 0.5mol/L ammonium hydrogen carbonate, cross 0.2 μm filter membrane 2 times, balance pillar.
3, filler is balanced: with mobile phase with 0.2ml/min flow rate 10min；
4, preparation of samples: with flowing phase dilution antigen to 0.2mg/mL；
5, sample introduction: 500 μ L samples is taken to inject injection port；
6, sample picks up: connecing within every 2 minutes pipe outflow sample, picks up 40 pipes altogether；
7, sample detection: the above-mentioned activity for picking up sample is detected with BioRad kit；
8, sample screens: the retentive activity highest period, (retention time was 40~44min, shows difference in the retention time
Detector shows no miscellaneous peak) sample up to galactomannans.
In the present embodiment between the selection and result of bacterial strain type, medium component and cultivation temperature shown in the following table 1-1.
The selection and result of table 1-1 bacterial strain type, medium component and cultivation temperature
Note: I value is the testing result of BioRad aspergillus galactomannans kit, and I value is higher to illustrate that aspergillus gala is sweet
It is stronger to reveal glycan activity.
As shown in table 1-1, compared to sabouraud culture medium, different Aspergillus is containing animal tissue's liquid, such as is added
Well-grown in the culture medium that ox lung tissue is crushed liquid or the two is all added, galactomannans is added in sterile cow's serum
Yield obviously increase, sterile cow's serum and ox lung tissue are especially added simultaneously being crushed liquid makes, the yield of galactomannans
It can reach 3 times or more of its yield in sabouraud culture medium；And the I value of prepared galactomannans also obviously mentions
Height illustrates to be remarkably reinforced using the activity of galactomannans prepared by culture medium provided in the present embodiment.
By taking strains A TCC13073 as an example, under 37 DEG C of condition of culture, the nothing that different proportion is added in sabouraud culture medium is tested
The influence of bacterium cow's serum and sterile ox lung tissue to galactomannans is prepared, as shown in table 1-2.
The sterile cow's serum of table 1-2 different proportion and sterile ox lung tissue
As shown in table 1-2, Aspergillus ATCC13073 is being crushed liquid containing different sterile cow's serums and sterile ox lung tissue
Middle yield and I value are higher, in the culture medium being especially combined with 10% sterile cow's serum and 5% sterile ox lung tissue, gala
The yield and the equal highest of I value of mannosan, can be used as the optimum proportioning in commercial process.
Below prepared by the culture medium of the 10% sterile ox lung tissue of sterile cow's serum+5% for galactomannans
It is tested.
The comparison of 2 antigen diluent of embodiment
With galactomannan prepared in the culture medium of the 10% sterile ox lung tissue of sterile cow's serum+5% in embodiment 1
Glycan is denoted as antigen B as antigen, and the galactomannans to generate in husky formula culture medium is denoted as antigen A as control,
Comparison addition antigen A, the reference material of antigen B and the dilution ratio variation tendency of positive sample.
Experimental method:
With the serum reference material and positive sample of negative serum difference doubling dilution addition antigen A, antigen B, BioRad is used
Aspergillus galactomannans kit detects its dilution ratio variation tendency.
Experimental procedure:
1, antigen A is diluted respectively with negative serum, B to concentration is 8,4,2,1,0.5,0.25,0ng/mL；
2, doubling dilution positive sample 1,2,3 is distinguished with negative serum；
3, sample process: being separately added into 300 μ l reference materials or positive sample in EP pipe, adds 100 μ l sample process
Liquid covers tightly pipe lid, mixes well, and heats 3 minutes in 100 DEG C of boiling water baths later, careful to take out, and is put into centrifuge,
10000g is centrifuged 10 minutes, and the supernatant after centrifugation is for detecting；
Sample-adding: 50 μ l enzyme labelled antibodies are added in the every hole of ELISA Plate, 50 μ l reference materials or positive sample supernatant are added, with envelope
Plate film seals orifice plate, is incubated for 90min in 37 DEG C of baking ovens；Liquid in hole is dried later, board-washing 5 times, is patted and is removed on blotting paper
Remove residual liquid；
Colour developing: 200 μ l developing solutions are added in the every hole of ELISA Plate, is incubated for 30min in 37 DEG C of baking ovens, is protected from light, do not have to sealing plate
Film seals orifice plate；
It terminates: 100 μ l terminate liquids is added in the every hole of ELISA Plate, Loading sequence is identical as developing solution, mixes well, in wavelength
450nm reads every hole OD value OD, then calculates positive sample concentration value with reference material dilution curve, and experimental result is shown in Table 2 Hes
Shown in table 3.
The extension rate and OD value of each sample of table 2
The extension rate and concentration of each sample of table 3
From table 2 and table 3: under the conditions of same concentrations, the reference material for adding antigen B is poly- with BioRad aspergillus galactomannan
The activity of sugared kit detection is higher, illustrates that the activity of antigen B under same concentrations is higher；With addition antigen B reference material it is dilute
The doubling dilution liquid that curve calculates positive sample is released, obtained concentration is in multiple proportion；And it is calculated with the reference material of addition antigen A
Obtained concentration has no multiple proportion, illustrates that antigen B is consistent with the antigen diluent curve in patients serum, antigen B and patient's blood
The structure of antigen in clear is more close.
3 antigen A of embodiment, the comparison of B immune serum
Test the potency and Sensitivity comparison of the immune gained serum of antigen A, antigen B.
Test method:
1, the potency of the immune gained serum of antigen A, B is detected respectively with ELISA method；
2, the enzyme labelled antibody in BioRad aspergillus galactomannans kit is replaced to verify serum pair with immune serum
The sensitivity of positive sample identification.
Experimental procedure:
Detect serum titer
1. antigen coat condition: the antigen of 100 μ L 50ng/mL, package amount 5ng/well, pH=being added into ELISA Plate
7.4,0.01M PBS buffer solution dilution, 80 DEG C；3h；
2. closing: closing uses 1wt%BSA-PBST solution, 100 μ l/well, 37 DEG C, 1h；
3. joining object stabilizer doubling dilution antibody since 1:10000 with lotus root, take 100 μ l dilutions that ELISA Plate hole is added,
37 DEG C of incubation 1h；
4. board-washing: board-washing is washed three times using 0.01M PBST washing lotion, 300 μ L/well；Stand 40s；
5. taking 100 μ l enzyme labelled antibodies that ELISA Plate, 37 DEG C of incubation 1h are added；
6. TMB100 μ L colour developing, 37 DEG C of incubation 15min are added；
7. 50 μ L of 2M sulfuric acid is added to terminate, microplate reader 450nm reading；
Detect antibody sensitivity
1. sample process
It is separately added into 300 μ l positive samples in EP pipe, adds 100 μ l sample treatment solutions, covers tightly pipe lid, it is sufficiently mixed
It is even, it is heated 3 minutes in 100 DEG C of boiling water baths later, it is careful to take out, it is put into centrifuge, is centrifuged 10 minutes in 10000g, after centrifugation
Supernatant for detecting；
2, sandwich antibody is prepared
Connect object stabilizer with lotus root and dilute immune serum in proportion: serum 1 (1:20000), serum 2 (1:20000), serum 3
(1:80000), serum 4 (1:160000), serum 5 (1:160000), serum 6 (1:160000) are configured to sandwich antibody 1-6.
3, it is loaded
The 50 above-mentioned sandwich antibodies of μ l (serum dilution) and BioRad enzyme labelled antibody are separately added into ELISA Plate hole, are added
50 μ l positive sample supernatants seal orifice plate with sealing plate film, are incubated for 90min in 37 DEG C of baking ovens；Liquid in hole, board-washing are dried later
5 times, removing residual liquid is patted on blotting paper；
100 μ l ELIAS secondary antibodies (for detecting) are added in the every hole of ELISA Plate for adding sandwich antibody 1-6, with sealing plate film sealing hole
Plate is incubated for 30min in 37 DEG C of baking ovens；Liquid in hole is dried later, and board-washing 5 times, removing residual liquid is patted on blotting paper；
The ELISA Plate of BioRad enzyme labelled antibody is added to be directly entered color operation；
4, it develops the color
200 μ l developing solutions are added in the every hole of ELISA Plate, is incubated for 30min in 37 DEG C of baking ovens, is protected from light, do not have to sealing plate film and seal
Orifice plate；
5, it terminates
100 μ l terminate liquids are added in the every hole of ELISA Plate, Loading sequence is identical as developing solution, mixes well, in wavelength 450nm
Each hole OD value OD is read, and calculates I value, experimental result is as shown in table 4 and table 5.
The OD value of each sample of table 4
The I value of each sample of table 5
By table 4 and table 5 it is found that the rabbit anteserum potency of immunizing antigen A is all low compared with immunizing antigen B；The rabbit blood of immunizing antigen A
The antigen in small part patients serum can only be identified clearly, and the rabbit anteserum of immunizing antigen B can identify in most of patients serum
Antigen.
Embodiment 4 adds the reference product examine blood serum sample comparison of antigen A, B
The cutoff Quality Control in BioRad aspergillus galactomannans kit is replaced with the reference material of addition antigen A, B
Product, respectively in the specificity and sensitivity of 3 laboratory proofing blood serum samples.
Experimental procedure
1, antigen A, B are added respectively in negative serum as reference material；
2, sample process
It is separately added into 300 μ l reference materials or blood serum sample in EP pipe, adds 100 μ l sample treatment solutions, covers tightly pipe lid,
It mixes well, is heated 3 minutes in 100 DEG C of boiling water baths later, it is careful to take out, it is put into centrifuge, is centrifuged 10 minutes in 10000g,
Supernatant after centrifugation is for detecting；
3, it is loaded
Following each steps are carried out respectively three different experiments rooms, in ELISA Plate 50 μ l enzyme labelled antibodies of every hole addition, then plus
Enter 50 μ l reference materials or Sample supernatants, seals orifice plate with sealing plate film, be incubated for 90min in 37 DEG C of baking ovens；Liquid in hole is dried later
Body board-washing 5 times, pats removing residual liquid on blotting paper；
4, it develops the color
200 μ l developing solutions are added in the every hole of ELISA Plate, is incubated for 30min in 37 DEG C of baking ovens, is protected from light, do not have to sealing plate film and seal
Orifice plate；
5 terminate
100 μ l terminate liquids are added in the every hole of ELISA Plate, Loading sequence is identical as developing solution, mixes well, in wavelength 450nm
Read every hole OD value OD, calculate I value, compare it is under three kinds of experimental situations as a result, its experiment the results are shown in Table 6.
The I value of not synantigen in 6 different experiments room of table
As shown in Table 6, it when adding the cutoff reference material of reference material substitution BioRad of antigen A, is used in different experiments room
Its resulting sample I value result of calculating is variant, and main cause is that detected value of the reference material under different experiments room environmental has change
Change, so as to cause coincidence rate reduction, and the reference material for adding antigen B is affected by environment smaller, it can be seen that utilizes 1 institute of embodiment
The galactomannan structure of preparation is stablized, and ambient enviroment is on it almost without influence.
The aspergillus galactomannans antigen of 5 the application culture of embodiment is preparing aspergillus IgG antibody detection kit (glue
Body gold method) in terms of application
Aspergillus IgG antibody detection kit (colloidal gold method) is used to detect the aspergillus IgG antibody in human serum, which adopts
With prize law colloidal gold immunochromatographimethod technology, the aspergillus IgG antibody in human serum sample is detected.Embedding is golden in advance in colloidal gold pad
Galactomannans is marked, mouse anti-human IgG antibodies is coated with respectively in detection line (T) and nature controlling line (C) and galactomannans is anti-
Body.The galactomannans antibody is made for aspergillus galactomannans antigen.
If detecting sample is the positive, aspergillus IgG antibody therein forms multiple in conjunction with colloid gold label galactomannans
Close object, chromatography effect under compound along paper slip move forward, after testing when line (T) with pre-coated mouse anti-human IgG antibodies
Reaction forms immune complex and shows red stripes, and free gold marks galactomannans then sweet in nature controlling line (C) and gala
Dew glycan antibody, which combines, shows red stripes.If detecting sample is feminine gender, immune complex not will form, it will not at detection line
There is band, only develops the color at nature controlling line (C).Nature controlling line (C) should all occur band when detecting sample, and nature controlling line (C) is shown
Red stripes be to determine the whether normal standard of chromatography process, while also as the inner quality standard of reagent.
It compares by the detection to 240 samples (including making a definite diagnosis, diagnose or compare equal samples) and with goldstandard,
The susceptibility of confirmed cases is 85.00%, specificity 99.21%, and total coincidence rate is 98.20%.The susceptibility of diagnosed case
It is 75.00%, specificity 99.17%, total coincidence rate is 93.25%, is analyzed through Kappa, and testing result and goldstandard determine
As a result height is consistent.Show that the kit has preferable sensibility and specificity, it is as a result high with clinically goldstandard coincidence rate,
More acurrate reliable inspection result can be provided, and that the operation is more convenient is easy, detect rapid sensitive.Pass through in the embodiment of the present application
Germfree animal tissue fluid is added in the medium, cultivation temperature is provided, so that the condition that aspergillus is grown in vitro is closer in people
The condition of tumor growth, so that gala is sweet in the galactomannans for being metabolized generation in vitro is closer to aspergillus infection person's body
Reveal glycan structures, so that the two is shown identical variation tendency in the detection, so that the aspergillus gala using in vitro culture is sweet
Dew glycan obtains antibody and the detection of the reagent the developed person's serum that is preferably applied to aspergillus infection.
In the reference material and aspergillus infection person serum that prepared aspergillus galactomannans is prepared in the embodiment of the present application
Galactomannans variation tendency it is consistent, the aspergillus antibody being able to detect in serum, sensibility, specificity it is very high；And
Prepared aspergillus galactomannans has very high immunogenicity in the present embodiment, special in serum with its immune acquisition
Antibody titer is very high and the person's serum that can identify aspergillus infection in galactomannans, clinical detection sensibility is higher, and special
It is anisotropic preferable.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, made any modification, equivalent replacement etc. be should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of aspergillus bacterium culture medium, which is characterized in that the culture medium includes germfree animal tissue fluid, and the culture medium is used for
Prepare galactomannans.
2. aspergillus bacterium culture medium as described in claim 1, which is characterized in that the germfree animal tissue fluid is sterile cow's serum
And/or sterile ox lung tissue grinds broken liquid.
3. aspergillus bacterium culture medium as described in claim 1, which is characterized in that the content of the sterile cow's serum be 5%~
20%, it is preferable that the content of the sterile cow's serum is 10%；The content that the sterile ox lung tissue grinds broken liquid is 3%
~15%, it is preferable that the content that the sterile ox lung tissue grinds broken liquid is 5%.
4. aspergillus bacterium culture medium as claimed in any one of claims 1-3, which is characterized in that the culture medium further includes grape
Sugar and peptone.
5. aspergillus bacterium culture medium as claimed in claim 4, which is characterized in that according to mass ratio meter, the culture medium includes
2.5% glucose, 1% peptone, 10% sterile cow's serum and 5% sterile ox lung tissue grind broken liquid.
6. a kind of method for cultivating Aspergillus, which is characterized in that trained using the culture medium of any of claims 1-5
It supports, it is preferable that the temperature of the culture is 34~40 DEG C.
7. a kind of galactomannans, which is characterized in that the galactomannans is using described in any one of claim 1-5
Culture medium, and/or be prepared using cultural method described in claim 6 or 7.
8. galactomannans as claimed in claim 7, which is characterized in that the I value of the galactomannans is more than or equal to
1.6, it is preferable that the I value is greater than 2.4, it is highly preferred that the I value is greater than 3.0.
9. the kit for detecting aspergillin infection, it includes galactomannans according to any one of claims 8 or comprising passing through
The antibody that galactomannans according to any one of claims 8 is prepared as antigen.
10. galactomannans according to any one of claims 8 is in preparing kit or genetic chip for detecting aspergillus infection
Application.
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