CN106399494B - The in vivo method of in situ detection analysis microbial interaction - Google Patents
The in vivo method of in situ detection analysis microbial interaction Download PDFInfo
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- CN106399494B CN106399494B CN201610807957.6A CN201610807957A CN106399494B CN 106399494 B CN106399494 B CN 106399494B CN 201610807957 A CN201610807957 A CN 201610807957A CN 106399494 B CN106399494 B CN 106399494B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
Abstract
A kind of method that the present invention discloses in vivo in situ detection analysis microbial interaction, including the complex matrix of single strain substrate and the gel state of culture substrate composition to be transferred in porous magnetic container hollow lumen, and enters in human body or the open organ of living animal to be resident in a manner of oral or deliver and be discharged after a certain period of time；Porous magnetic container is taken out from the excrement of discharge or open organ by magnetic force, the expression conditions that complex matrix tests and analyzes single culture are taken out after refrigerating；Objective microbe is allowed to complete interaction under living body in-situ condition, the plurality of target microorganism that can be placed on same environment in vivo distinguishes in humans and animals, the interaction of substance that objective microbe generates under condition of living body to other microorganisms in environment is not deadened yet, state and variation of the objective microbe under different natural conditions can be really captured, the test to interact between particular types microorganism is completed under humans and animals living body in-situ condition.
Description
Technical field
The present invention relates to microorganism field, in particular to a kind of side of in vivo in situ detection analysis microbial interaction
Method.
Background technique
Interaction type between microorganism is varied, most of to this research at present to carry out in the lab, can not
Reduction natural environmental condition completely, is also affected by human factors.The method usually to interact between research bacterium has bacterium to train altogether
Support method and Odontothrips loti etc..Bacterium, which co-cultures, to be referred to aseptically, and some specially appointed different microorganisms are in anaerobism
Or the mixed culture under aerobic condition.But when studying correlation between bacterium, bacterium mixing co-cultivation method can make not of the same race
Bacterium, which comes into full contact with, is but unable to judge accurately the number of different bacterium and extracting single culture genome DAN or RNA in culture solution,
As a result statistics and accurate analysis single culture gene expression are difficult, especially to close species in same category, inconvenience analysis experiment knot
Fruit.Odontothrips loti is that it is studied after spreading by Oxford cup to another by obtaining a kind of substance that bacterial activity generates in vitro
The inhibiting effect of kind bacterium, is unable to the interaction of microorganism in in-situ study living body body.
Therefore, human factor influence can be excluded by being badly in need of one kind, and objective microbe can be allowed complete under in-situ condition in vivo
At the method for interaction.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of internal in-situs while still alive to test and analyze microbial interaction
Method allows objective microbe to complete interaction under living body in-situ condition, can separate plurality of target microbiota,
The substance in living body in situ environment is not deadened between interacting under living body in-situ condition particular types microorganism, passes through detection
With its thalline quantity of analysis and changes in gene expression, microbial association is explored, objective microbe can be really captured and exist
State and variation in different people and animal body under in-situ condition, data are more accurate, as a result more accurate, operate it is easier, can be with
The test to interact between particular types microorganism is completed under humans and animals living body in-situ condition, excludes the dry of human factor
It disturbs, makes test data that there is great reliability and reference value.
The method of in vivo in situ detection analysis microbial interaction of the invention, comprising the following steps:
A. it is hollow the complex matrix for the gel state that single strain substrate and culture substrate form to be transferred to porous magnetic container
In inner cavity, and enter human body or animal body digestive tract environment in a manner of oral, it is resident to be discharged after a certain period of time；
B. porous magnetic container is chosen from the excrement of discharge by magnetic force, complex matrix is taken out after refrigerating and is tested and analyzed
The expression conditions of strain；
Further, the porous magnetic container of different single strain substrates will be respectively provided with simultaneously through human body or animal body alimentary canal
Environment；
Further, the porous magnetic vessel surface is with 1~3 × 105A/cm2Mode be distributed with aperture be 200~
The through-hole of 500nm；
Further, the porous magnetic container material quality is martensitic stain less steel, the porous magnetic container is long by 10~
250mm, diameter are 5~50mm；
Further, the porous magnetic container is made of the two parts up and down being threadedly coupled, and is arranged in interconnecting piece outer surface
There is sealing ring；
Further, the sealing ring material is high temperature resistant and corrosion resistant food grade silicone；
Further, it by single microorganism culture solution, aseptically mixes well to form gel with vivo environment matrix
The composite base plastid of state；
Further, composite base plastid is packed into porous stainless steel container, and sealed, and cover upper sealing ring, prevent inside and outside
The pollution of other non-targeted microorganisms；
Further, the porous stainless steel container equipped with objective microbe composite base plastid is taken orally and enters internal alimentary canal,
And resident a period of time；
Further, by porous magnetic container preservation in -80 DEG C or liquid nitrogen of discharge；
Further, aseptically, composite base is taken out after the porous magnetic container after refrigeration being cleaned and sterilized
Body is counted or is extracted genomic DNA or RNA, its gene expression of further analysis detection to microorganism in complex matrix；
Further, using sterile pH=8.0 is freezed, concentration is the phosphate wash porous magnetic container table of 0.1mol/L
Face sterilizes porous magnetic vessel surface with the sterile blotting paper of alcohol that concentration is 70~75% is moistened with.
Beneficial effects of the present invention: the method for living body in situ detection analysis microbial interaction of the invention allows target
Microorganism completes interaction under living body in-situ condition, can in vivo distinguish plurality of target microorganism in humans and animals
Come, does not also deaden the interaction of substance that objective microbe generates under condition of living body to other microorganisms in environment, pass through
Correlation between its particular number of detection and analysis and changes in gene expression microorganisms, can really capture objective microbe
State and variation under different natural conditions, data are more accurate, and as a result more accurate, operation is easier, can be in condition of living body
The lower test for completing to interact between particular types microorganism, excludes the interference of human factor, has test data great
Reliability and reference value.
Detailed description of the invention
The utility model is further described with reference to the accompanying drawings and examples:
Fig. 1 is that the ellipsoid of the utility model contains bacterium device structural schematic diagram；
Fig. 2 is that the cylindrical body of the utility model contains bacterium device structural schematic diagram；
Fig. 3 is that the sphere of the utility model contains bacterium device structural schematic diagram；
Fig. 4 is that the sub warhead body of the utility model contains bacterium device structural schematic diagram.
Specific embodiment
The method of the in vivo in situ detection analysis microbial interaction of the present embodiment, comprising the following steps:
A. it is hollow the complex matrix for the gel state that single strain substrate and culture substrate form to be transferred to porous magnetic container 1
In inner cavity, and enter human body or animal body digestive tract environment in a manner of oral or deliver, it is resident to be discharged or take after a certain period of time
Out；
B. porous magnetic container 1 is taken out from the excrement of discharge or open organ by magnetic force, is taken out after refrigerating compound
The thalline quantity and changes in gene expression of matrix detection and analysis strain；Porous magnetic container 1 needs sterilization treatment before use, will be more
Hole magnetism container 1 passes through resident after a certain period of time, the analysis in human body and animal open organ (enteron aisle, oral cavity, vagina, urethra etc.)
Micro organism quantity or extracting genomic DNA or RNA in thallus matrix composite body in porous magnetic container 1, carry out gene expression analysis
Analysis.The porous magnetic container 1 can also be moved into other natural environment (mud, soil of microbe survival by suitable density
Deng), in artificial fermentation's system (wine brewing, makes vinegar, ensilage etc. at koji-making), in the time of setting, using magnetic force from soil, grain
It is selected in the environment such as unstrained spirits, excrement, mud, tests and analyzes microbial interaction relationship.
In the present embodiment, the porous magnetic container 1 of different single strain substrates will be respectively provided with simultaneously through human body or animal body
Open organ environment；Different strain substrate and culture substrate homogenate is mixed to form after gel state and is respectively charged into porous magnetic appearance
In device 1, then enter human body or animal body in a manner of oral or deliver again, after the open organ of human body is resident a period of time, warp
It is discharged behind normal digestion channel or artificial taking-up is to be tested and analyzed.
In the present embodiment, 1 surface of porous magnetic container is with 1~3 × 105A/cm2Mode be distributed with aperture be 200
The through-hole 2 of~500nm；The quantity of through-hole 2 and the size in hole directly determine the using effect of porous magnetic container 1, Kong Tai great or
Intensively all objective microbe can not be effectively isolated very much, Kong Tai little or too sparse, but influence container in microorganism and
The contact of local environment state prevents microorganism from being completely disposed in the environment, influences the nature of microorganism, the container is not
It deadens diameter in natural environment and is less than the substance in 1 aperture of porous magnetic container to phase under field conditions (factors) particular types microorganism
The influence of interaction, makes microorganism be completely in nature.
In the present embodiment, 1 material of porous magnetic container is martensitic stain less steel, the porous magnetic container 1 long 10
~250mm, diameter are 5~50mm；Material, the size of porous magnetic container 1 ensure that it can carry out effective activity in human body
And circulation, while tissue is not influenced, and acid and alkali-resistance, corrosion-resistant even high temperature resistant, it is ensured that the microorganism in container is in certainly
Right state does not influence the activity of microorganism.
In the present embodiment, the porous magnetic container 1 is made of the two parts up and down being threadedly coupled or is not helicoid
Blind pipe open at one end is equipped with sealing ring 3 in interconnecting piece outer surface or blind pipe open end, and 3 material of sealing ring is high temperature resistant
With corrosion resistant food grade silicone.；Porous magnetic container 1 can be the structures such as spheroid, cylindrical body, sphere, sub warhead body,
By can two parts of internal and external threads twist form, 3 length of sealing ring is to cover the cyclic structure that diameter is 3mm~35mm, wide by 3~
20mm, material are high temperature resistant, corrosion resistant food grade silicone, can cover and form sealing at 1 screw thread twist of porous magnetic container, prevent
Only porous magnetic container 1 is added in environmental microorganism.
In the present embodiment, single microorganism culture solution aseptically mixes well to form gel with culture substrate
The complex matrix of state；Convenient for storage of the microorganism in porous magnetic container 1, bacteriogenic various substances can be by porous
Magnetic container 1 is diffused in system, realizes the direct contact between bacterium.
In the present embodiment, by discharge or the preservation in -80 DEG C or liquid nitrogen of porous magnetic container 1 taken out；Make answering for discharge
It is free from the influence of the external environment and lead to the variation again and effect of compound body to close matrix, make result also easily facilitate statistics and
Analysis.
In the present embodiment, using sterile pH=8.0 is freezed, the phosphate wash porous magnetic that concentration is 0.1mol/L holds
Composite base is taken out to 1 surface sterilization of porous magnetic container with the sterile blotting paper of alcohol that concentration is 70~75% is moistened in 1 surface of device
Body, is counted or is extracted genomic DNA to microorganism in complex matrix or RNA analysis detects its gene expression, it is ensured that detection
The accuracy of analysis excludes all disturbing factors.
Embodiment one
Study Escherichia coli, bifidobacterium bifidum, urine enterococcus and four kinds of lactobacillus acidophilus in Ba-Ma mini pig alimentary canal
The interaction of bacterium
1. by spheroid porous magnetic container 1, sealing ring 3 and the sealing-plug and auxiliary tool (tweezer of single bacterium kind analysis system
Son, pipettor head, delivery device) it sterilizes 30 minutes at 120 DEG C；
2. preparing microorganism pure culture liquid according to a conventional method:
Bacterial liquid culture medium is prepared, 121 DEG C sterilize 30 minutes；Pick them separately Escherichia coli, not tally bifid
Bacillus, urine enterococcus and lactobacillus acidophilus single bacterium drop down onto 5mL inoculum, culture value logarithmic phase,
Expand culture respectively to 50mL biomass；
3. preparing microorganism pure culture matrix: food matrix powder (40 mesh of granularity), matrix used sterilizes 30 points at 121 DEG C
Clock；
4. by Escherichia coli, bifidobacterium bifidum, urine enterococcus and lactobacillus acidophilus single colonie pure culture liquid, in sterile item
It under part, is mixed well respectively with food matrix, and forms the thallus substrate complex of gel state；
5. aseptically, gel state thallus substrate complex to be respectively charged into four kinds of spheroid porous magnetics of label
Container 1, twist have internal screw thread and externally threaded 1 two parts of porous magnetic container, and upper sleeve gasket is covered at screw thread twist；
6. spheroid porous magnetic container 1 is sent into Ba-Ma mini pig alimentary canal in a manner of oral with delivery device, disappearing
Change after being resident a period of time in road, excretes；Using magnetic separator from excrement spheroid porous magnetic container 1；
7. spheroid porous magnetic container 1 is immediately placed in sterile sampler bag, and it is put into preservation in -80 DEG C or liquid nitrogen.
Aseptically, 1 surface of porous magnetic container is cleaned with the sterile 0.1mol/L phosphate [pH=8.0] of freezing,
1 surface liquid of porous magnetic container is blotted with the sterile blotting paper of drying, with the sterile blotting paper for being moistened with the alcohol that concentration is 70%
To 1 surface sterilization of porous magnetic container, sealing shroud is removed, carefully unscrews porous magnetic container 1, takes out target thallus matrix composite
Body.
8. microorganism is counted or is extracted genomic DNA or RNA in pair thallus matrix composite body:
A) analysis detection is carried out to the DNA of extracting or RNA；
B) gene expression analysis is carried out to the DNA of extracting or RNA.
Embodiment two
Study the interaction of intravaginal bifidobacterium family, veillonella parvula and bacteroides capillosus
1. by bullet type porous magnetic container 1, sealing ring 3 and the sealing-plug and auxiliary tool (tweezer of single bacterium kind analysis system
Son, pipettor head, delivery device) it sterilizes 20 minutes at 120 DEG C；
2. preparing microorganism pure culture liquid according to a conventional method:
Bacterial liquid culture medium is prepared, 121 DEG C sterilize 15 minutes；Pick them separately bifidobacterium family, veillonella parvula and more
Hair bacteroid single bacterium drops down onto 5mL culture solution, culture value logarithmic phase, cultivates 15mL biomass expanding；
3. preparing microorganism pure culture matrix: the 15mL distilled water of three parts of 30% low melting point agar of addition of preparation, 121 DEG C go out
Bacterium 15 minutes；
4. low melting point agar temperature subject to sterilization is down to 30 DEG C~35 DEG C, respectively by bifidobacterium family, veillonella parvula
It is aseptically mixed well with 30% agar solution equal proportion of sterilizing with bacteroides capillosus pure culture liquid, and forms molten gel state
Thallus substrate complex；
5. aseptically, dissolved colloidal state thallus substrate complex to be separately added into the bullet type porous magnetic of three kinds of labels
Container 1 after forming gel state thallus complex in bullet type porous magnetic container 1, covers upper sleeve gasket in opening；
6. respectively by the bullet type porous magnetic equipped with bifidobacterium family, veillonella parvula and bacteroides capillosus thallus complex
Property 1 intravaginal administration device of container be sent into macaque female vagina in, after 3~5 days, using magnetic force from macaque intravaginal take out bullet type
Porous magnetic container 1；
7. bullet type porous magnetic container 1 is immediately placed in sterile sampler bag, and it is put into -80 DEG C or liquid nitrogen preservation.
8. aseptically, cleaning bullet type porous magnetic with the sterile 0.1mol/L phosphate [pH=8.0] of freezing to hold
1 surface liquid of porous magnetic container is blotted with the sterile blotting paper of drying in 1 surface of device, with the nothing for being moistened with the alcohol that concentration is 70%
Bacterium blotting paper inhales at examination sealing shroud outside porous magnetic container 11 surface sterilization of porous magnetic container with the sterile blotting paper of drying
Wall carefully removes sealing shroud with tweezers, takes out thallus substrate complex.
8. microorganism is counted or is extracted genomic DNA or RNA in pair thallus matrix composite body:
A) analysis detection is carried out to the DNA of extracting or RNA；
B) gene expression analysis is carried out to the DNA of extracting or RNA.
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to compared with
Good embodiment describes the invention in detail, those skilled in the art should understand that, it can be to skill of the invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the scope of the claims of invention.
Claims (8)
1. a kind of method of the in vivo in situ detection analysis microbial interaction for non-disease diagnosing and treating purpose,
It is characterized in that: the following steps are included:
A. the complex matrix for the gel state that single strain substrate and culture substrate form is transferred to porous magnetic container hollow lumen
In, and be discharged after entering human body or animal body digestive tract environment in a manner of oral；
B. porous magnetic container is chosen from the excrement of discharge by magnetic force, complex matrix is taken out after refrigerating and tests and analyzes strain
Expression conditions；The porous magnetic vessel surface is with 1~3 × 105A/cm2Mode be distributed with aperture be 200~
The through-hole of 500nm；The porous magnetic container material quality is martensitic stain less steel, 10~250mm of the porous magnetic container length, directly
Diameter is 5~50mm.
2. the in vivo in situ detection according to claim 1 for non-disease diagnosing and treating purpose analyzes microbial
The method of interaction, it is characterised in that: the porous magnetic containers of different single strain substrates will be respectively provided with simultaneously through human body or dynamic
Object digestive tract environment.
3. the in vivo in situ detection according to claim 2 for non-disease diagnosing and treating purpose analyzes microbial
The method of interaction, it is characterised in that: the porous magnetic container is made of the two parts up and down being threadedly coupled, outside interconnecting piece
Surface is arranged with sealing ring.
4. the in vivo in situ detection according to claim 3 for non-disease diagnosing and treating purpose analyzes microbial
The method of interaction, it is characterised in that: the sealing ring material is high temperature resistant and corrosion resistant food grade silicone.
5. the in vivo in situ detection according to claim 1 for non-disease diagnosing and treating purpose analyzes microbial
The method of interaction, it is characterised in that: by single microorganism culture solution, aseptically mix well to be formed with culture substrate
The complex matrix of gel state.
6. the in vivo in situ detection according to claim 5 for non-disease diagnosing and treating purpose analyzes microbial
The method of interaction, it is characterised in that: the porous magnetic container of discharge is resident after a certain period of time in the open organ of humans and animals,
Discharge or after taking out, immediately preservation in -80 DEG C or liquid nitrogen.
7. the in vivo in situ detection according to claim 6 for non-disease diagnosing and treating purpose analyzes microbial
The method of interaction, it is characterised in that: aseptically, taken after the porous magnetic container after refrigeration is cleaned and sterilized
Complex matrix out, is counted or is extracted genomic DNA to microorganism in complex matrix or RNA analysis detects its gene expression.
8. the in vivo in situ detection according to claim 7 for non-disease diagnosing and treating purpose analyzes microbial
The method of interaction, it is characterised in that: using sterile pH=8.0 are freezed, concentration is that the phosphate wash of 0.1 mol/L is more
Hole magnetism vessel surface sterilizes porous magnetic vessel surface with the sterile blotting paper of alcohol that concentration is 70~75% is moistened with.
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| CN101475930A (en) * | 2008-12-26 | 2009-07-08 | 中国科学院过程工程研究所 | Superparamagnetic nano particle in situ adsorptive separation-immobilized Rhodococcus biological desulphurization process |
| CN101629136B (en) * | 2009-08-06 | 2012-04-25 | 中国海洋大学 | High-efficient culture and sorting method and culture device of microorganisms |
| CN201587942U (en) * | 2009-12-21 | 2010-09-22 | 路域生态工程有限公司 | Microorganism gene detection analysis diagnostic apparatus |
| NZ607043A (en) * | 2010-08-04 | 2015-05-29 | Borody Thomas J | Compositions for fecal floral transplantation and methods for making and using them and devices for delivering them |
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| CN104007091A (en) * | 2013-02-26 | 2014-08-27 | 中国科学院天津工业生物技术研究所 | High-throughput detection system for microbe based on droplet microfluidic chip |
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