CN111440722A - Method for preparing free-flow isoelectric focusing electrophoresis microbial component by separation and application - Google Patents
Method for preparing free-flow isoelectric focusing electrophoresis microbial component by separation and application Download PDFInfo
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- CN111440722A CN111440722A CN202010139910.3A CN202010139910A CN111440722A CN 111440722 A CN111440722 A CN 111440722A CN 202010139910 A CN202010139910 A CN 202010139910A CN 111440722 A CN111440722 A CN 111440722A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/005—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor after treatment of microbial biomass not covered by C12N1/02 - C12N1/08
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
Abstract
The invention discloses a method for preparing free-flow isoelectric focusing electrophoresis microbial component separation and application, relating to the technical field of microbial component separation and comprising the following steps: A. removing human cells, cell debris, and mucin from the microbial sample; B. treating the microorganisms with an activating solution to convert them to an activated state suitable for separation; C. adding a carrier buffer solution into a free-flow isoelectric focusing electrophoresis apparatus to form a linear pH gradient in a separation chamber; D. and D, performing free-flow isoelectric focusing electrophoresis separation on the activated microorganisms obtained in the step B. The invention can separate the microbe group into microbe components according to the size of isoelectric point, is suitable for separating complex microbe groups from various sources, obviously reduces the complexity of the microbe group, reduces the amplification deviation of the sequencing technology caused by the interference of abundant microbes, increases the depth of microbe identification, and is beneficial to discovering new microbes in the complex microbe group.
Description
Technical Field
The invention belongs to the technical field of microbial component separation, and particularly relates to a free-flow isoelectric focusing electrophoresis microbial component separation preparation method and application.
Background
The microbiome refers to a collection of all microorganisms symbiotic in the same ecological environment, has the characteristics of complex composition, large number and the like (L ederberg and McCray 2001). the microbiome existing in human bodies symbioses with human bodies, the total number of genes encoded by the microorganisms exceeds 330 ten thousand, is about 100 times of the genes encoded by human bodies, plays a crucial role in human health, and is one of important factors for maintaining homeostasis in human bodies (Cameron, hugs et al 2015), and changes in the structural composition of the microorganisms can cause various diseases of human bodies, such as type ii diabetes (Qin, L i et al 2012), oral cancer (Mager, Haffajee et al 2005), lung cancer (Yan, yaet al 2015), pancreatic cancer (Farrell, Zhanget al.2012) and rheumatoid arthritis (Zhang, Zhang et al 2015).
Taking oral microorganisms as an example, since more than one third of the microorganisms in the oral cavity cannot be cultured in laboratory media (Chen, Yu et al 2010), current research methods for microbial community species composition are mainly culture-independent, such as 16S rRNA gene sequencing technology (16S-HTS) and metagenomic sequencing technology (WGS). since microbiome is a very complex sample, these microbiome analysis technologies are challenged, where microorganisms with higher relative abundance may bias and amplify redundancies, interfere with sequencing identification results, making the number of microorganisms identifiable in the sample less than the actual case (cross and Criddle 2003, Acinas, Sarma-rupavtamer et al 2005, Cao, Jiang et al 2014, L iu, Tao et al 2018), which affects overall identification of microbial groups and disease-related studies, and as the microbial preportioning of microorganisms does not develop suitable technologies, the microbial community is not reduced, and the microbial community content is reduced.
At present, the technology for pre-separating the microbial components is mainly capillary zone electrophoresis, which utilizes an electric field to separate the microbes with different migration rates into different components according to the charge-to-mass ratio, thereby achieving the purpose of reducing the complexity of the microbial components (Huge, Champion et al 2019). However, the method has the disadvantages that the method needs a culture means, has small flux, can not directly carry out sequencing identification on the separated microorganisms, thus leading to the loss of information of the microorganisms which can not be cultured, can not comprehensively analyze and identify the microorganism groups, establishes proper separation conditions and parameters to separate the microorganisms while generating no damage, and has difficulty in finding more microorganisms through separation.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for preparing a free-flow isoelectric focusing electrophoresis microbial component and its application. Can effectively increase the number of microorganisms identified in a complex microorganism group and is helpful for discovering new molecular diagnostic markers of diseases.
The purpose of the invention is realized by the following technical scheme: a method for preparing microbial component separation by free-flow isoelectric focusing electrophoresis comprises the following steps:
A. removing cells, cell debris and mucin from the microbiome sample;
freezing and centrifuging a microbiome sample to obtain a precipitate, wherein the precipitate comprises microorganisms, cells, cell debris and mucin;
carrying out several times of resuspension elution operations on the precipitate through a resuspension buffer solution, and filtering through a filter membrane to obtain a microorganism group; removing cells, cell debris and mucin to obtain a microbiome;
B. activating the microorganism group by the activating solution to obtain an activated microorganism group suitable for electrophoretic separation;
C. adding a carrier buffer solution into a free-flow isoelectric focusing electrophoresis apparatus to form a linear pH gradient in a separation chamber;
D. and D, performing free-flow isoelectric focusing electrophoresis separation on the activated microorganism group obtained in the step B.
Preferably, the resuspension elution operation in step a specifically includes: and (3) blowing and sucking the precipitate for 3 times or more by using the heavy suspension buffer solution to fully dissolve the precipitate in the heavy suspension buffer solution, carrying out vortex for 5-10 seconds on a vortex instrument, then carrying out refrigerated centrifugation, and discarding the supernatant to obtain the precipitate.
Preferably, the resuspension buffer is PBS buffer containing 0.5% Triton X-100.
Preferably, the refrigerated centrifuge specifically comprises: the microbiome samples were centrifuged for 5-10 minutes at 8000-.
Preferably, the filter membrane in step a comprises a polyvinylidene fluoride membrane with a pore size of 10 μm.
Preferably, the activation treatment in step B specifically includes: the microbiome was resuspended by the activation solution at 4 ℃ and treated with gentle shaking by a vortexer for 20 minutes to obtain an activated microbiome.
Preferably, the activation solution comprises 5mM CaCl2。
Preferably, the carrier buffer in step C comprises 0.8% -1.2% Ampalyte, 0.5% -1% Triton X-100, 1.1 g/L Ficoll, 250-300mM Mannitol, and the linear pH gradient is a linear gradient formed in the pH range of 2.5-10.5.
In the buffer solution component, excessive content of the Ampholyte can generate toxicity to microorganisms, and the separation efficiency can be influenced by too little content of the Ampholyte; the excessive content of Triton can cause damage to microorganisms, the insufficient content can affect the separation efficiency, and the Triton can be replaced by Tween; ficoll affects the flow rate of free flow too much, and forms turbulent flow too little to be beneficial to isoelectric focusing; mannitol, which is a substitute for Sorbitol, dies microorganisms with too high osmotic pressure and microorganisms with too low osmotic pressure.
Preferably, the free-flow isoelectric focusing electrophoretic separation in step D specifically comprises: the activated microorganism group is placed in an electrophoresis apparatus with a linear pH gradient, the temperature is kept at 4-6 ℃, the voltage is kept constant at 300V, the current is limited to 2-4mA, and the separation time is 1-1.5 hours.
Microorganisms die when the voltage is too high, and the separation efficiency is low when the voltage is too low; either too high or too low of a temperature can cause the microorganisms to decrease in separation efficiency; microorganisms die when the current is too high.
An application of a microorganism prepared by a free-flow isoelectric focusing electrophoresis microorganism component separation preparation method in microbiology and biomarkers.
In summary, compared with the prior art, the invention has the following beneficial effects:
the invention can effectively remove the microbiome interfered by human cells and mucin from the microorganism sample, and reduces the complexity of the microbiome and the redundant interference of high-abundance microorganisms to a sequencing technology through free flow isoelectric focusing electrophoresis separation, thereby identifying more types of microorganisms. The invention relates to a method for separating a microorganism sample into 32 different components by utilizing the characteristic that different microorganisms have different isoelectric points, thereby reducing the complexity of the microorganism group and greatly increasing the number of identified microorganisms. The technology can be used for pre-separation and subsequent research of all microorganism groups, more microorganisms can be found by combining a sequencing technology, the research of disease markers is facilitated, and the technology has wide universality.
Drawings
Other features, objects and advantages of the present invention will become more apparent upon reading of the following detailed description of non-limiting embodiments thereof, with reference to the accompanying drawings;
FIG. 1 is a schematic flow chart showing the steps of a method for separating and preparing a free-flow isoelectric focusing electrophoresis microbial pool according to examples 1 to 3 of the present invention;
FIG. 2 is a microscope photograph of microorganisms treated by the method for preparing free-flow isoelectric focusing electrophoresis microbial component separation according to comparative example 1 and example 1 of the present invention;
FIG. 3 is a linear pH gradient of 3 repeated experiments of a method for preparing free-flow isoelectric focusing electrophoresis microbial component separation according to example 1 of the present invention;
FIG. 4 is a schematic diagram showing the numbers of microorganisms identified by the free-flow isoelectric focusing electrophoresis microbial group separation preparation method according to example 1 and comparative example 2 of the present invention (3 experimental replicates);
FIG. 5 shows the differential microorganisms in saliva between lung cancer patients and normal persons.
Detailed Description
The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention. The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, which ranges of values are to be considered as specifically disclosed herein, the invention is described in detail below with reference to specific examples:
the present invention will be further described with reference to the following specific examples. However, they are exemplary embodiments of the present application, and the present invention is not limited thereto.
Example 1
A method for preparing free-flow isoelectric focusing electrophoresis microbial component separation is shown in figure 1, and comprises the following steps:
A. saliva of normal persons and lung cancer patients is collected at 5m L, and is subjected to refrigerated centrifugation at 4 ℃ and a centrifugal force of 10000g for 8 minutes to obtain a precipitate containing microorganisms, cells, cell debris and mucin.
PBS resuspension buffer was prepared with 0.5% Triton X-100. The sediment is resuspended by using a resuspension buffer solution, the sediment is repeatedly blown and sucked by a pipette suction head to be fully dissolved, 8-second vortex is carried out on a vortex instrument, then the centrifugation is carried out at 4 ℃ for 10000g and 5 minutes by centrifugal force, the supernatant is discarded, the operation is repeated for 3 times after the sediment is obtained, so that the microorganisms gathered in the biological membrane and in the mucin are eluted, and the human cells, cell fragments and the mucin are removed by combining a polyvinylidene fluoride filter membrane with the aperture of 10 mu m. As shown in FIG. 2, the microorganism sample eluted and filtered effectively removed human cells and cell debris, and the microorganism morphology was more dispersed and suitable for separation than the untreated sample.
B. Preparing an activation solution with the composition of 5mM CaCl2An aqueous solution. The salivary microorganisms were resuspended in activating solution at 4 ℃ and treated with gentle shaking on a vortexer for 20 minutes to convert them to an activated state suitable for isolation.
C. A carrier buffer comprising 1% Ampolyte, 0.5% Triton X-100, 1.1 g/L Ficoll, 250mM Mannitol, constant power 20W, constant temperature 4 ℃ and flow rate 1m L/min was added to a free flow isoelectric focusing electrophoresis apparatus and run for 1 hour to form a linear pH 3-10 gradient with an error of 0.5 in the separation chamber, as shown in FIG. 3.
D. And C, performing free-flow isoelectric focusing electrophoresis separation on the activated salivary microorganisms obtained in the step B, injecting a microorganism sample into a pH neutral port, adjusting the parameters of the instrument to be constant voltage of 5 ℃, 300V, current limiting of 4mA, flow rate of 1m L/min, and separating for 1 hour.
After the separated microorganisms are collected and refrigerated and centrifuged at 4 ℃ for 10 minutes under the centrifugal force of 10000g, the microorganism precipitates are taken to carry out 16S rRNA gene sequencing technology (16S-HTS), and microorganism samples are identified, and the result is shown in figure 4, the number of microorganism types identified by the microorganism samples subjected to the pre-separation technology is obviously increased compared with the microorganism types identified by the samples which are not processed, and original microorganism information is not lost.
Finally, the salivary microbiome of lung cancer patients was compared with that of normal persons to obtain significantly different microbial species, as shown in fig. 5 and table 1.
TABLE 1 microorganisms that differ significantly between lung cancer patients and normal persons
As can be seen from Table 1, through the differential analysis of salivary microorganisms between lung cancer patients and normal persons, 8 differential microorganisms were found, among which, Granulicatella and Abiotrophia were reported to be associated with lung cancer, indicating that the method is useful for developing molecular diagnostic techniques in which salivary microorganisms are associated with lung cancer.
Example 2
A method for preparing free-flow isoelectric focusing electrophoresis microbial component separation is shown in figure 1, and comprises the following steps:
A. saliva of normal and lung cancer patients was collected at 5m L, and subjected to refrigerated centrifugation at 4 ℃ for 10 minutes at 8000g centrifugal force to obtain a precipitate containing microorganisms, cells, cell debris and mucin.
PBS resuspension buffer was prepared with 0.5% Triton X-100. The sediment is resuspended by using a resuspension buffer solution, the sediment is repeatedly blown and sucked by a pipette suction head to be fully dissolved, the vortex is carried out on a vortex instrument for 10 seconds, then the freezing and centrifugation are carried out for 10 minutes at 4 ℃ under the centrifugal force of 8000g, the supernatant is discarded, the operation is repeated for 3 times after the sediment is obtained, so that the microorganisms gathered in the biological membrane and in the mucin are eluted, and the human cells, cell fragments and the mucin are removed by combining a polyvinylidene fluoride filter membrane with the aperture of 10 mu m. As shown in FIG. 2, the microorganism sample eluted and filtered effectively removed human cells and cell debris, and the microorganism morphology was more dispersed and suitable for separation than the untreated sample.
B. Preparing an activation solution with the composition of 5mM CaCl2An aqueous solution. The salivary microorganisms were resuspended in activating solution at 4 ℃ and treated with gentle shaking on a vortexer for 20 minutes to convert them to an activated state suitable for isolation.
C. A carrier buffer comprising 0.8% Amphylate, 0.8% Triton X-100, 1.1 g/L Ficoll, 270mM Mannitol, constant power 20W, constant temperature 4 deg.C, flow rate 1m L/min, was added to a free flow isoelectric focusing electrophoresis apparatus for 1 hour to produce a linear pH 3-10 gradient with an error of 0.5 in the separation chamber, as shown in FIG. 3.
D. And C, performing free-flow isoelectric focusing electrophoresis separation on the activated salivary microorganisms obtained in the step B, injecting a microorganism sample into a pH neutral port, adjusting the parameters of the instrument to constant voltage of 4 ℃, 300V, current limiting of 3mA, flow rate of 1m L/min, and separating for 1.5 hours.
The number of microbial species is significantly increased without losing the original microbial information.
Example 3
A method for preparing free-flow isoelectric focusing electrophoresis microbial component separation is shown in figure 1, and comprises the following steps:
A. saliva of normal and lung cancer patients was collected at 5m L, and subjected to refrigerated centrifugation at 12000g centrifugal force for 5 minutes at 4 ℃ to obtain a precipitate containing microorganisms, cells, cell debris and mucin.
PBS resuspension buffer was prepared with 0.5% Triton X-100. The sediment is resuspended by using a resuspension buffer solution, the sediment is repeatedly blown and sucked by a pipette suction head to be fully dissolved, 5 seconds of vortex is carried out on a vortex instrument, then, the centrifugal force of 12000g is carried out at 4 ℃ for 8 minutes, the supernatant is discarded, the operation is repeated for 3 times after the sediment is obtained, so that the microorganisms gathered in the biological membrane and in the mucin are eluted, and the human cells, cell fragments and the mucin are removed by combining a polyvinylidene fluoride filter membrane with the aperture of 10 mu m. As shown in FIG. 2, the microorganism sample eluted and filtered effectively removed human cells and cell debris, and the microorganism morphology was more dispersed and suitable for separation than the untreated sample.
B. Preparing an activation solution with the composition of 5mM CaCl2An aqueous solution. The salivary microorganisms were resuspended in activating solution at 4 ℃ and treated with gentle shaking on a vortexer for 20 minutes to convert them to an activated state suitable for isolation.
C. A carrier buffer solution comprising 1.2% Ampalyte, 1% Triton X-100, 1.1 g/L Ficoll, 300mM Mannitol, constant power of 20W, constant temperature of 4 ℃ and flow rate of 1m L/min was added to a free flow isoelectric focusing electrophoresis apparatus, and the operation was carried out for 1 hour, so that a linear pH 3-10 gradient with an error of 0.5 was formed in the separation chamber, as shown in FIG. 3.
D. And C, performing free-flow isoelectric focusing electrophoresis separation on the activated salivary microorganisms obtained in the step B, injecting a microorganism sample into a pH neutral port, adjusting the parameters of the instrument to be constant voltage of 6 ℃, 300V, current limit of 2mA, flow rate of 1m L/min and separation time of 1.2 hours.
The number of microbial species is significantly increased without losing the original microbial information.
Comparative example 1
A free flow isoelectric focusing electrophoresis microbial component separation preparation method is different from the example 1 in that saliva microbes are not processed in the step A, other steps are the same as the example 1, and the result is shown in figure 2, wherein figure 2 is a microbe microscopic picture, and the left picture is a saliva microbe sample processed in the comparative example 1, wherein human cells and cell fragments are contained, the microbe aggregation phenomenon is serious, and the separation and identification of a larger number of microbes are not facilitated; the right figure shows the microorganism samples treated in example 1, which effectively removed impurities such as human cells, and the microorganisms were dispersed and suitable for separation.
Comparative example 2
A method for preparing a free-flow isoelectric focusing electrophoresis microbial group separation, which is different from example 1, is characterized in that the number of microorganisms which are not subjected to free-flow isoelectric focusing electrophoresis separation is substantially higher than that of the microorganism sample treated in comparative example 2, as shown in FIG. 4, and almost all of the types of microorganisms which are not subjected to pre-separation are included, as other steps are the same as those of example 1 (3 experimental repetitions).
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.
Claims (10)
1. A method for preparing microbial component separation by free-flow isoelectric focusing electrophoresis is characterized by comprising the following steps:
A. removing cells, cell debris and mucin from the microbiome sample; the method comprises the following specific steps:
freezing and centrifuging a microbiome sample to obtain a precipitate, wherein the precipitate comprises microorganisms, cells, cell debris and mucin;
carrying out several times of resuspension elution operations on the precipitate through a resuspension buffer solution, and filtering through a filter membrane to obtain a microorganism group;
B. activating the microorganism group obtained in the step A by using an activating solution to obtain an activated microorganism group suitable for electrophoretic separation;
C. adding a carrier buffer solution into a free-flow isoelectric focusing electrophoresis apparatus to form a linear pH gradient in a separation chamber;
D. and D, performing free-flow isoelectric focusing electrophoresis separation on the activated microbiome obtained in the step B by using the free-flow isoelectric focusing electrophoresis apparatus in the step C.
2. The method for separating and preparing free-flow isoelectric focusing electrophoresis microbial components according to claim 1, wherein the resuspension elution operation in step a specifically comprises: and (3) blowing and sucking the precipitate for 3 times or more by using the heavy suspension buffer solution to fully dissolve the precipitate in the heavy suspension buffer solution, carrying out vortex for 5-10 seconds on a vortex instrument, then carrying out refrigerated centrifugation, and discarding the supernatant to obtain the precipitate.
3. The method for preparing microbial components based on free-flow isoelectric focusing electrophoresis according to claim 2, wherein the resuspension buffer is 0.5% Triton X-100 in PBS.
4. The method for preparing microbial components based on free-flow isoelectric focusing electrophoresis according to claim 2, wherein the refrigerated centrifugation specifically comprises: the microbiome samples were centrifuged for 5-10 minutes at 8000-.
5. The method for preparing a microbial composition based on free-flow isoelectric focusing electrophoresis according to claim 1 wherein step a the filter membrane comprises a polyvinylidene fluoride membrane with a 10 μm pore size.
6. The method for preparing microbial components based on free-flow isoelectric focusing electrophoresis according to claim 1, wherein the activation treatment in step B specifically comprises: the microbiome was resuspended by the activation solution at 4 ℃ and treated with gentle shaking by a vortexer for 20 minutes to obtain an activated microbiome.
7. The method for preparing a free-flow isoelectric focusing electrophoresis-based microbial component separation according to claim 6, wherein the activation solution comprises 5mM CaCl2。
8. The method for preparing a microorganism group separation based on free-flow isoelectric focusing electrophoresis according to claim 1, wherein the carrier buffer in step C comprises 0.8% -1.2% Ampolyte, 0.5% -1% Triton X-100, 1.1 g/L Ficoll, 250-300mM Mannitol, and the linear pH gradient is a linear gradient formed in the range of pH 2.5-10.5.
9. The method for preparing microbial components based on free-flow isoelectric focusing electrophoresis according to claim 1, wherein the free-flow isoelectric focusing electrophoresis separation in step D specifically comprises: the activated microorganism group is placed in a free flow isoelectric focusing electrophoresis apparatus with a linear pH gradient, the temperature is kept at 4-6 ℃, the voltage is kept at 300V, the current is limited to 2-4mA, and the separation time is 1-1.5 hours.
10. Use of the microorganism prepared by the method for preparing free-flow isoelectric focusing electrophoresis microbial component separation according to claim 1 in microbiology and biomarkers.
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| US5578460A (en) * | 1993-09-23 | 1996-11-26 | E. I. Du Pont De Nemours And Company | Electrophoretic method for the isolation and separation of microorganisms and cell populations |
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| CN107653243A (en) * | 2017-11-15 | 2018-02-02 | 中国农业科学院农业基因组研究所 | A kind of method that microorganism macro genome DNA is extracted from intestinal contents |
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