CN111440722A - A method for separation and preparation of microbiome by free flow isoelectric focusing electrophoresis and its application - Google Patents

Abstract

The invention discloses a method and application for the separation and preparation of a microbiome by free-flow isoelectric focusing electrophoresis, and relates to the technical field of microbiome separation, comprising the following steps: A. removing human cells, cell debris and mucin from a microbial sample; B. . Treat the microorganism with an activation solution to transform it into an activated state suitable for separation; C. Add a carrier buffer to the free-flow isoelectric focusing electrophoresis apparatus, so that a linear pH gradient is formed in the separation chamber; D. The obtained in step B Activated microorganisms are separated by free-flow isoelectric focusing electrophoresis. The isolated microorganisms are identified by gene sequencing, and the invention can separate and prepare the microorganisms into microbial components according to the size of the isoelectric point, which is suitable for the separation of complex microorganisms from various sources, and significantly reduces the complexity of the microorganisms and the sequencing technology. The amplification bias caused by the interference of high abundance microorganisms increases the depth of microbial identification and helps to discover new microorganisms in complex microbiomes.

Description

A method for separation and preparation of microbiome by free flow isoelectric focusing electrophoresis and its application

technical field

The invention belongs to the technical field of microbiome separation, and in particular relates to a method and application for the separation and preparation of microbiome by free-flow isoelectric focusing electrophoresis.

Background technique

Microbiome refers to the collection of all microorganisms symbiotic in the same ecological environment, which has the characteristics of complex composition and large number (Lederberg and McCray 2001). The microbiome that exists in the human body coexists with humans. The total number of genes encoded by these microorganisms exceeds 3.3 million, which is about 100 times the number of genes encoded by humans. It plays a vital role in human health and is important for maintaining the homeostasis of the human body One of the factors (Cameron, Huws et al. 2015), its structural composition changes may lead to a variety of human diseases, such as type 2 diabetes (Qin, Li et al. 2012), oral cancer (Mager, Haffajee et al. al. 2005), lung cancer (Yan, Yang et al. 2015), pancreatic cancer (Farrell, Zhang et al. 2012), and rheumatoid arthritis (Zhang, Zhang et al. 2015). Therefore, a comprehensive study of the human microbiome can provide new ideas for the diagnosis and treatment of many human diseases. At present, the research on the human microbiome and human health is still in its infancy. Due to the large variety and complex composition of the microbiome, it is difficult to analyze the microbiome in depth with the existing technology. Therefore, a new microbiome pre-isolation technology is needed to promote related research. .

Taking oral microbes as an example, since more than one-third of the microbes in the oral cavity cannot be cultured in laboratory culture medium (Chen, Yu et al. 2010), the current research methods for microbial community species composition are mainly culture-independent. type, such as 16S rRNA gene sequencing technology (16S-HTS) and metagenomic sequencing technology (WGS). Since the microbiome is a very complex sample, these microbiome analysis techniques all face challenges, in which microorganisms with relatively high relative abundance may bias PCR amplification and amplification redundancy, interfering with the sequencing identification results, so that the identification in the sample can be The number of microbes obtained is less than the actual situation (Crosby and Criddle 2003, Acinas, Sarma-Rupavtarm et al. 2005, Cao, Jiang et al. 2014, Liu, Tao et al. 2018), which affects the comprehensive identification of the microbiome and disease-related research. With the in-depth development of microbiome research, there is currently no suitable microbiome pre-isolation technology, which can divide complex microbial samples into different components, reduce the complexity of the microbiome, and reveal the microorganisms with lower content.

The current technology for pre-separation of the microbiome is mainly capillary zone electrophoresis, which uses an electric field to divide microorganisms with different migration rates into different components according to the size of the charge-to-mass ratio, so as to reduce the complexity of the microbiome (Huge, Champion). et al. 2019). However, its disadvantage is that it needs to use the means of culture, the throughput is small, and the isolated microorganisms cannot be directly sequenced and identified, which leads to the loss of information about the microorganisms that cannot be cultivated, and it is impossible to comprehensively analyze and identify the microbiome, establish appropriate separation conditions and The parameters make it possible to isolate and find more microorganisms through isolation without causing damage.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide a method and application for the separation and preparation of microorganisms by free-flow isoelectric focusing electrophoresis. It can effectively increase the number of microorganisms identified in the complex microbiome and help to discover new molecular diagnostic markers for diseases.

The object of the present invention is achieved through the following technical solutions: a method for the separation and preparation of microorganisms by free flow isoelectric focusing electrophoresis, comprising the following steps:

A. Removal of cells, cell debris, and mucins from microbiome samples;

Freezing and centrifuging the microbiome sample to obtain a precipitate, the precipitate includes microorganisms, cells, cell debris, and mucin;

Perform several re-suspension and elution operations on the sediment, and filter through a filter to obtain the microbiome; remove cells, cell debris and mucin to obtain the microbiome;

B. The activation solution activates the microbiome to obtain an activated microbiome suitable for electrophoretic separation;

C. Add carrier buffer to the free-flow isoelectric focusing electrophoresis apparatus, so that a linear pH gradient is formed in the separation chamber;

D. The activated microbiome obtained in step B is subjected to free-flow isoelectric focusing electrophoresis separation.

Preferably, the re-suspension and elution operation described in step A specifically includes: blowing and sucking the precipitate through the resuspension buffer three times or more, so that the precipitate is fully dissolved in the resuspension buffer, and performing 5- Vortex for 10 seconds, then freeze centrifugation and discard supernatant to obtain pellet.

Preferably, the resuspension buffer is a PBS buffer containing 0.5% Triton X-100.

Preferably, the frozen centrifugation specifically includes: centrifuging the microbiome sample at 4° C. under a centrifugal force of 8000-12000 g for 5-10 minutes.

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: resuspending the microbiome in an activation solution at 4° C. and gently shaking the microbiome with a vortexer for 20 minutes to obtain an activated microbiome.

Preferably, the activation solution includes 5mM CaCl ₂ .

Preferably, the carrier buffer in step C includes 0.8%-1.2%% Ampholyte, 0.5%-1% Triton X-100, 1.1g/L Ficoll, 250-300mM Mannitol; the linear pH gradient is: at pH Linear gradient formed in the range of 2.5-10.5.

In the buffer components, too much Ampholyte content will cause toxicity to microorganisms, too little content will affect the separation efficiency; too much Triton content will cause damage to microorganisms, too little will affect the separation efficiency, triton can be replaced by Tween; too much Ficoll affects Free flow velocity, too little to form turbulent flow is not conducive to isoelectric focusing; too much osmotic pressure of Mannitol increases the death of microorganisms, and too little osmotic pressure reduces the death of microorganisms, and Mannitol can be replaced by Sorbitol.

Preferably, the free-flow isoelectric focusing electrophoresis separation in step D specifically includes: placing the activated microbiome in an electrophoresis apparatus that has formed a linear pH gradient, at a constant temperature of 4-6°C, a constant voltage of 300V, and a current limit of 2-4mA, Separation time 1-1.5 hours.

If the voltage is too high, the microorganisms will die, and if the voltage is too low, the separation efficiency will be low; if the temperature is too high or too low, the separation efficiency of the microorganisms will decrease; if the current is too high, the microorganisms will die.

Application of microorganisms prepared by a method for separating and preparing microorganisms by free flow isoelectric focusing electrophoresis, and application of microorganisms prepared by the method for separating and preparing microorganisms by free flow isoelectric focusing electrophoresis in microbiology and biomarkers .

To sum up, compared with the prior art, the present invention has the following beneficial effects:

The present invention can effectively remove the microbiome disturbed by human cells and mucins from the microbial sample, and separate the microbiome through free flow isoelectric focusing electrophoresis, thereby reducing the complexity of the microbiome and the redundant interference of high-abundance microorganisms on the sequencing technology, thereby More species of microorganisms were identified. The invention relates to the use of the characteristics that different microorganisms have different isoelectric points, and the microorganism samples can be separated into 32 different components, thereby reducing the complexity of the microbiome and greatly increasing the number of identified microorganisms. This technology can be used for pre-isolation and subsequent research of all microbiomes. Combined with sequencing technology, more microorganisms can be discovered, which is helpful for disease marker research and has broad universality.

Description of drawings

Other features, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments with reference to the following drawings;

FIG. 1 is a schematic flowchart of steps of a method for separating and preparing microorganisms by free-flow isoelectric focusing electrophoresis in Examples 1-3 of the present invention;

Fig. 2 is a microscopic picture of microorganisms treated by a method for separating and preparing microorganisms by free-flow isoelectric focusing electrophoresis in Comparative Example 1 and Example 1 of the present invention;

3 is a linear pH gradient of three repeated experiments of a method for separating and preparing microorganisms by free-flow isoelectric focusing electrophoresis in Example 1 of the present invention;

4 is a schematic diagram of the number of microorganisms identified by a method for separating and preparing microorganisms by free flow isoelectric focusing electrophoresis in Example 1 and Comparative Example 2 of the present invention (repeated 3 experiments);

Figure 5 shows the microorganisms with differences in the saliva of lung cancer patients and normal people.

Detailed ways

The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. For those skilled in the art, on the premise of not departing from the concept of the present invention, several changes and improvements can also be made, which all belong to the protection scope of the present invention. The endpoints of ranges and any values disclosed herein are not limited to the precise ranges or values, which are to be understood to encompass values proximate to those ranges or values. For ranges of values, the endpoints of each range, the endpoints of each range and the individual point values, and the individual point values can be combined with each other to yield one or more new ranges of values that The scope should be considered to be specifically disclosed herein, and the present invention will be described in detail below in conjunction with specific embodiments:

The present invention will be further described below in conjunction with specific embodiments. However, they are exemplary embodiments of the present application, and the present invention is not limited thereto.

Example 1

A method for separating and preparing microbiome by free flow isoelectric focusing electrophoresis, the step flow chart is shown in Figure 1, and the specific steps are as follows:

A. Removal of human-derived cells, cellular debris, and mucins from saliva samples. Collect 5 mL of saliva from normal people and lung cancer patients, and then freeze and centrifuge at 4°C, centrifugal force 10,000 g, and 8 minutes to obtain a precipitate containing microorganisms, cells, cell debris and mucin.

Prepare 0.5% Triton X-100 in PBS resuspension buffer. Use resuspension buffer to resuspend the precipitate, repeatedly blow and suck the precipitate with a pipette tip to fully dissolve it, and vortex it on a vortexer for 8 seconds, then vortex it at 4°C, centrifugal force 10000g, 5 minutes of refrigerated centrifugation, discard the supernatant, and repeat the above operation 3 times after obtaining the precipitate, so as to elute the microorganisms aggregated in the biofilm and in the mucin, and combine with a polyvinylidene fluoride filter membrane with a pore size of 10 μm Removes human cells, cell debris and mucins. As shown in Figure 2, compared with the untreated sample, the microbial samples after elution and filtration effectively removed human-derived cells and cell debris, and the microbial morphology was more dispersed and suitable for separation.

B. The activation solution is configured, and its composition is a 5mM CaCl ₂ aqueous solution. The salivary microorganisms were resuspended in activation solution at 4°C and vortexed for 20 minutes to convert them to an activated state suitable for isolation.

C. Add carrier buffer to the free flow isoelectric focusing electrophoresis apparatus, the carrier buffer includes 1% Ampholyte, 0.5% Triton X-100, 1.1g/L Ficoll, 250mM Mannitol, constant power 20W, constant temperature 4°C, flow rate 1mL /min, run for 1 hour, so that a linear pH 3-10 gradient is formed in the separation chamber with an error of 0.5, as shown in Figure 3.

D. The activated saliva microorganisms obtained in step B are separated by free-flow isoelectric focusing electrophoresis, and the microorganism samples are injected into the pH neutral port. Time 1 hour.

The isolated microorganisms were collected, refrigerated and centrifuged at 4°C, centrifugal force 10000g, and 10 minutes, and the microbial precipitate was taken for 16S rRNA gene sequencing technology (16S-HTS) to identify the microbial samples. The results are shown in Figure 4. After pre-separation The number of microbial species identified in the technical microbial samples was significantly higher than that in the untreated samples without loss of original microbial information.

Finally, the saliva microbiome of lung cancer patients and normal people was compared, and microbial species with significant differences were obtained, as shown in Figure 5 and Table 1.

Table 1. Microorganisms that are significantly different between lung cancer patients and normal subjects

It can be seen from Table 1 that 8 differential microorganisms were found by analyzing the differences in salivary microorganisms between lung cancer patients and normal people, among which Granulicatella and Abiotrophia were reported to be related to lung cancer, indicating that this method is helpful for the development of molecular diagnosis of salivary microorganisms and lung cancer. technology.

Example 2

A method for separating and preparing microbiome by free flow isoelectric focusing electrophoresis, the step flow chart is shown in Figure 1, and the specific steps are as follows:

A. Removal of human-derived cells, cellular debris, and mucins from saliva samples. Collect 5 mL of saliva from normal people and lung cancer patients, and then freeze and centrifuge at 4° C., centrifugal force 8000 g for 10 minutes to obtain a precipitate containing microorganisms, cells, cell debris and mucin.

Prepare 0.5% Triton X-100 in PBS resuspension buffer. Use resuspension buffer to resuspend the precipitate, repeatedly blow and suck the precipitate with a pipette tip to fully dissolve it, and vortex on a vortexer for 10 seconds. 10 minutes of freezing and centrifugation, discard the supernatant, and repeat the above operation 3 times after obtaining the precipitate, so as to elute the microorganisms aggregated in the biofilm and in the mucin, and combine with a polyvinylidene fluoride filter membrane with a pore size of 10 μm Removes human cells, cell debris and mucins. As shown in Figure 2, compared with the untreated sample, the microbial samples after elution and filtration effectively removed human-derived cells and cell debris, and the microbial morphology was more dispersed and suitable for separation.

B. The activation solution is configured, and its composition is a 5mM CaCl ₂ aqueous solution. The salivary microorganisms were resuspended in activation solution at 4°C and vortexed for 20 minutes to convert them to an activated state suitable for isolation.

C. Add carrier buffer to the free flow isoelectric focusing electrophoresis apparatus, the carrier buffer includes 0.8% Ampholyte, 0.8% Triton X-100, 1.1g/L Ficoll, 270mM Mannitol, constant power 20W, constant temperature 4°C, flow rate 1mL /min, run for 1 hour, so that a linear pH 3-10 gradient is formed in the separation chamber with an error of 0.5, as shown in Figure 3.

D. The activated saliva microorganisms obtained in step B are separated by free-flow isoelectric focusing electrophoresis, and the microorganism samples are injected into the pH neutral port. Time 1.5 hours.

The number of microbial species increased significantly without losing the original microbial information.

Example 3

A method for separating and preparing microbiome by free flow isoelectric focusing electrophoresis, the step flow chart is shown in Figure 1, and the specific steps are as follows:

A. Removal of human-derived cells, cellular debris, and mucins from saliva samples. Collect 5 mL of saliva from normal people and patients with lung cancer, and obtain a precipitate containing microorganisms, cells, cell debris and mucin after refrigerated centrifugation at 4°C, centrifugal force 12,000 g, and 5 minutes.

Prepare 0.5% Triton X-100 in PBS resuspension buffer. Use resuspension buffer to resuspend the precipitate, repeatedly blow and suck the precipitate with a pipette tip to fully dissolve it, and vortex it on a vortexer for 5 seconds, then at 4°C, centrifugal force 12000g, 8 minutes of refrigerated centrifugation, discard the supernatant, and repeat the above operation 3 times after obtaining the precipitate, so as to elute the microorganisms aggregated in the biofilm and in the mucin, and combine with a polyvinylidene fluoride filter membrane with a pore size of 10 μm Removes human cells, cell debris and mucins. As shown in Figure 2, compared with the untreated sample, the microbial samples after elution and filtration effectively removed human-derived cells and cell debris, and the microbial morphology was more dispersed and suitable for separation.

B. The activation solution is configured, and its composition is a 5mM CaCl ₂ aqueous solution. The salivary microorganisms were resuspended in activation solution at 4°C and vortexed for 20 minutes to convert them to an activated state suitable for isolation.

C. Add carrier buffer to the free flow isoelectric focusing electrophoresis apparatus, the carrier buffer includes 1.2% Ampholyte, 1% Triton X-100, 1.1g/L Ficoll, 300mM Mannitol, constant power 20W, constant temperature 4°C, flow rate 1mL /min, run for 1 hour, so that a linear pH 3-10 gradient is formed in the separation chamber with an error of 0.5, as shown in Figure 3.

D. The activated saliva microorganisms obtained in step B are separated by free-flow isoelectric focusing electrophoresis, and the microorganism samples are injected into the pH neutral port. Time 1.2 hours.

The number of microbial species increased significantly without losing the original microbial information.

Comparative Example 1

A method for separation and preparation of microorganisms by free-flow isoelectric focusing electrophoresis is different from Example 1 in that the saliva microorganisms are not processed in step A, and other steps are the same as those in Example 1. The results are shown in Figure 2 and Figure 2 It is a microscopic picture of microorganisms. The left picture is the saliva microorganism sample treated in Comparative Example 1, which contains human cells and cell debris. The phenomenon of microbial aggregation is serious, which is not conducive to the isolation and identification of more microorganisms; the right picture is processed in Example 1. Microbial samples can effectively remove impurities such as human cells, and the microorganisms are relatively dispersed and suitable for separation.

Comparative Example 2

A method for separating and preparing microorganisms by free-flow isoelectric focusing electrophoresis, the difference from Example 1 is that for microorganisms that have not been separated by free-flow isoelectric focusing electrophoresis, other steps are the same as those in Example 1 (repeated 3 experiments) , the results are shown in Figure 4, the number of microorganisms treated in Example 1 is significantly higher than that of the microbial samples treated in Comparative Example 2, and almost all microbial species that have not been pre-separated are included.

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essential content of the present invention. The embodiments of the present application and features in the embodiments may be combined with each other arbitrarily, provided that there is no conflict.

Claims (10)

1. a method for free-flow isoelectric focusing electrophoresis microbiome separation and preparation, is characterized in that, comprises the steps: A. Remove cells, cell debris and mucin from the microbiome sample; the specific steps are: Freezing and centrifuging the microbiome sample to obtain a precipitate, the precipitate includes microorganisms, cells, cell debris, and mucin; The sediment is resuspended and eluted several times through the resuspension buffer, and the microbiome is obtained by filtration through a filter membrane; B, the microbiome obtained by the activation solution activation treatment step A to obtain an activated microbiome suitable for electrophoretic separation; C. Add carrier buffer to the free-flow isoelectric focusing electrophoresis apparatus, so that a linear pH gradient is formed in the separation chamber; D. The activated microbiome obtained in step B is separated by free flow isoelectric focusing electrophoresis using the free flow isoelectric focusing electrophoresis apparatus of step C. 2. The method for separating and preparing microorganisms by free-flow isoelectric focusing electrophoresis according to claim 1, characterized in that, the resuspending and elution operation described in step A specifically comprises: blowing and sucking sediment 3 times by resuspension buffer and above, the precipitate was fully dissolved in the resuspension buffer, vortexed on a vortexer for 5-10 seconds, then refrigerated and centrifuged, and the supernatant was discarded to obtain a precipitate. 3 . The method for separating and preparing microorganisms based on free-flow isoelectric focusing electrophoresis according to claim 2 , wherein the resuspension buffer is a PBS buffer of 0.5% Triton X-100. 4 . 4. The method for separating and preparing microbiome based on free-flow isoelectric focusing electrophoresis according to claim 2, wherein the refrigerated centrifugation specifically comprises: centrifuging the microbiome sample at 4°C and a centrifugal force of 8000-12000g for 5- 10 minutes. 5 . The method for separating and preparing microorganisms based on free-flow isoelectric focusing electrophoresis according to claim 1 , wherein the filter membrane in step A comprises a polyvinylidene fluoride membrane with a pore size of 10 μm. 6 . 6. The method for separating and preparing microbiome based on free-flow isoelectric focusing electrophoresis according to claim 1, wherein the activation treatment in step B specifically comprises: resuspending the microbiome in an activation solution at 4°C and vortexing the microbiome. Rotate the instrument with slight shaking for 20 minutes to obtain an activated microbiome. 7 . The method for separating and preparing microorganisms based on free-flow isoelectric focusing electrophoresis according to claim 6 , wherein the activation solution comprises 5 mM CaCl ₂ . 8 . 8. The method for separating and preparing microorganisms based on free-flow isoelectric focusing electrophoresis according to claim 1, wherein the carrier buffer in step C comprises 0.8%-1.2% Ampholyte, 0.5%-1% Triton X -100, 1.1 g/L Ficoll, 250-300 mM Mannitol; the linear pH gradient is: a linear gradient formed in the range of pH 2.5-10.5. 9. The method for separating and preparing microorganisms based on free-flow isoelectric focusing electrophoresis according to claim 1, wherein the free-flow isoelectric focusing electrophoresis separation described in step D specifically comprises: placing the activated microorganisms in the formed microbial group. In a free-flow isoelectric focusing electrophoresis apparatus with a linear pH gradient, the constant temperature is 4-6°C, the constant voltage is 300V, the current limit is 2-4mA, and the separation time is 1-1.5 hours. 10. The application of the microorganism prepared by the method for separating and preparing microorganisms by free-flow isoelectric focusing electrophoresis according to claim 1, wherein the method for separating and preparing microorganisms by free-flow isoelectric focusing electrophoresis is prepared by obtaining Applications of microbes in microbiome and biomarkers.

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