Immunomagnetic bead-based pre-microbiological detection broad-spectrum enrichment method and immunomagnetic bead reagent

Abstract

The invention discloses a biological immunomagnetic bead-based broad-spectrum enrichment method before microbiological detection, which comprises the following steps: preparing a plurality of different types of sample immunomagnetic beads, and modifying the surface of each sample immunomagnetic bead with an antibody or ligand corresponding to each bacterial genus; mixing different types of sample immunomagnetic beads according to different proportions to obtain a plurality of mixed immunomagnetic bead reagents; mixing each mixed immunomagnetic bead reagent with a biological sample containing an antigen, respectively incubating, and combining the immunomagnetic beads with the antigen; after the immunomagnetic beads combined with the antigens are separated from the sample, the antigen capture rate under each proportion is recorded to determine the optimal magnetic bead proportion; and preparing a magnetic bead reagent according to the optimal magnetic bead ratio so as to carry out broad-spectrum enrichment on bacteria. According to the biological immunomagnetic bead-based broad-spectrum enrichment method before microbiological detection disclosed by the invention, the ratio of different specific immunomagnetic beads is finely regulated and controlled, so that the method not only improves the capture efficiency and purity of bacteria, but also realizes broad-spectrum enrichment of various bacteria.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, and in particular relates to a broad-spectrum enrichment method before microbial detection based on biological immunomagnetic beads and an immunomagnetic bead reagent. Background Art

[0002] In microbiological research and applications, efficient and specific separation and enrichment of target microorganisms from complex samples is a crucial step.

[0003] In the existing technology, clinical microbial culture testing in samples takes a long time, and the microbial testing capacity conflicts with the clinical demand for rapid testing. In addition, bacteria are generally dispersed in the samples, which makes it possible for microbial culture testing to detect positive samples as negative results. False negatives will not only affect the test results but also delay the patient's treatment time. Summary of the invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides a method for broad-spectrum enrichment of microorganisms before detection based on biological immunomagnetic beads and an immunomagnetic bead reagent. The technical problem to be solved by the present invention is achieved by the following technical solutions:

[0005] Configuring a plurality of different types of sample immunomagnetic beads, and making the surface of each sample immunomagnetic bead modified with an antibody or ligand corresponding to each bacterial genus, wherein the bacterial genus of the sample immunomagnetic beads includes anaerobic bacteria and / or aerobic bacteria;

[0006] Mixing different types of sample immunomagnetic beads according to different ratios to obtain a number of mixed immunomagnetic bead reagents;

[0007] Each of the mixed immunomagnetic bead reagents is mixed with a biological sample containing an antigen, and then incubated separately to allow the immunomagnetic beads to bind to the antigen, wherein the binding of the immunomagnetic beads to the antigen includes specific adsorption binding and bacterial homology binding;

[0008] After separating the immunomagnetic beads bound to the antigen from the sample, the antigen capture rate under each ratio is recorded to determine the optimal magnetic bead ratio, wherein the optimal magnetic bead ratio includes the ratio of the sample to be enriched and the magnetic bead reagent and the content ratio of each bacterial genus in the magnetic bead reagent;

[0009] The magnetic bead reagent is configured according to the optimal magnetic bead ratio to perform broad-spectrum enrichment of bacteria.

[0010] In a specific embodiment, the ratio of the sample to be enriched and the magnetic bead reagent is: 50-500 μg of the magnetic bead reagent is configured for every 1 ml of the sample to be enriched.

[0011] In a specific embodiment, the aerobic bacteria include one or more of Escherichia, Staphylococcus, Klebsiella, Acinetobacter, Streptococcus, Pseudomonas, Enterococcus, and Proteus.

[0012] In a specific embodiment, when the aerobic bacteria include multiple genera, the mass percentage of Escherichia is 5% to 50%, the mass percentage of Staphylococcus is 5% to 25%, the mass percentage of Klebsiella is 3% to 15%, the mass percentage of Acinetobacter is 3% to 20%, the mass percentage of Streptococcus is 5% to 25%, the mass percentage of Pseudomonas is 5% to 25%, the mass percentage of Enterococcus is 5% to 25%, and the mass percentage of Proteus is 5% to 25%.

[0013] In a specific embodiment, the anaerobic bacteria include one or more of Prevotella, Peptiphilus, Peptostreptococcus, Veillonella, Bifidobacterium, Clostridium, Bacteroides, and Lactobacillus.

[0014] In a specific embodiment, when the anaerobic bacteria include multiple genera, the mass percentage of Prevotella is 5% to 30%, the mass percentage of Peptophilus is 5% to 25%, the mass percentage of Peptostreptococcus is 5% to 30%, the mass percentage of Veillonella is 5% to 25%, the mass percentage of Bifidobacterium is 5% to 30%, the mass percentage of Clostridium is 5% to 30%, the mass percentage of Bacteroides is 5% to 30%, and the mass percentage of Lactobacillus is 5% to 30%.

[0015] The present invention also provides an immunomagnetic bead reagent, comprising: 5% to 50% by mass of Escherichia, 5% to 25% by mass of Staphylococcus, 3% to 15% by mass of Klebsiella, 3% to 20% by mass of Acinetobacter, 5% to 25% by mass of Streptococcus, 5% to 25% by mass of Pseudomonas, 5% to 25% by mass of Enterococcus, and 5% to 25% by mass of Proteus.

[0016] The present invention also provides another immunomagnetic bead reagent, comprising: 5% to 30% by mass of Prevotella, 5% to 25% by mass of Peptophilus, 5% to 30% by mass of Peptostreptococcus, 5% to 25% by mass of Veillonella, 5% to 30% by mass of Bifidobacterium, 5% to 30% by mass of Clostridium, 5% to 30% by mass of Bacteroides, and 5% to 30% by mass of Lactobacillus.

[0017] Beneficial effects of the present invention:

[0018] The method for broad-spectrum enrichment of microorganisms before detection based on biological immunomagnetic beads of the present invention not only improves the capture efficiency and purity of bacteria, but also achieves broad-spectrum enrichment of multiple bacteria by finely regulating the ratio of immunomagnetic beads with different specificities.

[0019] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic flow chart of a method for broad-spectrum enrichment of microorganisms before detection based on biological immunomagnetic beads provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below with reference to specific embodiments, but the embodiments of the present invention are not limited thereto.

[0022] Embodiment 1

[0023] See also Figure 1 , Figure 1 The present invention provides a schematic flow chart of a method for broad-spectrum enrichment of microorganisms before detection based on biological immunomagnetic beads, including:

[0024] Step 1: configuring a plurality of different types of sample immunomagnetic beads, and modifying the surface of each sample immunomagnetic bead with antibodies or ligands corresponding to each bacterial genus;

[0025] When preparing the immunomagnetic beads in this embodiment, selection can be made according to clinical needs. This embodiment is illustrated by taking common clinical pathogens as an example. In this example, the bacterial genus of the sample immunomagnetic beads includes anaerobic bacteria and / or aerobic bacteria. For aerobic bacteria, for example, it can be Escherichia, Staphylococcus, Klebsiella, Acinetobacter, Streptococcus, Pseudomonas, Enterococcus or Proteus. For anaerobic bacteria, for example, it can be Prevotella, Peptophilus, Peptostreptococcus, Veillonella, Bifidobacterium, Clostridium, Bacteroides or Lactobacillus.

[0026] For other uncommon pathogens, corresponding immunomagnetic beads need to be prepared. In this embodiment, several different types of sample immunomagnetic beads are configured, that is, immunomagnetic beads corresponding to each genus of bacteria are configured, that is, Escherichia coli immunomagnetic beads, Staphylococcus immunomagnetic beads, etc., so that the surface of the immunomagnetic beads has corresponding antibodies or ligands.

[0027] Step 2: Mix different types of sample immunomagnetic beads according to different ratios to obtain a number of mixed immunomagnetic bead reagents;

[0028] Since the sample immunomagnetic beads obtained in step 1 are separate magnetic beads for each bacterial genus, different bacterial genera need to be mixed for broad-spectrum enrichment. However, since the specific adsorption degree and homology synergy degree of different bacteria are different, they will be affected by the mass ratio of the immunomagnetic beads of the bacterial genus. Based on this, in this step, each immunomagnetic bead is set with a series of different ratios and then mixed, so as to select the lower limit of the ratio and the optimal ratio.

[0029] Step 3, each of the mixed immunomagnetic bead reagents is mixed with a biological sample containing an antigen, and then incubated separately to allow the immunomagnetic beads to bind to the antigen, wherein the binding of the immunomagnetic beads to the antigen includes specific adsorption binding and bacterial homology binding;

[0030] It should be noted that some of the bacterial antigens currently used are expressed in different bacteria. The proteins expressed by the conserved sequences caused by biological evolution will make the specificity of the antibodies used poor, making it difficult to achieve the enrichment of a single bacterial species, resulting in the disadvantage of incomplete separation in existing separation technologies. However, in this application, there are multiple bacteria to be enriched, and the use of antibodies prepared by the above-mentioned homology between bacteria can make the magnetic beads enriched in different bacterial genera in a targeted manner, ensuring the specificity of each magnetic bead to a specific bacterial subpopulation and the selectivity between different genera.

[0031] In addition, in view of the homology between bacteria, this embodiment uses a combination of multiple magnetic beads, the surfaces of which are modified with different antibodies or ligands. Although each magnetic bead has higher specificity for a specific bacterial subpopulation, their synergistic effect can achieve broad-spectrum capture of multiple homologous bacteria.

[0032] Based on this, the present embodiment combines the above-mentioned multiple antibodies with each other, and realizes the broad-spectrum enrichment of the target microorganism in the sample by the selection and proportioning of magnetic beads. The enriched microbial sample has higher purity and richer species, which provides ideal materials for subsequent cultivation, identification or further analysis. Since there may be binding site conflicts when using different types of immunomagnetic beads, after mixing different immunomagnetic beads, the best ratio that can minimize the impact between different antigen antibodies can be found. By mixing different types of immunomagnetic beads and adjusting their ratios, the capture efficiency of each can be balanced and cross-reactions can be reduced. In order to better utilize the paramagnetism of magnetic beads, the present embodiment preferably adopts nanomagnetic beads, such as magnetic beads with a particle size less than 80nm, which can specifically be biological immune magnetic beads.

[0033] Step 4, after separating the immunomagnetic beads combined with antigen from the sample, record the antigen capture rate under each ratio to determine the best magnetic bead ratio, wherein the best magnetic bead ratio includes the ratio of the sample to be enriched and the magnetic bead reagent and the content ratio of each bacterial genus in the magnetic bead reagent. It should be noted that the antigen capture rate can be determined by means such as the quantity, quality, and specificity of the captured bacteria. This method can refer to the existing related technology, and this embodiment will not be repeated. Specifically, the ratio with the largest antigen capture rate is generally selected as the best magnetic bead ratio. When selecting, the antigen capture rate is generally positively correlated with the concentration of the bacterial genus in the magnetic bead reagent, and the higher the concentration, the higher the time cost and the economic cost, so it is not the higher the more practical. In practical applications, the critical value of the capture rate can be set according to the specific scene. When it is judged that the capture rate is greater than the critical value, it can be considered that the best magnetic bead ratio is not deliberately increased. The concentration of the bacterial genus.

[0034] Step 5: Configure magnetic bead reagents according to the optimal magnetic bead ratio to perform broad-spectrum enrichment of bacteria. Apply the magnetic bead reagents configured with the optimal magnetic bead ratio to the target sample, and achieve bacterial capture and enrichment through specific operating conditions. Use a magnetic field to separate the immunomagnetic beads bound to the target molecule from the sample, and perform subsequent purification and analysis.

[0035] In a specific embodiment, the ratio of the sample to be enriched and the magnetic bead reagent is: 50-500 μg of the magnetic bead reagent is configured for every 1 ml of the sample to be enriched.

[0036] In a specific embodiment, the aerobic bacteria include one or more of Escherichia, Staphylococcus, Klebsiella, Acinetobacter, Streptococcus, Pseudomonas, Enterococcus, and Proteus.

[0037] In a specific embodiment, when the aerobic bacteria include multiple genera, the mass percentage of Escherichia is 5% to 50%, the mass percentage of Staphylococcus is 5% to 25%, the mass percentage of Klebsiella is 3% to 15%, the mass percentage of Acinetobacter is 3% to 20%, the mass percentage of Streptococcus is 5% to 25%, the mass percentage of Pseudomonas is 5% to 25%, the mass percentage of Enterococcus is 5% to 25%, and the mass percentage of Proteus is 5% to 25%.

[0038] In a specific embodiment, the anaerobic bacteria include one or more of Prevotella, Peptiphilus, Peptostreptococcus, Veillonella, Bifidobacterium, Clostridium, Bacteroides, and Lactobacillus.

[0039] When the anaerobic bacteria include multiple genera, the mass percentage of Prevotella is 5% to 30%, the mass percentage of Peptophilus is 5% to 25%, the mass percentage of Peptostreptococcus is 5% to 30%, the mass percentage of Veillonella is 5% to 25%, the mass percentage of Bifidobacterium is 5% to 30%, the mass percentage of Clostridium is 5% to 30%, the mass percentage of Bacteroides is 5% to 30%, and the mass percentage of Lactobacillus is 5% to 30%.

[0040] In the specific implementation, this embodiment, for example, first configures 50μg, 100μg, 150μg, 200μg, 300μg, and 500ug of magnetic bead reagents per 1ml of sample to be enriched, wherein the magnetic bead reagent takes eight species of aerobic bacteria as an example, and the ratio of each genus is initially set to 3:1:1:1:1:1:1:1:1 in order. On this basis, the numerical ratio is adjusted up and down to calculate the capture rate to determine the enrichment effect of each genus. As mentioned above, the higher the concentration, the better. For example, when the enrichment effects of 100μg and 150μg are the same, this embodiment also needs to select a better enrichment effect between 100μg-150μg. If 100μg-150μg does not have a better enrichment effect, then 100μg with a smaller amount of reagent is selected as the optimal configuration.

[0041] Through the above ratio, the impact between different antigens and antibodies can be minimized, the capture efficiency of each can be balanced, and cross-reactions can be reduced.

[0042] This embodiment innovatively utilizes this specific adsorption property combined with the homology characteristics of bacteria to achieve a broad-spectrum enrichment method for target microorganisms in samples, which significantly improves the efficiency and accuracy of subsequent microbial culture or identification. Bacterial homology research is combined with the specific adsorption of immunomagnetic beads to find a way to improve the specificity of antigen-antibody reactions and broadly enrich bacteria in samples.

[0043] The method for broad-spectrum enrichment of microorganisms before detection based on biological immunomagnetic beads of the present invention not only improves the capture efficiency and purity of bacteria, but also achieves broad-spectrum enrichment of multiple bacteria by finely regulating the ratio of immunomagnetic beads with different specificities.

[0044] The present invention also provides an immunomagnetic bead reagent, comprising: 5% to 50% by mass of Escherichia, 5% to 25% by mass of Staphylococcus, 3% to 15% by mass of Klebsiella, 3% to 20% by mass of Acinetobacter, 5% to 25% by mass of Streptococcus, 5% to 25% by mass of Pseudomonas, 5% to 25% by mass of Enterococcus, and 5% to 25% by mass of Proteus.

[0045] The present invention also provides another immunomagnetic bead reagent, comprising: 5% to 30% by mass of Prevotella, 5% to 25% by mass of Peptophilus, 5% to 30% by mass of Peptostreptococcus, 5% to 25% by mass of Veillonella, 5% to 30% by mass of Bifidobacterium, 5% to 30% by mass of Clostridium, 5% to 30% by mass of Bacteroides, and 5% to 30% by mass of Lactobacillus.

[0046] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

[0047] Although the present application is described herein in conjunction with various embodiments, in the process of implementing the claimed application, those skilled in the art may understand and implement other variations of the disclosed embodiments by viewing the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other components or steps, and "one" or "an" does not exclude multiple situations. A single processor or other unit may implement several functions listed in a claim. Certain measures are recorded in different dependent claims, but this does not mean that these measures cannot be combined to produce good results.

[0048] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A method for broad-spectrum enrichment of microorganisms before detection based on immunomagnetic beads, characterized in that: include: Configuring a plurality of different types of sample immunomagnetic beads, and making the surface of each sample immunomagnetic bead modified with an antibody or ligand corresponding to each bacterial genus, wherein the bacterial genus of the sample immunomagnetic beads includes anaerobic bacteria and / or aerobic bacteria; Mixing different types of sample immunomagnetic beads according to different ratios to obtain a number of mixed immunomagnetic bead reagents; Each of the mixed immunomagnetic bead reagents is mixed with a biological sample containing an antigen, and then incubated separately to allow the immunomagnetic beads to bind to the antigen, wherein the binding of the immunomagnetic beads to the antigen includes specific adsorption binding and bacterial homology binding; After separating the immunomagnetic beads bound to the antigen from the sample, the antigen capture rate under each ratio is recorded to determine the optimal magnetic bead ratio, wherein the optimal magnetic bead ratio includes the ratio of the sample to be enriched and the magnetic bead reagent and the content ratio of each bacterial genus in the magnetic bead reagent; The magnetic bead reagent is configured according to the optimal magnetic bead ratio to perform broad-spectrum enrichment of bacteria.

2. The method for broad-spectrum enrichment of microorganisms before detection based on biological immunomagnetic beads according to claim 1, characterized in that: The ratio of the sample to be enriched and the magnetic bead reagent is: 50-500 μg of magnetic bead reagent per 1 ml of the sample to be enriched.

3. The method for broad-spectrum enrichment of microorganisms before detection based on immunomagnetic beads according to claim 1, characterized in that: The aerobic bacteria include one or more of Escherichia, Staphylococcus, Klebsiella, Acinetobacter, Streptococcus, Pseudomonas, Enterococcus, and Proteus.

4. The method for broad-spectrum enrichment of microorganisms before detection based on immunomagnetic beads according to claim 1, characterized in that: When the aerobic bacteria include multiple genera, the mass percentage of Escherichia is 5% to 50%, the mass percentage of Staphylococcus is 5% to 25%, the mass percentage of Klebsiella is 3% to 15%, the mass percentage of Acinetobacter is 3% to 20%, the mass percentage of Streptococcus is 5% to 25%, the mass percentage of Pseudomonas is 5% to 25%, the mass percentage of Enterococcus is 5% to 25%, and the mass percentage of Proteus is 5% to 25%.

5. The method for broad-spectrum enrichment of microorganisms before detection based on immunomagnetic beads according to claim 1, characterized in that: The anaerobic bacteria include one or more of Prevotella, Peptiphilus, Peptostreptococcus, Veillonella, Bifidobacterium, Clostridium, Bacteroides, and Lactobacillus.

6. The method for broad-spectrum enrichment of microorganisms before detection based on immunomagnetic beads according to claim 1, characterized in that: When the anaerobic bacteria include multiple genera, the mass percentage of Prevotella is 5% to 30%, the mass percentage of Peptophilus is 5% to 25%, the mass percentage of Peptostreptococcus is 5% to 30%, the mass percentage of Veillonella is 5% to 25%, the mass percentage of Bifidobacterium is 5% to 30%, the mass percentage of Clostridium is 5% to 30%, the mass percentage of Bacteroides is 5% to 30%, and the mass percentage of Lactobacillus is 5% to 30%.

7. An immunomagnetic bead reagent, characterized in that: include: The mass percentage of Escherichia is 5% to 50%, the mass percentage of Staphylococcus is 5% to 25%, the mass percentage of Klebsiella is 3% to 15%, the mass percentage of Acinetobacter is 3% to 20%, the mass percentage of Streptococcus is 5% to 25%, the mass percentage of Pseudomonas is 5% to 25%, the mass percentage of Enterococcus is 5% to 25%, and the mass percentage of Proteus is 5% to 25%.

8. An immunomagnetic bead reagent, characterized in that: include: The mass percentage of Prevotella is 5% to 30%, the mass percentage of Peptophilus is 5% to 25%, the mass percentage of Peptostreptococcus is 5% to 30%, the mass percentage of Veillonella is 5% to 25%, the mass percentage of Bifidobacterium is 5% to 30%, the mass percentage of Clostridium is 5% to 30%, the mass percentage of Bacteroides is 5% to 30%, and the mass percentage of Lactobacillus is 5% to 30%.

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