Chemical magnetic bead and kit, and use thereof in microbial enrichment and detection

Abstract

Specifically, the present invention relates to a chemical magnetic bead and a kit, and the use thereof in microbial enrichment and detection. By means of electrostatic adsorption and the utilization of the chemical magnetic bead surface-modified with a nitrogen-containing group, microorganisms can be efficiently enriched from a sample and are then used for subsequent microbial detection.

Description

A chemical magnetic bead, a reagent kit, and its application in microbial enrichment and detection. Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to a chemical magnetic bead and its application in microbial detection. Background Technology

[0002] Enriching and extracting bacteria from samples is a necessary step in understanding their biological activity.

[0003] Clinical microbiological staining testing refers to the process of staining microorganisms from patient samples using specific staining techniques for microscopic observation and identification. These staining methods are commonly used to detect pathogens such as bacteria, fungi, and viruses. After staining, the microorganisms exhibit specific morphological and color characteristics under a microscope, allowing clinicians to quickly identify and analyze the types of microorganisms in the sample based on these characteristics. Commonly used staining methods include Gram staining, acid-fast staining, and fluorescent staining. Different staining techniques help physicians observe the morphology and structure of microorganisms more clearly, facilitating diagnostic decisions.

[0004] Mass spectrometry is widely used for the identification and analysis of microorganisms. Currently, centrifugation is almost universally used for microbial enrichment, and multiple washing and re-centrifugation processes are often required to reduce interference from impurities. This process is time-consuming and does not meet the needs of rapid clinical response. Furthermore, even after multiple washing steps, trace amounts of residual protein impurities can still affect the accuracy of the test results. Currently reported rapid separation methods for positive body fluid samples include separating gel chromatography, lysis centrifugation, immunosorbent assay (IMA), and electrophoresis.

[0005] CN106191199A uses a combination of magnetic bead adsorption and gel method. This method is complicated to operate and can only make a preliminary judgment on whether the sample contains bacteria by visual inspection. It cannot be used for subsequent detection and analysis and cannot meet the extensive needs of bacterial detection and analysis.

[0006] Lysis centrifugation involves rupturing red blood cells, but this process is complex and time-consuming. Multiple centrifugations are required to obtain bacteria with lower impurity levels, limiting its market application. Immunosorbent assays utilize the specific adsorption of antigens and antibodies to adsorb bacteria, but this adsorption is extremely strong, requiring shearing and desorption of the adsorbed bacteria before subsequent detection. This method is not only costly and complex but also prone to bacterial death and fragmentation, hindering subsequent bacterial culture. Furthermore, immunosorbent assays cannot completely remove pathogens from the sample, easily leading to false negatives. Electrophoresis cannot be used for MALDI-TOF / MS identification, limiting its application.

[0007] Currently, the above methods cannot guarantee the purity of the isolated and purified bacteria, ensure the comprehensiveness of the enriched bacteria without causing missed detections, and improve the convenience of end-user use.

[0008] Patent CN116024206A, entitled "A Method and Application for Rapid Isolation and Enrichment of Foodborne Spores Based on Antibiotic-Magnetic Beads with Dual Recognition Effect," discloses a method for rapidly and efficiently enriching spores in complex microbial systems by mixing and incubating Van-Fe3O4 and Amp-Fe3O4 magnetic beads with the sample. However, the antibiotic magnetic beads used in this method primarily eliminate bacteria, thus not enriching them for microscopic observation. Patent CN104313130A, entitled "A Functionalized Magnetic Nanoparticle for Efficient Microbial Enrichment and its Preparation and Application," discloses a magnetic bead modification method, including modifying magnetic beads with vancomycin and blocking the remaining amino sites on the beads with carboxyl-activated polyethylene glycol. This patent enriches low concentrations of bacteria based on vancomycin's high affinity for bacterial cell walls; however, this technology cannot address the issue of minimal or no adsorption of impurities in the sample.

[0009] In conclusion, it is necessary to propose new improvements and strategies to address the shortcomings of existing technologies, and to develop a method that ensures both the purity of the isolated and purified bacteria and the comprehensiveness and effectiveness of the enriched bacteria. Summary of the Invention

[0010] The purpose of this invention is to provide a novel chemical magnetic bead and its application in the detection and enrichment of microorganisms, partially solving or alleviating the aforementioned shortcomings of the prior art. As can be seen from the background art, the chemical magnetic beads described in this invention are suitable for the enrichment of bacteria, rather than the enrichment of nucleic acid substances. The specific technical solution adopted in this invention is as follows.

[0011] A chemical magnetic bead, wherein the chemical magnetic bead refers to a magnetic bead with a nitrogen-containing group modified on its surface; including a surface modified with amino (-NH2) or imino (-NH-), preferably amino.

[0012] Furthermore, the surface of the chemical magnetic beads is modified with nitrogen-containing groups: -R-NH-R1;

[0013] Where R is the C that can be substituted by any choice. 1-10 Alkylene; R1 is H, optionally substituted C 1-20 alkyl, aromatic or The term "optionally substituted" means that any one or more hydrogen atoms on a group may or may not be substituted by any of the hydrogen atoms other than hydrogen atoms.

[0014] Where i is an integer from 2 to 5, q is an integer from 1 to 1000, and R2 is -NH- or -O-; the shape of the chemical magnetic beads includes non-spherical or near-spherical shapes.

[0015] Furthermore, R is the C that can be substituted by any choice. 1-7 Alkyl; preferably optionally substituted C 2-5 Alkyl; optional, substituent is C. 1-5 One or more of alkyl and aromatic groups, such as one or more of methyl, ethyl, propyl or phenyl.

[0016] Furthermore, i is an integer of 2 or 3, q ​​is an integer between 100 and 800, and / or R2 is -NH-.

[0017] Furthermore, q is any integer from 1 to 1000; as a preferred option, q = 5, 10, 50, 200, 300, 400, 600, 700 or 900.

[0018] Furthermore, R1 is an optional substitution of C. 1-10 Alkyl group, optional; substituents are C10 and C20. 1-5 One or more of alkyl and aromatic groups, such as one or more of methyl, ethyl, propyl or phenyl.

[0019] "-" represents a chemical bond between functional groups or atoms.

[0020] Understandably, R1 is a modifying group of amino or imino groups for magnetic beads.

[0021] It is understood that the chemical magnetic beads described in this invention use magnetic bead microparticles as a substrate, with nitrogen-containing groups coupled to their surface via coupling groups such as siloxane groups. Prior art literature Environ. Sci. Technol. 2010, 44, 7908–7913 SCHEME1 illustrates a chemical magnetic bead structure. Referring to the structure shown in Figure 1 of this invention, different structures of chemical magnetic beads as described in this invention can be obtained by changing the amino donor.

[0022] The present invention also relates to a reagent kit comprising the aforementioned chemical magnetic beads.

[0023] The chemical magnetic beads of the present invention have a particle size of less than 3000 nm, preferably 5 nm to 1000 nm.

[0024] In some embodiments, the particle size can be selected from 1nm, 5nm, 30nm, 100nm, 150nm, 450nm, 500nm, 800nm, 1μm or 3μm.

[0025] "Particle size" refers to the maximum size of chemical magnetic beads.

[0026] Furthermore, the shape of the chemical magnetic beads includes non-spherical or near-spherical shapes. It is understood that the non-spherical or near-spherical shapes of the chemical magnetic beads are irregular shapes.

[0027] The term "non-spherical or near-spherical" in this invention refers to magnetic beads with a non-regular spherical shape, the structure of which can be determined by microscopic observation and comparison with a regular sphere. The term "regular sphere" or "spherical" has a meaning well-known in the art; one interpretation is that, given a uniform material density, line segments passing through the center of gravity and intersecting the surface of an object have the same size. These line segments are called the centroid lines, and the diameter is referred to as the diameter of the sphere. Therefore, in this invention, such non-spherical or near-spherical magnetic beads refer to centroid lines with different sizes, wherein the ratio of the largest to the smallest centroid line is greater than 1.1, preferably greater than 1.15, 1.2, or 1.3.

[0028] The present invention can also provide another alternative, namely, the application of the above-mentioned chemical magnetic beads in the preparation of microbial enrichment reagents or in the enrichment of microorganisms. The amount of chemical magnetic beads used is 0.01-2 mg / mL relative to 1 mL of the sample to be tested; preferably 0.05-1.5 mg / mL. The volume of the sample to be tested in the present invention refers to the mixture formed by the sample to be tested and the binding buffer in the steps prior to the addition of the magnetic beads.

[0029] Furthermore, the microorganisms enriched by the chemical magnetic beads are used for detection;

[0030] Furthermore, the magnetic bead-microbe complex produced after the chemical magnetic beads enrich microorganisms is used for detection, including drug sensitivity analysis, staining test or mass spectrometry detection.

[0031] On the other hand, the chemical magnetic beads are used to prepare a kit, which is used for drug sensitivity analysis, staining detection or mass spectrometry analysis, wherein the mass spectrometry analysis is for the identification of bacterial species or drug resistance identification.

[0032] The chemical magnetic bead kit of the present invention includes other components in unit doses, including binding buffer, with unit doses of 1-1000 μL.

[0033] This invention utilizes binding buffer to simplify the original complex environment of the sample, further reducing the influence of impurities. Specifically, this can be achieved by centrifuging the sample and resuspending it in binding buffer, or by adding an excess of binding buffer to the sample to dilute its components and thus weaken the influence of its complex composition. This invention is not limited to any particular method, as long as it achieves the goal of simplifying the bacterial environment.

[0034] In some preferred embodiments, the binding buffer solution is a solution with a metal ion concentration of less than 10% by mass-volume ratio; preferably, the concentration of metal ions in the binding buffer solution is less than 2%.

[0035] Preferably, the binding buffer comprises one or more of pure water, buffer solution, metal salt solution or chelating agent.

[0036] The binding buffer has a pH below 13, and preferably, the pH of the binding buffer is 5-9.

[0037] Furthermore, the metal ion is a metal cation; preferably, the binding buffer is selected from those containing Na. + K + Ca 2+ Mg 2+ And / or EDTA solutions.

[0038] Preferably, the binding buffer is selected from those containing 0.2-2% Na. + K + Ca 2+ and / or Mg 2+ The buffer solution is selected from phosphate buffer, citrate buffer, carbonate buffer, acetate buffer, barbiturate buffer or Tris buffer; more preferably, a sodium chloride solution with a concentration of 0.1-10%.

[0039] In some embodiments of the present invention, the binding buffer is selected from water; or 0.9% NaCl; or a solution of 0.9% NaCl and 0.25M EDTA; or 0.9% NaCl, 1% SDS and 0.6mol / L NaOH; or 50mM BisTris-HCl pH=7.0; or 0.01M PBS pH=7.0; or 50mM acetate buffer solution pH=5.0; or 50mM BisTris-HCl pH=9.0.

[0040] The binding buffer of the present invention is mainly used to remove excess impurities in the sample, so that bacteria can survive in a simpler environment. Therefore, there are no particular restrictions on the composition of the binding buffer. Those skilled in the art can freely choose a suitable binding buffer based on simple components.

[0041] Furthermore, the reagents are prepared according to unit dosage, and the amount of chemical magnetic beads used is 0.01-2 mg / mL relative to 1 mL of the sample to be tested.

[0042] Furthermore, the kit also contains a variety of cleaning solutions, including: cleaning buffer I, with a unit dose of 2-1000 μL, wherein in some preferred embodiments, cleaning buffer I includes 1-8 M urea and a surfactant; and / or cleaning buffer II, with a unit dose of 2-1000 μL, wherein cleaning buffer II includes water and / or a surfactant.

[0043] Furthermore, the kit also includes a resuspension solution with a unit dose of 1-500 μL. In some preferred embodiments, the resuspension solution comprises water and / or sodium chloride solution.

[0044] As a preferred embodiment, the chemical magnetic beads are prepared at a unit dosage of 0.05-1.5 mg / mL.

[0045] This invention utilizes the strong interaction between positive and negative charges to achieve strong enrichment of bacteria by magnetic beads, forming a bacteria-magnetic bead complex. The magnetic beads do not require additional immunomodulation, making the operation simple and inexpensive.

[0046] Furthermore, when the enriched microorganisms are used for mass spectrometry analysis, the kit also includes a decomposition solution; the decomposition solution includes formic acid. Preferably, the concentration of the formic acid is 25%–100%.

[0047] The decomposition solution is used to break down bacteria and release bacterial proteins, which facilitates subsequent mass spectrometry identification.

[0048] Furthermore, the kit also includes a cleaning solution used to clean the bacterial-chemical magnetic bead complex, making the complex cleaner and improving subsequent identification results. This cleaning step is a standard procedure in the art, and those skilled in the art can choose appropriate cleaning reagents. The cleaning solution of this invention is selected from one or more of water, buffer solution, metal salt solution, chelating agent, dissociating salt, polycationic or polyanionic solution. Preferably, the dissociating salt is selected from one or more of guanidine hydrochloride, guanidine thiocyanate, urea, or lithium perchlorate; the polycationic solution is selected from spermine and / or polylysine; and the polyanionic solution is selected from dextran and / or polyacrylic acid.

[0049] Preferably, the concentration of the ionized salt is 1-10 M, and the concentration of the polycationic and / or polyanionic salt is 1-100 mM.

[0050] Preferably, the concentration of the metal salt solution in the cleaning solution is 0.2-2%, and the concentration of the chelating agent is 0.1-1M.

[0051] The "liquid salt" described in this invention is used to disrupt hydrogen bonds and hydrophobic interactions.

[0052] In some preferred embodiments, the chemical magnetic beads used for mass spectrometry analysis are irregular non-spherical or near-spherical.

[0053] The bacterial content in the sample is above 10 CFU / ml; preferably, the bacterial content in the sample is above 10 CFU / ml. 4 CFU / ml or higher.

[0054] For the enrichment of magnetic beads, 10 CFU / ml is sufficient to demonstrate its superiority, while 10 4 A bacterial count of CFU / ml can further improve the detection results.

[0055] Unless otherwise specified, "%" in this invention refers to a percentage by mass.

[0056] Another aspect of the present invention is to provide a method for enriching bacteria from a sample, comprising the following steps: (1) obtaining a bacterial sample; (2) adding the chemical magnetic beads to the bacterial sample and mixing well; (3) magnetizing the sample, discarding the supernatant, and obtaining a bacterial-magnetic bead complex.

[0057] Preferably, the bacterial sample is pretreated, for example, by centrifugation, before the addition of the chemical magnetic beads; more preferably, after centrifugation of the bacterial sample, the microorganisms (bacterial sample) are contacted with the binding buffer to resuspend the bacteria in the binding buffer.

[0058] Preferably, the enrichment method may further include a washing step, in which the bacteria-magnetic bead complex is washed with a washing solution.

[0059] On the other hand, the present invention discloses a sample analysis, including: the method and steps for obtaining bacteria from the sample as described above; and the use of a bacteria-magnetic bead complex for analysis.

[0060] Furthermore, the analysis can be any method capable of determining bacterial characteristics, such as detecting bacterial count, bacterial activity, and / or bacterial metabolic type. One approach is to directly analyze the bacterial-magnetic bead complex to determine bacterial characteristics; another approach is to use an eluent to elute the bacteria adsorbed on the chemical magnetic beads before analysis; yet another approach is to incubate the bacterial-magnetic bead complex in a substrate before analysis.

[0061] Analytical methods include, but are not limited to, indirectly determining the growth of bacteria under different concentrations of antibiotics by measuring bacterial weight, changes in metabolite concentration, and changes in the amount of nutrients consumed. This method can reflect information such as bacterial resistance, phenotype, and / or minimum inhibitory concentration (MIC). Alternatively, direct detection methods may be selected from staining methods, turbidimetric methods, KB methods, fluorescence microscopy, or direct mass spectrometry analysis of cultured bacteria.

[0062] The analytical methods include: i) culturing the bacterial-chemical magnetic bead complex or eluted bacteria in a substrate containing a specific substance and analyzing the specific substance and / or its derivatives in the substrate using mass spectrometry; or ii) culturing the bacterial-chemical magnetic bead complex or eluted bacteria in a substrate containing a specific substance and then measuring the bacteria using staining, turbidimetry, KB method, or microscopic imaging; or iii) culturing the bacterial-chemical magnetic bead complex or eluted bacteria in a substrate and then taking an appropriate amount of bacteria for mass spectrometry analysis; wherein the derivatives of the specific substance are substances produced after the specific substance is metabolized by the bacteria.

[0063] The method for indirectly determining bacterial growth under different concentrations of antibiotics includes, for example, culturing the bacteria in a substrate containing a specific substance and analyzing the specific substance and / or its derivatives in the substrate using mass spectrometry. The specific substance can be the antibiotic itself or other substances that reflect the growth characteristics of the bacteria in the presence of antibiotics. For example, the growth characteristics of the bacteria can be reflected by analyzing the presence of antibiotics or the production of their hydrolysates (antibiotic derivatives); or by adding radioactive or isotopic substances to the culture medium and reflecting the growth characteristics of the bacteria through the content of radioactive substances or isotopes in the bacterial metabolites; or, for example, reflecting the growth characteristics of the bacteria through changes in the concentration of bacterial metabolites.

[0064] "Substrate" refers to a substance that promotes bacterial growth or allows bacteria to survive for a certain period of time, such as broth or saline solution.

[0065] The purpose of using the "elution solution" described in this invention is to separate bacteria from chemical magnetic beads without causing the bacteria to die or rupture.

[0066] Furthermore, the metabolic activity of the bacteria is selected from turbidimetric metabolic activity, KB method metabolic activity, microscopic imaging metabolic activity, or direct mass spectrometry detection.

[0067] The experiment may require a sufficient bacterial load. To ensure the smooth progress of subsequent experiments, the bacterial content in the KB or turbidimetric samples should be 1*10⁻⁶. 8 CFU / ml (bacteria) or 1*10 6 CFU / ml (fungus) or higher. For microscopic imaging, the fungal content is 10. 2 CFU / ml or higher.

[0068] Staining methods are commonly used to detect pathogens such as bacteria, fungi, and viruses. After staining, microorganisms exhibit specific morphological and color characteristics under a microscope, allowing clinicians to quickly identify and analyze the types of microorganisms in a sample based on these characteristics.

[0069] Furthermore, the turbidity method uses a sensitive analysis medium for culturing bacteria; or the turbidity method uses a sensitive analysis plate, where the sensitive analysis is the metabolic status of bacteria under specific substances; or the turbidity method uses a combination of a sensitive analysis medium and a sensitive analysis plate.

[0070] Furthermore, the fluorescence microscopy method uses a sensitive analytical medium for culturing bacteria. The sensitive analytical medium contains a specific fluorescent substance, which is selected from calcofluor, fluorescent dyes, or reagents containing cell viability detection.

[0071] Furthermore, the fluorescent dye is selected from propidium iodide, DYPI, fluorescein diacetate, syto series dyes, sybr series dyes, or D-amino acid conjugated fluorescein, and the cell viability detection reagent is Prestoblue.

[0072] Furthermore, the KB method for metabolic activity utilizes susceptibility testing discs containing specific antimicrobial agents. These discs include β-lactams, aminoglycosides, macrolides, lincomycins, peptides, quinolones, sulfonamides, antituberculosis drugs, antifungal drugs, or other antibiotics.

[0073] Therefore, a specific substance can be an antibacterial drug as described above, or a substance that bacteria can metabolize and utilize or consume, or a substance produced by bacterial metabolism. Derivatives of a specific substance are substances produced by bacterial metabolism based on that specific substance; for example, when the specific substance is a β-lactam antibiotic, the hydrolysis products of the β-lactam antibiotic are derivatives of that specific substance. The number of bacteria or their activity can be indirectly measured by detecting changes in the specific substance or its derivatives.

[0074] Therefore, specifically, this invention also discloses a pretreatment method for bacterial analysis or identification, which specifically includes the following steps:

[0075] S10. After pretreatment of the bacterial sample, add it to the binding buffer;

[0076] S20. Add the chemical magnetic beads to the sample processed in step S10 and mix well;

[0077] S30. Magnetize the sample processed in step S20, discard the supernatant, and obtain the bacteria-magnetic bead complex.

[0078] S40. Add elution buffer to break up bacteria in the magnetic bead complex.

[0079] On the other hand, this invention discloses a method for mass spectrometry identification of bacteria, specifically including the following steps:

[0080] S10. After pretreatment of the bacterial sample, add it to the binding buffer;

[0081] S20. Add the chemical magnetic beads to the sample processed in step S10 and mix well;

[0082] S30. Magnetize the sample processed in step S20, discard the supernatant, and obtain the bacteria-magnetic bead complex.

[0083] S40. Add eluent to disrupt the bacteria in the bacterial-magnetic bead complex;

[0084] S50. Mass spectrometry detection of broken bacteria.

[0085] Furthermore, a cleaning step is included between steps S30 and S40, and the cleaning step is preferably performed more than once to reduce the influence of impurities; more preferably, the cleaning solution in the cleaning step is the same as the binding buffer component.

[0086] Furthermore, the bacterial sample is selected from samples such as blood, urine, bone marrow, cerebrospinal fluid, pleural effusion, ascites, pericardial fluid, joint fluid, hydrocele fluid, and bile obtained directly from an organism, or their culture samples.

[0087] The culture sample is a blood culture sample or a pure culture sample, which means that the above-mentioned puncture fluid containing bacteria, such as blood, urine, bone marrow, cerebrospinal fluid, pleural effusion, ascites, pericardial fluid, joint fluid, hydrocele fluid, and bile, is cultured in a blood culture bottle.

[0088] The "blood" sample referred to in this invention includes whole blood or processed blood samples, such as serum, plasma, or extracts containing blood cells. Preferably, the sample to be tested is blood or a culture sample thereof.

[0089] To reduce interference from somatic cells in detection and analysis, such as interference from blood cells in blood samples or blood culture samples, a preferred approach is that the pretreatment includes centrifugation and / or the addition of a lysis agent.

[0090] The blood cell lysis step achieves its purpose by adding a blood cell lysis agent. It is understood that the lysis solution only lyses blood cells without damaging the bacterial cell wall or cell membrane. The lysis agent is one or more of surfactants, saponins, or hemolysins. The surfactant is selected from cationic surfactants, nonionic surfactants, amphoteric surfactants, and / or anionic surfactants. Specific examples include quaternary ammonium salts, alkyl glycosides, alkyl sulfates, alkyl sulfonates, Tween series, deoxycholates, and Triton series.

[0091] In this invention, "bacteria" and "microorganisms" have the same meaning, referring to bacteria, fungi, or a mixture of both.

[0092] On the other hand, the present invention discloses the application of the chemical magnetic beads in the pretreatment of bacteria for mass spectrometry identification, wherein the chemical magnetic beads are used to enrich bacteria in bacterial samples.

[0093] In another aspect, the present invention discloses the use of the chemical magnetic beads in enriching bacteria in samples or in the preparation of analytical reagents for detecting bacterial metabolic activity.

[0094] In another aspect, the present invention discloses the use of the chemical magnetic beads and binding buffer in enriching bacteria in samples.

[0095] The mass spectrometers in this invention include one of the following: MALDI-TOF-MS, MALDI-FT-MS, MALDI-FT-ICR-MS, and MALDI triple quadrupole mass spectrometer, with MALDI-TOF-MS being the most typical.

[0096] Beneficial technical effects:

[0097] This invention utilizes electrostatic interaction to bind positively charged magnetic beads with negatively charged microorganisms to form a complex. This method effectively enriches microorganisms in samples. The magnetic bead enrichment technology significantly improves the sensitivity of clinical microbial staining tests and the accuracy of mass spectrometry results. The kit uses a binding buffer to reduce the adsorption of non-bacterial substances by the chemical magnetic beads, improving the enrichment effect and the effectiveness of identification and analysis. By optimizing the surface modification characteristics of the magnetic beads and / or changing the particle size, morphology, or dosage, it is possible to achieve concentrations as low as 10² to 10⁻⁶ microorganisms per milliliter of sample. 3 Even with a small number of microorganisms, the presence of microorganisms can still be detected, reducing impurity adsorption and improving identification accuracy. Related experimental data show that the kit containing these magnetic beads demonstrates good performance in accuracy, sensitivity, and clinical applications, providing a faster and more reliable technical means for microbial detection. Attached Figure Description

[0098] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0099] Figure 1 is a schematic diagram of a chemical magnetic bead structure according to the present invention, wherein the black irregular object is a magnetic bead, and the figure shows multiple different centroid line dimensions of the irregular chemical magnetic bead.

[0100] Figure 2 is a scanning electron microscope image of the spherical amino magnetic beads prepared in one embodiment of the present invention;

[0101] Figure 3 is a scanning electron microscope image of the novel chemical magnetic beads prepared according to one embodiment of the present invention;

[0102] Figure 4 is a scanning electron microscope image of another novel chemical magnetic bead prepared according to one embodiment of the present invention. Detailed Implementation

[0103] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0104] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0105] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0106] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4%, more typically + / -3%, more typically + / -2%, even more typically + / -1%, and even more typically + / -0.5%.

[0107] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0108] Example 1

[0109] This embodiment provides an example of the preparation and testing of magnetic beads.

[0110] 1. Preparation of spherical amino magnetic beads

[0111] S01: 1g of spherical magnetic particles with a diameter of about 100nm were added to a mixed solution of 100g ethanol, 25g water, 6g ammonia and 1g TEOS. The mixture was reacted at room temperature for 6h, filtered and dried to obtain the treated magnetic beads.

[0112] S02: 1g of the treated magnetic beads were added to a premixed solution of 100g ethanol, 1g amino donor and 6g ammonia water, reacted at room temperature for 6h, filtered and dried to prepare amino magnetic beads 1-2 and 4-9 with one pretreatment (see Table 1).

[0113] S03: 1g of magnetic beads treated with S01 were added to a mixture of 100g ethanol and 1g KH560 and reacted at 50℃ for 12h to obtain magnetic beads with secondary pretreatment; then the magnetic beads with secondary pretreatment were mixed with 1g PEI (degree of polymerization 800) and 25g water and reacted at 80℃ for 12h. After filtration and drying, magnetic beads 3 were prepared, and the scanning electron microscope is shown in Figure 2.

[0114] 2. Preparation of non-spherical or near-spherical magnetic beads

[0115] S01: Dissolve 50g of ferric chloride hexahydrate and 18g of ferrous chloride tetrahydrate in 800g of water, remove dissolved oxygen by nitrogen purging, add 200g of ammonia water and mix, heat at 70℃ for 3h and wash with water until neutral to prepare magnetic microparticles.

[0116] S02: Add 1g of magnetic microparticles to a mixture of 100g of ethanol, 25g of water, 6g of ammonia and 1g of TEOS and react for 6h to obtain pretreated magnetic beads.

[0117] S03: Take 1g of magnetic beads prepared from S02, react them with a solution containing 100g ethanol, 1g amino donor and 6g ammonia at room temperature for 6h, filter and dry to prepare novel chemical magnetic beads 10 and 11. The scanning electron microscope images of the novel chemical magnetic beads 10 and 11 are shown in Figures 3 and 4, respectively. The structural schematic diagram of the chemical magnetic beads is shown in Figure 1. The magnetic beads are non-spherical, that is, the ratio of the maximum centroidal dimension D to the minimum centroidal dimension L is not equal to 1.

[0118] 3. Testing

[0119] (1) Culture Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC29213) and Candida albicans (ATCC14503) in concentrated broth, and centrifuge an appropriate amount of bacterial suspension.

[0120] (2) Add 0.9% sodium chloride bound buffer back to the precipitate, according to 10 3 Prepare various bacterial suspensions at a concentration of 1 / mL; take 1 mL of each suspension and add 0.1 mg of magnetic beads, discard the supernatant and resuspend in 30 μL of physiological saline.

[0121] (3) Take 10 μL and add it to a glass slide for fixation and staining. Observe and count under a microscope, calculate the enrichment rate of different bacteria, and then calculate the average enrichment of the three bacteria. The results are shown in Table 1.

[0122] Table 1 Information on different amino donors and magnetic beads

[0123] *In magnetic bead 3, i = 2, q = 800, and R2 is -NH-

[0124] As can be seen from Table 1, the number of carbon chains of the R group has a certain impact on the enrichment rate; however, the enrichment effects of magnetic beads 2, 3 and 5 show that the type of amino or imino modifying groups in the magnetic beads has no significant impact on the enrichment effect; compared with non-spherical or near-spherical magnetic beads (i.e. irregular magnetic beads), irregular magnetic beads have a higher average adsorption rate for microorganisms under the same chain length.

[0125] Example 2

[0126] This embodiment provides an example of smear staining detection.

[0127] 1. Preparation of Escherichia coli with OD0.5 (approximately 10 μL) 8 (cFU / mL), Staphylococcus aureus (approximately 10) 8 Candida albicans (approximately 10 cells / mL), Candida albicans 6 1 mL each of bacterial suspension (number of bacteria / mL).

[0128] 2. Perform 10-fold serial dilutions to prepare a concentration of 10. 7 10 6 10 5 10 4 10 3 10 2 Samples of Escherichia coli and Staphylococcus aureus bacterial suspensions at concentrations of 10 CFU / mL, and samples of Escherichia coli and Staphylococcus aureus bacterial suspensions at concentrations of 10 CFU / mL. 5 10 4 10 3 10 2 1 mL of Candida albicans suspension (number of cells / mL).

[0129] 3. Take 10 μL of the diluted bacterial suspension and add it to the first glass slide for fixation and staining, then observe under a microscope.

[0130] 4. Determine the detection limit for each bacteria by smear staining.

[0131] 5. Add 1 mg of No. 4 amino magnetic beads to each of the bacterial suspension samples diluted in step (2) for enrichment, discard the supernatant and resuspend in 30 uL of physiological saline.

[0132] 6. Take 10 μL of each and add it to the second glass slide for fixation and staining, then observe under a microscope.

[0133] 7. Determine the detection limit of each bacteria after magnetic bead enrichment by smear staining, where “○” indicates that it can be detected and “●” indicates that it cannot be detected. The results are shown in Table 2.

[0134] Table 2 Detection Limit Results

[0135] Experimental conclusion: The detection limit for each bacterium after magnetic bead enrichment by smear staining was 10. 3 The concentration of microorganisms per mL of amino magnetic beads effectively reduces the number of microorganisms required for smear observation and shortens the growth time. The No. 4 spherical magnetic beads used in the above experiment effectively enriched microorganisms. Understandably, using non-spherical or near-spherical magnetic beads would have yielded better enrichment results in the above experiment.

[0136] Example 3

[0137] This embodiment provides an example of the optimal concentration for enriching various bacteria with magnetic beads.

[0138] 1. Preparation of 10 3 20 mL of bacterial suspensions of Escherichia coli, Staphylococcus aureus, and Candida albicans at a concentration of CFU / mL.

[0139] 2. Take 1 mL of each bacterial suspension, add No. 4 magnetic beads to each, discard the supernatant, and resuspend in 30 μL of physiological saline.

[0140] 3. Take 10 μL of each and add it to a glass slide for fixation and staining, then observe under a microscope.

[0141] 4. Determine the observation effect of bacteria enriched at different magnetic bead dosages on smears.

[0142] Following the above method, the magnetic beads described in this invention were replaced with commercially available amino chemical magnetic beads (Suzhou Weidu Biotechnology Co., Ltd. MA0308N450nm) for testing.

[0143] The amount of each magnetic bead used and the observation results are shown in Table 3. In the table, “○” indicates that the outline of the bacteria is easy to observe and easy to count, “◎” indicates that the outline and number of bacteria can be observed, and “●” indicates that it is difficult to observe the outline and number of each bacteria completely.

[0144] Table 3. Results of microbial enrichment at different magnetic bead dosages

[0145] A comparison between the chemical magnetic beads described in this invention and commercially available amino magnetic beads revealed that the amount of magnetic beads used affects the direct observation results. Too many magnetic beads interfere with the detection field of view, while too few affect the enrichment efficiency. The optimal amount of magnetic beads relative to the sample volume is 0.01-3 mg / mL, preferably 0.05-2 mg / mL. Within the particle size range described in this invention, the chemical magnetic beads described in this invention and other commercially available amino magnetic beads still showed considerable beneficial effects, indicating a generally consistent application of amino magnetic beads.

[0146] Example 4

[0147] This embodiment provides an example of mass spectrometry detection after magnetic bead enrichment.

[0148] After enriching the three types of microorganisms with magnetic beads 4, 10 and 11 from Table 1, mass spectrometry analysis was performed. The specific operation steps are as follows.

[0149] 1. Culture Escherichia coli, Staphylococcus aureus, and Candida albicans in concentrated broth, and centrifuge an appropriate amount.

[0150] 2. Add pure water to the precipitate to form 10 5 Take 1 ml of bacterial suspension per mL, add 0.1 mg of magnetic beads, and stir rapidly for 15 seconds.

[0151] 3. After magnetization, discard the supernatant to obtain the magnetic bead-microbe complex.

[0152] 4. Add 50 μL of formic acid solution (70%) to the complex to lyse the microorganism.

[0153] 5. After drying the lysate, 1 μL of matrix solution was added, and after a second drying, it was identified using a MALDI-TOF microbial mass spectrometer (MALDI-TOF-MS, EXS2600, Zhongyuan Huiji Biotechnology Co., Ltd.). According to the mass spectrometry scoring rules, a score greater than 1.7 was considered reliable. The results are shown in Table 4.

[0154] Table 4 Mass spectrometry identification results

[0155] Experimental conclusions: Based on the adsorption rates in Table 1, it can be seen that the adsorption rates of regular and irregular magnetic beads are basically the same. However, the results of mass spectrometry identification (Table 4) show that the detection accuracy of microorganisms enriched by irregular magnetic beads is significantly higher than that of irregular magnetic beads. This indicates that irregular magnetic beads have a weaker adsorption force for impurities such as proteins, and thus exhibit a higher detection accuracy.

[0156] Example 5

[0157] The binding buffer was a 0.9% NaCl solution.

[0158] The sample was a positive blood culture sample (10 ml of sterile defibrinated sheep blood and 1000 CFU of ATCC25922 strain were added to a blood culture bottle, and the sample was cultured on a blood culture instrument until it was reported as positive). The specific operation was as follows:

[0159] S10. After centrifuging the positive blood culture sample, resuspend it in the binding buffer, add 1 mg of magnetic beads per 1 ml, and mix well;

[0160] S20. Magnetize the sample processed in step S10, discard the supernatant, and obtain the bacteria-magnetic bead complex.

[0161] S30. Clean the bacterial-magnetic bead complex;

[0162] S40. Incubate the supernatant and the bacterial-magnetic bead complex separately on blood agar plates.

[0163] In this embodiment, the cleaning solution and the binding buffer have the same composition in the cleaning step, and the amount of different magnetic beads added is kept constant in this embodiment.

[0164] Simultaneously, a comparative experiment was set up: blood culture samples were not centrifuged and resuspended in binding buffer, but chemical magnetic beads were added directly to positive samples, and the bacterial-magnetic bead complex and sample supernatant were cultured separately in plates.

[0165] The results are shown in Table 5 below. The data in Table 5 were obtained from the statistical analysis of the growth status of bacteria on the plates.

[0166] Table 5. Adsorption effect of magnetic beads in this invention

[0167] As can be seen from Table 5, the adsorption efficiency of magnetic beads for bacteria is greatly reduced without the treatment of binding buffer. This is mainly because the solution of the sample itself is more complex, which will interfere with the process of magnetic beads adsorbing bacteria.

[0168] Following the above method, when magnetic beads are added and then binding buffer is added, it can be observed that the percentage of magnetic beads adsorbed is basically the same as that without the addition of binding buffer, but still significantly lower than the percentage of adsorption when binding buffer is added and resuspended before the addition of magnetic beads. This indicates that the order of adding binding buffer also has a direct impact on the adsorption effect of magnetic beads.

[0169] Example 7

[0170] This embodiment demonstrates the effect of magnetic beads of different particle sizes on bacterial adsorption. Following the preparation method of Example 1, different sizes of nanomaterials were selected to prepare magnetic nanobeads of different sizes. The amino donor was 3-aminopropyltriethoxysilane. The adsorption effect of magnetic beads of different particle sizes was tested according to Example 6.

[0171] The adsorption results of magnetic beads of different sizes on bacteria are shown in Table 6:

[0172] Table 6. Enrichment effect of magnetic beads of different sizes

[0173] It can be seen that the enrichment effect of magnetic beads with a particle size of less than 3μm is good, and the enrichment effect is best when the particle size of magnetic beads is between 5nm and 1μm.

[0174] Example 8

[0175] This example verifies the effect of different binding buffer components. The sample was a positive blood culture sample, and the magnetic beads used in this example were No. 4 chemical magnetic beads. The operation method was the same as in Example 5.

[0176] This invention does not have any special requirements for the binding buffer. The main purpose is to replace the complex components of the sample itself and provide bacteria with a simple environment for survival. The formulations of all binding buffers in this embodiment are shown in Table 7.

[0177] This embodiment lists some of the available binding buffers, but it should be noted that the binding buffers described in this invention are not limited to this embodiment, and those skilled in the art can make corresponding adjustments according to their needs.

[0178] In another embodiment, the metal ion content of the binding buffer is less than 10%, more preferably less than 2%; preferably a metal cation, including Na+, K+, Ca. 2 + and / or Mg 2 +.

[0179] Table 7 Buffer formulation

[0180] Experimental results show that the ion content, type, and pH environment in the binding buffer have a certain impact on the adsorption of magnetic beads. For positively charged magnetic beads adsorbing negatively charged bacteria, although the adsorption effect in an alkaline environment with a pH higher than 13 (Formula 3) can still meet the needs of subsequent detection and analysis, the adsorption effect has decreased significantly. Therefore, the pH of the adsorption environment for positively charged magnetic beads should be lower than 13, preferably pH 5-9, and more preferably around pH 7. Preferably, the binding buffer should contain at most two types of cations, more preferably at most one type, to reduce the influence of cations in the binding buffer on the adsorption process.

[0181] Example 9: Effect of Different Detection Samples

[0182] The specific steps are as follows:

[0183] S10. Add the lysing agent saponin to the bacterial sample and centrifuge after a period of time; resuspend the precipitate in 0.9% NaCl binding buffer, add chemical magnetic beads, and mix well;

[0184] S20. Magnetize the sample processed in step S10, discard the supernatant, and obtain the bacteria-magnetic bead complex.

[0185] S30. Clean the bacterial-magnetic bead complex;

[0186] S40. After culturing the supernatant and bacteria-magnetic beads on blood agar plates for a period of time, the magnetic beads were counted.

[0187] The enrichment effects of different samples are shown in Table 8.

[0188] Table 8. Enrichment effect of the present invention on different samples

[0189] The detection method described in this invention is applicable to various types of samples, and is particularly suitable for blood samples.

[0190] Example 10: Mass Spectrometry Detection of Blood Culture Samples

[0191] In this embodiment, the samples selected were positive blood culture samples, and the mass spectrometer was selected from Zhongyuan Huiji EXS3000.

[0192] Magnetic beads: 100nm amino magnetic beads (purchased from JK-01-009-100 by JK Biotech).

[0193] Binding buffer: 0.9% NaCl;

[0194] Sample: 10 ml of sterile defibrinated sheep blood and about 1000 CFU of bacterial strain were added to a blood culture bottle and cultured on a blood culture instrument until positive.

[0195] The implementation method is as follows:

[0196] S10. Centrifuge the sample, discard the supernatant, and resuspend it in binding buffer;

[0197] S20. Add magnetic beads to the sample processed in step S10 and mix well;

[0198] S30. Magnetize the sample processed in step S20, discard the supernatant, and obtain the bacteria-magnetic bead complex.

[0199] S40. Clean the bacterial-magnetic bead complex;

[0200] S50. Add eluent to disrupt the bacteria in the bacterial-magnetic bead complex;

[0201] S60. Mass Spectrometry Detection

[0202] The solution of this invention can be selected from manual operation or mechanical operation. Compared with manual operation, mechanical operation is more efficient and precise and less polluting. Manual operation can meet the requirements of mass spectrometry identification, but the effect of mechanical operation is better than manual operation. Therefore, this embodiment shows the results of mechanical operation.

[0203] The specific steps for operating the machine are as follows:

[0204] S10. Using a 1mL syringe, draw 1mL of the sample (positive blood culture sample) into a 1.5mL centrifuge tube, centrifuge at 13000rpm for 2min, and discard the supernatant;

[0205] S20. Add 500 μL of binding buffer to the precipitate, shake to mix, and then add to the first column of the deep plate of the blood culture positive sample magnetic bead pretreatment kit.

[0206] S30. Place the deep well plate into the semi-automatic nucleic acid extractor (EXS3000, Zhongyuan Huiji Biotechnology Co., Ltd.), and start the magnetic bead pretreatment procedure for blood culture positive samples according to the instructions. The pretreatment procedure includes magnetic attraction, washing, and elution steps.

[0207] S40. After the program finishes running, use a pipette to add 1 μL of eluent to the target site on the MALDI-TOF / MS target plate and wait for it to dry.

[0208] S50. After drying, add 1 μL of matrix solution and wait for it to dry.

[0209] S60. After drying, place the target plate into a MALDI-TOF / MS, select the blood culture positive sample spectral acquisition program and the corresponding database for identification.

[0210] In some embodiments, step S20 may include a pyrolysis step, wherein the reagents in the pyrolysis step include, but are not limited to, the use of surfactants.

[0211] In this embodiment, the cleaning step is performed twice, and the cleaning solution is a 0.9% NaCl solution.

[0212] In this embodiment, the bacteria in the blood culture positive sample were artificially added to demonstrate the universality of the magnetic beads in adsorbing bacteria.

[0213] In some other implementations, lysis buffer may be added to the centrifuge tube to dissolve blood cells in positive blood culture samples, further reducing interference from the sample itself.

[0214] The matrix solution and elution buffer were obtained from the mass spectrometry system sample processing matrix solution kit of Zhongyuan Huiji Co., Ltd.

[0215] According to the mass spectrometry product instructions, a score of "2" or higher in the mass spectrometry output results indicates that the identification results are reliable. The results show that the identification results of Enterobacter aerogenes, Staphylococcus intermedia, Staphylococcus epidermidis, Acinetobacter baumannii, Staphylococcus aureus, and Enterobacter cloacae all scored above 2 for 28 different strains, indicating that the enrichment method and subsequent mass spectrometry identification method of the present invention can accurately detect bacteria to the species level and are reliable.

[0216] Furthermore, after replacing the samples with bone marrow culture, cerebrospinal fluid culture, pleural effusion culture, ascites culture, pericardial fluid culture, synovial fluid culture, hydrocele culture, bile culture, midstream urine, and blood samples, mass spectrometry identification can still accurately identify the bacteria to the species level in the samples.

[0217] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0218] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A kit for microbial detection, characterized in that, The kit contains chemical magnetic beads with amino or imino surfaces modified on them; wherein the particle size of the chemical magnetic beads is 1 nm to 3000 nm, preferably 5 nm to 1000 nm.

2. The kit according to claim 1, characterized in that, The chemical magnetic beads can be non-spherical or near-spherical in shape.

3. The kit according to claim 1 or 2, characterized in that, The kit also contains a binding buffer, which is used to replace the reagent components in the bacterial sample itself.

4. The kit according to claim 3, characterized in that, The binding buffer solution is a solution with a metal ion concentration of less than 2% by mass-volume ratio, and / or a solution with a pH of less than 13.

5. The kit according to claim 4, characterized in that, The binding buffer solution is selected from pure water, buffer solution, metal salt solution, or chelating agent solution; preferably, the buffer solution and metal salt solution contain 0.2-2% Na. + K + Ca 2+ and / or Mg 2+ The chelating agent solution contains 0.1-1M EDTA solution, and the buffer solution is selected from phosphate buffer, citrate buffer, carbonate buffer, acetate buffer, barbiturate buffer or Tris buffer.

6. The kit according to any one of claims 1-5, characterized in that, The kit also contains a decomposition solution for cleaving bacteria to release bacterial proteins; preferably, the decomposition solution includes formic acid.

7. The kit according to any one of claims 1-6, characterized in that, The kit also includes a cleaning solution; preferably, the cleaning solution is selected from one or more of water, buffer solution, metal salt solution, chelating agent solution, ionizing salt solution, polycationic or polyanionic solution.

8. The kit according to any one of claims 1-7, characterized in that, The surface of the chemical magnetic beads is modified with the following nitrogen-containing group: -R-NH-R1; Where R is the C that can be substituted by any choice. 1-10 Alkylene; R1 is H, or optionally substituted C 1-20 alkyl, aromatic or i is an integer from 2 to 5, q is an integer from 1 to 1000, and R2 is -NH- or -O-.

9. The reagent kit as described in claim 8, characterized in that, R is C, which can be substituted by any choice. 1-7 Alkylene; and / or i is 2 or 3; and / or q is an integer from 100 to 800; and / or R2 is -NH-.

10. The application of chemical magnetic beads in microbial enrichment, characterized in that, The surface of the chemical magnetic beads is modified with amino or imino groups; the amount of chemical magnetic beads used is 0.01-2 mg / mL relative to 1 mL of the sample to be tested; preferably 0.05-1.5 mg / mL.

11. The application as described in claim 10, characterized in that, The particle size of the chemical magnetic beads is 1 nm to 3000 nm, preferably 5 nm to 1000 nm.

12. The application as described in claim 10 or 11, characterized in that, The chemical magnetic beads can be non-spherical or near-spherical in shape.

13. The application as described in any one of claims 10-12, characterized in that, The sample to be tested is a bacterial fluid or its culture sample obtained directly from an organism; preferably, the sample to be tested is blood, a blood culture sample, or a pure culture sample.

14. The application as described in any one of claims 10-13, characterized in that, Microorganisms enriched by chemical magnetic beads are used for drug sensitivity analysis, staining analysis, or mass spectrometry detection.

15. A method for enriching bacteria from a bacterial sample, comprising the following steps: (1) obtaining a bacterial sample; (2) adding chemical magnetic beads to the sample and mixing well; (3) subjecting the sample to magnetic ablation treatment, discarding the supernatant, and obtaining a bacteria-magnetic bead complex; wherein, The surface of the chemical magnetic beads is modified with amino or imino groups, and the particle size of the chemical magnetic beads is 1 nm to 3000 nm; preferably, the particle size is 5 nm to 1000 nm.

16. The method as described in claim 15, characterized in that, Before adding the chemical magnetic beads, the microorganisms are contacted with a binding buffer solution, wherein the binding buffer solution is a solution with a metal ion content of less than 2%; preferably, the binding buffer solution is a solution with a pH of less than 13.

17. The method as described in claim 15 or 16, characterized in that, The chemical magnetic beads include non-spherical or near-spherical shapes.

18. An analytical method for bacterial samples, characterized in that, The analytical method includes: the method for enriching bacteria according to any one of claims 15-17, and the use of the bacteria-magnetic bead complex for analysis.

19. The analytical method as described in claim 18, characterized in that, The methods for using the bacterial-magnetic bead complex for analysis are as follows: the bacterial-magnetic bead complex is analyzed to determine the characteristics of the bacteria, or the bacterial-magnetic bead complex is placed in a substrate and cultured before analysis.

20. The analytical method as described in claim 19, characterized in that, Using bacterial-magnetic bead complexes for analysis includes one or more of the following methods: i) culturing the bacterial-magnetic bead complex or eluted bacteria in a substrate containing a specific substance and analyzing the specific substance and / or its derivatives in the substrate using mass spectrometry; or ii) culturing the bacterial-magnetic bead complex or eluted bacteria in a substrate containing a specific substance and then measuring the bacteria using staining, turbidimetry, KB method, or microscopic imaging; or iii) culturing the bacterial-magnetic bead complex or eluted bacteria in a substrate and then taking an appropriate amount of bacteria for mass spectrometry analysis. Derivatives of a specific substance are substances produced after a specific substance is metabolized by bacteria.

21. The analytical method according to any one of claims 18-20, characterized in that, Before analysis, the bacteria-magnetic bead complex or the eluted bacteria should be washed with a cleaning solution.

22. The analytical method according to any one of claims 18-21, characterized in that, The analytical method also includes a pretreatment step of centrifuging the sample and / or adding a lysis agent.

Images