Sample treatment kit and application thereof

Abstract

The invention relates to the field of biological detection, in particular to a positive sample pretreatment kit and application thereof. The method comprises the following steps: providing a simple survival environment for bacteria by utilizing incubation liquid, and then utilizing the interaction of opposite electric properties between chemical magnetic beads and the bacteria, that is, when the bacteria in a positive sample are negatively charged, the chemical magnetic beads are positive magnetic beads; or when the bacteria in the positive sample are positively charged and the chemical magnetic beads are negative electromagnetic beads, the bacteria-magnetic bead compound is obtained, the operation is simple, the cost is low, the enrichment efficiency is high, the impurity interference resistance is high, the activity state of the enriched bacteria is not changed, the method can be used for subsequent mass spectrum identification and metabolic activity analysis, the application range is wide, and the method is suitable for popularization and application. Various in-vitro samples can be universally enriched, and the method has great significance on the development of the IVD field.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a sample processing kit and application thereof. Background Art

[0002] Under normal circumstances, body fluids in the human body are sterile, so they are called "sterile body fluids". Clinically, when sterile body fluids are infected, the symptoms of the human body are often more severe and the disease progresses rapidly. If it is not diagnosed quickly and treated correctly, the mortality rate is extremely high. Generally, diagnosis requires obtaining infected body fluids in vitro, but because the initial body fluid infection often does not reach the concentration required for smear positivity, culture of infected body fluids (aseptic humoral culture) is still a necessary method.

[0003] Cultivation of post-infection body fluids refers to the artificial culture method of inoculating freshly isolated positive body fluid samples on nutrient culture media, and allowing bacteria with high nutritional requirements to grow and reproduce under certain temperature, humidity and other conditions, and then identifying them, so as to determine the species of pathogens. When identifying and detecting bacteria, it is necessary to separate the bacteria from the sample. When identifying bacteria by mass spectrometry, the sample must not contain substances that interfere with MALDI-TOF / MS identification and the amount of bacterial samples must be sufficient to obtain reliable identification.

[0004] The currently reported methods for rapid separation of positive body fluid samples include separation gel method, lysis centrifugation method, immunoadsorption and electrophoresis. The separation gel method is complicated to operate, with large bacterial loss during operation and low positive rate, and cannot be widely used in clinical practice. CN106191199A adopts a combination of magnetic bead adsorption and gel method. The method is complicated to operate, and can only be used to preliminarily judge whether the sample contains bacteria by naked eyes, and subsequent detection and analysis cannot be performed, which is difficult to meet the extensive needs of bacterial detection and analysis. The lysis centrifugation rule breaks red blood cells, which is complicated to operate and time-consuming. In order to obtain bacteria with less impurity content, multiple centrifugation is required, and the market application level is not high. The immunoadsorption rule utilizes the specific adsorption of antigen-antibody to achieve the adsorption of bacteria, but the specific adsorption force is extremely strong, and the bacteria after adsorption need to be sheared and desorbed before subsequent detection can be performed. This method is not only costly but also complicated in process, and it is very easy to cause the death and fragmentation of bacteria, which is not conducive to the subsequent bacterial culture. In addition, immunoadsorption cannot completely obtain the pathogens in the sample, which is very easy to cause missed detection; the electrophoresis rule cannot be used for MALDI-TOF / MS identification, and its application is limited.

[0005] Therefore, there is an urgent need to develop a separation method that is simple to operate, short in time, low in cost, can adsorb comprehensive bacteria and can be widely used, so as to provide more possibilities for the analysis and detection of bacteria. Summary of the invention

[0006] In order to solve the above problems and obtain a separation method that is simple to operate, short in time, low in cost and can be widely used, the present invention proposes a pretreatment kit for positive samples, which is used to adsorb and enrich bacteria in the sample rather than to extract nucleic acid substances in the sample.

[0007] The pre-treatment kit contains chemical magnetic beads and incubation solution, and the positive sample is a bacteria-containing sample;

[0008] When the bacteria in the positive sample are negatively charged, the chemical magnetic beads are positive magnetic beads;

[0009] Or when the bacteria in the positive sample are positively charged, the chemical magnetic beads are negatively charged magnetic beads.

[0010] The "chemical magnetic beads" mentioned in the present invention refer to magnetic beads whose surfaces are modified with chemical groups. The surfaces of the magnetic beads may also be modified with immune reactants such as antigens or antibodies.

[0011] The incubation solution is used to replace the reagent components in the bacteria-containing sample.

[0012] Preferably, the surface of the chemical magnetic beads is modified with positively charged groups, such as nitrogen-containing groups.

[0013] Preferably, the nitrogen-containing group includes an amino group or an ammonium group, such as a primary amino group, a secondary amino group, a tertiary amino group or a quaternary ammonium group; more preferably, an aliphatic amine group, an aromatic amine group; such as a pyrrolyl group or a pyridyl group.

[0014] Negative magnetic beads are magnetic beads with negative charge on their surface, for example, modified with electronegative groups; the electronegative groups include carboxyl groups, alcohol / silanol groups, sulfonic acid groups or phosphoric acid groups.

[0015] In one embodiment, the incubation solution is used to replace the reagent components in the bacteria-containing sample. For example, the bacteria-containing sample is centrifuged, the supernatant is discarded, and the sample is resuspended in the incubation solution to simplify the bacterial environment and make the chemical magnetic beads more rapid and efficient in enriching the bacteria.

[0016] The "replacement" method can be to centrifuge the sample and then resuspend it in the incubation solution, or to add an excess of incubation solution to the sample to dilute the components of the sample, thereby weakening the impact of its own complex components. The present invention is not limited to the method as long as the purpose of simplifying the bacterial environment can be achieved.

[0017] The present invention utilizes the strong interaction between positive and negative charges to achieve strong enrichment of bacteria by magnetic beads and form a bacteria-magnetic bead complex, which is simple to operate and low in cost. The samples selected by the present invention are positive sterile body fluids or culture samples thereof, which are more complex than pure culture samples or aqueous solution samples of bacteria. Therefore, the pretreatment process must achieve good enrichment effect, and the pretreatment kit must have high stress resistance.

[0018] Therefore, the present invention utilizes incubation solution replacement to simplify the original complex environment of the sample, thereby improving the stress resistance of the pretreatment kit.

[0019] The "amino magnetic beads" described in the present invention are magnetic beads whose surface is modified with nitrogen-containing groups (-N-), the "imidazole magnetic beads" are magnetic beads whose surface is modified with imidazole groups, the "carboxyl magnetic beads" are magnetic beads whose surface is modified with carboxyl groups, the "hydroxyl magnetic beads" are magnetic beads whose surface is modified with hydroxyl groups, and the "sulfonic acid magnetic beads" are magnetic beads whose surface is modified with sulfonic acid groups.

[0020] The "blood sample" mentioned in the present invention includes whole blood or processed blood samples, such as serum, plasma, extracts containing blood cells or cultures containing blood.

[0021] The terms “about” or “approximately” as used herein represent a measurement error within ±5%.

[0022] Preferably, the particle size of the positive electromagnetic beads or negative electromagnetic beads is less than 3000 nm, and preferably, the particle size is 5 nm to 1000 nm.

[0023] In some embodiments, the particle size is selected from 1 nm, 5 nm, 30 nm, 100 nm, 150 nm, 450 nm, 500 nm, 1 μm or 3 μm.

[0024] Preferably, the content of bacteria in the positive sample is above 10 CFU / ml; preferably, the content of bacteria in the positive sample is above 10 4 CFU / ml and above.

[0025] For the enrichment of magnetic beads, 10 CFU / ml is sufficient to show its superiority, while 10 4 The bacterial amount of CFU / ml can further enhance stress resistance.

[0026] The bacteria-containing sample is selected from puncture fluid samples or culture samples thereof such as blood, urine, bone marrow, cerebrospinal fluid, pleural effusion, ascites, pericardial fluid, joint fluid, hydrocele and bile containing bacteria.

[0027] Preferably, the culture sample is a blood culture sample, that is, the above-mentioned bacteria-containing blood, bone marrow, cerebrospinal fluid, pleural effusion, ascites, pericardial fluid, joint fluid, hydrocele and bile puncture fluid are expanded and cultured in a blood culture bottle.

[0028] More preferably, the bacteria-containing sample is blood.

[0029] The bacteria are selected from fungi, bacteria or a mixture thereof.

[0030] Furthermore, the metal ion content of the incubation solution is below 10%; preferably, the metal ion content of the incubation solution is below 2%.

[0031] Unless otherwise specified, "%" in the present invention refers to mass percentage.

[0032] Preferably, the incubation solution includes pure water, a buffer solution, a metal salt solution or a chelating agent.

[0033] The pH of the incubation solution is lower than 13, preferably, the pH of the incubation solution is 5-9.

[0034] Preferably, the metal ion is a metal cation.

[0035] Preferably, the incubation solution is selected from + , K + , Ca + Mg + and / or EDTA solution.

[0036] Preferably, the metal ion is selected from at most two of sodium ion, potassium ion, magnesium ion or calcium ion; more preferably at most one.

[0037] Preferably, the incubation solution is selected from a + , K + , Ca + and / or Mg + or a solution containing 0.1-1M EDTA; the buffer is selected from a phosphate buffer solution, a citric acid buffer solution, a carbonate buffer solution, an acetate buffer solution, a barbituric acid buffer solution or a Tris (tris(hydroxymethylaminomethane)) buffer solution.

[0038] In some embodiments of the present invention, the incubation solution is, for example, 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 / LNaOH; 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.

[0039] The incubation solution of the present invention is mainly used to remove excess impurities in the sample and allow the bacteria to survive in a simpler environment. Therefore, there is no special restriction on the composition of the incubation solution. Those skilled in the art can freely select a suitable incubation solution based on simple ingredients.

[0040] Another aspect of the present invention discloses the use of the chemical magnetic beads for enriching bacteria in positive samples.

[0041] The positive sample is selected from puncture fluid samples or culture samples thereof such as blood, urine, bone marrow, cerebrospinal fluid, pleural effusion, ascites, pericardial fluid, joint fluid, hydrocele and bile containing bacteria.

[0042] More preferably, the bacteria-containing sample is blood.

[0043] Another aspect of the present invention discloses the enrichment effect of the chemical magnetic beads and incubation solution on bacteria in positive samples.

[0044] In another aspect, the present invention discloses a method for enriching bacteria in a positive sample, comprising the following steps: (1) pre-treating the positive sample and then placing it in an incubation solution; (2) adding the chemical magnetic beads and mixing; (3) magnetically absorbing and discarding the supernatant to obtain a bacteria-magnetic bead complex;

[0045] Preferably, the enrichment method may further include a washing step.

[0046] Since most of the samples to be analyzed and processed contain blood, in order to further avoid contamination of blood cells, the sample detection pretreatment kit of the present invention may also include a lysing solution for blood samples, and the lysing solution includes a surfactant, saponin or hemolysin. It should be understood that the lysing solution has a lysing effect on blood cells but cannot lyse bacteria, wherein the surfactant is selected from a cationic surfactant, a nonionic surfactant, an amphoteric surfactant and / or an anionic surfactant. Specific examples include quaternary ammonium salts, alkyl glycosides, alkyl sulfates, alkyl sulfonates, Tween series, deoxycholate, and Triton series.

[0047] Thus, optionally, pre-treatment comprises centrifuging the sample and / or treating it with a lysis buffer.

[0048] Therefore, on the other hand, the present invention discloses a metabolic activity kit for bacteria in a positive sample, wherein the metabolic activity kit comprises the components of the metabolic activity pre-treatment kit described above.

[0049] Furthermore, the metabolic activity kit also contains a drug-sensitive culture medium, and the drug-sensitive culture medium is used to culture bacteria;

[0050] Or the metabolic activity kit further comprises a drug sensitivity plate, and the drug sensitivity plate is used for bacterial resistance analysis;

[0051] Or the metabolic activity kit further comprises a combination of a drug sensitivity culture medium and a drug sensitivity plate;

[0052] Or the metabolic activity kit further comprises a drug-susceptibility paper sheet.

[0053] Furthermore, the drug-sensitive plate or drug-sensitive paper sheet contains more than one antimicrobial drug.

[0054] Furthermore, the antibacterial drugs include β-lactams, aminoglycosides, macrolides, lincomycins, peptides, quinolones, sulfonamides, anti-tuberculosis drugs, antifungal drugs or other antibiotics.

[0055] Furthermore, the antibacterial drug is selected from penicillin G, piperacillin-tazobactam, flucloxacillin, mezlocillin, cefazolin; cefuroxime; ceftazidime, ceftriaxone, ceftriaxone-tazobactam, cefoperazone, cefoperazone-sulbactam, cefotaxime, cefepime, cefoxitin, cefmetazole, cefminox, aztreonam; imipenem, meropenem, ertapenem, ciprofloxacin, Ofloxacin, levofloxacin, moxifloxacin, vancomycin, teicoplanin, linezolid, tigecycline, gentamicin, amikacin, erythromycin, clindamycin, azithromycin, tetracycline, minocycline, trimethoprim-sulfamethoxazole, metronidazole, isoniazid, rifampicin, ethambutol, pyrazinamide, fluconazole, itraconazole, caspofungin, voriconazole, amphotericin B, posaconazole, etc.

[0056] Furthermore, the drug-sensitive culture medium contains or does not contain a fluorescent substance, and the fluorescent substance is selected from a fluorescent brightener (calcofluor), a fluorescent dye, or a cell viability detection reagent.

[0057] 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.

[0058] On the other hand, the present invention discloses a method for obtaining bacteria from a sample, which specifically comprises the following steps:

[0059] S10. Sample pretreatment;

[0060] S20. Add the chemical magnetic beads to the pretreated sample and mix well;

[0061] S30. Perform magnetic absorption, discard the supernatant, and obtain the bacteria-magnetic bead complex;

[0062] Preferably, the step S30 further includes a cleaning step, and the cleaning step is performed more than once, that is:

[0063] S10. Sample preprocessing;

[0064] S20. Add chemical magnetic beads to the pretreated sample and mix well;

[0065] S30. Perform magnetic absorption, discard the supernatant, and obtain the bacteria-magnetic bead complex;

[0066] S40. Washing the bacteria-magnetic bead complex.

[0067] Preferably, the reagents in the washing step include: one or more of water, buffer, metal salt solution, chelating agent, chaotropic salt, polycation or polyanion. Preferably, the chaotropic salt is selected from guanidine hydrochloride, guanidine thiocyanate, urea or lithium perchlorate; the polycation is selected from spermine or polylysine; the polyanion is selected from dextran or polyacrylic acid.

[0068] Furthermore, the pretreatment includes centrifuging and / or pre-treating the sample with a lysate; preferably, it includes treatment with a lysate. When the pretreatment includes centrifugation, the pre-treatment step also includes resuspending the bacteria in an incubation solution, and the incubation solution has the composition described above.

[0069] In another aspect, the present invention discloses a sample analysis method, characterized in that it comprises the following steps:

[0070] A method for obtaining bacteria from a sample as described above; and

[0071] The bacteria-magnetic bead complex is used for culture analysis or the bacteria adsorbed on the chemical magnetic beads are eluted with an elution solution and then used for culture analysis.

[0072] The purpose of using the "elution solution" described in the present invention is to separate the bacteria from the chemical magnetic beads without causing the death or rupture of the bacteria.

[0073] Furthermore, the sample is a blood sample, and the purpose of the pretreatment is to eliminate the influence of blood cells on subsequent bacterial enrichment.

[0074] Furthermore, the analysis method further comprises a washing step, that is, washing the bacteria-chemical magnetic bead complex with a washing solution before the bacterial culture analysis. Furthermore, the washing solution in the washing step has the same components as the incubation solution.

[0075] Furthermore, the culture analysis can be any method that can detect the number of bacteria, including but not limited to indirectly measuring the growth of bacteria under different concentrations of antibiotics by measuring bacterial weight, changes in metabolite concentrations, changes in the amount of nutrients consumed, etc.; or a direct detection method is selected from turbidity method, KB method, fluorescence microscopy method or directly performing mass spectrometry analysis on the cultured bacteria.

[0076] The method for indirectly determining the growth of bacteria under the action of different concentrations of antibiotics, specifically, for example, culturing in a substrate containing a specific substance and analyzing the specific substance and / or its derivatives in the substrate by mass spectrometry. The specific substance can be the antibiotic itself or other substances that can reflect the growth characteristics of bacteria in the presence of antibiotics. For example, the growth characteristics of bacteria can be reflected by analyzing the presence of antibiotics or the production of their hydrolyzates (antibiotic derivatives), or by adding radioactive or isotopic substances to the culture medium, and reflecting the growth characteristics of bacteria by the content of radioactive substances or isotopes in the metabolites of the bacteria; for another example, the growth characteristics of bacteria can be reflected by changes in the concentration of bacterial metabolites.

[0077] Furthermore, the metabolic activity of the bacteria is selected from turbidity metabolic activity, KB metabolic activity, microscopic photography metabolic activity or direct mass spectrometry detection.

[0078] The experiment may require sufficient bacterial content. In order to smoothly proceed with subsequent experiments, the bacterial content in the KB or turbidity method samples is 1*10 8 CFU / ml (bacteria) or 1*10 6 CFU / ml (fungus) or more. For microscopic photography, the content of bacteria is 10 2 CFU / ml and above.

[0079] Furthermore, the turbidity method uses a sensitive analytical medium, and the sensitive medium is used to culture bacteria;

[0080] Or the turbidity method uses a sensitive analysis plate, and the sensitive analysis is to measure the metabolism of bacteria under specific substances;

[0081] Or the turbidity method uses a combination of a sensitive assay medium and a sensitive assay plate.

[0082] Furthermore, the fluorescence microscopy method uses a sensitive analysis medium, which is used to culture bacteria. The sensitive analysis medium contains a specific fluorescent substance, which is selected from fluorescent brighteners (calcofluor), fluorescent dyes or cell viability detection reagents.

[0083] 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.

[0084] Furthermore, the KB method metabolic activity uses drug-sensitive paper sheets, which contain specific antibacterial drugs, including β-lactams, aminoglycosides, macrolides, lincomycins, peptides, quinolones, sulfonamides, anti-tuberculosis drugs, antifungal drugs or other antibiotics.

[0085] Therefore, the specific substance can be an antibacterial drug as described above, or a substance that can be metabolized or consumed by bacteria, as well as a substance produced by bacterial metabolism. The derivative of the specific substance is a substance produced by the metabolism of the specific substance based on bacteria. For example, when the specific substance is a β-lactam antibiotic, the hydrolysis product of the β-lactam antibiotic is a derivative of the specific substance. The number of bacteria or their activity can be indirectly measured by detecting changes in the specific substance or its derivative.

[0086] In addition, mass spectrometry can also be used to analyze the cultured bacteria. The mass spectrometry analysis method is related to the selection of mass spectrometry type, and MALDI-TOF is preferred.

[0087] On the other hand, the present invention discloses the use of the chemical magnetic beads in the preparation of analytical reagents for detecting bacterial metabolic activity, and the chemical magnetic beads are used for enriching bacteria.

[0088] Furthermore, the metabolic activity is determined by mass spectrometry analysis of the metabolism of specific substrates by bacteria in the sample to be tested.

[0089] The preferred sample is a blood sample, and more preferably, the blood sample is pre-treated with a blood cell lysing solution.

[0090] Furthermore, the particle size of the chemical magnetic beads is less than 3 μm, and the preferred particle size of the magnetic beads is 5 nm-1 μm.

[0091] The "metabolic activity" of the present invention refers to the growth of microorganisms in a substrate, for example, the difference in the ability of the microorganisms to decompose the substrate or produce specific substances.

[0092] Beneficial effects:

[0093] The present invention creatively applies chemical magnetic beads to enrich bacteria in positive samples, and uses incubation solution to provide a simple living environment for the bacteria, so that the interaction between the magnetic beads and the bacteria is stronger due to opposite electrical properties, and there are no excessive impurities in the reaction environment. Efficient enrichment of bacteria can be achieved through simple operation and low cost, and the entire enrichment process has strong resistance to impurity interference. The activity state of the enriched bacteria will not change, and can be used for subsequent mass spectrometry identification and metabolic activity.

[0094] The method of the present invention can fully enrich different bacteria in a sample at one time, and since the complex formed by electrostatic adsorption does not affect the activity of the bacteria, it is beneficial to the re-cultivation and subsequent analysis of the bacteria.

[0095] The present invention creatively applies chemical magnetic beads to the metabolic analysis of bacteria. Compared with immunomagnetic beads, chemical magnetic beads are low in cost and easy to obtain. In addition, the present invention uses chemical magnetic beads for direct enrichment, which is simple to operate and has universal applicability for the enrichment of bacteria. The enrichment is efficient and gentle, will not destroy the activity of the bacteria, and shortens the bacterial culture cycle.

[0096] The present invention proposes for the first time an analytical method for directly culturing bacteria adsorbed by chemical magnetic beads in a specific substrate and then conducting mass spectrometry identification. This analysis does not require desorption of the bacteria, and only requires the culture supernatant to perform mass spectrometry analysis and complete the identification of the metabolic characteristics of the bacteria. Compared with existing mass spectrometry methods for analyzing bacterial metabolic activity, the time is significantly shortened and there is less interference.

[0097] The use of chemical magnetic beads in combination with cell lysis buffer can not only eliminate the influence of blood cells on bacterial analysis and avoid multiple centrifugation operations, but magnetic separation is more convenient and efficient than centrifugal separation. Therefore, the kit and analysis method of the present invention can realize the rapid analysis of bacterial characteristics in samples, and further combine other data and vital signs information to indirectly provide important parameters for clinical drug use. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1 : Experimental results of magnetic bead adsorption effect in Example 2. DETAILED DESCRIPTION

[0099] The kit of the present invention and its preparation method and application will be described in further detail below in conjunction with specific examples. It should be understood that the following examples are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies realized based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0100] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0101] Example 1 Magnetic Bead Preparation

[0102] A. Synthesis of amino magnetic beads

[0103] (1) Synthesis of amino magnetic beads 1

[0104] 46g of ferric chloride hexahydrate and 16g of ferrous chloride tetrahydrate were dissolved in 400g of water, and then 120g of ammonia water was added, and then the temperature was raised to 70°C and reacted for 2h; after cooling, magnetic separation was performed, and the clear solution was washed with water until the pH value was neutral; then the magnetic particles were dispersed in 400g of water, 400g of ethanol was added, and 10g of aminosilane reagent [γ-aminopropyltriethoxysilane (brand A-1100, KH-550)] was added, and the reaction was carried out at 70°C for 5h, and after cooling, magnetic separation was performed, and the ethanol was washed 3 times and the water was washed 3 times to obtain amino magnetic beads 1. The particle size of the amino magnetic beads was determined to be about 80nm by SEM.

[0105] (2) Synthesis of amino magnetic beads 2

[0106] 46g of ferric chloride hexahydrate and 16g of ferrous chloride tetrahydrate were dissolved in 400g of water, and then 120g of ammonia water was added, and then the temperature was raised to 70°C and reacted for 2h; after cooling, magnetic separation was performed, and then vacuum drying was performed to obtain magnetic particle powder, and then the magnetic particle powder was dispersed in 400g of isopropanol, and then polyaminoalkyltrialkoxysilane (Y-5691) was added, and then 50g of water was added, and the reaction was carried out at 70°C for 5h, and after cooling, magnetic separation was performed, and ethanol washing was performed 3 times, and water washing was performed 3 times to obtain amino magnetic beads 2. The particle size of the amino magnetic beads was determined to be about 200nm by SEM.

[0107] (3) Synthesis of amino magnetic beads 3

[0108] 6g of ferric chloride hexahydrate was dissolved in 30mL of ethylene glycol, 0.6g of sodium acetate and 1g of sodium citrate were dissolved in 30mL of ethylene glycol, the two were mixed, loaded into a 100mL hydrothermal reactor, and reacted at 200°C for 15h; after the reaction was completed, magnetic separation was performed, and the particles were washed with ethanol and water three times in sequence to obtain magnetic particles; the magnetic particles were dispersed in 80mL of ethanol, and then 20mL of water and 8mL of ammonia water were added, and then 0.5g of tetraethyl orthosilicate was added, and the reaction was carried out for 6h; after the reaction was completed, the particles were washed with ethanol and water three times in sequence to obtain magnetic beads; the magnetic beads were dispersed in 40mL of ethanol, and then 40mL of water was added, and then γ-aminopropyltrimethoxysilane was added, and the reaction was carried out for 6h. After the reaction was completed, the particles were washed with ethanol and water three times in sequence to obtain amino magnetic beads 3. The particle size of the amino magnetic beads was determined to be about 2μm by SEM.

[0109] (4) Synthesis of amino magnetic beads 4

[0110] 10 g of commercial carboxyl magnetic beads (Dynabeads TM5g of polyethyleneimine was dissolved in 400mL of the same MES buffer (4-morpholineethanesulfonic acid) (20mM, pH=5.5); 5g of polyethyleneimine was dissolved in 400mL of the same MES buffer, the two were mixed, and then 2g of EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) (dissolved in 100mL of MES) was added, and the reaction was carried out for 5h. After the reaction was completed, the amino magnetic beads 4 were separated by magnetic separation and washed with water for 4 times to obtain the amino magnetic beads 4. The particle size of the amino magnetic beads was determined by SEM to be about 1μm.

[0111] (5) Amino magnetic beads 5

[0112] Purchased from Nanjing Dongna Biotechnology Co., Ltd. (Mag9002), particle size 100nm.

[0113] B. Synthesis of Imidazole-Based Magnetic Beads

[0114] Synthesis of Imidazole-based Magnetic Beads

[0115] 1. Synthesis of magnetic core

[0116] 120mL ethylene glycol, 6g ferric chloride hexahydrate, and 9.72g anhydrous sodium acetate were mixed and dissolved by ultrasonication. The mixture was placed in a 200° oven and reacted for 12h. The mixture was then magnetically separated, washed with ethanol 3 times, washed with water 3 times, and dried in vacuo to obtain a solid powder.

[0117] 2. Preparation of transition layer

[0118] Step 1: Disperse 10g of magnetic core in 400g of ethanol, add 100g of water and 20g of acetic acid, then add 5g of tetramethyl orthosilicate, and react at 50° for 10h; after the reaction is completed, perform magnetic separation, wash with ethanol and water three times in sequence, and obtain silica-coated magnetic particles;

[0119] Step 2: Disperse the magnetic particles in step 1 in 400 g of ethanol, add 1 g of KH570, and then react at 50° for 12 h; after the reaction is completed, perform magnetic separation, and wash with and water three times in sequence to obtain KH570-modified magnetic particles;

[0120] 3. Preparation of hydrophobic layer

[0121] Step 3: The magnetic particles obtained in step 2 are dispersed in 800g PVA DMSO (dimethyl sulfoxide) solution (1% wt), and then 0.5g azobisisobutyronitrile is added. After nitrogen replacement, the temperature is raised to 70°, and then a mixed solution of 5g N-vinyl imidazole, 0.5g N,N'-methylenebisacrylamide and 20g DMSO is dripped in. The dripping is completed in 30 minutes, and the reaction is continued for 20 hours; after cooling to room temperature, magnetic separation is performed, and the mixture is washed with ethanol and water three times in sequence to obtain imidazole-coated magnetic particles.

[0122] C. Carboxyl magnetic beads

[0123] (1) Carboxyl magnetic beads were purchased from Thermo Fisher Scientific (Dynabeads TM 65012), Suzhou Weidu Biotechnology Co., Ltd. (CMP0600CA), Xiamen PuriMag Biotechnology Co., Ltd. (PuriMag GS-COOH) and Suzhou Nanovita Biotechnology Co., Ltd. (DS415_MS500-COOH), which were named as Carboxyl Magnetic Beads 1, Carboxyl Magnetic Beads 2, Carboxyl Magnetic Beads 3 and Carboxyl Magnetic Beads 4, respectively.

[0124] Example 2 Magnetic Bead Adsorption Effect

[0125] The incubation solution was 0.9% NaCl solution.

[0126] The sample is a blood culture positive sample (10 ml of sterile defibrinated sheep blood and 1000 CFU ATCC25922 strain are added to the blood culture bottle and cultured on a blood culture instrument until positive). The specific operation is as follows:

[0127] S10. Centrifuge the blood culture positive sample, resuspend it in the incubation solution, add the magnetic beads, and mix well;

[0128] S20. Magnetic aspiration of the sample treated in step (1) is performed, and the supernatant is discarded to obtain a bacteria-magnetic bead complex;

[0129] S30. Washing the bacteria-magnetic bead complex;

[0130] S40. Culture the bacteria-magnetic bead complex on blood agar plate culture medium.

[0131] The cleaning solution in the cleaning step described in this embodiment has the same composition as the incubation solution, and the amount of different magnetic beads added in the embodiment remains constant.

[0132] In actual operation, in order to prepare for subsequent metabolic activity or mass spectrometry identification, the bacterial content in the positive blood culture sample is usually large, and direct coating can only be analyzed qualitatively, but not quantitatively. Therefore, in order to facilitate counting and statistical adsorption effects from the plate, this embodiment requires dilution of the positive blood culture sample, and the bacterial concentration after dilution is approximately: 1000 CFU / ml.

[0133] If the bacterial content in the sample is less than 10 CFU / ml, or the sample after culture is below 1000 CFU / ml, no dilution is required and it can be directly verified.

[0134] The results are shown in Table 1 and Figure 1 As shown in Table 1, the data from Figure 1 The statistics show that the statistical methods of the subsequent embodiments are consistent, and are all obtained from the growth conditions of the bacteria on the plate.

[0135] Table 1 Magnetic beads adsorption effect

[0136]

[0137]

[0138] As can be seen from Table 1, for negatively charged bacteria, positive magnetic beads (amino magnetic beads, imidazole magnetic beads) have a good adsorption effect, while negatively charged carboxyl magnetic beads have a poor adsorption effect.

[0139] In other embodiments, since the bacteria themselves carry a large amount of positively charged substances, such as Haemophilus influenzae with a polysaccharide capsule, the bacteria can be adsorbed more efficiently using negatively charged chemical magnetic beads in the incubation solution (0.9% NaCl) compared to positively charged chemical magnetic beads. Experiments have shown that the adsorption rates of the four carboxyl chemical magnetic beads all exceeded 90%.

[0140] Comparative Example 1

[0141] The sample is a blood culture positive sample (10 ml of sterile defibrinated sheep blood and 1000 CFU ATCC25922 strain are added to the blood culture bottle and cultured on a blood culture instrument until positive), without adding incubation fluid. The specific operation is as follows:

[0142] S10. Add magnetic beads to the blood culture positive sample and mix well;

[0143] S20. Magnetic aspiration is performed on the sample treated in step S10, and the supernatant is discarded to obtain a bacteria-magnetic bead complex;

[0144] S30. Washing the bacteria-magnetic bead complex;

[0145] S40. Culture the bacteria-magnetic beads on blood agar plate medium.

[0146] The results are shown in Table 2 below:

[0147] Table 2 Magnetic bead adsorption effect without incubation solution

[0148]

[0149] It can be seen from Table 2 that the adsorption efficiency of magnetic beads on bacteria will be greatly reduced if they are not treated with incubation solution. This is mainly because the solution of the sample itself is relatively complex, which will interfere with the process of magnetic beads adsorbing bacteria.

[0150] According to the above method, when the incubation solution is added after the magnetic beads, it can be observed that the magnetic bead adsorption percentage is basically the same as that without adding the incubation solution, and the adsorption percentage is still significantly lower than that of resuspending by adding the incubation solution before adding the magnetic beads, indicating that the order of adding the incubation solution also has a direct impact on the magnetic bead adsorption effect.

[0151] Example 3 Effect of magnetic beads of different particle sizes on bacterial adsorption

[0152] This example uses amino magnetic beads as an example, the sample is consistent with that in Example 2, and 0.9% NaCl is selected as the incubation solution.

[0153] Prepare amino magnetic beads with different particle sizes: 1nm, 5nm, 30nm, 150nm, 500nm, 1μm and 3μm.

[0154] Among them, 1nm and 5nm are obtained by the synthesis method of amino magnetic beads 5 in Example 1, 30nm and 150nm are obtained by the synthesis method of amino magnetic beads 3, and 500nm is obtained by the synthesis method of amino magnetic beads 1. Those skilled in the art can obtain the required particle size by adjusting the addition ratio of each compound and / or using the corresponding method;

[0155] 1 μm amino magnetic beads were purchased from Beijing Beike New Materials Technology Co., Ltd.; 3 μm amino magnetic beads were purchased from Beijing Zhongke Keyou Technology Co., Ltd.

[0156] The results of bacterial adsorption by magnetic beads of different particle sizes are shown in Table 3:

[0157] Table 3 Enrichment effect of magnetic beads with different particle sizes

[0158]

[0159] It can be seen that the enrichment effect of magnetic beads is good when the particle size is below 3μm, among which the enrichment effect is best when the particle size is 5nm-1μm.

[0160] Example 4 Study on the composition of incubation solution

[0161] The sample in this example is a blood culture positive sample. The magnetic beads used in this example are 100 nm amino magnetic beads. The operation method is consistent with that in Example 1.

[0162] The present invention has no special requirements for the incubation solution. The main purpose is to replace the complex components of the sample itself and provide the bacteria with a simple survivable environment. The formulas of all incubation solutions in this embodiment are shown in Table 4 (per liter as an example), and the effects of each incubation solution on adsorption enrichment are shown in Table 5.

[0163] Table 4 Incubation solution formula

[0164]

[0165]

[0166] Table 5 Effects of different incubation solutions on magnetic bead enrichment

[0167]

[0168] It can be seen from the contents shown in Tables 1 and 5 that the ion content, type and pH environment in the incubation solution have a certain influence on the adsorption of magnetic beads. For the adsorption of negatively charged bacteria by positively charged magnetic beads, although the adsorption effect of the alkaline environment with a pH higher than 13 (Formula 3) can still meet the needs of subsequent detection and analysis, the adsorption effect (average adsorption percentage twice) has been significantly reduced, so the pH of the adsorption environment of chemical magnetic beads should be lower than 13, preferably pH 5-9, and more preferably pH 7; preferably, the number of cations in the incubation solution should be at most two, and more preferably at most one to reduce the influence of cations in the incubation solution on the adsorption process.

[0169] Example 5 Effect of different test samples

[0170] The magnetic beads used in this example are 100 nm amino magnetic beads (obtained by referring to the synthesis method of amino magnetic beads 3), and the incubation solution is: 0.9% NaCl

[0171] The specific operations are as follows:

[0172] S10. Resuspend the positive sample in the incubation solution, add magnetic beads, and mix well;

[0173] S20. Magnetic aspiration is performed on the sample treated in step S10, and the supernatant is discarded to obtain a bacteria-magnetic bead complex;

[0174] S30. Washing the bacteria-magnetic bead complex;

[0175] S40. Cultivate the bacteria-magnetic beads on a blood agar plate medium;

[0176] The bacteria-containing bone marrow culture samples, cerebrospinal fluid culture samples, pleural effusion culture samples, ascites culture samples, pericardial fluid culture samples, joint fluid culture samples, hydrocele culture samples, and bile culture samples are samples of bacteria-containing bone marrow, cerebrospinal fluid, pleural effusion, ascites, pericardial fluid, joint fluid, hydrocele, bile, and blood samples cultured in blood culture bottles. The purpose of culture is to amplify the number of bacteria to facilitate subsequent mass spectrometry identification. Therefore, the present invention does not particularly limit the culture medium of the bacteria-containing samples. The blood culture bottle culture sample is selected in this embodiment for better comparison with the aforementioned embodiment. The blood sample is pretreated with SDS cell lysis solution after culture, and the enrichment effects of different samples are shown in Table 6.

[0177] Table 6 Enrichment effect of different samples of the present invention

[0178]

[0179] It can be seen that the detection method of the present invention is applicable to various types of samples, and is particularly applicable to blood samples.

[0180] Comparative Example 2 No incubation solution

[0181] The method used in this comparative example 2 is the same as that in Example 5, except that no incubation solution is used for treatment. The specific results are shown in Table 7.

[0182] Table 7 Enrichment effect of different samples without incubation solution

[0183]

[0184] It can be seen that without adding incubation solution, the sample environment has a great interference with the adsorption of magnetic beads, which greatly weakens the effect of magnetic beads in enriching bacteria.

[0185] Through the above verification, it can be known that the chemical magnetic beads of the present invention have an excellent enrichment effect on microorganisms, and the enriched bacteria-chemical magnetic beads are further used for metabolic activity.

[0186] The metabolic activity can be any method that can detect the number of bacteria, including but not limited to indirectly measuring the growth of bacteria under different concentrations of antibiotics by measuring bacterial weight, changes in metabolite concentrations, changes in the amount of nutrients consumed, etc.; or the metabolic activity is selected from direct detection methods such as turbidity metabolic activity, KB metabolic activity or fluorescence microscopy metabolic activity.

[0187] In some embodiments, turbidimetry, KB method or fluorescence microscopy can be selected.

[0188] The fluorescence microscopy photography method is as follows:

[0189] S10. Centrifuge 1 ml of the positive blood culture sample, discard the supernatant, and resuspend in the incubation solution;

[0190] S20. Add magnetic beads and mix well;

[0191] S30. Absorb the magnetic beads, discard the supernatant, and obtain the bacteria-magnetic bead complex;

[0192] S40. Washing bacteria-magnetic bead complex;

[0193] S50. Discard the supernatant, add physiological saline and drug sensitivity culture medium (containing fluorescent substances) to the magnetic beads, mix well and add to the microscopic photography drug sensitivity plate, and perform metabolic activity using the microscopic photography metabolic activity process. The software can automatically read the minimum inhibitory concentration (MIC) and RIS results.

[0194] Example 6 Turbidimetric drug sensitivity test 1

[0195] In this embodiment, the blood culture positive samples were selected by adding 10 ml of sterile defibrinated sheep blood and 1000 CFU of strains into a blood culture bottle and culturing them on a blood culture instrument until positive. The strains were Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Streptococcus pneumoniae. Metabolic activity was measured using BioMerieux VITEK2 and its supporting reagents.

[0196] Amino magnetic beads were purchased from Suzhou Weidu Biotechnology Co., Ltd. (MA0308N 450nm).

[0197] The incubation solution was 0.9% NaCl.

[0198] A. Excluding the cleaning step, the detection steps are as follows:

[0199] S10. Centrifuge 1 mL of positive blood culture sample, discard the supernatant, and resuspend in incubation solution;

[0200] S20. Add magnetic beads and mix well;

[0201] S30. Absorb the magnetic beads, discard the supernatant, and obtain the bacteria-magnetic bead complex;

[0202] S40. After culturing the bacteria-magnetic bead complex, the bacterial metabolic activity is examined.

[0203] The specific operation of metabolic activity analysis is as follows: Take 1-50μL bacteria-magnetic bead complex and add it to 3ml 0.45% saline. Fill and seal the drug sensitivity card, then load it on the instrument incubator and operate the drug sensitivity system. The instrument monitors the bacterial growth in each well within the specified time. The test results after the incubation period are shown in Table 8-Table 10:

[0204] Table 8 Growth of Enterococcus faecalis and Staphylococcus aureus

[0205]

[0206]

[0207] Table 9 Growth of Escherichia coli and Pseudomonas aeruginosa

[0208]

[0209]

[0210] Table 10 Growth of Streptococcus pneumoniae

[0211]

[0212]

[0213] It can be seen that for the five quality control bacteria, the three replicates were all within the quality control range, but the repeatability was not very good.

[0214] B. Contains a cleaning step, and the cleaning solution is 0.9% NaCl solution.

[0215] S10. Centrifuge 1 ml of the positive blood culture sample, discard the supernatant, and resuspend in the incubation solution;

[0216] S20. Add magnetic beads and mix well;

[0217] S30. Absorb the magnetic beads, discard the supernatant, and obtain the bacteria-magnetic bead complex;

[0218] S40. Washing bacteria-magnetic bead complex;

[0219] S50. The washed bacteria-magnetic bead complex is used for culture analysis to detect bacterial metabolic activity;

[0220] The specific operations of sample composition and metabolic activity were the same as those in A above, and the test results are shown in Tables 11-13.

[0221] Table 11 Growth of Enterococcus faecalis and Staphylococcus aureus

[0222]

[0223]

[0224]

[0225] Table 12 Growth of Escherichia coli and Pseudomonas aeruginosa

[0226]

[0227]

[0228]

[0229] Table 13 Growth of Streptococcus pneumoniae

[0230]

[0231] It can be seen that for the five quality control bacteria, after chemical magnetic bead adsorption and washing, the three repetitions were all within the quality control range and had good repeatability.

[0232] C. No incubation solution resuspension step

[0233] S10. Centrifuge 1 ml of blood culture positive sample, add magnetic beads, and mix well;

[0234] S20. Absorb the magnetic beads, discard the supernatant, and obtain the bacteria-magnetic bead complex;

[0235] S30. The washed bacteria-magnetic bead complex is used for culture analysis to check the metabolic activity of the bacteria;

[0236] The specific operations of the sample composition and metabolic activity are the same as those in A above, and the test results are shown in Tables 14-16.

[0237] Table 14 Growth of Enterococcus faecalis and Staphylococcus aureus

[0238]

[0239]

[0240] Table 15 Growth of Escherichia coli and Pseudomonas aeruginosa

[0241]

[0242]

[0243] Table 16 Growth of Streptococcus pneumoniae

[0244]

[0245]

[0246] It can be seen that for the five quality control bacteria, accurate MIC results could not be obtained after three repetitions.

[0247] Combining the above three groups of experiments A, B and C, it can be seen that metabolic activity can be achieved after enrichment with chemical magnetic beads without adding cleaning solution, but the detection effect will be better after adding cleaning solution to wash the bacteria-magnetic bead complex and remove excess impurities. However, if no incubation solution is added, the accuracy of metabolic activity detection decreases, so adding incubation solution is preferred.

[0248] For subsequent experiments, the kit should include an incubation solution and a washing solution.

[0249] Example 7 Turbidimetric drug sensitivity test 2

[0250] This example replaces the metabolic activity system and provides another turbidity metabolic activity method. The metabolic activity uses the Deere NMIC-150 metabolic activity system and its supporting reagents. The rest of the operations are consistent with Example 6B.

[0251] Take 1μL to 50μL of bacteria-magnetic bead complex in 15mL of drug sensitivity broth, mix well and add to the drug sensitivity analysis plate, 100μL per well. After culturing according to the requirements of the semi-automatic or fully automatic drug sensitivity culture system, the software can automatically read the MIC and RIS results. The test results are shown in Table 17-Table 21.

[0252] Table 17 ATCC25922 test results

[0253]

[0254]

[0255]

[0256] Table 18 ATCC27853 test results

[0257]

[0258]

[0259] Table 19ATCC29213 test results

[0260]

[0261]

[0262] Table 20ATCC29212 test results

[0263]

[0264]

[0265] Table 21ATCC49619 test results

[0266]

[0267]

[0268] It can be seen from the contents of Tables 17 to 21 that the turbidimetric metabolic activity shown in this embodiment has good repeatability. Combining Example 6 and Example 7, it can be seen that the pre-analysis treatment method of the present invention is suitable for different turbidimetric metabolic activities.

[0269] The present invention is not limited to the above two turbidimetric metabolic activities, and those skilled in the art can select a suitable turbidimetric metabolic activity method according to their needs.

[0270] Example 8 K-B method drug sensitivity test

[0271] This example is mainly to explore the differences between different metabolic activity methods, so the samples, magnetic beads, incubation solution, and cleaning solution are all consistent with Example 6B.

[0272] S10. Centrifuge 1 ml of the positive blood culture sample, discard the supernatant, and resuspend in the incubation solution;

[0273] S20. Add magnetic beads and mix well;

[0274] S30. Absorb the magnetic beads, discard the supernatant, and obtain the bacteria-magnetic bead complex;

[0275] S40. Washing bacteria-magnetic bead complex;

[0276] S50. Spread the bacteria-magnetic bead complex on the MH plate and stick a drug-sensitive paper according to the KB method metabolic activity process, culture it in an incubator, and perform metabolic activity and data reading according to the requirements of CLSIM100.

[0277] The specific operation of step S50 is as follows:

[0278] Dip a sterile cotton swab into the bacteria-magnetic bead suspension, and rotate it over the liquid several times close to the inner wall of the test tube to remove excess liquid.

[0279] Use a swab to scratch the entire agar surface, from the top to the bottom of the plate, and spread it evenly. Repeat this process twice (three times in total), rotating the plate about 60 degrees each time to ensure that the inoculated bacteria are evenly distributed each time. Finally, apply a circle around the edge of the agar plate to prevent the bacterial suspension from flowing and to ensure that the bacterial suspension is spread to every corner of the entire plate.

[0280] The plate coated with the bacterial solution can be placed at room temperature for 3-5 minutes to allow the agar to absorb excess surface moisture before placing the paper containing the drug.

[0281] Depending on the test bacteria, according to the pre-set grouping, the antimicrobial drug paper discs are attached to the agar surface inoculated with bacteria. Each paper disc must be pressed down to ensure complete contact with the agar surface. Whether the paper discs are placed individually or with a paper disc distribution device, the paper discs must be evenly distributed, the distance between the center of the circle of two paper discs is not less than 24mm, and the edge of the paper disc is not less than 15mm from the edge of the agar. For a 90mm MH plate, no more than 5 paper discs are placed. Since some drugs diffuse almost instantly, once the paper disc contacts the agar surface, it cannot be moved. If the position is not good, a new paper disc can be placed at another position on the agar.

[0282] After placing the paper disc, invert the MH agar in the incubator within 15 minutes and incubate for 16-18 hours (the Haemophilus influenzae, Neisseria gonorrhoeae, Neisseria meningitidis and Streptococcus KB paper disc test plates should be placed in an environment containing 5% CO2 for incubation).

[0283] When testing the sensitivity of oxacillin and cefoxitin to Staphylococcus, the incubation time must reach 24h to meet the sensitivity to Staphylococcus aureus and Enterococcus spp.

[0284] Usually, after 16-18 hours of incubation, the MH plate with the paper is placed on a black non-reflective background, and the diameter of the inhibition zone (the diameter of the complete inhibition zone) is measured with a vernier caliper. The diameter can be read to the nearest millimeter (read an integer) from the back of the plate using reflected light and naked eye observation. The area without obvious growth visible to the naked eye is the edge of the inhibition zone. In general, tiny colonies that can only be observed under a magnifying glass at the edge of the inhibition zone can be ignored.

[0285] The inhibition ring on the MH plate is a uniform circle, and the test strain shows a fused growth moss on the entire plate. If a single colony grows sparsely, it means that the concentration of the bacterial solution prepared during inoculation is too dilute. It is necessary to find the possible cause and test again.

[0286] The results are shown in Tables 22 and 23.

[0287] Table 22 ATCC25922 test results by K-B method

[0288]

[0289]

[0290] Table 23 K-B method ATCC27853 test results

[0291]

[0292]

[0293] The results showed that all antibiotics tested for the two quality control bacteria were within the quality control range and the metabolic activity results were accurate.

[0294] Example 9 Mass spectrometry experiment

[0295] The effectiveness of chemical magnetic beads for bacterial adsorption has been verified, and the efficiency of direct bacterial susceptibility analysis has also been verified. The effectiveness of indirect detection and analysis of bacterial metabolic activity through bacterial metabolic specific substances or their derivatives is further verified. The steps are as follows:

[0296] 1. Add 10 ml of sterile defibrinated sheep blood and 1000 CFU of resistant bacteria to the positive blood culture bottle, and add 10 ml of sterile defibrinated sheep blood and 1000 CFU of sensitive bacteria to the negative blood culture bottle, and culture them until positive;

[0297] 2. Add 200 μL SDS lysis buffer to 1 mL of positive blood culture sample, lyse for 4 min at room temperature, centrifuge, discard the supernatant, and resuspend in 0.9% NaCl incubation solution;

[0298] 3. Add magnetic beads (MA0308N 450nm, Suzhou Weidu Biotechnology Co., Ltd.) and mix well;

[0299] 4. Absorb the magnet, discard the supernatant, and obtain the bacteria-magnetic bead complex;

[0300] 5. Prepare three cephalosporin test tube solutions: dissolve 1 μg of cephalosporin in 50 μL of dissolution and hydrolysis buffer (ammonium bicarbonate buffer) and mix using a vortex meter for 30 seconds;

[0301] 6. Add 2 μg of bacteria-magnetic bead complex to two of the cephalosporin solutions respectively, and the uninoculated sample serves as a blank control;

[0302] 7. Incubate the test tube at a constant temperature of 35±2℃ for 40 minutes;

[0303] 8. Remove the culture tube from the incubator and centrifuge at 12000 rpm for 5 minutes;

[0304] 9. Take 1 μL of supernatant from each test tube, add it to the sample target plate and dry it in a thermostatic metal bath at 40°C;

[0305] 10. Cover each with 1 μL of matrix solution and dry in a thermostatic metal bath at 40°C.

[0306] 11. Add 1 μL of calibrant to the target plate and dry in a thermostatic metal bath at 40 °C. The calibration system for mass spectrometry is in the range of 0-1000 molecular weight.

[0307] 12. Place the sample target plate into the mass spectrometer (MALDI-TOF-MS, EXS2600 Zhongyuan Huiji Biological Co., Ltd.) and system it according to the mass spectrometer operation manual;

[0308] 13. After calibration, use the Antibiotic Hydrolysis Analysis Software to analyze the results.

[0309] The mass spectrum of β-lactam drugs (cephalosporin) has significant characteristic peaks at 396±1m / z and 555±1m / z. When the specific substance cephalosporin is co-cultured with the bacteria-magnetic bead complex, the disappearance of 396±1m / z and 555±1m / z indicates that the specific substance has been hydrolyzed.

[0310] Further based on the results of mass spectrometry analysis, the results determined by antibiotic hydrolysis analysis software showed that positive samples had cephalosporin resistance, while negative samples did not have cephalosporin resistance. The measurement results were consistent with the sample information.

[0311] The present invention demonstrates the feasibility and reliability of directly culturing and analyzing the bacteria-magnetic bead complex after enriching the bacteria with chemical magnetic beads. This method avoids the streaking culture process, and the culture substrate can be directly used for mass spectrometry analysis, which significantly shortens the analysis time of bacterial metabolic activity. This indicates that the method of the present invention can be further used in mass spectrometry detection such as drug sensitivity analysis and bacterial species analysis.

[0312] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A blood sample processing kit, characterized in that: The kit comprises: blood cell lysing solution, chemical magnetic beads and incubation solution; wherein the surface of the chemical magnetic beads is modified with positively charged groups or negatively charged groups, and the incubation solution is used to replace the reagent components in the bacteria-containing sample.

2. The kit according to claim 1, characterized in that The positively charged groups include nitrogen-containing groups, and the negatively charged groups include carboxyl groups, alcoholic hydroxyl groups, silanol groups, sulfonic acid groups or phosphoric acid groups; preferably, the nitrogen-containing groups include amino groups or ammonium groups.

3. The kit according to claim 1 or 2, characterized in that The blood cell lysis solution comprises a surfactant, saponin and / or hemolysin.

4. The kit according to any one of claims 1 to 3, characterized in that The incubation solution is a solution with a metal ion content of less than 2%; preferably, the pH of the incubation solution is lower than 13, and more preferably pH 5-9.

5. The kit according to any one of claims 1 to 4, characterized in that The metal ions in the incubation solution include at most two of sodium ions, potassium ions, magnesium ions or calcium ions.

6. A method for obtaining bacteria from a sample, characterized in that: The sample is a blood sample, and the method comprises the following steps: S10. Pre-treat the sample to eliminate the influence of blood cells; S20. Add chemical magnetic beads to the pretreated sample and mix well; S30. Perform magnetic absorption, discard the supernatant, and obtain the bacteria-chemical magnetic bead complex; The surface of the chemical magnetic beads is modified with positively charged groups or negatively charged groups.

7. The method according to claim 6, characterized in that The pretreatment comprises centrifugation and / or treatment with a lysate; preferably comprises treatment with a lysate.

8. The analysis method according to claim 6 or 7, characterized in that The method further comprises: before adding the chemical magnetic beads, replacing the reagent components in the bacteria-containing sample with an incubation solution. Preferably, the incubation solution is a solution with a metal ion content of less than 2%; more preferably, the pH value of the incubation solution is lower than 13.

9. A method for analyzing bacterial metabolic activity, characterized in that: The analysis method comprises: the method for obtaining bacteria from a sample according to any one of claims 6 to 8, and step S40: culturing and analyzing the bacteria-chemical magnetic bead complex or using a desorption solution to elute the bacteria adsorbed on the chemical magnetic beads and then using the bacteria for culturing and analysis; Preferably, the culture and analysis include: i) placing the bacteria-chemical magnetic bead complex or the eluted bacteria in a substrate of a specific substance for culture and analyzing the specific substance and / or its derivatives in the substrate by mass spectrometry; or ii) placing the bacteria-chemical magnetic bead complex or the eluted bacteria in a substrate of a specific substance for culture and then measuring the bacteria by turbidity, KB method or microscopic photography; iii) placing the bacteria-chemical magnetic bead complex or the eluted bacteria in a substrate for culture and then taking an appropriate amount of the bacteria for mass spectrometry analysis; The derivatives of specific substances are substances produced after the specific substances are metabolized by bacterial enzymes.

10. The analysis method according to claim 9, characterized in that Before culturing, use a cleaning solution to clean the bacteria-chemical magnetic bead complex or the eluted bacteria.

11. The use of chemical magnetic beads in the preparation of analytical reagents for detecting bacterial metabolic activity, characterized in that: The chemical magnetic beads are used for enriching bacteria, and the surface of the chemical magnetic beads is modified with positively charged groups or negatively charged groups; the particle size of the chemical magnetic beads is less than 3 μm, and the preferred particle size of the magnetic beads is 5 nm-1 μm.

12. The use according to claim 11, characterized in that The sample used for the metabolic activity analysis is a blood sample.

13. The use according to claim 12, characterized in that The bacterial metabolic activity is the drug sensitivity of the bacteria.

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