Pretreatment kit for positive sample and application thereof

Abstract

The invention relates to the field of biological detection, in particular to a pretreatment kit for a positive sample containing chemical magnetic beads and application of the pretreatment kit. According to the method, the chemical magnetic beads are creatively applied to mass spectrum identification, and the strong adsorption capacity of the chemical magnetic beads to bacteria is utilized, so that the cost can be saved, and the pretreatment time of mass spectrum detection is greatly shortened. Meanwhile, an incubation solution is used for replacing a sample, so that the environment of bacteria is simpler, efficient adsorption of the magnetic beads to the bacteria is more facilitated, a bacteria-chemical magnetic bead compound can be extracted through simple magnetic adsorption, then the bacteria on the bacteria-chemical magnetic bead compound are eluted and detected, the purity of detected mycoprotein is very high, species identification can be achieved through mass spectrum, and the method is suitable for large-scale popularization and application. A new thought is provided for mass spectrum microorganism detection.

Description

Technical Field

[0001] The invention relates to the field of biological detection, and in particular to a pre-treatment kit for positive samples and applications thereof. Background Art

[0002] Enriching and extracting bacteria from positive samples is a necessary step to understand the biological activity of bacteria. Especially for microbial mass spectrometry analysis, it is necessary to reduce other biological substances besides bacteria to ensure the accuracy of mass spectrometry information. For example, human proteins or blood cell residues will cause great interference to mass spectrometry analysis. Currently reported methods for rapid separation of positive body fluid samples include separation gel method, lysis centrifugation method, immunoadsorption and electrophoresis.

[0003] The separation gel method uses a gel (such as agar) containing specific ingredients as a culture medium, and separates bacteria from a mixed sample through a specific operating procedure to form a single colony, which is complex for subsequent identification, purification and research. The bacterial loss during this operation is large, the positive rate is low, and it cannot be widely used clinically. CN106191199A uses a combination of magnetic bead adsorption and gel method. This method is complicated to operate, and can only preliminarily judge whether the sample contains bacteria by the naked eye. Subsequent detection and analysis cannot be performed, and it is difficult to meet the widespread demand for bacterial detection and analysis.

[0004] Lysis centrifugation method breaks red blood cells, which is complicated and time-consuming. In order to obtain bacteria with less impurities, multiple centrifugation is required, and the market application is not high. Immunoadsorption method uses the specific adsorption of antigen-antibody to achieve bacterial adsorption, but the specific adsorption force is extremely strong, and the adsorbed bacteria need to be sheared and desorbed before subsequent detection. This method is not only costly and complicated, but also very easy to cause bacterial death and fragmentation, which is not conducive to subsequent bacterial cultivation. In addition, immunoadsorption cannot completely obtain the pathogens in the sample, which is very easy to cause missed detection; electrophoresis method cannot be used for MALDI-TOF / MS identification, and its application is limited.

[0005] At present, the above methods cannot guarantee the purity of separation and purification, ensure the comprehensiveness of enriched bacteria without missing detection, and also improve the convenience of terminal use. Summary of the invention

[0006] In order to solve the above problems, obtain a separation method that is simple to operate, short in time, low in cost and can be widely used, and the bacteria enriched by this method can also be used in high-demand mass spectrometry analysis processes. The present invention proposes a pretreatment kit for positive samples.

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

[0011] Preferably, the surface of the chemical magnetic beads is modified with positively charged groups, such as nitrogen-containing groups; or electronegative groups;

[0012] 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; further preferably, an aliphatic amine group, an aromatic amine group; such as a pyrrolyl group or a pyridyl group, and the electronegative group includes a carboxyl group, an alcohol / silanol group, a sulfonic acid group or a phosphoric acid group.

[0013] 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.

[0014] 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.

[0015] The present invention utilizes the strong interaction between positive and negative charges to achieve strong enrichment of bacteria by magnetic beads and form bacteria-magnetic bead complexes. The magnetic beads do not need additional immune modification, etc., and the operation is simple and the cost is low. 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 effects, and the pretreatment kit must have high stress resistance.

[0016] 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.

[0017] 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.

[0018] Preferably, the surface of the positive magnetic beads is modified with nitrogen-containing groups;

[0019] More preferably, the surface modification of the positive electromagnetic beads includes primary amine groups, secondary amino groups, tertiary amino groups or quaternary ammonium groups; further preferably, fatty amine groups, aromatic amine groups; such as pyrrolyl groups or pyridyl groups, and the surface modification of the negative electromagnetic beads includes carboxyl groups, alcohol / silanol groups, sulfonic acid groups or phosphate groups.

[0020] Preferably, the positive electromagnetic beads are selected from amino magnetic beads or imidazole magnetic beads, and the negative electromagnetic beads are selected from carboxyl magnetic beads, hydroxyl magnetic beads or sulfonic acid magnetic beads.

[0021] 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.

[0022] 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.

[0023] The "about" and "approximately" in the present invention represent a measurement error within ±5%. Preferably, the particle size of the positive electromagnetic beads or negative electromagnetic beads is less than 3000nm, preferably, the particle size is 5nm to 1000nm.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

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

[0032] The pH of the incubation solution is lower than 13, preferably, the pH of the incubation solution is 5-9.

[0033] Preferably, the metal ion is a metal cation.

[0034] Preferably, the incubation solution is selected from a solution containing Na+, K+, Ca+, Mg+ and / or EDTA.

[0035] Preferably, the incubation solution is selected from a solution containing 0.2-2% Na+, 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.

[0036] Furthermore, the kit includes chemical magnetic beads, incubation solution and elution solution.

[0037] The eluent comprises formic acid. Preferably, the concentration of the formic acid is 25-100%.

[0038] The eluent is used to disrupt bacteria and release bacterial proteins, which is convenient for subsequent mass spectrometry identification.

[0039] Unless otherwise specified, "%" in the present invention refers to mass percentage.

[0040] In some embodiments of the present invention, the incubation solution 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.

[0041] 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.

[0042] Furthermore, the kit also includes a cleaning solution, which is used to clean the bacteria-chemical magnetic bead complex, making the bacteria-chemical magnetic bead complex cleaner, so that the subsequent mass spectrometry identification effect can be better. The cleaning step is a routine operation selection in the field, and those skilled in the art can select a suitable cleaning reagent. The cleaning solution of the present invention is selected from one or more of water, buffer, metal salt solution, chelating agent, chaotropic salt, polycation or polyanion solution. Preferably, the chaotropic salt is selected from one or more of guanidine hydrochloride, guanidine thiocyanate, urea or lithium perchlorate; the polycation is selected from spermine and / or polylysine; the polyanion is selected from dextran and / or polyacrylic acid.

[0043] Preferably, the concentration of the chaotropic salt is 1-10 M, and the concentration of the polycation and / or polyanion is 1-100 mM.

[0044] 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.

[0045] The "chaotropic salt" described in the present invention is used to destroy hydrogen bonds and hydrophobic interactions.

[0046] On the other hand, the present invention discloses a pretreatment method for mass spectrometry identification of bacteria, characterized in that it comprises the above-mentioned pretreatment kit, and specifically comprises the following steps:

[0047] S10. Pre-treat the positive sample and add it to the incubation solution;

[0048] S20. Add the chemical magnetic beads to the sample treated in step S10 and mix well;

[0049] S30. Magnetic aspiration is performed on the sample treated in step S20, and the supernatant is discarded to obtain a bacteria-magnetic bead complex;

[0050] S40. Add the elution solution to disrupt the bacteria in the bacteria-magnetic bead complex.

[0051] Pretreatment may include centrifugation and / or blood cell lysis steps.

[0052] On the other hand, the present invention discloses a method for mass spectrometry identification of bacteria, characterized in that it specifically comprises the following steps:

[0053] S10. Pre-treat the positive sample and add it to the incubation solution;

[0054] S20. Add the chemical magnetic beads to the sample treated in step S10 and mix well;

[0055] S30. Magnetic aspiration is performed on the sample treated in step S20, and the supernatant is discarded to obtain a bacteria-magnetic bead complex;

[0056] S40. adding the eluent to disrupt the bacteria in the bacteria-magnetic bead complex;

[0057] S50. Mass spectrometry detection of disrupted bacteria.

[0058] Furthermore, a washing step is further included between steps S30 and S40, and the washing step is preferably performed more than once to reduce the influence of impurities; more preferably, the washing solution in the washing step has the same component as the incubation solution.

[0059] Furthermore, 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.

[0060] Preferably, the culture sample is a blood culture sample, that is, the above-mentioned bacteria-containing blood, urine, 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.

[0061] When the culture sample is a blood culture sample, in order to reduce the interference of blood cells, the blood cell lysis step achieves the corresponding purpose by adding a blood cell lysing agent. It can be understood that the lysing solution only lyses blood cells without destroying the cell wall or cell membrane of bacteria. The lysing agent is, for example, a surfactant, saponin or hemolysin.

[0062] Preferably, the bacteria are bacteria, fungi or a mixture of the two.

[0063] On the other hand, the present invention discloses the application of the chemical magnetic beads in the pre-treatment of mass spectrometry identification of bacteria, wherein the chemical magnetic beads are used to enrich bacteria in positive samples.

[0064] Another aspect of the present invention discloses the use of the chemical magnetic beads for enriching bacteria in positive samples.

[0065] Another aspect of the present invention discloses the enrichment effect of the chemical magnetic beads and incubation solution on bacteria in positive samples.

[0066] In another aspect, the present invention discloses a method for enriching bacteria in a positive sample, comprising the following steps: (1) resuspending the positive sample in the incubation solution after centrifugation; (2) adding the chemical magnetic beads and mixing; (3) magnetically aspirating and discarding the supernatant to obtain a bacteria-magnetic bead complex;

[0067] Preferably, the enrichment method may further include a washing step.

[0068] Beneficial effects:

[0069] (1) 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, and makes the interaction between the magnetic beads and the bacteria stronger due to opposite electrical properties. In addition, there are no excessive impurities in the reaction environment, and the efficient enrichment of bacteria can be achieved through simple operation and low cost. The entire enrichment process has strong resistance to impurity interference, and the activity state of the enriched bacteria will not change, which can be used for subsequent mass spectrometry identification and drug sensitivity testing.

[0070] (2) The present invention innovatively applies chemical magnetic beads to mass spectrometry identification and utilizes the strong adsorption ability of chemical magnetic beads of a specific particle size to bacteria, which not only saves costs but also greatly shortens the pre-processing time of mass spectrometry detection.

[0071] (3) After the magnetic beads adsorb bacteria, the bacteria-magnetic bead complex is cleaned with a cleaning solution, which helps to reduce interfering substances, improve the purity of protein detection, and further realize the identification of bacterial species by mass spectrometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 : Experimental results of magnetic bead adsorption effect in Example 2. DETAILED DESCRIPTION

[0073] 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.

[0074] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0075] Example 1 Magnetic Bead Preparation

[0076] A. Synthesis of amino magnetic beads

[0077] (1) Synthesis of amino magnetic beads 1

[0078] 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.

[0079] (2) Synthesis of amino magnetic beads 2

[0080] 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.

[0081] (3) Synthesis of amino magnetic beads 3

[0082] 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.

[0083] (4) Synthesis of amino magnetic beads 4

[0084] 10 g of commercial carboxyl magnetic beads (Dynabeads TM 5g 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.

[0085] (5) Amino magnetic beads 5

[0086] Purchased from Nanjing Dongna Biotechnology Co., Ltd. (Mag9002), particle size 100nm.

[0087] B. Synthesis of Imidazole-Based Magnetic Beads

[0088] Synthesis of Imidazole-based Magnetic Beads

[0089] 1. Synthesis of magnetic core

[0090] 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.

[0091] 2. Preparation of transition layer

[0092] 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;

[0093] 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;

[0094] 3. Preparation of hydrophobic layer

[0095] 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.

[0096] C. Carboxyl magnetic beads

[0097] (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.

[0098] Example 2 Magnetic Bead Adsorption Effect

[0099] The incubation solution was 0.9% NaCl solution.

[0100] 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:

[0101] S10. Centrifuge the blood culture positive sample, resuspend it in the incubation solution, add the magnetic beads, and mix well;

[0102] S20. Magnetic aspiration is performed on the sample treated in step S10, and the supernatant is discarded to obtain a bacteria-magnetic bead complex;

[0103] S30. washing the bacteria-magnetic bead complex;

[0104] S40. Culture the bacteria-magnetic bead complex on blood agar plate culture medium.

[0105] 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.

[0106] In actual operation, in preparation for subsequent drug sensitivity testing 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.

[0107] 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.

[0108] 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.

[0109] Table 1 Adsorption effect of magnetic beads of the present invention

[0110]

[0111]

[0112] 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.

[0113] 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%.

[0114] Comparative Example 1

[0115] 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:

[0116] S10. Add magnetic beads to the blood culture positive sample and mix well;

[0117] S20. Magnetic aspiration of the sample treated in step (1) is performed, and the supernatant is discarded to obtain a bacteria-magnetic bead complex;

[0118] S30. washing the bacteria-magnetic bead complex;

[0119] S40. Cultivate the bacteria-magnetic beads on blood agar plate medium

[0120] The results are shown in Table 2 below:

[0121] Table 2 Magnetic bead adsorption effect without incubation solution

[0122]

[0123]

[0124] 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.

[0125] 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.

[0126] Example 3 Effect of magnetic beads of different particle sizes on bacterial adsorption

[0127] 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.

[0128] Prepare amino magnetic beads with different particle sizes: 1nm, 5nm, 30nm, 150nm, 500nm, 1μm and 3μm.

[0129] 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 adopting the corresponding method;

[0130] 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.

[0131] The results of bacterial adsorption by magnetic beads of different particle sizes are shown in Table 3:

[0132] Table 3 Enrichment effect of magnetic beads with different particle sizes

[0133]

[0134]

[0135] 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.

[0136] Example 4 Study on the composition of incubation solution

[0137] 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.

[0138] 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, and the effects of each incubation solution on adsorption enrichment are shown in Table 5.

[0139] This embodiment lists some available incubation solutions, but it should be noted that the incubation solution of the present invention is not limited to this embodiment, and those skilled in the art can make corresponding adjustments according to needs.

[0140] In another embodiment, the metal ion content of the incubation solution is less than 10%, more preferably less than 2%; preferably, metal cations include Na + , K + , Ca + and / or Mg + .

[0141] Table 4 Incubation solution formula

[0142]

[0143] Table 5 Effects of different incubation solutions on magnetic bead enrichment

[0144]

[0145]

[0146] 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 positively charged 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.

[0147] Example 5 Effect of different test samples

[0148] 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)

[0149] Incubation solution: 0.9% NaCl

[0150] The specific operations are as follows:

[0151] S10. Resuspend the positive sample in the incubation solution, add magnetic beads, and mix well;

[0152] S20. Magnetic aspiration is performed on the sample treated in step S10, and the supernatant is discarded to obtain a bacteria-magnetic bead complex;

[0153] S30. washing the bacteria-magnetic bead complex;

[0154] S40. Cultivate the bacteria-magnetic beads on a blood agar plate medium;

[0155] The bacteria-containing bone marrow culture sample, cerebrospinal fluid culture sample, pleural effusion culture sample, ascites culture sample, pericardial fluid culture sample, joint fluid culture sample, hydrocele culture sample, and bile culture sample are samples of bacteria-containing bone marrow, cerebrospinal fluid, pleural effusion, ascites, pericardial fluid, joint fluid, hydrocele, bile and blood samples after being cultured in blood culture bottles. The purpose of culture is to amplify the number of bacteria to facilitate the needs of subsequent mass spectrometry identification. Therefore, the present invention has no special restrictions on 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, wherein the blood sample is pretreated with SDS cell lysis solution, and the enrichment effects of different samples are shown in Table 6.

[0156] Table 6 Enrichment effect of different samples of the present invention

[0157]

[0158] 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.

[0159] Comparative Example 2 No incubation solution

[0160] 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.

[0161] Table 7 Enrichment effect of different samples without incubation solution

[0162]

[0163]

[0164] 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.

[0165] Example 7 Mass spectrometry detection of blood culture samples

[0166] In this embodiment, the samples selected were blood culture positive samples, and the mass spectrometer was selected from Zhongyuan Huiji EXS3000.

[0167] Magnetic beads: 100 nm amino magnetic beads (purchased from JK-01-009-100 from Chico Biotechnology).

[0168] Incubation medium: 0.9% NaCl;

[0169] Sample: Add 10 ml of sterile defibrinated sheep blood and about 1000 CFU of the strain into a blood culture bottle and culture on a blood culture instrument until positive.

[0170] The implementation method is as follows:

[0171] S10. Centrifuge the sample, discard the supernatant, and resuspend in the incubation solution;

[0172] S20. Add magnetic beads to the sample treated in step S10 and mix well;

[0173] S30. Magnetic aspiration is performed on the sample treated in step S20, and the supernatant is discarded to obtain a bacteria-magnetic bead complex;

[0174] S40. Washing the bacteria-magnetic bead complex;

[0175] S50. Adding the eluent to disrupt the bacteria in the bacteria-magnetic bead complex;

[0176] S60. Mass spectrometry

[0177] The scheme of the present invention can be selected from manual operation or mechanical operation. Compared with manual operation, mechanical operation has higher efficiency and precision and less pollution. Manual operation can meet the requirements of mass spectrometry identification, but the effect of mechanical operation will be better than manual operation, so this embodiment shows the result of mechanical operation.

[0178] The specific steps of mechanical operation are as follows:

[0179] S10. Use a 1 mL syringe to draw 1 mL of the sample (blood culture positive sample) into a 1.5 mL centrifuge tube and centrifuge at 13,000 rpm for 2 min, then discard the supernatant;

[0180] S20. Add 500 μL of incubation solution to the precipitate, shake and mix, and add it to the first column of the deep-well plate of the blood culture positive sample magnetic bead pretreatment kit.

[0181] S30. Place the deep-well plate into a 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 absorption (Move Magnetic), washing (washing) and elution (Elution) steps.

[0182] S40. After the program is finished, use a pipette to draw 1 μL of eluent and drop it onto the target spot on the MALDI-TOF / MS target plate and wait for it to dry.

[0183] S50. After drying, add 1 μL of the matrix solution and wait for it to dry.

[0184] S60. After drying, place the target plate into MALDI-TOF / MS and select the blood culture positive sample spectrum acquisition program and the corresponding database for identification.

[0185] In some embodiments, step S20 may include a lysis step, and the reagents in the lysis step include but are not limited to the use of a surfactant.

[0186] The cleaning step in this embodiment is 2 times, and the cleaning solution is 0.9% NaCl solution

[0187] In this example, the bacteria in the positive blood culture samples were artificially added in order to prove the universality of the magnetic beads in adsorbing bacteria.

[0188] In some other embodiments, a lysis solution may be added to the centrifuge tube to dissolve the blood cells in the blood culture positive sample, thereby further reducing the interference of the sample itself.

[0189] The matrix solution and eluent are from the mass spectrometry system sample processing matrix solution kit of Zhongyuan Huiji Co., Ltd.

[0190] According to the mass spectrometry product manual, when the mass spectrometry output result scores "2" or above, the identification result can be considered reliable. The test results are shown in Table 8:

[0191] Table 8 Mass spectrometry identification results of blood culture positive samples

[0192]

[0193]

[0194] As can be seen from Table 8, the identification results scores for the 28 different strains were all above 2, indicating that the bacterial enrichment method and subsequent mass spectrometry identification method of the present invention can accurately detect bacteria to the species level and are reliable.

[0195] Example 9: Replacement of test sample

[0196] The specific operation method is the same as that in Example 8, except that the test samples are replaced with bacterial bone marrow culture samples, cerebrospinal fluid culture samples, pleural effusion culture samples, ascites culture samples, pericardial fluid culture samples, synovial fluid culture samples, hydrocele culture samples, bile culture samples, midstream urine and blood samples. The identification results are shown in Table 9 below.

[0197] Table 9 Test results of different culture samples

[0198]

[0199]

[0200] It can be seen that changing the test sample does not affect the detection effect, and mass spectrometry identification can accurately identify the bacteria contained in the sample to the species.

[0201] Example 10: No cleaning solution added

[0202] The detection method of this embodiment is consistent with that of Embodiment 8, only the mass spectrometry pretreatment procedure is adjusted, and the cleaning step is not performed. The detection results are shown in Table 10.

[0203] Table 10 Mass spectrometry identification results of blood culture positive samples

[0204]

[0205] It can be seen that all scores are above 2, and all bacteria can be accurately detected to the species level, but the scores are lower than those in Example 8, indicating that the cleaning step can improve the identification accuracy to a certain extent, but its impact is significantly smaller than that brought by the incubation solution.

[0206] 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, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A pre-treatment kit for positive samples, characterized in that: The pre-treatment kit contains chemical magnetic beads, and the positive sample is a bacteria-containing sample; The chemical magnetic beads are magnetic beads with positively charged groups or negatively charged groups modified on the surface; Wherein, the particle size of the chemical magnetic beads is 1nm to 3000nm, preferably, the particle size is 5nm to 1000nm.

2. The pretreatment kit according to claim 1, characterized in that 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.

3. The pretreatment kit according to claim 1, characterized in that The magnetic beads with positively charged groups are selected from amino magnetic beads or imidazole magnetic beads; the magnetic beads with negatively charged groups are selected from carboxyl magnetic beads, hydroxyl magnetic beads or sulfonic acid magnetic beads.

4. The pretreatment kit according to any one of claims 1 to 3, characterized in that The kit also contains an incubation solution, which is used to replace the reagent components in the bacteria-containing sample; Preferably, the incubation solution is a solution with a metal ion content of less than 2%, and more preferably, the pH of the incubation solution is less than 13.

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

6. The pretreatment kit according to any one of claims 1 to 5, characterized in that The kit also contains an elution solution for disrupting bacteria to release bacterial proteins; preferably, the elution solution includes formic acid.

7. The pretreatment kit according to any one of claims 1 to 6, characterized in that The kit further comprises a cleaning solution; preferably, the cleaning solution is selected from one or more of water, buffer, metal salt solution, chelating agent solution, chaotropic salt solution, polycation or polyanion solution; more preferably, the chaotropic salt is selected from one or more of guanidine hydrochloride, guanidine thiocyanate, urea or lithium perchlorate solution, the polycation solution is selected from spermine and / or polylysine solution, and the polyanion solution is selected from dextran and / or polyacrylic acid solution; more preferably, the concentration of the chaotropic salt is 1-10M, the concentration of the polycation and / or polyanion is 1-100mM, the concentration of the metal salt solution is 0.2-2%, and the concentration of the chelating agent is 0.1-1M.

8. A method for enriching bacteria in a positive sample, comprising the following steps: S10. Using the incubation solution to replace the reagent components in the bacteria-containing sample; S20. Add chemical magnetic beads to the sample and mix well; S30. Magnetic aspiration is performed on the sample, and the supernatant is discarded to obtain a bacteria-magnetic bead complex; Preferably, the enrichment method further comprises a chemical magnetic bead washing step; wherein, The chemical magnetic beads are selected from positively charged amino magnetic beads and imidazole magnetic beads or negatively charged carboxyl magnetic beads, hydroxyl magnetic beads and sulfonic acid magnetic beads. More preferably, the incubation solution has a metal ion content of less than 2%. Most preferably, the pH of the incubation solution is lower than 13.

9. The enrichment method according to claim 8, characterized in that The particle size of the chemical magnetic beads is 1 nm to 3000 nm, preferably, the particle size is 5 nm to 1000 nm.

10. A method for identifying bacteria by mass spectrometry, characterized in that: The steps include: S10. After pre-treating the positive sample, add the incubation solution; S20. Add chemical magnetic beads to the sample in step S10 and mix well; S30. Magnetic aspiration is performed on the sample treated in step S20, and the supernatant is discarded to obtain a bacteria-magnetic bead complex; S40. adding the eluent to disrupt the bacteria in the bacteria-magnetic bead complex; S50. Mass spectrometry detection of broken bacteria; Wherein, the chemical magnetic beads are selected from positively charged amino magnetic beads or imidazole magnetic beads, or negatively charged carboxyl magnetic beads, hydroxyl magnetic beads or sulfonic acid magnetic beads; Preferably, the incubation solution is a solution with a metal ion content of less than 2%; more preferably, the pH of the incubation solution is lower than 13.

11. The method according to claim 10, characterized in that The method further comprises a chemical magnetic bead washing step; preferably, the washing step is performed more than once; More preferably, the cleaning solution is selected from water, buffer, metal salt solution, chelating agent solution, chaotropic salt solution, polycation or polyanion solution; most preferably, the chaotropic salt is selected from guanidine hydrochloride, guanidine thiocyanate, urea or lithium perchlorate solution, the polycation solution is selected from spermine or polylysine solution, and the polyanion solution is selected from dextran or polyacrylic acid solution.

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