Kanamycin magnetic bead as well as preparation method and application thereof

Abstract

The invention discloses a kanamycin magnetic bead as well as a preparation method and application thereof, and belongs to the technical field of biological analysis. The kanamycin magnetic beads obtained after coupling modification of silicon hydroxyl magnetic beads and kanamycin through a silane coupling agent GPTMS can form hydrogen bonds with small grooves of pathogenic bacteria DNA to adsorb nucleic acid, the extraction rate is high, and the kanamycin magnetic beads have a certain splitting action on pathogenic bacteria; the kanamycin magnetic beads and the lysozyme are adopted for combined extraction of pathogenic bacteria DNA in blood, bacteria can be rapidly and massively split on the premise of avoiding human blood cell splitting, nucleic acid of pathogenic bacteria can be accurately extracted, interference of host nucleic acid is reduced, and the accuracy of a detection result is ensured.

Description

Technical Field

[0001] The present invention relates to the field of bioanalysis technology, and more particularly to kanamycin magnetic beads and a preparation method and application thereof. Background Art

[0002] Bloodstream infection refers to a systemic inflammatory response syndrome caused by pathogenic microorganisms such as bacteria and fungi invading the bloodstream. Pathogenic microorganisms exist transiently, intermittently or persistently in the circulating blood, causing damage to all organs of the body, such as heart valves and joints. In severe cases, it can lead to shock, multiple organ failure, disseminated intravascular coagulation, and even death. In recent years, due to the widespread use of invasive equipment and treatments such as intravenous catheter placement, mechanical ventilation, and parenteral administration, as well as the abuse of immunosuppressants and a large number of antibacterial drugs, the incidence of bloodstream infection has increased year by year. In addition, since bloodstream infection not only has a high mortality rate but also prolongs hospitalization time and increases hospitalization costs, it has attracted more and more attention from doctors, patients and researchers.

[0003] The most common cause of bloodstream infection is bacteria, such as Escherichia coli, Staphylococcus aureus and Pneumococcus. At present, blood culture is still the main reference method for bloodstream infection detection, but it is not an ideal gold standard due to its significant disadvantages such as low sensitivity and long time consumption. With the development of molecular biology technology, molecular biology diagnostic techniques represented by polymerase chain reaction (PCR) and second-generation sequencing technology have begun to be applied to the microbial detection of bloodstream infection. Molecular biology-based diagnostic methods have significant advantages such as fast speed, high sensitivity and small sample size.

[0004] However, all commercial kits on the market for enriching pathogens from whole blood require selective lysis of human blood cells. These methods will lyse red blood cells and white blood cells, while bacteria and fungi remain intact due to the protection of the cell wall, and then centrifuge to achieve pathogen enrichment and DNA extraction. However, the lysis of blood cells will release a large amount of mitochondrial and genomic DNA as interference. If DNase is used for treatment, there is a risk of reducing the concentration of pathogen DNA and increasing the complexity of the operation. In addition, inhibitors in the blood cell matrix, such as heparin, immunoglobulins, or iron associated with hemoglobin and lactoferrin, will also reduce the sensitivity of PCR detection.

[0005] Therefore, quickly separating and enriching pathogenic microorganisms from whole blood and extracting nucleic acids that can be used for molecular biological detection is of great significance for improving the detection efficiency of bloodstream infection pathogens, providing timely guidance to doctors for reasonable and targeted medication and treatment, and at the same time gaining golden treatment time for patients, thereby increasing their survival rate. Summary of the invention

[0006] In view of the above problems, the present invention provides a new method for extracting DNA of multiple pathogens in blood with simple operation, low cost and high sensitivity. The present invention can avoid the interference of a large amount of host nucleic acid, extract the DNA of pathogens in blood in a targeted manner, and improve the accuracy of detection.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The invention discloses kanamycin magnetic beads, which are obtained by coupling and modifying silanol magnetic beads and kanamycin through a silane coupling agent GPTMS.

[0009] Another object of the present invention is to provide a method for preparing kanamycin magnetic beads, comprising the following steps:

[0010] (1) adding kanamycin and silane coupling agent GPTMS to water in proportion, mixing by ultrasonic treatment, and incubating in a metal bath to obtain a GPTMS-kanamycin solution;

[0011] (2) After mixing the silicon hydroxyl magnetic beads with the piranha solution in a certain proportion, the beads are immersed in a metal bath, and the immersed magnetic beads are washed with deionized water until the pH value is 7, and then dried until completely dry;

[0012] (3) The magnetic beads dried in step (2) are mixed with the GPTMS-kanamycin solution in proportion, and the mixture is soaked. The soaked magnetic beads are washed with deionized water to remove the unreacted GPTMS-kanamycin solution, and the beads are dried until completely dry. Deionized water is added to resuspend the beads, and kanamycin magnetic beads are obtained.

[0013] The epoxy group of the GPTMS coupling agent ((3-epoxypropyl)methyldioxysilane) was covalently linked to the amino group of kanamycin, and then the Si atom of GPTMS was linked to the Si-OH bond of the silanol magnetic beads to form a Si-O-Si bond.

[0014] Preferably, the ultrasonic treatment in step (1) is performed at 50 Hz for 5-30 min.

[0015] Preferably, the ratio of the water, kanamycin and the silane coupling agent GPTMS in step (1) is 1 ml: 3 mg: 5 μl. By setting the ratio of the water, kanamycin and the silane coupling agent, the coupling agent and kanamycin can be effectively coupled.

[0016] Preferably, the metal bath incubation condition in step (1) is: incubation at 30-50° C. for 0.5-5 h. The metal bath incubation is conducive to the combination of kanamycin solution and GPTMS.

[0017] Preferably, the concentration of the silicon hydroxy magnetic beads in step (2) is 10-100 mg / mL; the volume ratio of H2SO4 to H2O2 in the piranha solution is 2-3:1; and the volume ratio of the silicon hydroxy magnetic beads to the piranha solution is 1:1-5. Infiltration of the silicon hydroxy magnetic beads with the piranha solution can expose more hydroxyl groups.

[0018] Preferably, the metal bath infiltration in step (2) is carried out at a temperature of 60-90° C. for 4-10 minutes. The metal bath infiltration is advantageous in exposing more hydroxyl groups for better coupling with GPTMS.

[0019] Preferably, the amount of the GPTMS-kanamycin solution added in step (3) is 3 times the volume of the hydroxy magnetic beads in step (1); and the infiltration conditions are: infiltration at room temperature for 6-24 hours.

[0020] Preferably, the drying temperature in step (2) and step (3) is 56°C.

[0021] Another object of the present invention is to provide the use of the kanamycin magnetic beads in the extraction and / or detection of DNA of multiple pathogens in blood.

[0022] Another object of the present invention is to provide the use of the kanamycin magnetic beads in preparing a kit for extracting DNA of multiple pathogens in blood.

[0023] Another object of the present invention is to provide a kit for extracting DNA of multiple pathogens in blood, comprising lysozyme, washing solution I, washing solution II, eluent and kanamycin magnetic beads.

[0024] The results showed that both kanamycin magnetic beads and lysozyme could lyse bacteria and had no effect on blood cells. Moreover, the nucleic acid quality obtained by combining kanamycin magnetic beads and lysozyme for bacterial lysis was the best.

[0025] Preferably, the washing solution I is 0.6 mol / L guanidine hydrochloride + 100 mmol / L Tris-Hcl + 30% (w / w) ethanol solution; the washing solution II is 0.6 mol / L guanidine hydrochloride + 100 mmol / L Tris-Hcl + 30% (w / w) ethanol solution.

[0026] Preferably, the elution solution is a 0.1% NH3·H2O solution. The 0.1% NH3·H2O solution can well separate the kanamycin magnetic beads from the DNA of the pathogen and has no effect on the subsequent amplification.

[0027] Through the above technical scheme, it can be known that the present invention discloses a kanamycin magnetic bead and its preparation method and application. The kanamycin magnetic beads obtained by coupling and modifying silanol magnetic beads and kanamycin through the silane coupling agent GPTMS can adsorb nucleic acids by forming hydrogen bonds with the minor groove of pathogenic bacteria DNA, with a high extraction rate and a certain lysis effect on pathogenic bacteria; kanamycin magnetic beads and lysozyme are used to jointly extract pathogenic bacteria DNA in blood, which can quickly and massively lyse bacteria under the premise of avoiding human blood cell lysis, and can accurately extract nucleic acids of pathogens, reduce interference of host nucleic acids, and ensure the accuracy of the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : Comparative analysis of infrared spectra of silicon hydroxy magnetic beads and kanamycin magnetic beads, a: kanamycin magnetic beads, b: silicon hydroxy magnetic beads;

[0029] Figure 2 : The amount of nucleic acid adsorbed by kanamycin magnetic beads prepared in Example 1 and the determination of the adsorption efficiency;

[0030] Figure 3A :Statistical diagram of the elution efficiency of kanamycin magnetic beads and DNA by different elution buffers;

[0031] Figure 3B : Fluorescence curves of PCR amplification after nucleic acid was eluted with different eluents;

[0032] Figure 4A : Agarose gel electrophoresis of washing solution screening for nucleic acid extraction using kanamycin magnetic beads, M: 150000bp Marker, 1: washing solution a, 2: washing solution b, 3: washing solution c, 4: washing solution d, 5: washing solution e;

[0033] Figure 4B : Agarose gel electrophoresis diagram of optimized washing solution concentration for nucleic acid extraction using kanamycin magnetic beads, M: 150000bp Marker, 1: washing solution d-ethanol, 2: washing solution d-0.1, 3: washing solution d-0.2, 4: washing solution d-0.4, 5: washing solution d-0.6, 6: washing solution d-1.2, 7: washing solution d-2.4;

[0034] Figure 5A : The results of kanamycin magnetic beads and lysozyme treating Escherichia coli and blood cells for 10, 20, and 45 minutes respectively (600× magnification of the microscope);

[0035] Figure 5B:Detection diagram of the lysis and extraction ability of lysozyme and kanamycin magnetic beads on bacteria, M: Maeker, 1: 95℃, 5min high temperature lysis + silanol magnetic beads extraction, 2: kanamycin magnetic beads lysis for 20min + kanamycin magnetic beads extraction, 3: kanamycin magnetic beads and lysozyme lysis bacteria co-lysis for 20min + kanamycin magnetic beads extraction;

[0036] Fig. 6A : Fluorescence curves of PCR reaction after blood samples with different E. coli concentrations were treated with kanamycin magnetic beads extraction system, 1:10 6 cfu / mL, 2:10 5 cfu / mL, 3:10 4 cfu / mL, 4:10 3 cfu / mL, 5:10 2 cfu / mL, 6:10 1 cfu / mL, 7:10 0 cfu / mL, 8: NTC negative control;

[0037] Figure 6B : The relationship between the Ct value of the PCR reaction and the logarithm (lgC) of the E. coli concentration after the blood samples with different E. coli concentrations were treated with the kanamycin magnetic bead extraction system;

[0038] Figure 7: PCR amplification test results of the target extract obtained after the kanamycin magnetic bead extraction system was used to extract nucleic acids from blood samples infected with multiple pathogens. Fig. 7A :E. coli detection, Figure 7B :Klebsiella pneumoniae detection, Figure 7C :Enterococcus faecalis detection, Fig.7D : Staphylococcus aureus detection, Fig. 7E : Pseudomonas aeruginosa detection. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] The reagents involved in the embodiments of the present invention are all purchased from commercial channels, and the methods not mentioned are conventional experimental methods and will not be described in detail here.

[0041] The following reagents are illustrative:

[0042] Silane magnetic beads (particle size 300 nm, concentration 50 mg / mL, solvent 0.05% sodium azide aqueous solution) were purchased from Wuxi Biomag Biotechnology Co., Ltd.

[0043] DNA extraction kit (B518725-0050) was purchased from Shanghai Sangon Biotech Co., Ltd., China.

[0044] Escherichia coli (E. coli, ATCC8739), Klebsiella pneumoniae (K. pneumoniae, CMCC46117), Enterococcus faecalis (E. faecalis, AC12861), Staphylococcus aureus (S. aureus, ATCC6538) and Pseudomonas aeruginosa (P. aeruginosa, ATCC10145) can all be purchased through commercial channels.

[0045] Blood: Blood was obtained from the tail or eye of healthy BALB / c mice.

[0046] Kanamycin and lysozyme (A610308) were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.

[0047] Example 1 Preparation of Kanamycin Magnetic Beads

[0048] (1) Weigh 3 mg of kanamycin powder and dissolve it in 1 ml of water. Add 5 μl of silane coupling agent GPTMS, place it in an ultrasonic cleaner, treat it with ultrasound at 50 Hz for 5 min, and then incubate it in a metal bath at 30° C. for 0.5 h to obtain a GPTMS-kanamycin solution.

[0049] (2) Take 50 μl of 50 mg / mL hydroxy magnetic beads (300 nm) in a 1 ml centrifuge tube, add 100 μl of piranha solution (H2SO4:H2O2=2:1), soak in a 60°C metal bath for 10 min, remove the piranha solution from the tube, repeatedly wash the soaked magnetic beads with deionized water to a pH of 7, move into an oven, and bake at 56°C until completely dry.

[0050] (3) Take out the dried magnetic beads, add 150 μl of GPTMS-kanamycin solution, soak at room temperature for 6 h, wash the soaked magnetic beads with deionized water to remove the unreacted GPTMS-kanamycin solution, move them into an oven, bake them at 56°C until completely dry, and add deionized water to a total volume of 50 μl to obtain kanamycin magnetic beads.

[0051] The product prepared above was analyzed by infrared spectroscopy with hydroxy magnetic beads. Figure 1 ,Depend on Figure 1 It can be seen that the spectral lines corresponding to the above products are at 1600, 1384, 1095, 780, 467 cm-1 relative to the spectral lines of hydroxyl magnetic beads.-1 There is an obvious absorption peak at the position, indicating that the kanamycin magnetic beads are successfully prepared by the present invention.

[0052] Example 2 Determination of nucleic acid extraction efficiency of kanamycin magnetic beads

[0053] After extracting the genomic DNA of Escherichia coli using a DNA extraction kit, 200, 400, 600, 800, 1000, 1500, 2000, 3000, and 4000 ng of DNA samples with different mass gradients were prepared, and 200 μg of kanamycin magnetic beads (prepared by the method in Example 1) were added respectively, and adsorbed for 5 minutes at room temperature (vortex oscillation was performed for 2.5 minutes during the period to fully mix the kanamycin magnetic beads and the DNA sample). After the end, the remaining DNA after the kanamycin magnetic beads were adsorbed was measured using a QuickDrop ultra-micro spectrophotometer. The capture amount and capture rate of kanamycin magnetic beads on DNA were calculated according to the following formula, and the results are shown in Figure 2 .

[0054] Capture amount = total amount of sample DNA - amount of DNA remaining;

[0055] Capture rate = capture amount ÷ total amount of sample DNA.

[0056] Depend on Figure 2 It can be seen that when the DNA amount of the sample is between 200ng and 1000ng, the capture efficiency of kanamycin magnetic beads for DNA can be maintained above 80%, and the maximum adsorption amount of DNA by 200μg magnetic beads is 1400ng, which shows its efficient DNA adsorption ability.

[0057] Example 3 Optimization of nucleic acid extraction system

[0058] 1. Eluate screening

[0059] The genomic DNA of Escherichia coli was extracted using a DNA extraction kit and prepared to a concentration of 50 ng / μL for later use.

[0060] Take 5 centrifuge tubes numbered 1-5, add 10 μl of Escherichia coli genomic DNA (50 ng / μL) and 100 ng of kanamycin magnetic beads (prepared by the method in Example 1) to each test tube, vortex and mix at room temperature, then incubate for 2 min, place on a magnetic stand for magnetic separation, and use a QuickDrop ultra-micro spectrophotometer to measure the amount of DNA in the supernatant, which is the amount of remaining DNA in the supernatant after adsorption;

[0061] After the measurement, the supernatant was discarded, 30 μl of TE buffer was added to test tube No. 1, 30 μl of 1×PBS was added to test tube No. 2, 30 μl of 10×PBS was added to test tube No. 3, 30 μl of 0.1% NH3·H2O was added to test tube No. 4, and 30 μl of DEPC water was added to test tube No. 5. The samples were vortexed and incubated for 5 min at room temperature, and placed on a magnetic stand for magnetic separation. The supernatant obtained was the eluted nucleic acid. The DNA of the eluted supernatant was measured using a QuickDrop ultra-micro spectrophotometer. The elution efficiency was calculated according to the following formula. The results are shown in Figure 3A .

[0062] Elution efficiency = the amount of DNA in the eluted supernatant / (the total amount of sample DNA - the amount of DNA remaining in the supernatant after adsorption).

[0063] from Figure 3A It can be seen that the elution efficiency of DNA by TE buffer, 1×PBS and DEPC water is less than 20%, and the elution efficiency of 10×PBS is 80%; while the elution efficiency of DNA by 0.1% NH3·H2O is the highest, with an elution efficiency of 88%.

[0064] Using E. coli-F (5'-AATAAGAGCAGGCAAGCTAT-3', SEQ ID No.1) and E. coli-R (5'-ATGTTGGAACCGTAAGAGAA-3', SEQ ID No.2) as primers, the eluted nucleic acid was amplified by PCR. The PCR amplification system (20 μl) was: 10×taqbuffer 2 μl, dNTP 0.4 μl at a concentration of 10 mmol / L, 2×Evagreen 0.5 μl, upstream and downstream primers 0.4 μl each at a concentration of 10 μmol / L, Taq enzyme (5 U / μL) 0.2 μl, amplification template 2 μl, and DEPC water supplemented to 20 μl; the PCR reaction program was: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 10 s, annealing at 60°C for 10 s, and extension at 72°C for 30 s, for a total of 40 cycles.

[0065] Set up a positive control: In the PCR amplification system, use 2 μl of nucleic acid eluted with TE as template DNA;

[0066] Set up a negative control: In the PCR amplification system, replace the template DNA with 2 μl of DEPC water.

[0067] PCR amplification curve is shown in Figure 3B ,Depend on Figure 3B It can be seen that 10×PBS inhibits PCR amplification, and 0.1% NH3·H2O has no effect on PCR, so 0.1% NH3·H2O was finally selected as the elution solution.

[0068] 2. Screening of washing solution composition

[0069] Prepare washing solutions of different compositions according to the following formula and set aside.

[0070] Washing solution a: 70 mmol / L NaCl + 30 mmol / L 3-morpholinepropanesulfonic acid + 50% (w / w) ethanol solution;

[0071] Washing solution b: ① 60% (w / w) PEG-6000 + 100 mmol / L NaCl solution, ② 70% (w / w) ethanol solution;

[0072] Washing solution c: 40% (w / w) PEG-6000 + 100 mmol / L NaCl + 50% (w / w) ethanol solution;

[0073] Washing solution d: ① 1 mol / L guanidine hydrochloride + 100 mmol / L Tris-HCl + 30% (w / w) ethanol solution, ② 1 mol / L guanidine hydrochloride + 100 mmol / L Tris-HCl + 80% (w / w) ethanol solution;

[0074] Washing solution e: 75% (w / w) ethanol solution.

[0075] Take 5 centrifuge tubes numbered a, b, c, d, and e, and add 1 ml (1×10 7 coli liquid (200 μl, 200 μl lysozyme (A610308) with a concentration of 20 mg / ml) and 200 μg of kanamycin magnetic beads (prepared by the method of Example 1) were added, vortexed at room temperature for mixing and then allowed to stand for 20 min, magnetic separation was performed on a magnetic rack and the supernatant was discarded, 500 μl of washing solution with corresponding letters were added respectively, vortexed for 5 s and the supernatant was discarded, the magnetic beads were washed twice (when the solutions used for the two washes were different, ① indicated the solution used for the first wash, and ② indicated the solution used for the second wash), the supernatant was discarded, the lid was opened and allowed to stand for 8 min, 30 μl of 0.1% NH3·H2O was added as an eluent, vortexed and incubated for 5 min at room temperature, and placed on a magnetic rack for magnetic separation, and the supernatant obtained was the eluted nucleic acid.

[0076] The eluted nucleic acid was amplified by PCR using the method of Example 5.1. The electrophoresis results are shown in Figure 4A .

[0077] Depend on Figure 4AIt can be seen that among the 5 lanes, the band of lane 4 (corresponding to washing solution d) is the widest and brightest in color, indicating that after the kanamycin magnetic beads extract the nucleic acid, on the basis of the same eluent (both 0.1% NH3·H2O), when the first wash uses 1 mol / L guanidine hydrochloride + 100 mmol / L Tris-Hcl + 30% (w / w) ethanol solution, and the second wash uses 1 mol / L guanidine hydrochloride + 100 mmol / L Tris-Hcl + 80% (w / w) ethanol solution, the purity of the nucleic acid obtained after elution with the eluent is the highest and the amount extracted is the largest.

[0078] 3. Optimization of washing solution concentration

[0079] In order to obtain purer DNA, the concentrations of the above-screened washing solution d (①1 mol / L guanidine hydrochloride + 100 mmol / L Tris-Hcl + 30% (w / w) ethanol solution, ②1 mol / L guanidine hydrochloride + 100 mmol / L Tris-Hcl + 80% (w / w) ethanol solution) were optimized, specifically: the concentration of guanidine hydrochloride was set to a concentration gradient of 0.1 mol / L, 0.2 mol / L, 0.4 mol / L, 0.6 mol / L, 1.2 mol / L, and 2.4 mol / L, and the concentrations of the remaining components were the same as those in the above-screening of the washing solution composition. The prepared washing solutions of different concentrations are as follows:

[0080] Washing solution d-0.1: ① 0.1 mol / L guanidine hydrochloride + 100 mmol / L Tris-Hcl + 30% (w / w) ethanol solution, ② 0.1 mol / L guanidine hydrochloride + 100 mmol / L Tris-Hcl + 80% (w / w) ethanol solution;

[0081] Washing solution d-0.2: ① 0.2 mol / L guanidine hydrochloride + 100 mmol / L Tris-Hcl + 30% (w / w) ethanol solution, ② 0.2 mol / L guanidine hydrochloride + 100 mmol / L Tris-Hcl + 80% (w / w) ethanol solution;

[0082] Washing solution d-0.4: ① 0.4 mol / L guanidine hydrochloride + 100 mmol / L Tris-Hcl + 30% (w / w) ethanol solution, ② 0.4 mol / L guanidine hydrochloride + 100 mmol / L Tris-Hcl + 80% (w / w) ethanol solution;

[0083] Washing solution d-0.6: ① 0.6 mol / L guanidine hydrochloride + 100 mmol / L Tris-Hcl + 30% (w / w) ethanol solution, ② 0.6 mol / L guanidine hydrochloride + 100 mmol / L Tris-Hcl + 80% (w / w) ethanol solution;

[0084] Washing solution d-1.2: ① 1.2 mol / L guanidine hydrochloride + 100 mmol / L Tris-HCl + 30% (w / w) ethanol solution, ② 1.2 mol / L guanidine hydrochloride + 100 mmol / L Tris-HCl + 80% (w / w) ethanol solution;

[0085] Washing solution d-2.4: ① 2.4 mol / L guanidine hydrochloride + 100 mmol / L Tris-Hcl + 30% (w / w) ethanol solution, ② 2.4 mol / L guanidine hydrochloride + 100 mmol / L Tris-Hcl + 80% (w / w) ethanol solution.

[0086] The blank control group washing solution d-ethanol was set up: ① 30% (w / w) ethanol solution, ② 80% (w / w) ethanol solution.

[0087] Take 7 centrifuge tubes and add 1 ml of E. coli solution (1×10 7 cfu / mL), 200 μl of 20 mg / ml lysozyme solution (A610308) and 200 μg of kanamycin magnetic beads (prepared by the method of Example 1), vortexed and mixed at room temperature, and then incubated for 20 min. After magnetic separation on a magnetic rack, the supernatant was discarded, and 500 μl of the above-mentioned washing solution was added respectively. After vortexing for 5 s, the supernatant was discarded. The magnetic beads were washed twice (① was the solution used for the first washing, and ② was the solution used for the second washing), the supernatant was discarded, the lid was opened and allowed to stand for 8 min, and 30 μl of 0.1% NH3·H2O was added as an eluent. The mixture was vortexed and incubated for 5 min at room temperature. The mixture was placed on a magnetic rack for magnetic separation, and the supernatant obtained was the eluted nucleic acid.

[0088] The eluted nucleic acid was amplified by PCR using the method of Example 5.1. The electrophoresis results are shown in Figure 4B .

[0089] Depend on Figure 4B It can be seen that among the 7 lanes, the band of lane 5 (corresponding to washing solution d-0.6) is the widest and brightest in color, indicating that after the kanamycin magnetic beads extract the nucleic acid, on the basis of the same elution solution (both 0.1% NH3·H2O), the Tris-Hcl and ethanol content of the washing solution remain unchanged during the two washes, and when the concentration of guanidine hydrochloride is 0.6 mol / L, the nucleic acid obtained after elution with the elution solution has the highest purity and the largest amount.

[0090] In summary, after the kanamycin magnetic beads were used to extract nucleic acid, the first washing solution was 0.6 mol / L guanidine hydrochloride + 100 mmol / L Tris-Hcl + 30% (w / w) ethanol solution, and the second washing solution was 0.6 mol / L guanidine hydrochloride + 100 mmol / L Tris-Hcl + 80% (w / w) ethanol solution. They were named washing solution I and washing solution II, respectively, for subsequent experiments.

[0091] Example 4 Detection of the lysis performance of kanamycin magnetic beads on pathogens and tissue cells in blood

[0092] With a concentration of 10 7 The following experiments were performed using Escherichia coli with a concentration of 10 cfu / mL as the bacterial liquid sample and blood from the tail vein of healthy BALB / c mice as the blood sample.

[0093] 1. Use kanamycin magnetic beads and lysozyme to treat Escherichia coli bacterial liquid samples and blood samples respectively, specifically:

[0094] Take 12 centrifuge tubes and divide them into four groups: A, B, C, and D. Add 500 μl of E. coli solution (1×10 7 cfu / mL), then add 10 μl of 50 mg / mL kanamycin magnetic beads (prepared by the method of Example 1), mix by inversion (avoid violent shaking), and incubate at room temperature for 10 min, 20 min, and 45 min respectively;

[0095] Add 500 μl of E. coli solution (10 7 cfu / mL), then add 100 μl of 20 mg / mL lysozyme solution (A610308), mix by inversion (avoid violent shaking), and incubate at room temperature for 10 min, 20 min, and 45 min respectively;

[0096] An equal amount of untreated E. coli liquid was used as the negative control group of groups A and B, and the E. coli liquid heated at 95°C for 5 min was used as the positive control group of groups A and B. After the treatment of groups A, B and the control group, 10 μl of the mixed liquid was taken from each group and dropped on a glass slide. After Gram staining, the samples were observed under a microscope at a magnification of 600×.

[0097] After adding 100 μl of blood sample to each centrifuge tube of group C, 10 μl of 50 mg / mL kanamycin magnetic beads (prepared by the method of Example 1) were added, and after inversion to mix (avoid violent shaking), they were incubated at room temperature for 10 min, 20 min, and 45 min respectively;

[0098] After adding 100 μl of blood sample to each centrifuge tube of group D, add 100 μl of 20 mg / mL lysozyme solution (A610308), mix by inversion (avoid violent shaking), and incubate at room temperature for 10 min, 20 min, and 45 min, respectively;

[0099] An equal amount of untreated blood samples was used as the negative control group for groups C and D. After the treatment of groups C, D and the control group, 10 μl of each sample was dropped on a glass slide and observed under a microscope at a magnification of 600×.

[0100] The above observations are shown in 5A. Figure 5A It can be seen that kanamycin magnetic beads and lysozyme can completely lyse bacteria within 20 minutes, but do not lyse blood cells, indicating that kanamycin magnetic beads have a lytic effect on Escherichia coli, and neither kanamycin magnetic beads nor lysozyme cause damage to blood cells.

[0101] 2. Equal amounts of hydroxy magnetic beads and kanamycin magnetic beads were used to extract nucleic acids from Escherichia coli bacterial samples under different lysis conditions, specifically:

[0102] ① Add 200 μl of E. coli solution (1×10 7 cfu / mL) at 12000rpm for 2min, discard the supernatant, add 200μl DEPC water, and heat and lyse at 95℃ for 5min; add 2μl silicon hydroxy magnetic beads (50mg / ml), vortex and oscillate at room temperature, incubate for 5min, place on a magnetic stand for magnetic separation, discard the supernatant, add 500μl washing solution I, vortex and oscillate for 5s, discard the supernatant, add 500μl washing solution II, vortex and oscillate for 5s, discard the supernatant; open the lid and let stand for 8min, add 30μl TE buffer, vortex and incubate for 5min at room temperature, place on a magnetic stand for magnetic separation, take the supernatant, and obtain the target extract.

[0103] ②Take 200 μl of E. coli solution (1×10 7 cfu / mL) in a centrifuge tube, add 4 μl of 50 mg / mL kanamycin magnetic beads (prepared by the method in Example 1), vortex and oscillate at room temperature, then incubate for 20 min, place on a magnetic stand for magnetic separation, discard the supernatant, add 500 μl of washing solution I, vortex and oscillate for 5 s, discard the supernatant, then add 500 μl of washing solution II, vortex and oscillate for 5 s, discard the supernatant; open the cover and let stand for 8 min, add 30 μl of TE buffer, vortex and incubate for 5 min at room temperature, place on a magnetic stand for magnetic separation, take the supernatant, and obtain the target extract.

[0104] ③Take 200 μl of E. coli solution (10 7cfu / mL) in a centrifuge tube, add 4 μl of 50 mg / mL kanamycin magnetic beads (prepared by the method of Example 1) and 40 μl of lysozyme solution (20 mg / ml) in sequence, incubate at room temperature for 20 min, add 500 μl of washing solution I, vortex and oscillate for 5 seconds, discard the supernatant, add 500 μl of washing solution II, vortex and oscillate for 5 seconds, discard the supernatant; open the lid and let it stand for 8 min, add 30 μl of 0.1% NH3·H2O solution, vortex and oscillate at room temperature for 5 min, place on a magnetic stand for magnetic separation, take the supernatant, and obtain the target extract.

[0105] The supernatant obtained from ①②③ was subjected to PCR amplification detection (primers, amplification system and procedure were the same as in Example 5.1). The electrophoresis results were as follows: Figure 5B As shown, lanes 1, 2, and 3 all have bands at the same position, and the band in lane 3 (corresponding to ③) is the widest and brightest, followed by lane 1 (corresponding to ①), and finally lane 2 (corresponding to ②). This shows that for Escherichia coli with the same bacterial content, the amount of DNA extracted using silicon hydroxyl magnetic beads is much lower than the amount of DNA extracted using the combined method of kanamycin magnetic beads and lysozyme. This indicates that the effect of extracting DNA using kanamycin magnetic beads and lysozyme is better than that of silicon hydroxyl magnetic beads, and kanamycin magnetic beads can extract nucleic acids without external lysis conditions, which also shows that kanamycin magnetic beads have a certain bacterial lysis ability.

[0106] Therefore, the final kanamycin magnetic bead extraction system is: lysozyme, kanamycin magnetic beads, washing solution I, washing solution II and elution solution (0.1% NH3·H2O).

[0107] Example 5 Determination of the detection limit of Escherichia coli in blood by kanamycin magnetic bead extraction

[0108] Take 100 μl of E. coli solution with different concentration gradients and add it to 900 μl of blood to prepare a solution with an E. coli content of 10 6 cfu / mL, 10 5 cfu / mL, 10 4 cfu / mL, 10 3 cfu / mL, 10 2 cfu / mL, 10 1 cfu / mL, 10 0cfu / mL of blood samples, 1000 μl each, and the kanamycin magnetic bead extraction system was used to extract DNA from the above blood samples, specifically: 200 μl of lysozyme solution (20 mg / ml) and 10 μl of kanamycin magnetic beads with a concentration of 50 mg / mL (prepared by the method in Example 1) were added to each blood sample, vortexed and mixed at room temperature, and then incubated for 20 minutes, placed on a magnetic rack for magnetic separation, and then the supernatant was discarded, 500 μl of washing solution I was added, vortexed for 5 seconds, and then the supernatant was discarded, and then 500 μl of washing solution II was added, and the supernatant was discarded after vortexing for 5 seconds; the lid was opened and allowed to stand for 8 minutes, 30 μl of 0.1% NH3·H2O was added, and the mixture was vortexed and incubated for 5 minutes at room temperature, and then placed on a magnetic rack for magnetic separation, and the supernatant was taken to obtain the target extract.

[0109] Using E. coli-F (5'-AATAAGAGCAGGCAAGCTAT-3', SEQ ID No.1) and E. coli-R (5'-ATGTTGGAACCGTAAGAGAA-3', SEQ ID No.2) as primers, PCR amplification was performed on the target extract obtained by the above extraction. The PCR amplification system (20μl) was: 10×taq buffer 2μl, dNTP 0.4μl at a concentration of 10mmol / L, 2×Evagreen 0.5μl, upstream and downstream primers 0.4μl each at a concentration of 10μmol / L, Taq enzyme (5U / μL) 0.2μl, target extract 2μl, DEPC water supplemented to 20μl; PCR reaction program was: 95℃ pre-denaturation for 2min; 95℃ denaturation for 10s, 60℃ annealing for 10s, 72℃ extension for 30s, for a total of 40 cycles.

[0110] The target extract in the PCR amplification system was replaced with the same volume of water as a negative control group (denoted as NTC), and PCR amplification was performed according to the above method.

[0111] PCR amplification curve Fig. 6A As shown in the figure, in the blood samples, as the concentration of E. coli increased from 1×10 6 cfu / mL to 1 cfu / mL, the peak time of the signal just appeared kept moving forward, and the two were in direct proportion.

[0112] according to Fig. 6A The relationship between the Ct value of the PCR amplification reaction and the logarithm of the concentration of Escherichia coli in the blood sample (lgC) was further obtained (see Figure 6B ), and the regression equation is obtained: Ct = -2.299*1gC + 35.231, R 2=0.9852. It can be seen that the Ct value of the above PCR amplification reaction increases linearly with the increase of the negative logarithm of the concentration of Escherichia coli in the blood sample. This shows that the use of the kanamycin magnetic beads of the present invention to extract Escherichia coli DNA from blood does not affect the stability of PCR amplification detection.

[0113] Example 6: Using kanamycin magnetic bead extraction system to extract nucleic acid from blood samples infected with multiple pathogens

[0114] Blood containing Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, and Klebsiella pneumoniae, each of which has a concentration of 100 cfu / mL, is used as a blood sample infected with multiple pathogens. 200 μl of lysozyme solution (20 mg / ml) and 10 μl of kanamycin magnetic beads (prepared by the method in Example 1) with a concentration of 50 mg / mL are added to 1 mL of the blood sample, vortexed and mixed at room temperature, and then allowed to stand for 20 minutes, placed on a magnetic stand for magnetic separation, and then the supernatant is discarded. 500 μl of washing solution I is added, and the supernatant is discarded after vortexing for 5 seconds. 500 μl of washing solution II is added, and the supernatant is discarded after vortexing for 5 seconds; the cover is opened and allowed to stand for 8 minutes, 30 μl of 0.1% NH3·H2O is added, and the mixture is vortexed and incubated for 5 minutes at room temperature, and the mixture is placed on a magnetic stand for magnetic separation, and the supernatant is taken to obtain the target extract.

[0115] Using 2 μl of the target extract as a DNA template, the following primers were used to perform PCR amplification on the target extract:

[0116] Using E. coli-F (5'-AATAAGAGCAGGCAAGCTAT-3', SEQ ID No. 1) and E. coli-R (5'-ATGTTGGAACCGTAAGAGAA-3', SEQ ID No. 2) as primers, the extract was tested for the presence of E. coli DNA;

[0117] Using K. Pneumoniae -F (5'-ATGGCCGGGCATGGTACTTC-3', SEQ ID No. 3) and K. Pneumoniae -R (5'-ACCGGAGGTGATGTTTTCGGT-3', SEQ ID No. 4) as primers, the extract was tested for the presence of K. pneumoniae DNA;

[0118] Using E. faecalis -F (5'-TGAAACTCAAAGGAATTGACGG-3', SEQ ID No. 5) and E. faecalis -R (5'-CTCACGACACGAGCTGACGAC-3', SEQ ID No. 6) as primers, the extract was tested for the presence of Enterococcus faecalis DNA;

[0119] Using S.Aureu-F (5'-CGAGCATCATGGCTTTACAACT-3', SEQ ID No. 7) and S.Aureu-R (5'-AATCCAGCACGCTCTTCAGTTT-3', SEQ ID No. 8) as primers, the extract was tested for the presence of Staphylococcus aureus DNA;

[0120] Using P. aeruginosa -F (5'-AAGTACGAAGGCGGTCTGAA-3', SEQ ID No. 9) and P. aeruginosa -R (5'-GTTGTTGGTGAAGCAGAGCA-3', SEQ ID No. 10) as primers, the extract was tested for the presence of P. aeruginosa DNA;

[0121] The amplification system of the PCR reaction with different primers is the same, which is: 10×taqbuffer 2μl, dNTP with a concentration of 10mmol / L 0.4μl, 2×Evagreen 0.5μl, upstream and downstream primers with a concentration of 10μmol / L 0.4μl each, Taq enzyme (5U / μL) 0.2μl, amplification template 2μl, and DEPC water supplemented to 20μl;

[0122] The PCR amplification program was as follows: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 10 s, annealing at 60°C for 10 s, and extension at 72°C for 30 s, for a total of 40 cycles;

[0123] The PCR amplification system without template DNA was used as the negative control group. The amplification curves of the above PCR were shown in Fig. 7A -E. Figure 7A-7E Obvious amplification curves appeared in both of them, indicating that the kanamycin magnetic beads and the extraction system provided by the present invention can not only extract Escherichia coli ( Fig. 7A ), and can also extract Klebsiella pneumoniae from blood samples infected with multiple pathogens ( Figure 7B ), Enterococcus faecalis ( Figure 7C ), Staphylococcus aureus ( Fig.7D ) and Pseudomonas aeruginosa ( Fig. 7E ), can be applied to the extraction and detection of different types of single pathogenic microorganisms or mixed pathogenic bacteria.

[0124] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0125] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A kanamycin magnetic bead, characterized in that: The kanamycin magnetic beads are obtained by coupling and modifying silanol magnetic beads and kanamycin through a silane coupling agent GPTMS.

2. A method for preparing kanamycin magnetic beads, characterized in that: The following steps are involved: (1) adding kanamycin and silane coupling agent GPTMS to water in proportion, mixing by ultrasonic treatment, and incubating in a metal bath to obtain a GPTMS-kanamycin solution; (2) After mixing the silicon hydroxyl magnetic beads with the piranha solution in a certain proportion, the beads are immersed in a metal bath, and the immersed magnetic beads are washed with deionized water until the pH value is 7, and then dried until completely dry; (3) The magnetic beads dried in step (2) are mixed with the GPTMS-kanamycin solution in proportion, and the mixture is soaked. The soaked magnetic beads are washed with deionized water to remove the unreacted GPTMS-kanamycin solution, and the beads are dried until completely dry. Deionized water is added to resuspend the beads, and kanamycin magnetic beads are obtained.

3. The method for preparing kanamycin magnetic beads according to claim 2, characterized in that: In step (1), the ratio of water, kanamycin and silane coupling agent GPTMS is 1 ml: 0.5-10 mg: 1-10 μl; The conditions for the metal bath incubation are: incubation at 30-50° C. for 0.5-5 h.

4. The method for preparing kanamycin magnetic beads according to claim 2, characterized in that: The concentration of the silanol magnetic beads in step (2) is 10-100 mg / mL; The volume ratio of H2SO4 to H2O2 in the piranha solution is 2-3:1; The volume ratio of silanol magnetic beads to piranha solution was 1:1-5; The conditions of the metal bath infiltration are: infiltration at a temperature of 60-90° C. for 4-10 minutes.

5. The method for preparing kanamycin magnetic beads according to claim 2, characterized in that: The amount of the GPTMS-kanamycin solution added in step (3) is 3 times the volume of the hydroxy magnetic beads in step (1); The infiltration conditions are: infiltration at room temperature for 6-24 hours.

6. Use of the kanamycin magnetic beads according to claim 1 or the kanamycin magnetic beads obtained by the method according to any one of claims 2 to 5 in the extraction and / or detection of DNA of multiple pathogens in blood.

7. Use of the kanamycin magnetic beads according to claim 1 or the kanamycin magnetic beads obtained by the method according to any one of claims 2 to 5 in preparing a kit for extracting DNA of multiple pathogens in blood.

8. A kit for extracting DNA of multiple pathogens in blood, characterized in that: The invention comprises lysozyme, washing solution I, washing solution II, eluting solution and the kanamycin magnetic beads according to claim 1.

9. The extraction kit according to claim 7, characterized in that The washing solution I is 0.6 mol / L guanidine hydrochloride + 100 mmol / L Tris-Hcl + 30% (w / w) ethanol solution; The washing solution II is 0.6 mol / L guanidine hydrochloride + 100 mmol / L Tris-Hcl + 30% (w / w) ethanol solution.

10. The extraction kit according to claim 7, characterized in that The eluent is 0.1% NH3·H2O solution.

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