Detection method for verifying bacterial separation and enrichment efficiency of immune nano magnetic beads
By labeling bacteria with specific fluorescent probes and using immune nanomagnetic beads for enrichment separation, the problems of long time, harsh conditions and poor capture efficiency in the prior art are solved, and efficient and accurate bacterial isolation and enrichment effects are achieved.
Patent Information
- Application Number
- CN202510228448.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-26
- Publication Date
- 2025-04-22
AI Technical Summary
The prior art has problems such as long time consumption, harsh conditions and poor magnetic bead capture efficiency in the process of bacterial isolation and enrichment.
The target bacteria were labeled with a specific fluorescent probe and enriched and isolated using immune nanomagnetic beads. Capture efficiency was calculated by constant temperature oscillation incubation and magnetic field separation technology combined with confocal microscopy to observe and count fluorescently labeled bacteria.
It improves the specificity and capture efficiency of bacterial isolation, reduces detection time and laboratory infection risk, and is suitable for laboratory research and clinical diagnosis.
Smart Images
Figure CN119859667A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the fields of clinical auxiliary detection and biotechnology, and in particular to a detection method for verifying the efficiency of bacterial separation and enrichment by immune nanomagnetic beads. Background Art
[0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis, which ranks first in mortality rate among infectious diseases worldwide. Mycobacterium tuberculosis is the main pathogen that causes tuberculosis. Its highly variable characteristics make it a major challenge to global public health. It is extremely necessary to study a technology for efficiently enriching and separating Mycobacterium tuberculosis in samples.
[0003] Currently, immunomagnetic bead separation technology is widely used in the field of medical research to separate and enrich specific pathogens, such as bacteria and viruses, thereby speeding up the diagnosis process and improving the accuracy of detection. Its advantages lie in its high specificity, high sensitivity, rapid separation and easy operation.
[0004] However, the existing technology still has the following shortcomings: 1. Traditional bacterial isolation and culture methods take days or even weeks to obtain results, which leads to delays in diagnosis and treatment; 2. Bacteria require specific culture conditions, such as a low oxygen environment and a specific culture medium, which places high demands on laboratory facilities and technical levels; 3. Separation technology based on immunomagnetic beads still has limitations. The relative surface area of micron-sized magnetic beads is small, resulting in poor capture efficiency.
[0005] Therefore, the existing technologies for verifying the efficiency of bacterial separation and enrichment still need to be further improved. Summary of the invention
[0006] In view of this, and in view of the shortcomings of the prior art, the present invention proposes a detection method for verifying the efficiency of bacterial separation and enrichment by immune nanomagnetic beads, which solves the technical problems of the prior art that bacterial separation takes a long time, the conditions are harsh, and the magnetic bead capture efficiency is poor.
[0007] The technical solution of the present invention is achieved in this way:
[0008] The present invention provides a detection method for verifying the efficiency of bacterial separation and enrichment by immune nanomagnetic beads, comprising the following steps:
[0009] S1, use specific fluorescent probes to label target bacteria, dilute the labeled target bacteria by 10 1 -10 7 Multiple samples with different concentration gradients between times;
[0010] S2, pre-treat the immune nanoparticles, and then add 10 1-10 7 The samples with different concentrations are mixed thoroughly; the mixed samples are placed in a constant temperature oscillation device for incubation; after the incubation, the magnetic field separation technology is used to separate the immune nanomagnetic beads bound to the target bacteria from the unbound substances, and the separated substances are processed separately;
[0011] S3, prepare gel blocks, take samples and evenly distribute them on the surface of the gel blocks, cover them with coverslips after drying, seal the slides, and observe the enrichment and separation effects under a microscope; observe and count the number of fluorescently labeled bacteria in a specific field of view under a microscope, and calculate the capture efficiency of the immune nanomagnetic beads for the target bacteria based on the counting results.
[0012] On the basis of this technical solution, further preferably, the specific fluorescent probe in step S1 comprises fluorescent d-amino acids (FDAAs), ExBT or Ac3GlcNAz-1-P, which is used for labeling the cell wall of living bacteria.
[0013] On the basis of this technical solution, it is further preferred that the sufficient mixing in step S2 comprises the mixing process comprising the rotation speed of the oscillating device being 1000-2000 r / min and the time being 10-20 s.
[0014] On the basis of this technical solution, it is further preferred that the incubation conditions in step S2 are 37° C., an oscillation speed of 1200 r / min, and an incubation time of 20-30 min.
[0015] On the basis of this technical solution, it is further preferred that magnetic field separation technology is used to separate the immune nanomagnetic beads bound to the target bacteria from the unbound substances, specifically including the use of magnetic separation equipment for magnetic removal.
[0016] On the basis of this technical solution, it is further preferred that the surface of the immune nanomagnetic beads is modified with an antibody conjugate, and the antibody conjugate can specifically recognize a specific epitope of the target bacteria.
[0017] On the basis of this technical solution, it is further preferred to prepare a plurality of samples with different concentration gradients, specifically including: for each concentration gradient sample, preparing a sample group for control, obtaining supernatant and binding reaction with immune nanomagnetic beads respectively.
[0018] On the basis of this technical solution, it is further preferred to separate the immune nanomagnetic beads bound to the target bacteria from the unbound substances using magnetic field separation technology, specifically including: separating the immune nanomagnetic beads bound to the target bacteria from the unbound substances using magnetic field separation technology, obtaining a supernatant sample from the unbound substances, and separately processing the obtained supernatant sample and the immune nanomagnetic bead sample bound to the target bacteria.
[0019] On the basis of this technical solution, further preferably, the calculation formula of the capture efficiency is: capture efficiency = (control sample bacterial number - supernatant sample bacterial number) / control sample bacterial number.
[0020] On the basis of this technical solution, further preferably, the microscope is a confocal microscope.
[0021] The present invention provides a detection method for verifying the efficiency of bacterial separation and enrichment by immune nanomagnetic beads, which has the following beneficial effects compared with the prior art:
[0022] (1) The use of fluorescent probes to specifically label bacteria combined with specific antibodies on nanomagnetic beads improves the specificity of separation and reduces the adsorption of non-target bacteria or impurities, thereby improving the accuracy of detection; the nanomagnetic beads have a larger specific surface area and improve the capture efficiency, making it possible to effectively enrich Mycobacterium tuberculosis even when the number of Mycobacterium tuberculosis in the sample is small;
[0023] (2) It avoids the aerosols that may be generated in traditional centrifugation methods, reduces the risk of laboratory infection, and improves the safety of experimental operations; the use of confocal microscopy can visually observe the results of the separation of Mycobacterium tuberculosis, which is helpful for statistical evaluation of the separation effect; this method is not only suitable for laboratory research, but also for clinical diagnosis, which helps to improve the diagnostic efficiency and accuracy of tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0025] Figure 1 This is a flow chart of the detection method for verifying the efficiency of bacterial separation and enrichment of immune nanomagnetic beads as described in Example 1 of the present invention;
[0026] Figure 2 This is a schematic diagram of the adsorption effect of bacterial magnetic beads of different concentrations in Example 2 of the present invention;
[0027] Figure 3 This is a fluorescence magnified image of bacteria adsorbed by nanomagnetic beads in Example 2 of the present invention. DETAILED DESCRIPTION
[0028] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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.
[0029] The present invention provides a detection method for verifying the efficiency of bacterial separation and enrichment by immunonanomagnetic beads, which utilizes nanoimmunomagnetic beads to enrich and separate bacteria, directly observes and quantitatively analyzes the enrichment effect; and combines confocal microscopy to directly observe and calculate the capture efficiency, thereby enabling a faster and more intuitive evaluation of the enrichment effect and reducing the detection time.
[0030] In a preferred embodiment, H37Ra (Mycobacterium tuberculosis H37Ra) is selected for this experimental verification. It is an attenuated Mycobacterium tuberculosis strain that is widely used to study the immune response mechanism of tuberculosis. Compared with its toxic counterpart H37Rv, H37Ra has similar immunogenicity, but its toxicity to humans is extremely weak, so it is considered relatively safe for the host. This makes H37Ra an important tool strain for studying the immune response mechanism of Mycobacterium tuberculosis infection.
[0031] Specifically, this method is not only applicable to Mycobacterium tuberculosis, but can also be extended to the enrichment and separation of other bacteria by adjusting fluorescent probes and antibodies.
[0032] In a preferred embodiment, nanomagnetic beads are selected for verification experiments, which are biogenic nanocrystal magnetic beads (BNCM), which are single magnetic domain, cubic octahedral single crystals with a particle size of 50nm. The core component is Fe3O4 or Fe3S4, which is coated with a phospholipid, protein or glycoprotein biomembrane and coupled with LAM antibodies.
[0033] In a preferred embodiment, a fluorescent marker is selected in the validation experiment, and a clinically validated AIE probe that can specifically bind to the bacterial wall of Mycobacterium tuberculosis is selected.
[0034] In a preferred embodiment, the magnetic sorting equipment is selected from DynaMagTM-2REF 12321D L0T4619002 of Thermo Fisher Scientific, the imaging instrument is a Leica Stellars 5 confocal microscope, and the vortex mixer is selected from XH-D model of Wuxi Jerian Instrument Equipment Co., Ltd.
[0035] Example 1 A detection method for verifying the efficiency of bacterial separation and enrichment by immune nanomagnetic beads,
[0036] like Figure 1As shown, the following steps are included:
[0037] S1, use specific fluorescent probes to label the target bacteria, and prepare the labeled target bacteria into multiple samples with different concentration gradients; specifically, the labeled target bacteria are set to a specific concentration gradient, which helps to evaluate the changes in enrichment efficiency at different concentrations, thereby optimizing the enrichment conditions.
[0038] S2, taking the immune nano-magnetic beads for pretreatment, then adding samples of different concentrations and mixing them thoroughly; placing the mixed samples in a constant temperature oscillation device for incubation; after the incubation, using magnetic field separation technology to separate the immune nano-magnetic beads bound to the target bacteria from the unbound substances, and treating the separated substances separately;
[0039] S3, prepare gel blocks, take samples and evenly distribute them on the surface of the gel blocks, cover them with coverslips after drying, seal the slides, and observe the enrichment and separation effects under a microscope; observe and count the number of fluorescently labeled bacteria in a specific field of view under a microscope, and calculate the capture efficiency of the immune nanomagnetic beads for the target bacteria based on the counting results.
[0040] In a preferred embodiment, specifically, the specific fluorescent probe in step S1 comprises fluorescent d-amino acids (FDAAs), ExBT or Ac3GlcNAz-1-P, which is used for labeling the cell wall of living bacteria.
[0041] In a preferred embodiment, specifically, the sufficient mixing in step S2 comprises the mixing process of using an oscillating device at a speed of 1000-2000 r / min for a time of 10-20 s.
[0042] More specifically, the mixing speed should not be too long to avoid damaging the bacteria, thus ensuring sufficient contact between the sample and the nanomagnetic beads and improving the enrichment efficiency.
[0043] In a preferred embodiment, specifically, the incubation conditions in step S2 are 37° C., an oscillation speed of 1200 r / min, and an incubation time of 20-30 min. Accurate temperature and mixing speed control are required to ensure the stability and repeatability of the enrichment process.
[0044] In a preferred embodiment, specifically, magnetic field separation technology is used to separate the immune nanomagnetic beads bound to the target bacteria from the unbound substances, specifically including the use of magnetic separation equipment for magnetic removal.
[0045] In a preferred embodiment, specifically, the surface of the immune nanomagnetic beads is modified with an antibody conjugate, and the antibody conjugate can specifically recognize a specific epitope of the target bacteria.
[0046] In a preferred embodiment, specifically, a plurality of samples with different concentration gradients are prepared, and specifically also include: for each sample with a concentration gradient, a sample group for control, obtaining supernatant, and binding reaction with immune nanomagnetic beads is prepared respectively.
[0047] In a preferred embodiment, specifically, the magnetic field separation technology is used to separate the immune nanomagnetic beads bound to the target bacteria from the unbound substances, specifically including: using the magnetic field separation technology to separate the immune nanomagnetic beads bound to the target bacteria from the unbound substances, obtaining a supernatant sample from the unbound substances, and processing the obtained supernatant sample and the immune nanomagnetic bead sample bound to the target bacteria separately.
[0048] In a preferred embodiment, specifically, the capture efficiency is calculated as follows: capture efficiency = (control sample bacterial count - supernatant sample bacterial count) / control sample bacterial count.
[0049] In a preferred embodiment, specifically, the microscope is a confocal microscope.
[0050] Example 2
[0051] A detection method for verifying the efficiency of bacterial separation and enrichment by immunonanomagnetic beads comprises the following steps:
[0052] S1, using fluorescent probe AIE to specifically label Mycobacterium tuberculosis, the labeled bacteria were set to a concentration gradient of [10 6 ,10 5 ,10 4 ,10 3 ,10 2 ]CFU / mL; prepare 5 tubes of precipitate, 5 tubes of supernatant and 5 tubes of control, and mark them;
[0053] S2, take 40uL of enriched magnetic beads and place them in the 5 precipitation group EP tubes of the experimental group, use the magnetic sorting equipment to remove the supernatant by magnetic suction; add 100uL of each concentration of the sample to be enriched to the precipitation group EP tube, use the vortex equipment to mix for 15s; place the EP tube on the constant temperature mixing equipment, set the incubation temperature to 37°C, adjust the mixing speed to 1200r / min, so that the enriched magnetic beads are evenly dispersed in the solution, and set the incubation time to 30min; precise temperature and mixing speed control to ensure the stability and repeatability of the enrichment process. The magnetic suction time is 5min, take 100uL of supernatant to the EP tube, resuspend the remaining precipitate with 100uL 4% paraformaldehyde, and use 100uL PBS to wash the precipitate, avoiding blowing;
[0054] Specifically, the labeling of the precipitate, supernatant and control tubes is unique and distinguishable, ensuring the traceability of the experiment and the accuracy of the results.
[0055] S3, prepare agarose gel blocks, take 10uL of each group of samples, evenly distribute them on the surface of the gel blocks, cover with coverslips after drying, seal the slides, and observe the enrichment and separation effects under a microscope. The results of the separation of Mycobacterium tuberculosis were visualized using a Leica Stellars 5 confocal microscope, which provided intuitive evidence for evaluating the enrichment and separation effects.
[0056] The results are as follows Figure 2 , 3 As shown, Figure 2 The adsorption effect of magnetic beads for bacteria of different concentrations. Figure 2 It can be seen that even if the bacterial concentration is as low as 102CFU / mL, the nanomagnetic beads can still capture fluorescently labeled bacteria, indicating that this detection method verifies the capture efficiency is high; Figure 3 This is a fluorescence magnified image of bacteria adsorbed on magnetic beads. It can be seen that the fluorescently labeled bacteria and the magnetic beads are well combined, indicating that the nanomagnetic beads can effectively capture the fluorescent bacteria.
[0057] In summary, the present invention provides a detection method for verifying the efficiency of bacterial separation and enrichment by immune nanomagnetic beads. Compared with the prior art, chemical reagents may need to be used for staining during the enrichment process, and chemical modification and staining are required to verify the enrichment effect, and the operation is relatively complicated; the present invention mainly uses fluorescent labeling and magnetic bead separation, and uses fluorescent probes to label bacteria, which can directly observe and quantitatively analyze the enrichment effect. In addition, the use of chemical reagents is reduced, and the operation is safer.
[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A detection method for verifying the efficiency of bacterial separation and enrichment by immune nanomagnetic beads, characterized in that: The following steps are involved: S1, use specific fluorescent probes to label target bacteria, dilute the labeled target bacteria by 10 1 -10 7 Multiple samples with different concentration gradients between times; S2, pre-treat the immune nanoparticles, and then add 10 1 -10 7 The samples with different concentrations are mixed thoroughly; the mixed samples are placed in a constant temperature oscillation device for incubation; after the incubation, the magnetic field separation technology is used to separate the immune nanomagnetic beads bound to the target bacteria from the unbound substances, and the separated substances are processed separately; S3, prepare gel blocks, take samples and evenly distribute them on the surface of the gel blocks, cover them with coverslips after drying, seal the slides, and observe the enrichment and separation effects under a microscope; observe and count the number of fluorescently labeled bacteria in a specific field of view under a microscope, and calculate the capture efficiency of the immune nanomagnetic beads for the target bacteria based on the counting results.
2. The detection method for verifying the efficiency of bacterial separation and enrichment by immune nanomagnetic beads as claimed in claim 1, characterized in that: The specific fluorescent probe in step S1 comprises fluorescent d-amino acids (FDAAs), ExBT or Ac3GlcNAz-1-P, which is used for labeling the cell wall of living bacteria.
3. The detection method for verifying the efficiency of bacterial separation and enrichment by immune nanomagnetic beads as claimed in claim 1, characterized in that: The thorough mixing in step S2 includes the mixing process of the oscillating device at a speed of 1000-2000 r / min for a time of 10-20 s.
4. The detection method for verifying the efficiency of bacterial separation and enrichment by immune nanomagnetic beads as claimed in claim 1, characterized in that: The incubation conditions in step S2 are 37° C., an oscillation speed of 1200 r / min, and an incubation time of 20-30 min.
5. The detection method for verifying the efficiency of bacterial separation and enrichment by immune nanomagnetic beads as claimed in claim 1, characterized in that: Magnetic field separation technology is used to separate the immune nanomagnetic beads bound to the target bacteria from the unbound substances, specifically including the use of magnetic sorting equipment for magnetic removal.
6. The detection method for verifying the efficiency of bacterial separation and enrichment by immune nanomagnetic beads as claimed in claim 1, characterized in that: The surface of the immune nanomagnetic beads is modified with antibody conjugates, and the antibody conjugates can specifically recognize specific epitopes of target bacteria.
7. The detection method for verifying the efficiency of bacterial separation and enrichment by immune nanomagnetic beads as claimed in claim 1, characterized in that: The samples are prepared into a plurality of different concentration gradients, and specifically include: for each concentration gradient sample, a sample group for control, obtaining supernatant, and binding reaction with immune nanomagnetic beads is prepared respectively.
8. The detection method for verifying the efficiency of bacterial separation and enrichment by immune nanomagnetic beads as claimed in claim 1, characterized in that: The method utilizes magnetic field separation technology to separate the immune nano-magnetic beads bound to the target bacteria from the unbound substances, specifically comprising: utilizing magnetic field separation technology to separate the immune nano-magnetic beads bound to the target bacteria from the unbound substances, obtaining a supernatant sample from the unbound substances, and separately processing the obtained supernatant sample and the immune nano-magnetic bead sample bound to the target bacteria.
9. The detection method for verifying the efficiency of bacterial separation and enrichment by immune nanomagnetic beads as claimed in claim 1, characterized in that: The calculation formula of the capture efficiency is: capture efficiency = (control sample bacterial number - supernatant sample bacterial number) / control sample bacterial number.
10. The detection method for verifying the efficiency of bacterial separation and enrichment by immune nanomagnetic beads as claimed in claim 1, characterized in that: The microscope was a confocal microscope.