Application of polyhydroxy cationic quaternary ammonium salt magnetic beads constructed by amino epoxy ring opening in PCR (polymerase chain reaction) enrichment detection of pathogen aerosol

Abstract

The invention discloses application of polyhydroxy cation quaternary ammonium salt magnetic beads constructed by amino epoxy ring opening in pathogenic aerosol PCR (polymerase chain reaction) enrichment detection, epoxy quaternary ammonium salt modified ferroferric oxide magnetic beads are used for enriching pathogenic microorganisms, and the application method comprises the following steps: preparing the epoxy quaternary ammonium salt modified ferroferric oxide magnetic beads into a solution with the concentration of 0.01-1mg / mL; the magnetic bead solution is added into a cyclone cup matched with a wet-wall cyclone aerosol sampler, a cyclone sampling device of the aerosol sampler is started, a detection device is started for collection, and the collected sample is pathogen aerosol. The air pathogen aerosol is efficiently adsorbed and desorbed through the polyhydroxy cation quaternary ammonium salt magnetic beads, efficient enrichment of the pathogen aerosol is achieved, then PCR high-sensitivity detection is achieved, and the method is of great importance to prevention and control of pathogen propagation.

Description

Technical Field

[0001] The invention belongs to the field of pathogen aerosol PCR detection and relates to the application of polyhydroxy cationic quaternary ammonium salt magnetic beads constructed by amino-epoxy ring opening in pathogen aerosol PCR enrichment detection. Background Art

[0002] Air sampling and microbial detection are important links in aerosol detection, so efficient sampling and high-sensitivity detection methods are the guarantee for effective detection of microbial aerosols.

[0003] There are many methods for sampling air microbial aerosols, such as natural precipitation, solid impact, liquid impact, centrifugation, cyclone, and electrostatic adsorption. Liquid phase cyclone aerosol samplers are usually used to collect viral aerosols, and molecular detection can be performed directly after sampling without culture counting. Compared with other samplers, it is simple to operate and can effectively collect aerosol particles of various sizes. At present, we mainly use reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) to detect COVID-19 in clinical practice. With the occurrence and mutation of COVID-19, false negative results will occur from time to time during the detection process, so it is advocated to use more sensitive and accurate diagnostic techniques.

[0004] Since the concentration of pathogen aerosols in the air is relatively low, their detection requires high efficiency in each step. Many groups interested in improving the sensitivity of viral genome detection have tried to concentrate the virus. Ultracentrifugation of pathogenic microorganisms has been shown to be a superior concentration method, but it is very time-consuming and not suitable for screening purposes. Polyethylene glycol precipitation technology is simple and easy to concentrate pathogenic microorganisms, but excessive polyethylene glycol extracted will hinder the PCR reaction. Genome capture has advantages for concentrating specific viral genomes, but fixed probes for each virus must be prepared to capture multiple viruses. Therefore, it is of great significance to find a broader, more efficient, simple and feasible method for concentrating pathogens to improve the sensitivity of pathogen aerosol PCR detection.

[0005] An alternative to traditional methods is the use of magnetic beads coated with virus-specific antibodies, which can effectively bind to the virus and promote its concentration and collection through a magnetic field. Specific antibody magnetic beads are a potential method to improve the enrichment of pathogen aerosols. However, antibodies are specific and cannot meet the complex diversity of aerosol detection. Therefore, the development of a broad-spectrum and efficient pathogen aerosol enrichment system based on magnetic beads can achieve high-sensitivity detection of multiple pathogen aerosols while improving the sensitivity of PCR detection. Pathogenic aerosols, including airborne bacteria, fungi, viruses and their related substances such as endotoxins, all exhibit negative charge. Quaternary ammonium salts are a group with a strong positive charge that exhibits a positive charge in almost all pH ranges. Therefore, rapid adsorption of pathogenic aerosols can be achieved by magnetic beads with quaternary ammonium groups. Then, by adding an appropriate eluent, the pathogenic aerosol can be converted to an electrically neutral state, enabling it to be separated from the adsorbent into the eluent, thereby achieving efficient enrichment. In addition, there are other negatively charged harmful substances in the air, such as endotoxins, which will also produce electrostatic binding with positively charged magnetic beads, thereby reducing the efficiency of pathogen aerosol enrichment. A common strategy to reduce the adsorption of negatively charged impurities is to modify the surface with hydroxyl groups. For example, polyethylene glycol (PEG) can form a hydration layer on the surface of the material through hydrogen bonds, exerting a spatial repulsive force on harmful substances. However, PEG is expensive and requires strict storage conditions, so it is not suitable for large-scale sample preparation. Therefore, it is of great practicality and significance to prepare polyhydroxy cationic quaternary ammonium salt magnetic beads by a simple and convenient method for efficient enrichment of pathogen aerosols and achieve high-sensitivity PCR detection. Summary of the invention

[0006] In view of this, the present invention provides an application of polyhydroxy cationic quaternary ammonium salt magnetic beads constructed by amino-epoxy ring opening in pathogen aerosol PCR enrichment detection. The present invention specifically provides the following technical solutions:

[0007] 1. Application of polyhydroxy cationic quaternary ammonium salt magnetic beads constructed by amino-epoxy ring opening in PCR enrichment detection of pathogenic aerosols, wherein ferroferric oxide magnetic beads modified by epoxy quaternary ammonium salts are used to enrich pathogenic microorganisms, and the method of use is as follows: the ferroferric oxide magnetic beads modified by epoxy quaternary ammonium salts are prepared into a solution with a concentration of 0.01-1 mg / mL, the magnetic bead solution is added to the cyclone cup of a wet-wall cyclone aerosol sampler, the cyclone sampling device of the aerosol sampler is activated, and the detection device is turned on for collection, and the collected sample is a pathogenic aerosol.

[0008] Furthermore, the epoxy quaternary ammonium salt-modified ferroferric oxide magnetic beads are the product of an "amino-epoxy" ring-opening reaction between epoxy quaternary ammonium salt and amino ferroferric oxide magnetic beads, and the mass ratio of epoxy quaternary ammonium salt to amino ferroferric oxide magnetic beads is 0.5:1 to 0.8:1.

[0009] Furthermore, the epoxy quaternary ammonium salt is a reaction product of a small molecule of a primary amine group and a halogen alkane containing an epoxy group, wherein the molar ratio of the amino group on the small molecule of the primary amine group to the epoxy group on the halogen alkane containing an epoxy group is 1:1.

[0010] Furthermore, the small molecule of the primary amine group is N,N-dimethylethanolamine.

[0011] Furthermore, the halogen alkane containing an epoxy group is epichlorohydrin.

[0012] Further, the preparation method of the ferroferric oxide magnetic beads modified by epoxy quaternary ammonium salt is:

[0013] 1) Small molecules of primary amine groups react with halogenated alkanes containing epoxy groups to obtain epoxy quaternary ammonium salts;

[0014] 2) subjecting the epoxy quaternary ammonium salt to an aminoferric oxide magnetic bead for an amine-epoxy ring-opening reaction to obtain a product.

[0015] Furthermore, the reaction temperature of step 1) is 25-70° C., and the reaction time is 12 h to 48 h.

[0016] Furthermore, the reaction solvent of step 2) is dimethyl sulfoxide, the reaction temperature is 40-70° C., and the reaction time is 24 h-72 h.

[0017] Furthermore, the pathogenic microorganisms in the pathogenic aerosol are influenza virus, porcine coronavirus, Staphylococcus aureus, adenovirus, Staphylococcus aureus, Haemophilus influenzae, Streptococcus pneumoniae or Klebsiella pneumoniae.

[0018] Furthermore, the enrichment efficiency of Klebsiella pneumoniae was increased by 2-1000 times, and the enrichment efficiency of Staphylococcus aureus was increased by 2-1000 times.

[0019] The beneficial effects of the present invention are that the concentration of pathogenic microorganisms in the air is relatively low, and the existing cyclone sampling cannot efficiently enrich the pathogenic microorganisms in the air, and often produces false negative results. The present application prepares polyhydroxy cationic quaternary ammonium salt magnetic beads by a simple and convenient method, and achieves efficient enrichment of pathogenic aerosols through the efficient adsorption and desorption of air pathogenic aerosols by polyhydroxy cationic quaternary ammonium salt magnetic beads, thereby achieving highly sensitive PCR detection, which is crucial to preventing and controlling the spread of pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings:

[0021] Figure 1 The particle size potential diagram of raw material magnetic beads and polyhydroxy cationic quaternary ammonium salt magnetic beads.

[0022] Figure 2 XPS spectra of raw material magnetic beads and polyhydroxy cationic quaternary ammonium salt magnetic beads.

[0023] Figure 3 This is a graph showing the adsorption performance of polyhydroxy cationic quaternary ammonium salt magnetic beads on bacteria.

[0024] Figure 4 To simulate the scene, the adsorption and desorption performance results of polyhydroxy cationic quaternary ammonium salt magnetic beads on different concentrations of Klebsiella pneumoniae are shown.

[0025] Figure 5 To simulate the scene, the adsorption and desorption performance results of polyhydroxy cationic quaternary ammonium salt magnetic beads on different concentrations of Staphylococcus aureus are shown.

[0026] Figure 6 The preparation route of polyhydroxy cationic quaternary ammonium salt magnetic beads. DETAILED DESCRIPTION

[0027] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings.

[0028] Figure 6 This is a preparation route for polyhydroxy cationic quaternary ammonium salt magnetic beads. First, epoxy quaternary ammonium salt is prepared by substitution reaction, and then amino magnetic beads are quaternized by amino epoxy ring opening reaction, and quaternary ammonium salt is introduced on the raw material magnetic beads. At the same time, amino epoxy ring opening can introduce hydroxyl groups on the magnetic beads. Polyhydroxy cationic quaternary ammonium salt magnetic beads can enrich pathogen aerosols in a broad spectrum and efficiently, and improve the detection sensitivity of PCR.

[0029] Example 1

[0030] 1. Preparation of polyhydroxy cationic quaternary ammonium salt magnetic beads

[0031] (1) Synthesis of epoxy quaternary ammonium salt

[0032] Add N,N-dimethylethanolamine (DMEA) into a round-bottom flask, and slowly drop epichlorohydrin (ECH) in an ice bath. The molar ratio of N,N-dimethylethanol to epichlorohydrin is 1:1. Stir the reaction for 12 hours until the magnetic stirrer can no longer stir, thereby obtaining epoxy quaternary ammonium salt.

[0033] (2) Preparation of polyhydroxy cationic quaternary ammonium salt magnetic beads by amino-epoxy ring-opening method

[0034] The epoxy quaternary ammonium salt (0.05 mol) of step (1) and amino magnetic beads (10 mg / mL) were subjected to amino-epoxy ring-opening reaction under nitrogen protection at 70° C. After the reaction was completed, the lower precipitate was obtained by centrifugation, washed twice with deionized water, and freeze-dried to obtain polyhydroxy cationic quaternary ammonium salt magnetic beads.

[0035] 2. Characterization of Polyhydroxy Cationic Quaternary Ammonium Salt Magnetic Beads

[0036] The obtained polyhydroxy cationic quaternary ammonium salt magnetic beads were prepared into a solution, and the particle size and potential of the magnetic beads before and after modification were detected by a nanometer particle size and potential measuring instrument. 2 mg of the obtained polyhydroxy cationic quaternary ammonium salt magnetic beads were weighed and the chemical bonds of the magnetic beads before and after modification were detected by X-ray photoelectron spectroscopy (XPS) technology.

[0037] Figure 1 The particle size potential of the raw magnetic beads and the polyhydroxy cationic quaternary ammonium salt magnetic beads. It can be seen from the figure that the modified polyhydroxy cationic quaternary ammonium salt magnetic beads have enhanced potential and reduced particle size, proving that the quaternary ammonium salt modification is successful.

[0038] Figure 2 The XPS spectra of raw magnetic beads and polyhydroxy cationic quaternary ammonium salt magnetic beads are shown in Figure 1. + The ratio of the peak area of ​​CN, C=N, and CNO to the peak area is 44%:56%. The modified polyhydroxy cationic quaternary ammonium salt magnetic beads NH4 + The ratio of the peak area of ​​CN, C=N, and CNO is 55%:45%, and the polyhydroxy cationic quaternary ammonium salt magnetic beads NH4 + The increase in the peak area ratio proves that the quaternary ammonium salt grafting rate has increased. The ratio of the peak area of ​​the raw material magnetic beads C=O=C, C=OH, COH to the peak area of ​​the metal oxygen is 57%:43%, and the ratio of the peak area of ​​the modified polyhydroxy cationic quaternary ammonium salt magnetic beads C=O=C, C=OH, COH to the peak area of ​​the metal oxygen is 68%:32%. The peak area ratio of the modified polyhydroxy cationic quaternary ammonium salt magnetic beads C=O=C, C=OH, COH has increased, proving that hydroxyl groups have been introduced through the amino-epoxy ring-opening reaction.

[0039] Example 2 Application of polyhydroxycation quaternary ammonium salt magnetic beads in pathogen aerosol PCR enrichment detection

[0040] The ferroferric oxide magnetic beads modified by epoxy quaternary ammonium salt were prepared into a solution with a concentration of 0.05 mg / mL, and the magnetic bead solution was added to the cyclone cup of the wet wall cyclone aerosol sampler (SA-S120M), the cyclone sampling device of the aerosol sampler was enabled, and the detection device was turned on for collection to collect pathogenic aerosols. Subsequently, the polyhydroxy cationic quaternary ammonium salt magnetic beads were adsorbed to the bottom of the cyclone cup with a magnet, the supernatant was discarded, and 1 mL of sodium heparin was added to dissociate the pathogenic microorganisms from the magnetic beads. The 1 mL sodium heparin supernatant was completely sucked out, the bottom bacteria were collected by centrifugation, and the pathogenic microorganism genomic DNA extraction kit (TIANGEN) was used for lysis to obtain the genomic DNA of the pathogenic microorganism.

[0041] DNA concentration was measured by micro-spectrophotometer and subsequent detection was performed. PCR amplification reaction was performed using TaKaRa Ex Taq HS, and the intensity of PCR amplification fluorescence signal was detected by using Probe. PCR reaction solution was prepared on ice according to the instructions of RR390A enzyme (Takara), and each system was 20μL: 10μL of Premix Ex Taq (ProbeqPCR) (2X), 1μL of primer-F (10μM), primer-R (10μM) and probe (FAM-BHQ1), 1μL of ROX dye, and 4μL of sterile water were added to each PCR tube, and 2μL of collected bacterial DNA was added to each tube and three sets of parallel were set up. The prepared PCR reaction solution was set up in the two-step program in Step One Plus Real-Time PCR System according to the instructions of RR390A enzyme (Takara). Finally, the obtained data was analyzed and the amplification curve was drawn.

[0042] Test Example 1 Test on the adsorption and desorption capacity of pathogenic microorganisms

[0043] First, prepare the magnetic bead material at a concentration of 0.05 mg / mL, incubate Staphylococcus aureus and Klebsiella pneumoniae in LB (lysate broth) medium, incubate in a shaker at 37°C for 12 hours, and then read the OD using a micro-spectrophotometer. 600 to the logarithmic growth phase. Then dilute with saline to an initial bacterial concentration of 1×10 4 CFU / mL. A raw material magnetic bead group, a polyhydroxy cationic quaternary ammonium salt magnetic bead group, and a blank control group were set up. The polyhydroxy cationic quaternary ammonium salt magnetic beads were prepared with physiological saline to a concentration of 0.02 mg / mL and 1×10 4 Take 5 mL of pathogenic microorganism liquid with CFU / mL, vortex for 3 minutes, adsorb on the magnet for 1 minute, take 50 μL of supernatant each time and evenly spread it on the solid culture medium, and culture it in a suitable environment for 12 hours. Use a digital camera to take pictures and record the agar plate, count the number of colonies on the agar plate, and calculate the adsorption rate.

[0044] After magnet adsorption, the supernatant was completely aspirated, 1 mL of 1% heparin sodium was added, and then the supernatant was evenly vortexed and adsorbed by the magnet. Then 50 μL of the supernatant was evenly spread on the solid culture medium. The blank control group was directly 5 mL of bacterial solution and then 5 mL of physiological saline was added. After vortexing, the same amount was spread on the plate. And cultured in a suitable environment for 12 hours. Use a digital camera to take pictures and record the agar plate and desorb and count the number of colonies on the agar plate.

[0045] Figure 3 The figure shows the results of the adsorption performance of polyhydroxy cationic quaternary ammonium salt magnetic beads on bacteria. Figure 3(Top) It can be seen that for Klebsiella pneumoniae, the adsorption rate of raw magnetic beads and polyhydroxy cationic quaternary ammonium salt magnetic beads is higher than 80%. During dissociation, the colony count of the raw magnetic beads after dissociation is lower than that of the group without adding magnetic beads, while the colony count of the polyhydroxy cationic quaternary ammonium salt magnetic beads is twice that of the control group without adding magnetic beads, indicating that the polyhydroxy cationic quaternary ammonium salt magnetic beads have better bacterial enrichment performance.

[0046] from Figure 3 (Below) It can be seen that for Staphylococcus aureus, the adsorption rate of raw magnetic beads and polyhydroxy cationic quaternary ammonium salt magnetic beads is higher than 80%. During dissociation, the colony count of the raw magnetic beads after dissociation is lower than that of the group without adding magnetic beads, while the colony count of the polyhydroxy cationic quaternary ammonium salt magnetic beads is twice that of the control group without adding magnetic beads, indicating that the polyhydroxy cationic quaternary ammonium salt magnetic beads have better bacterial enrichment performance.

[0047] Test Example 2: Test on the adsorption and desorption capacity of pathogenic microorganisms in simulated field

[0048] The bioaerosol sampling and detection evaluation cabin was operated at a wind speed of 20 m / min, and the convection fan was used at 90% power to purify the evaluation cabin. After purification, the sampler to be tested and the center of the wind tunnel were used for sampling until the end of the test. Polyhydroxy cationic quaternary ammonium salt magnetic beads were prepared into 0.05 and 0.01 mg / mL physiological saline solutions, and the bacterial solution was diluted to a concentration of 1×10 5 CFU / mL and 1×10 7 CFU / mL, add 10mL of magnetic bead solution to the cyclone cup each time, enable the cyclone sampling device of the aerosol sampler, set the sampling time to 7min, and turn on the detection device for collection. The pathogen aerosol particle injection speed is 0.2mm / min, and the aerosol generation time is 10min. The detected aerosol particles are 600-750 / cm 3 After sampling, the evaluation cabin was closed and purified. After purification, the sampling device was taken out, the magnetic beads were adsorbed with a magnet, and 200 μL of the supernatant was evenly spread on the solid culture medium. The incubator temperature was set to 37°C. A digital camera was used to take pictures of the agar plate, and the number of colonies on the agar plate was counted and the adsorption performance was statistically analyzed.

[0049] After the magnetic beads are adsorbed with a magnet, all the supernatant is sucked out, 1 mL of 1% heparin sodium is added, and then the mixture is shaken evenly and adsorbed with a magnet. Then 50 μL of the supernatant is evenly spread on the solid culture medium and cultured in an incubator for 12 hours. The incubator temperature is set to 37°C. A digital camera is used to take photos and record the agar plate, count the number of colonies on the agar plate, and calculate the desorption rate.

[0050] Figure 4 To simulate the scene, the adsorption and desorption performance results of polyhydroxy cationic quaternary ammonium salt magnetic beads on Klebsiella pneumoniae aerosols of different concentrations are shown. Figure 4It can be seen that for the generator 10 5 When the number of CFU / mL of Klebsiella pneumoniae was 10, the control group without magnetic beads did not collect any bacteria, and the polyhydroxy cationic quaternary ammonium salt magnetic beads collected 157600 CFU / mL of bacteria at 0.01 mg / mL; 7 When the concentration of Klebsiella pneumoniae was 0.01 mg / mL, the control group without magnetic beads collected 200 CFU / mL of Klebsiella pneumoniae, and the polyhydroxy cationic quaternary ammonium magnetic beads collected 200,800 CFU / mL of Klebsiella pneumoniae at 0.01 mg / mL. The enrichment efficiency was increased by 1000 times, which can greatly improve the sensitivity of subsequent aerosol PCR detection.

[0051] Figure 5 To simulate the scene, the adsorption and desorption performance results of polyhydroxy cationic quaternary ammonium salt magnetic beads on white grape ball aerosols of different concentrations are shown. Figure 5 It can be seen that for the generator 10 5 CFU / mL of Staphylococcus aureus, no bacteria were collected in the control group without magnetic beads, and 380800 CFU / mL of bacteria were collected at 0.01 mg / mL of polyhydroxy cationic quaternary ammonium salt magnetic beads; 7 When the CFU / mL of white Staphylococcus aureus was detected, the control group without magnetic beads collected 42100 CFU / mL of white Staphylococcus aureus, and the polyhydroxy cationic quaternary ammonium salt magnetic beads collected 200800 CFU / mL of white Staphylococcus aureus at 0.05 mg / mL, which can greatly improve the sensitivity of subsequent aerosol PCR detection.

[0052] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. An application of polyhydroxy cationic quaternary ammonium salt magnetic beads constructed by amino-epoxy ring opening in pathogen aerosol PCR enrichment detection, characterized in that: Polyhydroxy cationic quaternary ammonium salt magnetic beads are used to enrich pathogenic microorganisms. The method of use is: prepare ferroferric oxide magnetic beads modified by epoxy quaternary ammonium salt into a solution with a concentration of 0.01-1 mg / mL, add the magnetic bead solution into the cyclone cup of the wet-wall cyclone aerosol sampler, activate the cyclone sampling device of the aerosol sampler, turn on the detection device for collection, and the collected sample is a pathogenic aerosol.

2. The application of polyhydroxy cationic quaternary ammonium salt magnetic beads constructed by amino-epoxy ring opening according to claim 1 in pathogen aerosol PCR enrichment detection is characterized in that: The epoxy quaternary ammonium salt modified ferroferric oxide magnetic beads are the product of an "amino-epoxy" ring-opening reaction between epoxy quaternary ammonium salt and amino ferroferric oxide magnetic beads, and the mass ratio of the epoxy quaternary ammonium salt to the amino ferroferric oxide magnetic beads is 0.5:1 to 0.8:

1.

3. The application of polyhydroxy cationic quaternary ammonium salt magnetic beads constructed by amino-epoxy ring opening according to claim 1 in pathogen aerosol PCR enrichment detection is characterized in that: The epoxy quaternary ammonium salt is a reaction product of a small molecule of a primary amine group and a halogen alkane containing an epoxy group, wherein the molar ratio of the amino group on the small molecule of the primary amine group to the epoxy group on the halogen alkane containing an epoxy group is 1:

1.

4. The use of polyhydroxy cationic quaternary ammonium salt magnetic beads constructed by amino-epoxy ring opening according to claim 3 in pathogen aerosol PCR enrichment detection, characterized in that: The small molecule of the primary amine group is N,N-dimethylethanolamine.

5. The use of polyhydroxy cationic quaternary ammonium salt magnetic beads constructed by amino-epoxy ring opening according to claim 3 in pathogen aerosol PCR enrichment detection, characterized in that: The halogen alkane containing epoxy group is epichlorohydrin.

6. The use of polyhydroxy cationic quaternary ammonium salt magnetic beads constructed by amino-epoxy ring opening according to claim 1 in pathogen aerosol PCR enrichment detection, characterized in that: The preparation method of the ferroferric oxide magnetic beads modified by epoxy quaternary ammonium salt is as follows: 1) Small molecules of primary amine groups react with halogenated alkanes containing epoxy groups to obtain epoxy quaternary ammonium salts; 2) subjecting the epoxy quaternary ammonium salt to an aminoferric oxide magnetic bead for an amine-epoxy ring-opening reaction to obtain a product.

7. The application of the polyhydroxy cationic quaternary ammonium salt magnetic beads constructed by amino-epoxy ring opening in pathogen aerosol PCR enrichment detection according to claim 6 is characterized in that: The reaction temperature of step 1) is 25-70° C., and the reaction time is 12 h to 48 h.

8. The use of the polyhydroxy cationic quaternary ammonium salt magnetic beads constructed by amino-epoxy ring opening according to claim 6 in pathogen aerosol PCR enrichment detection, characterized in that: The reaction solvent of step 2) is dimethyl sulfoxide, the reaction temperature is 40-70° C., and the reaction time is 24-72 hours.

9. The use of polyhydroxy cationic quaternary ammonium salt magnetic beads constructed by amino-epoxy ring opening according to claim 1 in pathogen aerosol PCR enrichment detection, characterized in that: The pathogenic microorganisms in the pathogenic aerosol are influenza virus, porcine coronavirus, Staphylococcus aureus, adenovirus, Staphylococcus aureus, Haemophilus influenzae, Streptococcus pneumoniae or Klebsiella pneumoniae.

10. The use of polyhydroxy cationic quaternary ammonium salt magnetic beads constructed by amino-epoxy ring opening according to claim 9 in pathogen aerosol PCR enrichment detection, characterized in that: The enrichment efficiency of Klebsiella pneumoniae was increased by 2-1000 times, and the enrichment efficiency of Staphylococcus aureus was increased by 2-1000 times.

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