Pathogenic nucleic acid extraction method based on reverse nucleic acid grabbing

Abstract

The invention discloses a pathogenic nucleic acid extraction method based on reverse nucleic acid grabbing, which comprises the following steps: adding a sephadex microsphere solution with the surface modified with functional molecules and a lysis buffer into a bacterial liquid containing pathogenic bacteria, oscillating, inserting filter paper with negative electricity, incubating, taking out the filter paper, centrifuging, and taking out supernatant to obtain purified pathogenic nucleic acid, the functional molecules are o-phthalaldehyde, phenylboronic acid and hydrophobic molecules, the lysate contains lauryl sodium sulfate, disodium hydrogen phosphate and ethylenediamine tetraacetic acid, and the pH value of the lysate is 10.0-10.5. The method has the advantages of simplicity and convenience in operation, high extraction speed, high nucleic acid quality, high nucleic acid extraction rate and the like, and can provide a high-quality nucleic acid sample for molecular diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid extraction technology, and relates to a method for extracting pathogenic nucleic acids based on reverse nucleic acid capture, and more particularly to a rapid method for extracting pathogenic nucleic acids based on dextran gel microspheres that capture non-nucleic acid substances such as proteins, carbohydrates, lipids and small molecules and repel nucleic acid substances. Background Technology

[0002] Nucleic acid extraction and purification is a key step in the amplification and detection of nucleic acids in pathogenic microorganisms such as bacteria and viruses, enabling molecular diagnosis. The quality of the extracted nucleic acid is a key factor affecting the accuracy of molecular diagnosis. Nucleic acid extraction and purification based on magnetic bead adsorption is currently the mainstream nucleic acid extraction technology, which generally includes four key steps: lysis, adsorption, washing, and elution. (1) The sample is mixed with lysis buffer to rupture the cell membrane and release the internal nucleic acid; (2) The lysed sample is mixed with magnetic beads, and the affinity groups on the surface of the magnetic beads specifically bind to the nucleic acid; (3) The magnetic beads are washed with washing buffer to remove non-specifically adsorbed impurity molecules; (4) The binding between the magnetic beads and the nucleic acid is broken with elution buffer, and the magnetic beads are separated from the elution solution to obtain a supernatant containing purified nucleic acid. This method can obtain pathogenic nucleic acids with high purity, providing high-quality templates for subsequent molecular biology research. However, the entire process of this technology takes a long time, generally 15-25 minutes, and both adsorption and elution processes will cause nucleic acid loss, affecting the nucleic acid recovery rate. To further meet the demand for faster detection, existing technologies have developed several magnetic bead extraction methods that simplify the four steps of nucleic acid extraction. However, increasing the extraction speed makes nucleic acid loss more likely. Therefore, it is necessary to further develop high-quality, rapid nucleic acid extraction methods to improve the efficiency of molecular diagnostics, shorten the detection cycle, and enhance the accuracy and convenience of clinical diagnosis. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a pathogen nucleic acid extraction method based on reverse nucleic acid grasping that is simple to operate, fast to extract, high in quality and high in extraction rate.

[0004] To solve the above-mentioned technical problems, the present invention adopts the following technical solution.

[0005] A method for extracting pathogenic nucleic acid based on reverse nucleic acid grasping includes the following steps: adding a solution of dextran gel microspheres with functional molecules modified on the surface and a lysis buffer to a bacterial culture containing pathogenic bacteria; after shaking, inserting a negatively charged filter paper, incubating, removing the filter paper, centrifuging, and collecting the supernatant to obtain purified pathogenic nucleic acid; wherein the functional molecules include o-phthalaldehyde, phenylboronic acid, and hydrophobic molecules (or hydrophobic groups); the lysis buffer contains sodium dodecyl sulfate, disodium hydrogen phosphate, and ethylenediaminetetraacetic acid; and the pH value of the lysis buffer is 10.0–10.5.

[0006] The preferred method for pathogen nucleic acid extraction based on reverse nucleic acid grasping described above involves the following preparation process of the dextran gel microsphere solution with functional molecules modified on the surface: At room temperature, dextran gel powder is soaked in water and stirred continuously for 24-48 hours or boiled for 1-2 hours to allow the gel to fully swell until its volume no longer changes. The resulting swollen dextran gel microspheres are then mixed with three silanizing agents with different functional groups: o-phthalaldehyde-PEG-Silane, phenylboronic acid-PEG-Silane, and C18-PEG-Silane. After coupling reaction at room temperature, the supernatant is removed by centrifugation to obtain the dextran gel microsphere solution with functional molecules modified on the surface.

[0007] In the aforementioned method for pathogen nucleic acid extraction based on reverse nucleic acid capture, preferably, during the preparation of the dextran gel microsphere solution with surface-modified functional molecules, the ratio of the number of swollen dextran gel microspheres to the number of molecules of phthalaldehyde-polyethylene glycol-silane is 1:10. 12 ~10 14 The ratio of the number of molecules of the swollen dextran gel microspheres to that of the phenylboronic acid-polyethylene glycol-silane is 1:10. 12 ~10 14 The ratio of the number of molecules of the swollen dextran gel microspheres to the number of molecules of the octadecyl-polyethylene glycol-silane is 1:10. 12 ~10 14 .

[0008] In the above-mentioned pathogen nucleic acid extraction method based on reverse nucleic acid grasping, preferably, the dextran gel powder has a separation range of molecular weight <700, molecular weight 1000-5000, and molecular weight 1500-30000; and the particle size range of the dextran gel powder is 40 micrometers to 120 micrometers.

[0009] In the above-mentioned method for pathogen nucleic acid extraction based on reverse nucleic acid grasping, preferably, the preparation process of the lysis buffer is as follows: sodium dodecyl sulfate, disodium hydrogen phosphate, and ethylenediaminetetraacetic acid are dissolved in water, and sodium hydroxide is added dropwise to adjust the pH to 10.0-10.5 to obtain the lysis buffer. The mass fraction of sodium dodecyl sulfate in the lysis buffer is 0.02%-1%, the concentration of disodium hydrogen phosphate is 20 mmol / L-100 mmol / L, and the concentration of ethylenediaminetetraacetic acid is 0.5 mmol / L-2 mmol / L.

[0010] In the above-mentioned pathogen nucleic acid extraction method based on reverse nucleic acid grasping, preferably, the volume ratio of the bacterial solution containing pathogens, the dextran gel microsphere solution with functional molecules modified on the surface, and the lysis buffer is 2-6:1:2-6.

[0011] In the above-mentioned pathogen nucleic acid extraction method based on reverse nucleic acid grasping, preferably, the oscillation time is 30s to 60s, the incubation time is 4min to 8min, the centrifugation time is 30s to 60s, and the centrifugation speed is 1500 rpm to 3000 rpm.

[0012] In the above-mentioned method for extracting pathogenic nucleic acid based on reverse nucleic acid grasping, preferably, the preparation process of the bacterial solution containing pathogenic bacteria is as follows: the pathogenic bacteria taken from the temperature of -20℃ to -80℃ are thawed at room temperature, the bacterial solution is picked and separated by streak plating, and after being cultured at 35℃ to 37℃ for 24 h to 36 h, single colonies are picked, and the picked single colonies are dispersed in physiological saline to obtain the bacterial solution containing pathogenic bacteria.

[0013] In the above-mentioned pathogen nucleic acid extraction method based on reverse nucleic acid grasping, preferably, the pathogen includes Escherichia coli or Staphylococcus aureus.

[0014] In this invention, the dextran gel microspheres are commercially available cross-linked dextran gel microspheres that swell in water but do not dissolve. The degree of cross-linking of the dextran gel microspheres is inversely proportional to the gel pore size; the higher the degree of cross-linking, the smaller the gel pore size. Dextran gels are currently commonly used as stationary phases in chromatographic analysis. This invention utilizes dextran gel microspheres for reverse nucleic acid capture. The internal pore size of the dextran gel microspheres achieves both nucleic acid repulsion and small molecule capture. Pathogenic nucleic acids have very large molecular weights, typically ranging from tens of thousands to millions, thus preventing nucleic acid molecules from entering the gel microspheres. However, various small molecules in the nucleic acid extraction system, including surfactants, sugars, and lipids, can enter the gel and be captured.

[0015] In this invention, the functional molecules modified on the surface of dextran gel microspheres include phthalaldehyde, phenylboronic acid, and hydrophobic groups (alkyl, phenyl, etc.). Due to the presence of phenylboronic acid, the surface of the dextran gel microspheres carries a negative charge when the nucleic acid extraction system is alkaline, thus repelling negatively charged nucleic acid molecules. Phthalate captures proteins through cross-linking reactions with amino groups on proteins, while phenylboronic acid reacts with hydroxyl groups in diols to form phenylboronic esters, which are used to capture carbohydrates. The hydrophobic molecules non-specifically adsorb and capture proteins, carbohydrates, lipids, and other molecules through hydrophobic interactions. The surface modification of the dextran gel microspheres involves silanization reactions between hydroxyl groups and silane groups on the microsphere surface, modifying the microsphere surface with functional molecules. Polyethylene glycol is used as a "molecular arm" between the functional molecules and the gel microsphere surface, reducing steric hindrance and enhancing the functional molecules' ability to capture various substances.

[0016] In this invention, the nucleic acid extraction process includes pathogen lysis and the capture of non-nucleic acid substances. The core components of the pathogen lysis buffer used in this invention are sodium dodecyl sulfate (SDS), a disodium hydrogen phosphate-sodium hydroxide buffer system, and the solution is kept alkaline. SDS is an anionic surfactant that can rapidly destroy the cell membrane surface of pathogens under alkaline conditions. The alkaline environment also allows the surface of dextran gel microspheres to carry a negative charge, and it is also conducive to the cross-linking reaction between phthalaldehyde and amino groups and the reaction between phenylboronic acid and the hydroxyl groups of diols. However, excessive alkalinity may affect the downstream nucleic acid amplification reaction.

[0017] In this invention, the pathogen nucleic acid extraction process also involves a concentration process to increase the concentration of the nucleic acid sample. For existing magnetic bead nucleic acid extraction and purification methods that include four steps—lysis, adsorption, washing, and elution—concentration can be achieved by increasing the added sample volume and decreasing the elution volume. The method of this invention uses filter paper to absorb water to achieve the sample nucleic acid concentration process. Using negatively charged filter paper can repel nucleic acids, achieving water absorption without causing nucleic acid loss. The filter paper material can be commercially available Waterman Fusion 5 filter paper with a negatively charged surface, and the volume of water absorbed can be adjusted by the size of the filter paper.

[0018] like Figure 1 The figure shows a schematic diagram of the principle of the pathogen nucleic acid extraction method based on reverse nucleic acid capture of the present invention. As can be seen from the figure, after the surface-modified functionalized dextran gel microspheres are mixed and incubated with bacterial lysate, the functional molecules such as phthalaldehyde, phenylboronic acid and alkyl groups on the surface of the microspheres and the pore structure inside the microspheres can efficiently capture impurities such as proteins, lipids, sugars and small molecules, thereby obtaining purified nucleic acids.

[0019] In this invention, the nucleic acid extraction process involves mixing and incubating the pathogen sample with lysis buffer and dextran gel microspheres, and then placing the sample in filter paper to absorb water for 4-8 minutes. After centrifugation at low speed for about 30 seconds, the supernatant is collected to obtain the purified nucleic acid sample. The entire nucleic acid extraction process can be completed in one step in just 8-10 minutes.

[0020] In this invention, the room temperature is typically between 18°C ​​and 25°C.

[0021] Compared with the prior art, the advantages of the present invention are as follows: This invention provides a rapid pathogen nucleic acid extraction method based on reverse nucleic acid capture using dextran gel microspheres. It utilizes the characteristic that small molecules can enter the interior of cross-linked dextran gel microspheres to achieve the capture of small molecules, while nucleic acid molecules, due to their large size, cannot enter the microspheres. This invention achieves the capture of proteins, carbohydrates, lipids, and other substances while repelling nucleic acids by functionally modifying the surface of the dextran gel microspheres. This invention allows for the capture of impurities such as proteins, carbohydrates, and lipids (i.e., small molecules) in complex systems in a single step, leaving only nucleic acids, thus completing the nucleic acid extraction process.

[0022] This method eliminates the need for four steps: lysis, adsorption, washing, and elution. It is simple to operate, fast to extract, and produces high-quality nucleic acid with a high recovery rate. Pathogen lysis and reverse nucleic acid capture can be performed simultaneously, requiring minimal equipment; only a micro-oscillator and centrifuge are needed. Nucleic acid extraction is rapid, completing the entire process in one step in 8-10 minutes, providing high-quality nucleic acid samples for molecular diagnostics. Conventional nucleic acid extraction methods based on magnetic bead adsorption and elution processes often result in nucleic acid loss during adsorption and elution, typically achieving an extraction rate of only 70%-80%. The method of this invention, through the selection of the internal pore size of dextran gel microspheres and the control of surface properties, prevents nucleic acids from entering the microspheres and from being adsorbed onto the surface, achieving a nucleic acid recovery rate generally exceeding 90%. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the principle of the pathogen nucleic acid extraction method based on reverse nucleic acid grasping of the present invention.

[0024] Figure 2 The images show the LAMP amplification results after nucleic acid extraction from Escherichia coli (A) in Example 1 and Staphylococcus aureus (B) in Example 2 of this invention.

[0025] Figure 3 The images show the LAMP amplification results of nucleic acid extraction from Escherichia coli (A) under different pH lysis buffer formulations in Example 3 and Staphylococcus aureus (B) under different pH lysis buffer formulations in Example 4 of this invention.

[0026] Figure 4 The images show the LAMP amplification results after nucleic acid extraction from Escherichia coli (A) using dextran gel microspheres modified with different functional molecules in Example 5 of this invention, and from Staphylococcus aureus (B) using dextran gel microspheres modified with different functional molecules in Example 6 of this invention.

[0027] Figure 5 The images show the LAMP amplification results of Escherichia coli (A) and Staphylococcus aureus (B) after nucleic acid extraction following different magnetic beads / microspheres were co-modified with phthalaldehyde / phenylboronic acid / alkyl groups.

[0028] Figure 6 The image shows the LAMP amplification results of different concentrations of E. coli nucleic acid recovered using the method of the present invention (A) and the commercial magnetic bead nucleic acid extraction kit (B).

[0029] Figure 7 This image shows the LAMP amplification results of the human endogenous gene GAPDH after nucleic acid extraction from oral and pharyngeal swab samples using the method of this invention.

[0030] Figure 8 This image shows the LAMP amplification results of E. coli nucleic acid (A) and human endogenous gene GAPDH (B) after nucleic acid extraction from E. coli-labeled oral and pharyngeal swab samples using the method of this invention.

[0031] Figure 9 This is a comparison of the LAMP amplification results of Escherichia coli after nucleic acid extraction using the method of the present invention (A) and the commercial magnetic bead nucleic acid extraction reagent (B). Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention. Unless otherwise specified, the materials and instruments used in the following embodiments are all commercially available. In all embodiments, real-time fluorescence loop-mediated isothermal amplification (LAMP) was used to detect nucleic acids. The total volume of the LAMP amplification reaction buffer system was 25 μL, and the components are shown in Table 1. The LAMP amplification reaction procedure was as follows: A CFX Connect™ real-time PCR system (purchased from BioVolkswagen, USA) was used. Rad (Radio Technologies) performed rapid amplification for 40 cycles at 63°C and 1 min / cycle, collecting fluorescence signals once every other cycle and plotting fluorescence curves over time.

[0033] Table 1. Composition of LAMP amplification reaction buffer system

[0034] Example 1 A method for pathogen nucleic acid extraction based on reverse nucleic acid grasping according to the present invention, specifically for extracting nucleic acid from Escherichia coli samples, includes the following steps: The standard strain of *Escherichia coli* (ATCC 25922, purchased from Beijing Biowell Biotechnology Co., Ltd.) was taken from the -80℃ freezer, thawed at room temperature, and a loopful of bacterial culture was picked and streaked onto a blood agar plate for isolation. After incubation at 36℃ for 24 hours, the bacterial plate (blood agar plate medium, purchased from Guangdong Huankai Microbial Technology Co., Ltd.) was removed, and single colonies were picked in a biosafety cabinet. One single colony was picked and evenly dispersed in 3 mL of physiological saline, transferred to a calibration tube, and the bacterial concentration was determined on a bacterial turbidimeter (WGZ-XT, Hangzhou Qiwei Instrument Co., Ltd.). Take 200 μL of Escherichia coli bacterial culture into a centrifuge tube with a bacterial concentration of 5000 CFU / mL, add 50 μL of dextran gel microsphere solution with surface modified functional molecules and 200 μL of lysis buffer, place it on a small vortex shaker and shake for 60 s, then insert a negatively charged filter paper strip into the centrifuge tube and incubate for 5 min, remove the filter paper strip, place the centrifuge tube in a microcentrifuge and centrifuge at 1500 rpm for 30 s, and take out the supernatant to obtain 50 μL of purified nucleic acid sample.

[0035] In this embodiment, the preparation process of the dextran gel microsphere solution with functional molecules on the surface is as follows: At room temperature, the dextran gel powder is soaked in water and stirred continuously for 36 hours or boiled for 1 hour to allow the gel to fully swell and its volume no longer changes. The resulting swollen dextran gel microspheres are mixed with three silanizing agents with different functional groups. The three silanizing agents with different functional groups are phthalaldehyde-polyethylene glycol-silane, phenylboronic acid-polyethylene glycol-silane, and octadecyl-polyethylene glycol-silane. After coupling reaction at room temperature, the supernatant is removed by centrifugation to obtain the dextran gel microsphere solution with functional molecules on the surface.

[0036] In this embodiment, during the preparation of the dextran gel microsphere solution with functional molecules on its surface, the ratio of the number of swollen dextran gel microspheres to the number of phthalaldehyde-polyethylene glycol-silane molecules is 1:10. 13 The ratio of the number of dextran gel microspheres to the number of molecules of phenylboronic acid-polyethylene glycol-silane after swelling is 1:10. 13 The ratio of the number of dextran gel microspheres to octadecyl-polyethylene glycol-silane molecules after swelling is 1:10. 13 .

[0037] In this embodiment, the dextran gel powder has a molecular weight range of 1000 to 5000 and a particle size range of 40 micrometers to 120 micrometers.

[0038] In this embodiment, the preparation process of the lysis buffer is as follows: Sodium dodecyl sulfate, disodium hydrogen phosphate and ethylenediaminetetraacetic acid are dissolved in water, and sodium hydroxide is added dropwise to adjust the pH to 10.0 to obtain the lysis buffer. The mass fraction of sodium dodecyl sulfate in the lysis buffer is 0.1%, the concentration of disodium hydrogen phosphate is 50 mmol / L, and the concentration of ethylenediaminetetraacetic acid (EDTA) is 1 mmol / L.

[0039] In this embodiment, the filter paper strips are FUSION 5 filter paper from Whatman, cut into 5cm × 1cm pieces.

[0040] Five μL of the extracted purified nucleic acid sample was taken for LAMP amplification (buffer system components are shown in Table 1). Primers were designed based on the specific conserved sequence of E. coli, including: outer primers F3 and B3, and inner primers FIP and BIP, as shown in Table 2. Figure 2 (A) is a diagram of LAMP amplification results after nucleic acid extraction from Escherichia coli. As can be seen from the diagram, the method of the present invention extracts nucleic acid from the bacterial culture of Escherichia coli and detects the specific genes of the corresponding bacteria. The method of the present invention is simple to operate and can extract nucleic acid quickly and efficiently.

[0041] Example 2 A method for pathogen nucleic acid extraction based on reverse nucleic acid grasping according to the present invention, specifically for extracting nucleic acid from Staphylococcus aureus samples, includes the following steps: The standard strain of Staphylococcus aureus (ATCC 29213, purchased from Beijing Biowell Biotechnology Co., Ltd.) was taken out from the -80℃ freezer, thawed at room temperature, and a loopful of bacterial solution was picked and streaked onto a blood agar plate for isolation. After incubation at 36 ℃ for 24 h, the bacterial plate (blood agar plate medium, purchased from Guangdong Huankai Microbial Technology Co., Ltd.) was taken out and single colony picking was performed in the biosafety cabinet. One single colony was picked and evenly dispersed in 3 mL of physiological saline, transferred to a calibration tube, and the bacterial concentration was determined on a bacterial turbidimeter (WGZ-XT, Hangzhou Qiwei Instrument Co., Ltd.). Take 200 μL of Staphylococcus aureus bacterial culture into a centrifuge tube with a bacterial concentration of 5000 CFU / mL, add 50 μL of dextran gel microsphere solution with surface modified functional molecules and 200 μL of lysis buffer, place it on a small vortex shaker and shake for 60 s, then insert a negatively charged filter paper strip into the centrifuge tube and incubate for 5 min, remove the filter paper strip, place the centrifuge tube in a microcentrifuge and centrifuge at 1500 rpm for 30 s, and take out the supernatant to obtain 50 μL of purified nucleic acid sample.

[0042] In this embodiment, the preparation process of the dextran gel microsphere solution with functional molecules on the surface (including the raw material ratio and characteristics of the dextran gel powder), the preparation process of the lysis solution, and the filter paper strips are all the same as in Example 1.

[0043] Five μL of the extracted purified nucleic acid sample was taken for LAMP amplification (buffer system components are shown in Table 1). Primers were designed based on the Staphylococcus aureus-specific conserved sequence, including: outer primers F3 and B3, and inner primers FIP and BIP, as shown in Table 2. Figure 2 (B) is a diagram of LAMP amplification results after nucleic acid extraction from Staphylococcus aureus. As can be seen from the diagram, the method of the present invention extracts nucleic acid from the bacterial culture of Staphylococcus aureus and detects the specific genes of the corresponding bacteria. The method of the present invention is simple to operate and can extract nucleic acid quickly and efficiently.

[0044] Table 2 Primer sequence listing for Escherichia coli and Staphylococcus aureus

[0045] Example 3 Different pH values ​​(7.0, 8.0, 9.0, 9.5, 10.0, 10.5, and 11.0) were selected for lysis buffer formulations, and nucleic acid extraction and LAMP detection were performed on Escherichia coli samples (Ec) following the same procedure as in Example 1. Figure 3 The amplification results in (A) show that the nucleic acid amplification effect is good and the difference is not significant when the pH value of the lysis buffer is 10 and 10.5. It can be seen that the pH value of the lysis buffer is optimally controlled at 10-10.5 in the method of the present invention. Too low a pH value will cause nucleic acid to be adsorbed onto the surface of dextran microspheres, resulting in nucleic acid loss. Too high a pH value may inhibit the subsequent nucleic acid amplification reaction.

[0046] Example 4 Different pH lysis buffer formulations (7.0, 8.0, 9.0, 9.5, 10.0, 10.5, and 11.0, respectively) were selected, and nucleic acid extraction and LAMP detection were performed on Staphylococcus aureus samples (Sa) following the same procedure as in Example 2. Figure 3 The amplification results in (B) show that the nucleic acid amplification effect is good and the difference is not significant when the pH value of the lysis buffer is 10 and 10.5. It can be seen that the pH value of the lysis buffer is optimally controlled at 10-10.5 in the method of the present invention. Too low a pH value will cause nucleic acid to be adsorbed onto the surface of dextran microspheres, resulting in nucleic acid loss. Too high a pH value may inhibit the subsequent nucleic acid amplification reaction.

[0047] Example 5 Different surface-modifying molecules were selected for dextran gel microspheres to extract nucleic acids and perform LAMP detection on E. coli. The dextran gel microspheres with different surface-modifying functional molecules were phthalaldehyde-modified, phenylboronic acid-modified, alkyl (specifically octadecyl)-modified, phthalaldehyde / phenylboronic acid co-modified, phthalaldehyde / alkyl co-modified, and phthalaldehyde / phenylboronic acid / alkyl co-modified. Nucleic acid extraction and LAMP detection were performed on E. coli samples following the same procedure as in Example 1. Figure 4 The amplification results in (A) show that the different functional molecules selected by the method of the present invention have a synergistic capture effect on the removal of proteins, carbohydrates and lipids. The best nucleic acid extraction effect is achieved when multiple types of functional molecules are used to modify dextran gel microspheres at the same time.

[0048] Example 6 Different surface-modifying molecules were selected for dextran gel microspheres to extract nucleic acids and perform LAMP detection on Staphylococcus aureus. The dextran gel microspheres with different functional molecule surface modifications included phthalaldehyde-modified dextran gel microspheres, phenylboronic acid-modified dextran gel microspheres, alkyl (specifically octadecyl)-modified dextran gel microspheres, phthalaldehyde / phenylboronic acid co-modified dextran gel microspheres, phthalaldehyde / alkyl co-modified dextran gel microspheres, and phthalaldehyde / phenylboronic acid / alkyl co-modified dextran gel microspheres. Following the same procedure as in Example 2, nucleic acid extraction and LAMP detection were performed on Staphylococcus aureus samples. Figure 4 The amplification results in (B) show that the different functional molecules selected by the method of the present invention have a synergistic capture effect on the removal of proteins, carbohydrates and lipids. The best nucleic acid extraction effect is achieved when multiple types of functional molecules are used to modify dextran gel microspheres at the same time.

[0049] Comparative Experiment 1 The surfaces of silanol magnetic beads and hydroxyl polystyrene microspheres were co-modified with phthalaldehyde / phenylboronic acid / alkyl groups, respectively, and their nucleic acid extraction effects were compared with those of the phthalaldehyde / phenylboronic acid / alkyl co-modified dextran gel microspheres in Example 5. Nucleic acid extraction and LAMP detection were performed on E. coli samples following the same procedure as in Example 1. Figure 5 The amplification results in (A) show that, compared with solid microspheres, the internal pore structure of the dextran gel microspheres of the present invention allows small molecule inhibitors to enter and be captured, effectively eliminating their interference with nucleic acid amplification reactions.

[0050] Comparative Experiment 2 The surfaces of silanol magnetic beads and hydroxyl polystyrene microspheres were co-modified with phthalaldehyde / phenylboronic acid / alkyl groups, respectively, and their nucleic acid extraction effects were compared with those of the phthalaldehyde / phenylboronic acid / alkyl co-modified dextran gel microspheres in Example 6. Nucleic acid extraction and LAMP detection were performed on Staphylococcus aureus samples following the same procedure as in Example 2. Figure 5 The amplification results in (B) show that, compared with solid microspheres, the internal pore structure of the dextran gel microspheres of the present invention allows small molecule inhibitors to enter and be captured, effectively eliminating their interference with nucleic acid amplification reactions.

[0051] Performance Test 1: Recovery Rate Evaluation of E. coli Nucleic Acid Samples Following the product instructions, nucleic acid was extracted from E. coli solution samples using a commercial magnetic bead nucleic acid extraction kit (MEG-100), and the extracted nucleic acid was quantitatively detected using a Qubit fluorometer. 200 μL of E. coli nucleic acid at different concentrations (see Table 3) was recovered using both the commercial magnetic bead nucleic acid extraction kit (MEG-100) and the pathogen nucleic acid extraction method based on reverse nucleic acid grasping of this invention, with a recovery volume of 50 μL. The recovery process of this invention is similar to that in Example 1: 200 μL of nucleic acid was placed in a centrifuge tube, 50 μL of dextran gel microsphere solution and 200 μL of lysis buffer were added, and the tube was vortexed for 2 min. A filter paper strip was then inserted into the centrifuge tube and incubated for 5 min. The filter paper strip was then removed, and the centrifuge tube was centrifuged at 1500 rpm for 30 s in a microcentrifuge. The supernatant was collected to obtain 50 μL of recovered nucleic acid sample. The nucleic acids recovered by the two methods were quantitatively detected using a Qubit fluorometer, and the E. coli nucleic acids recovered by the two methods were amplified and detected using LAMP. The total volume of the LAMP amplification reaction buffer system was 25 μL, and the composition is shown in Table 1. As can be seen from Table 3, the pathogen nucleic acid extraction method based on reverse nucleic acid grasping of the present invention has a better nucleic acid recovery rate than commercially available magnetic bead nucleic acid extraction kits. Figure 6 The LAMP amplification results in (A) and (B) also show that the nucleic acid samples recovered by the method of the present invention have an earlier peak elution time.

[0052] Table 3. Evaluation of nucleic acid recovery rates for two nucleic acid extraction methods using a Qubit fluorometer.

[0053] Example 7: Nucleic acid extraction from oral and pharyngeal swab samples and detection of human endogenous gene GAPDH. Using a sterile medical cotton swab, swab the inside of the cheek and rub for about 10 seconds to collect secretions from the oral pharynx. Then, place the swab into a centrifuge tube containing 250 μL of liquid (50 μL of dextran gel microsphere solution with surface-modified functional molecules, the same as in Example 1, and 200 μL of lysis buffer). Stir for 10 seconds, remove the swab, and then place the centrifuge tube on a small vortex mixer and vortex for 2 min. Next, insert a negatively charged filter paper strip into the centrifuge tube and incubate for 5 min. Remove the filter paper strip, and centrifuge the centrifuge tube at 1500 rpm for 30 s. Collect the supernatant to obtain 50 μL of purified nucleic acid sample. Take 5 μL of the extracted nucleic acid sample for LAMP amplification (buffer system components are shown in Table 1). Primers were designed based on the specific conserved sequence of the human endogenous gene GAPDH. Primers included: outer primers B3 and F3, and inner primers FIP and BIP, as shown in Table 4. Figure 7 As can be seen, the method of the present invention detects the human endogenous gene GAPDH after extracting nucleic acid from oral pharyngeal swab samples. The method of the present invention is simple to operate and can extract nucleic acid quickly and efficiently.

[0054] Table 4 Primer sequence listing for human endogenous gene GAPDH

[0055] Example 8: Nucleic acid extraction and detection of Escherichia coli-labeled oral and pharyngeal swab samples Using a sterile medical cotton swab, wipe the inside of the cheek and rub for 10 seconds to collect secretions from the oral pharynx. Then, place the swab into a centrifuge tube containing 250 μL of liquid (50 μL of dextran gel microspheres with functional molecules modified on the surface, the same as in Example 1, and 200 μL of lysis buffer). Stir for 10 seconds, then remove the swab. Next, add 50 μL of *E. coli* bacterial suspension (5000 CFU / mL), and then vortex the centrifuge tube for 2 min. Afterward, insert a negatively charged filter paper strip into the centrifuge tube and incubate for 5 min. Remove the filter paper strip, and centrifuge the centrifuge tube at 1500 rpm for 30 s. Collect the supernatant to obtain 50 μL of purified nucleic acid sample. Take 5 μL of the extracted nucleic acid sample for LAMP amplification reactions of *E. coli* nucleic acid or human endogenous gene GAPDH (buffer system components are shown in Table 1). Figure 8 As can be seen from (A) and (B), the method of the present invention can simultaneously detect Escherichia coli and human endogenous gene GAPDH after extracting nucleic acid from oral pharyngeal swab samples spiked with Escherichia coli. The method of the present invention is simple to operate and can extract nucleic acid quickly and efficiently.

[0056] Performance Test 2: Comparison of Different Nucleic Acid Extraction Methods The inside of the cheek was wiped with a sterile medical cotton swab and rubbed for 10 seconds. The swab was then placed in a sample preservation solution containing different concentrations of E. coli, shaken for 10 seconds, and then removed to obtain a series of oral pharyngeal swab samples spiked with different concentrations of E. coli (200 CFU / mL, 500 CFU / mL, 1000 CFU / mL, 2000 CFU / mL, and 5000 CFU / mL). Nucleic acid extraction was performed on the same samples using the method of this invention and an existing magnetic bead extraction kit (MEG). The magnetic bead extraction kit was purchased from Yaneng Biotechnology (Shenzhen) Co., Ltd. The extraction effect was compared using LAMP amplification. The method of this invention involves taking 200 μL of E. coli-spiked oral pharyngeal swab sample, 50 μL of dextran gel microsphere solution with functional molecules on the surface, and 200 μL of lysis buffer and mixing them together in a centrifuge tube. The dextran gel microsphere solution with functional molecules on the surface and the lysis buffer are the same as in Example 1. The centrifuge tube is placed on a small vortex mixer and shaken for 2 min. Then, a negatively charged filter paper strip is inserted into the centrifuge tube and incubated for 5 min. After removing the filter paper strip, the centrifuge tube is placed in a microcentrifuge and centrifuged at 1500 rpm for 30 s. The supernatant is collected to obtain 50 μL of purified nucleic acid sample. Using a magnetic bead extraction kit, 200 μL of E. coli-spiked oral pharyngeal swab sample is taken, and then 200 μL of lysis buffer is added according to the product instructions. The sample is subjected to 95°C high-temperature lysis, room-temperature nucleic acid binding, room-temperature impurity washing, and 65°C nucleic acid elution steps to finally obtain 50 μL of purified nucleic acid sample. For the nucleic acids extracted by the two methods, 5 μL of sample was taken for LAMP amplification reaction (the components of the buffer system are shown in Table 1). Figure 9 (A) Shows that the LAMP amplification reaction using nucleic acid extracted by the method of the present invention as a template has an earlier peak elution time than Figure 9 (B) shows a commercially available kit. The nucleic acid extraction time required by this method is approximately 10 minutes, while that of the commercially available kit is approximately 20 minutes.

[0057] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A method for extracting pathogenic nucleic acid based on reverse nucleic acid grabbing, characterized by, Includes the following steps: A solution of dextran gel microspheres with surface-modified functional molecules and a lysis buffer were added to the bacterial culture containing pathogenic bacteria. After shaking, the mixture was inserted into a negatively charged filter paper and incubated. After removing the filter paper, the mixture was centrifuged, and the supernatant was collected to obtain purified pathogenic nucleic acid. The functional molecules included o-phthalaldehyde, phenylboronic acid, and hydrophobic molecules. The lysis buffer contained sodium dodecyl sulfate, disodium hydrogen phosphate, and ethylenediaminetetraacetic acid. The pH of the lysis buffer was 10.0–10.

5.

2. The reverse nucleic acid grab-based pathogenic nucleic acid extraction method according to claim 1, characterized by, The preparation process of the surface-modified dextran gel microsphere solution is as follows: At room temperature, the dextran gel powder is soaked in water and stirred continuously for 24h to 48h or boiled for 1h to 2h to allow the gel to fully swell and its volume no longer changes. The resulting swollen dextran gel microspheres are mixed with three silanizing agents with different functional groups, including phthalaldehyde-polyethylene glycol-silane, phenylboronic acid-polyethylene glycol-silane, and octadecyl-polyethylene glycol-silane. After coupling reaction at room temperature, the supernatant is removed by centrifugation to obtain the surface-modified dextran gel microsphere solution.

3. The reverse nucleic acid grab-based pathogenic nucleic acid extraction method according to claim 2, characterized by, The ratio of the number of molecules of the swollen dextran gel microspheres to the o-phthaldehyde-polyethylene glycol-silane is 1:10 12 ~ 10 14 The ratio of the number of molecules of the swollen dextran gel microspheres to the benzene boronic acid-polyethylene glycol-silane is 1:10 12 ~ 10 14 The ratio of the number of molecules of the swollen dextran gel microspheres to the octadecyl-polyethylene glycol-silane is 1:10 12 ~ 10 14 .

4. The reverse nucleic acid grab-based pathogenic nucleic acid extraction method according to claim 2, characterized by, The dextran gel powder has a molecular weight range of <700, 1000-5000, or 1500-30000; and a particle size range of 40 micrometers to 120 micrometers.

5. The reverse nucleic acid grab-based pathogenic nucleic acid extraction method according to any one of claims 1 to 4, characterized by, The lysis buffer is prepared as follows: sodium dodecyl sulfate, disodium hydrogen phosphate, and ethylenediaminetetraacetic acid are dissolved in water, and sodium hydroxide is added dropwise to adjust the pH to 10.0–10.5 to obtain the lysis buffer. The mass fraction of sodium dodecyl sulfate in the lysis buffer is 0.02%–1%, the concentration of disodium hydrogen phosphate is 20 mmol / L–100 mmol / L, and the concentration of ethylenediaminetetraacetic acid is 0.5 mmol / L–2 mmol / L.

6. The reverse nucleic acid grab-based pathogenic nucleic acid extraction method according to any one of claims 1 to 4, characterized by, The volume ratio of the bacterial solution containing pathogens, the dextran gel microsphere solution with functional molecules modified on the surface, and the lysis buffer is 2-6:1:2-6.

7. The reverse nucleic acid grab-based pathogenic nucleic acid extraction method according to any one of claims 1 to 4, characterized by, The oscillation time is 30s to 60s, the incubation time is 4min to 8min, the centrifugation time is 30s to 60s, and the centrifugation speed is 1500 rpm to 3000 rpm.

8. The reverse nucleic acid grab-based pathogenic nucleic acid extraction method according to any one of claims 1 to 4, characterized by, The preparation process of the bacterial suspension containing pathogens is as follows: pathogens taken from temperatures of -20℃ to -80℃ are thawed at room temperature, bacterial suspension is picked and streaked on plates for separation, and after being cultured at 35℃ to 37℃ for 24 h to 36 h, single colonies are picked and dispersed in physiological saline to obtain bacterial suspension containing pathogens.

9. The reverse nucleic acid grab-based pathogenic nucleic acid extraction method according to any one of claims 1 to 4, characterized by, The pathogens include Escherichia coli or Staphylococcus aureus.

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