A method for extracting extracellular DNA from microbial aggregates using a combination of glycosidases and proteases
Patent Information
- Application Number
- CN202611343424.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-09-01
- Publication Date
- 2026-10-27
Smart Images
Figure CN122979295A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of environmental DNA extraction technology, specifically relating to a method for extracting extracellular DNA from microbial aggregates by using a combination of glycosidase and protease. Background Technology
[0002] Environmental DNA (eDNA), including intracellular DNA (iDNA) and extracellular DNA (exDNA), is a valuable archive recording the characteristics and evolution of microbial communities, especially in aquatic environments. EDNA also carries a wealth of genetic information, such as antibiotic resistance genes (ARGs), posing a significant risk to public health. A considerable portion of exDNA is tightly bound to ubiquitous aquatic microbial aggregates (AMAs) rather than existing freely in the environment. This means that exDNA often mixes with iDNA, especially when using a 0.22 μm filter membrane for separation, where overlap is particularly pronounced. This poses a significant challenge to the efficient extraction of exDNA, thus affecting the accuracy of iDNA and exDNA microbiome analysis in aquatic environments.
[0003] Existing extracellular DNA isolation and extraction methods (such as chemical treatment and protease hydrolysis) all have significant limitations when processing dense aquatic microbial aggregates. Traditional chemical methods use phosphate buffer as a chemical competitor, attempting to release exDNA through anion bridge adsorption competition. However, due to the steric hindrance and permeability defects of the chemical dispersant, the actual effect is limited. Studies such as nano-infrared imaging have shown that exDNA is mainly intertwined with a highly branched long-chain extracellular polysaccharide backbone in three-dimensional space. This dense polysaccharide network generates huge steric hindrance, making it difficult for exogenous chemical reagents to penetrate and contact the tightly bound exDNA deep within the network, resulting in extremely low extraction efficiency.
[0004] In terms of enzymatic digestion methods, the most widely used method currently is to rely on trypsin or proteinase K. However, proteases can only loosen the outer protein structure and make the aggregates porous, but they cannot cut the main chain and branch connections of the extracellular polysaccharide. The tight spatial interweaving and physical binding between the extracellular polysaccharide network and exDNA still exist. Therefore, proteolytic digestion alone is very limited in releasing tightly bound exDNA.
[0005] Therefore, there is an urgent need for a method that can simultaneously cut the polysaccharide backbone, branching links, and protein chains in microbial aggregates to completely break the binding of exDNA by polysaccharides and protein backbones, thereby achieving efficient extraction of exDNA that is intertwined with polysaccharides and protein backbones. Summary of the Invention
[0006] The purpose of this invention is to overcome the shortcomings of the prior art and to provide a method for extracting extracellular DNA from microbial aggregates by using a combination of glycosidase and protease.
[0007] The specific technical solution adopted in this invention is as follows:
[0008] This invention provides a method for extracting extracellular DNA from microbial aggregates using a combination of glycosidase and protease, as detailed below:
[0009] S1: The chelating agent is used to pre-treat the sample containing microbial aggregates to initially break down the ion bridging effect in the microbial aggregates;
[0010] S2: Add protease to the pretreated sample to carry out an enzymatic hydrolysis reaction to destroy the protein backbone in the microbial aggregate; after the enzymatic hydrolysis reaction is terminated, use a filter membrane for the first filtration separation to obtain a primary separation filtrate containing extracellular DNA and membrane residue, respectively.
[0011] S3: The membrane residue obtained in step S2 is treated with glycosidase to sever the main chain and branch connections of the polysaccharide backbone in the microbial aggregate; after the reaction, a second filtration separation is performed using a new filter membrane, and the secondary separation filtrate containing extracellular DNA is retained;
[0012] S4: Combine the primary and secondary separation filtrates and concentrate them to extract extracellular DNA from the concentrated filtrate.
[0013] Preferably, the chelating agent in step S1 is a buffer solution of ethylenediaminetetraacetic acid or sodium pyrophosphate.
[0014] Preferably, the concentration of the chelating agent in step S1 is 5-10 mM, and the pretreatment reaction time is 3-10 minutes.
[0015] Preferably, the protease used in step S2 is trypsin or proteinase K.
[0016] Preferably, the final concentration of the protease in step S2 is 1~2.5 mg / mL; and the enzymatic hydrolysis reaction time is 5~15 minutes.
[0017] Preferably, the glycosidase mentioned in step S3 is one or more combinations of α-amylase, β-amylase, glucosylamylase, glycosylpeptidase F, or pullulanase.
[0018] Preferably, the total enzyme activity concentration of the glycosidase in step S3 is controlled within the range of 500~2000 U / mL.
[0019] Preferably, the glycosidase treatment temperature in step S3 is 40℃~60℃, and the treatment time is 20~40 minutes.
[0020] Preferably, the filter membrane used in steps S2 and S3 is a polycarbonate membrane with a diameter of 0.22 μm.
[0021] Preferably, extracellular DNA is extracted in step S4 using a free DNA extraction kit.
[0022] Compared with the prior art, the present invention has the following advantages:
[0023] (1) This invention employs a combined treatment of glycosidase and protease. By cleaving the polysaccharide backbone, branching connections, and protein chains in microbial aggregates, it completely disrupts the binding of extracellular DNA by the polysaccharide and protein backbone, transforming the dense microbial aggregate structure into a loose state, thereby fully releasing the intertwined extracellular DNA within. Experimental data show that, compared to the traditional method using only protease, this invention can increase the extracellular DNA extraction yield by up to 97.6%.
[0024] (2) This invention ensures effective separation of intracellular DNA and extracellular DNA through two filtration separation and filtrate merging operations, avoiding contamination and interference from intracellular DNA. This invention does not cause significant intracellular DNA leakage, nor does it interfere with the fragment length distribution of extracellular DNA, thus ensuring the accuracy and reproducibility of downstream molecular biology analyses.
[0025] (3) The present invention uses enzymatic hydrolysis to replace physical crushing or strong chemical lysis methods. It does not require complex equipment, the reaction conditions are mild, and the glycosidases and proteases used are commercially available conventional enzyme preparations. The cost is controllable and it is easy to promote and implement in conventional molecular biology laboratories. It is suitable for daily monitoring and analysis of large batches of environmental samples. Attached Figure Description
[0026] Figure 1 A comparison chart showing the content of extracellular DNA extracted using different treatment methods. Detailed Implementation
[0027] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly, provided that there is no mutual conflict.
[0028] In the following examples and comparative examples, the samples to be extracted were transparent extracellular polymeric particles obtained by passing environmental river water through a membrane.
[0029] The cell-free DNA extraction kits used in the following examples and comparative examples were blood cell-free circulating nucleic acid extraction kits (magnetic bead method) purchased from Wuxi Baimaige Biotechnology Co., Ltd.
[0030] Example 1
[0031] This embodiment employs a method for extracting extracellular DNA from microbial aggregates using a combination of α-amylase and trypsin, as detailed below:
[0032] (1) Take an appropriate amount of the sample to be extracted, add 10 mL of phosphate buffer solution containing 5 mM EDTA as a chelating agent, wash for 5 minutes to initially break down the ion bridging effect in the microbial aggregates.
[0033] (2) Add trypsin solution to the washed sample to a final concentration of 0.125 mg / mL and hydrolyze for 5 minutes. Then add an equal volume of trypsin inhibitor solution to terminate the enzymatic reaction. Use a 0.22 μm polycarbonate membrane for constant pressure filtration at low temperature to obtain membrane residue and a primary separation filtrate containing extracellular DNA.
[0034] (3) The residue on the membrane was extracted a second time using α-amylase solution, and the total enzyme activity concentration was controlled at 1000 U / mL. The membrane was treated at 50℃ for 30 minutes (determined based on known enzyme information and preliminary experiments). After treatment, the membrane was filtered again using a new 0.22 μm polycarbonate membrane under the same low temperature and constant pressure conditions to obtain a secondary separation filtrate containing extracellular DNA.
[0035] (4) Combine the primary separation filtrate obtained in step (2) and the secondary separation filtrate obtained in step (3), and place them in a freeze dryer for lyophilization and concentration. Then, use a free DNA extraction kit (magnetic bead method) to extract extracellular DNA from the above concentrated filtrate according to the manufacturer's instructions.
[0036] Example 2
[0037] This embodiment employs a method for extracting extracellular DNA from microbial aggregates using a combination of β-amylase and trypsin, as detailed below:
[0038] (1) Take an appropriate amount of the sample to be extracted, add 10 mL of phosphate buffer solution containing 5 mM EDTA, wash for 5 minutes to initially break down the ion bridging in the microbial aggregates.
[0039] (2) Add trypsin solution to the washed sample to a final concentration of 0.125 mg / mL and hydrolyze for 5 minutes. Then add an equal volume of trypsin inhibitor solution to terminate the enzymatic reaction. Use a 0.22 μm polycarbonate membrane for constant pressure filtration at low temperature to obtain membrane residue and a primary separation filtrate containing extracellular DNA.
[0040] (3) The residue on the membrane was extracted a second time using β-amylase solution, and the total enzyme activity concentration was controlled at 1000 U / mL. The membrane was treated at 50℃ for 30 minutes (determined based on known enzyme information and preliminary experiments). After treatment, the membrane was filtered again using a new 0.22 μm polycarbonate membrane under the same low temperature and constant pressure conditions to obtain a secondary separation filtrate containing extracellular DNA.
[0041] (4) Combine the primary separation filtrate obtained in step (2) and the secondary separation filtrate obtained in step (3), and place them in a freeze dryer for lyophilization and concentration. Then, use a free DNA extraction kit (magnetic bead method) to extract extracellular DNA from the above concentrated filtrate according to the manufacturer's instructions.
[0042] Example 3
[0043] This embodiment employs a method of extracting extracellular DNA from microbial aggregates using a combination of glucoamylase and trypsin, as detailed below:
[0044] (1) Take an appropriate amount of the sample to be extracted, add 10 mL of phosphate buffer solution containing 5 mM EDTA, wash for 5 minutes to initially break down the ion bridging in the microbial aggregates.
[0045] (2) Add trypsin solution to the washed sample to a final concentration of 0.125 mg / mL and hydrolyze for 5 minutes. Then add an equal volume of trypsin inhibitor solution to terminate the enzymatic reaction. Use a 0.22 μm polycarbonate membrane for constant pressure filtration at low temperature to obtain membrane residue and a primary separation filtrate containing extracellular DNA.
[0046] (3) The residue on the membrane was extracted a second time using a glucoamylase solution, with the total enzyme activity concentration controlled at 1000 U / mL, and treated at 50℃ (determined based on known enzyme information and preliminary experiments) for 30 minutes. After treatment, the residue was filtered again using a new 0.22 μm polycarbonate membrane under the same low temperature and constant pressure conditions to obtain a secondary separation filtrate containing extracellular DNA.
[0047] (4) Combine the primary separation filtrate obtained in step (2) and the secondary separation filtrate obtained in step (3), and place them in a freeze dryer for lyophilization and concentration. Then, use a free DNA extraction kit (magnetic bead method) to extract extracellular DNA from the above concentrated filtrate according to the manufacturer's instructions.
[0048] Example 4
[0049] This embodiment employs a method for extracting extracellular DNA from microbial aggregates using a combination of glycosylpeptidase F and trypsin, as detailed below:
[0050] (1) Take an appropriate amount of the sample to be extracted, add 10 mL of phosphate buffer solution containing 5 mM EDTA, wash for 5 minutes to initially break down the ion bridging in the microbial aggregates.
[0051] (2) Add trypsin solution to the washed sample to a final concentration of 0.125 mg / mL and hydrolyze for 5 minutes. Then add an equal volume of trypsin inhibitor solution to terminate the enzymatic reaction. Use a 0.22 μm polycarbonate membrane for constant pressure filtration at low temperature to obtain membrane residue and a primary separation filtrate containing extracellular DNA.
[0052] (3) The residue on the membrane was extracted a second time using glycosylpeptidase F solution, and the total enzyme activity concentration was controlled at 1000 U / mL. The membrane was treated at 50℃ for 30 minutes (determined based on known enzyme information and preliminary experiments). After treatment, the membrane was filtered again using a new 0.22 μm polycarbonate membrane under the same low temperature and constant pressure conditions to obtain a secondary separation filtrate containing extracellular DNA.
[0053] (4) Combine the primary separation filtrate obtained in step (2) and the secondary separation filtrate obtained in step (3), and place them in a freeze dryer for lyophilization and concentration. Then, use a free DNA extraction kit (magnetic bead method) to extract extracellular DNA from the above concentrated filtrate according to the manufacturer's instructions.
[0054] Example 5
[0055] This embodiment employs a method of extracting extracellular DNA from microbial aggregates using pullulanase and trypsin in combination, as detailed below:
[0056] (1) Take an appropriate amount of the sample to be extracted, add 10 mL of phosphate buffer solution containing 5 mM EDTA, wash for 5 minutes to initially break down the ion bridging in the microbial aggregates.
[0057] (2) Add trypsin solution to the washed sample to a final concentration of 0.125 mg / mL and hydrolyze for 5 minutes. Then add an equal volume of trypsin inhibitor solution to terminate the enzymatic reaction. Use a 0.22 μm polycarbonate membrane for constant pressure filtration at low temperature to obtain membrane residue and a primary separation filtrate containing extracellular DNA.
[0058] (3) Pullulanase solution was used to perform a second extraction on the residue on the membrane, and the total enzyme activity concentration was controlled at 1000 U / mL. The mixture was treated at 50℃ for 30 minutes (determined based on known enzyme information and preliminary experiments). After treatment, the residue was filtered again using a new 0.22 μm polycarbonate membrane under the same low temperature and constant pressure conditions to obtain a secondary separation filtrate containing extracellular DNA.
[0059] (4) Combine the primary separation filtrate obtained in step (2) and the secondary separation filtrate obtained in step (3), and place them in a freeze dryer for lyophilization and concentration. Then, use a free DNA extraction kit (magnetic bead method) to extract extracellular DNA from the above concentrated filtrate according to the manufacturer's instructions.
[0060] Example 6
[0061] This embodiment employs a method for extracting extracellular DNA from microbial aggregates using a combination of α-amylase, β-amylase, and trypsin, as detailed below:
[0062] (1) Take an appropriate amount of the sample to be extracted, add 10 mL of phosphate buffer solution containing 5 mM EDTA, wash for 5 minutes to initially break down the ion bridging in the microbial aggregates.
[0063] (2) Add trypsin solution to the washed sample to a final concentration of 0.125 mg / mL and hydrolyze for 5 minutes. Then add an equal volume of trypsin inhibitor solution to terminate the enzymatic reaction. Use a 0.22 μm polycarbonate membrane for constant pressure filtration at low temperature to obtain membrane residue and a primary separation filtrate containing extracellular DNA.
[0064] (3) The residue on the membrane was extracted a second time using a mixed solution of α-amylase and β-amylase, with the total enzyme activity concentration controlled at 1000 U / mL. The mixture was treated at 50℃ for 30 minutes (determined based on known enzyme information and preliminary experiments). After treatment, the residue was filtered again using a new 0.22 μm polycarbonate membrane under the same low temperature and constant pressure conditions to obtain a secondary separation filtrate containing extracellular DNA.
[0065] (4) Combine the primary separation filtrate obtained in step (2) and the secondary separation filtrate obtained in step (3), and place them in a freeze dryer for lyophilization and concentration. Then, use a free DNA extraction kit (magnetic bead method) to extract extracellular DNA from the above concentrated filtrate according to the manufacturer's instructions.
[0066] Example 7
[0067] This embodiment employs a method for extracting extracellular DNA from microbial aggregates using pullulanase, glucoamylase, and trypsin in combination, as detailed below:
[0068] (1) Take an appropriate amount of the sample to be extracted, add 10 mL of phosphate buffer solution containing 5 mM EDTA, wash for 5 minutes to initially break down the ion bridging in the microbial aggregates.
[0069] (2) Add trypsin solution to the washed sample to a final concentration of 0.125 mg / mL and hydrolyze for 5 minutes. Then add an equal volume of trypsin inhibitor solution to terminate the enzymatic reaction. Use a 0.22 μm polycarbonate membrane for constant pressure filtration at low temperature to obtain membrane residue and a primary separation filtrate containing extracellular DNA.
[0070] (3) The residue on the membrane was extracted a second time using a pullulanase + glucoamylase mixed solution, and the total enzyme activity concentration was controlled at 1000 U / mL. The membrane was treated at 50℃ for 30 minutes (determined based on known enzyme information and preliminary experiments). After treatment, the membrane was filtered again using a new 0.22 μm polycarbonate membrane under the same low temperature and constant pressure conditions to obtain a secondary separation filtrate containing extracellular DNA.
[0071] (4) Combine the primary separation filtrate obtained in step (2) and the secondary separation filtrate obtained in step (3), and place them in a freeze dryer for lyophilization and concentration. Then, use a free DNA extraction kit (magnetic bead method) to extract extracellular DNA from the above concentrated filtrate according to the manufacturer's instructions.
[0072] Example 8
[0073] This embodiment employs a method for extracting extracellular DNA from microbial aggregates using a combination of α-amylase, pullulanase, glucoamylase, and trypsin, as detailed below:
[0074] (1) Take an appropriate amount of the sample to be extracted, add 10 mL of phosphate buffer solution containing 5 mM EDTA, wash for 5 minutes to initially break down the ion bridging in the microbial aggregates.
[0075] (2) Add trypsin solution to the washed sample to a final concentration of 0.125 mg / mL and hydrolyze for 5 minutes. Then add an equal volume of trypsin inhibitor solution to terminate the enzymatic reaction. Use a 0.22 μm polycarbonate membrane for constant pressure filtration at low temperature to obtain membrane residue and a primary separation filtrate containing extracellular DNA.
[0076] (3) The residue on the membrane was extracted a second time using a mixed solution of α-amylase, pullulanase, and glucoamylase, with the total enzyme activity concentration controlled at 1000 U / mL. The mixture was treated at 50℃ for 30 minutes (determined based on known enzyme information and preliminary experiments). After treatment, the residue was filtered again using a new 0.22 μm polycarbonate membrane under the same low temperature and constant pressure conditions to obtain a secondary separation filtrate containing extracellular DNA.
[0077] (4) Combine the primary separation filtrate obtained in step (2) and the secondary separation filtrate obtained in step (3), and place them in a freeze dryer for lyophilization and concentration. Then, use a free DNA extraction kit (magnetic bead method) to extract extracellular DNA from the above concentrated filtrate according to the manufacturer's instructions.
[0078] Example 9
[0079] This embodiment employs a method for extracting extracellular DNA from microbial aggregates using a combination of α-amylase, pullulanase, and trypsin, as detailed below:
[0080] (1) Take an appropriate amount of the sample to be extracted, add 10 mL of phosphate buffer solution containing 5 mM EDTA, wash for 5 minutes to initially break down the ion bridging in the microbial aggregates.
[0081] (2) Add trypsin solution to the washed sample to a final concentration of 0.125 mg / mL and hydrolyze for 5 minutes. Then add an equal volume of trypsin inhibitor solution to terminate the enzymatic reaction. Use a 0.22 μm polycarbonate membrane for constant pressure filtration at low temperature to obtain membrane residue and a primary separation filtrate containing extracellular DNA.
[0082] (3) The residue on the membrane was extracted a second time using a mixed solution of α-amylase and pullulanase, with the total enzyme activity concentration controlled at 1000 U / mL, and treated at 50℃ for 30 minutes (determined based on known enzyme information and preliminary experiments). After treatment, the residue was filtered again using a new 0.22 μm polycarbonate membrane under the same low temperature and constant pressure conditions to obtain a secondary separation filtrate containing extracellular DNA.
[0083] (4) Combine the primary separation filtrate obtained in step (2) and the secondary separation filtrate obtained in step (3), and place them in a freeze dryer for lyophilization and concentration. Then, use a free DNA extraction kit (magnetic bead method) to extract extracellular DNA from the above concentrated filtrate according to the manufacturer's instructions.
[0084] Comparative Example
[0085] This comparative example provides a method for extracting extracellular DNA from microbial aggregates using only trypsin, as detailed below:
[0086] (1) Take an appropriate amount of the sample to be extracted, add 10 mL of phosphate buffer solution containing 5 mM EDTA, wash for 5 minutes to initially break down the ion bridging in the microbial aggregates.
[0087] (2) Add trypsin solution to the washed sample to a final concentration of 0.125 mg / mL and hydrolyze for 5 minutes. Then add an equal volume of trypsin inhibitor solution to terminate the enzymatic reaction. Filter under constant pressure using a 0.22 μm polycarbonate membrane at low temperature to obtain a separation filtrate containing extracellular DNA.
[0088] (3) The separation filtrate obtained in step (2) was placed in a freeze dryer for lyophilization and concentration. Then, extracellular DNA was extracted from the concentrated filtrate using a free DNA extraction kit (magnetic bead method) according to the manufacturer's instructions.
[0089] To verify the concentration of extracellular DNA extracted in each example and comparative example, the extracellular DNA extracted in Examples 1-9 and the comparative example was analyzed using an ultra-micro spectrophotometer (Nano-300). The results are as follows: Figure 1 As shown.
[0090] according to Figure 1 It can be seen that the extracellular DNA content obtained in the comparative example was 9.90±0.30 μg / L. The extracellular DNA content obtained in Example 1 was 12.12±0.25 μg / L; the extracellular DNA content obtained in Example 2 was 9.30±0.60 μg / L; the extracellular DNA content obtained in Example 3 was 10.82±0.15 μg / L; the extracellular DNA content obtained in Example 4 was 10.50±0.50 μg / L; the extracellular DNA content obtained in Example 5 was 12.85±0.08 μg / L; the extracellular DNA content obtained in Example 6 was 12.72±0.35 μg / L; the extracellular DNA content obtained in Example 7 was 14.95±0.97 μg / L; the extracellular DNA content obtained in Example 8 was 19.60±2.07 μg / L; and the extracellular DNA content obtained in Example 9 was 16.30±0.76 μg / L.
[0091] Compared with the comparative examples, the use of α-amylase alone for secondary separation in Example 1 increased the content of extracted extracellular DNA by 22.42%; Example 5 by 29.80%; Example 6 by 28.48%; Example 7 by 51.01%; Example 8 by 97.60%; and Example 9 by 64.20%. Furthermore, fluorescence microscopy staining of the membrane residue after secondary extraction and separation revealed that the sample contained almost entirely intracellular DNA, indicating good separation.
[0092] In summary, the extraction method provided by this invention releases extracellular DNA from microbial aggregates. The combined use of α-amylase + pullulanase + glucoamylase yields the best extraction effect, followed by the combined use of α-amylase + pullulanase. This method can accurately extract exDNA from microbial aggregates, overcoming the low extraction efficiency caused by neglecting the polysaccharide backbone in existing chelating agent and protease methods. It is of great value for accurately assessing environmental DNA, gene transfer, and microbial community structure in aquatic ecosystems.
[0093] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.
Claims
1. A method for extracting extracellular DNA from microbial aggregates using a combination of glycosidase and protease, characterized in that, Specifically as follows: S1: The chelating agent is used to pre-treat the sample containing microbial aggregates to initially break down the ion bridging effect in the microbial aggregates; S2: Add protease to the pretreated sample to carry out an enzymatic hydrolysis reaction to destroy the protein backbone in the microbial aggregate; after the enzymatic hydrolysis reaction is terminated, use a filter membrane for the first filtration separation to obtain a primary separation filtrate containing extracellular DNA and membrane residue, respectively. S3: The membrane residue obtained in step S2 is treated with glycosidase to sever the main chain and branch connections of the polysaccharide backbone in the microbial aggregate; after the reaction, a second filtration separation is performed using a new filter membrane, and the secondary separation filtrate containing extracellular DNA is retained; S4: Combine the primary and secondary separation filtrates and concentrate them to extract extracellular DNA from the concentrated filtrate.
2. The method for extracting extracellular DNA from microbial aggregates using a combination of glycosidase and protease according to claim 1, characterized in that, The chelating agent used in step S1 is a buffer solution of ethylenediaminetetraacetic acid or sodium pyrophosphate.
3. The method for extracting extracellular DNA from microbial aggregates using a combination of glycosidase and protease according to claim 1, characterized in that, The concentration of the chelating agent in step S1 is 5-10 mM, and the pretreatment reaction time is 3-10 minutes.
4. The method for extracting extracellular DNA from microbial aggregates using a combination of glycosidase and protease according to claim 1, characterized in that, The protease used in step S2 is either trypsin or proteinase K.
5. The method for extracting extracellular DNA from microbial aggregates using a combination of glycosidase and protease according to claim 1, characterized in that, The final concentration of the protease in step S2 is 1~2.5 mg / mL; the enzymatic hydrolysis reaction time is 5~15 minutes.
6. The method for extracting extracellular DNA from microbial aggregates using a combination of glycosidase and protease according to claim 1, characterized in that, The glycosidase mentioned in step S3 is one or more combinations of α-amylase, β-amylase, glucosylamylase, glycosylpeptidase F, or pullulanase.
7. The method for extracting extracellular DNA from microbial aggregates using a combination of glycosidase and protease according to claim 1, characterized in that, The total enzyme activity concentration of the glycosidase in step S3 is controlled within the range of 500~2000 U / mL.
8. The method for extracting extracellular DNA from microbial aggregates using a combination of glycosidase and protease according to claim 1, characterized in that, The glycosidase treatment in step S3 is carried out at a temperature of 40℃~60℃ for 20~40 minutes.
9. The method for extracting extracellular DNA from microbial aggregates using a combination of glycosidase and protease according to claim 1, characterized in that, The filter membranes used in steps S2 and S3 are polycarbonate membranes with a diameter of 0.22 μm.
10. The method for extracting extracellular DNA from microbial aggregates using a combination of glycosidase and protease according to claim 1, characterized in that, In step S4, extracellular DNA is extracted using a free DNA extraction kit.