Efficient collection and extraction method of air eDNA and application

Abstract

The application discloses a kind of efficient collection and extraction method and application of air eDNA.The method uses glass fiber filter as sampling filter, uses medium-flow particulate matter sampler and TSP sampling head to carry out sampling, uses dry ice to carry out sample preservation transportation, uses PBS buffer and low-temperature low-speed centrifugal elution DNA, uses PES filter to enrich and precipitate particulate matter, and PES filter is cut into small pieces to improve DNA extraction efficiency.The environmental DNA extracted by the application has higher concentration and good integrity, and the DNA extracted by the method can obtain good amplification results using universal primers of different species for amplification, which is convenient for subsequent eDNA macro-barcode technology to analyze species diversity.

Description

Technical Field

[0001] This invention belongs to the field of atmospheric environmental quality assessment technology, specifically a method and application for efficient collection and extraction of airborne eDNA. Background Technology

[0002] Environmental DNA (eDNA) technology enables the classification and identification of biological species by sampling, extracting, and analyzing residual DNA fragments in environmental samples, without the need for pre-separation of complete microbial, animal, or plant samples. As an emerging biomonitoring method, airborne eDNA technology has matured and expanded into various application areas such as ecological monitoring and biodiversity assessment, thanks to advancements in detecting plant pollen from the air and successfully capturing mammalian eDNA. Particularly in the detection of cryptic and invasive species, its high efficiency and non-invasive nature significantly overcome the limitations of traditional monitoring methods.

[0003] The core challenge of airborne eDNA extraction lies in the extremely low concentration and high degradation rate of DNA in the air, making it difficult to obtain high-quality DNA during collection and extraction, thus posing a significant challenge to this work. DNA in air samples is mainly attached to particulate matter such as microorganisms, pollen, and skin flakes, but its concentration is extremely low and its distribution is uneven. The amount of DNA eluted from collection filters or adsorbent materials is usually very small, possibly only a few nanograms or even less. Furthermore, airborne DNA fragments are easily degraded by ultraviolet light and nucleases, resulting in short and incomplete extracted DNA fragments. The quality of DNA extraction directly affects the subsequent PCR amplification and sequencing results.

[0004] Airborne eDNA technology enables efficient monitoring of ecosystem biodiversity by collecting and analyzing suspended DNA fragments (such as dander, pollen, and microorganisms) in the air. This technology offers advantages such as non-invasiveness and low disturbance, allowing for continuous operation and revealing patterns of species migration and seasonal variation. Compared to traditional manual survey methods that are labor-intensive and difficult to implement at high frequencies on large scales, airborne eDNA technology can capture massive amounts of species information more quickly and comprehensively, thereby driving a shift in ecological conservation paradigms from single-species management to holistic system monitoring. This technology is expected to become the "new standard" for future ecological monitoring, providing a scientific basis for the formulation of conservation strategies. Summary of the Invention

[0005] Technical problem solved: In view of the shortcomings of existing air eDNA sampling and extraction technologies, this invention provides a highly efficient method for collecting and extracting air eDNA, which solves the problem of obtaining high-quality DNA in the current process of collecting and extracting eDNA from the atmospheric environment.

[0006] The first objective of this invention is to provide a highly efficient method for collecting and extracting airborne eDNA, comprising the following steps:

[0007] a. Sampling filter membrane preparation: Use sterile glass fiber filter membrane;

[0008] b. Air sample collection;

[0009] c. Sampling filter membrane transportation and storage: Store and transport at -80℃ to -70℃;

[0010] d. Pretreatment of sampling filter membrane: Immerse the sampling filter membrane in PBS buffer;

[0011] e. Elution and collection: Centrifuge the mixture of PBS buffer and sampling filter membrane at 3-5℃ and 100-300 g for 2-4 hours to elute the precipitated particulate matter;

[0012] f. Filtration and enrichment: After resuspending the solution from step e, filter it under vacuum and enrich the precipitated particulate matter using a PES filter membrane;

[0013] g. Divide the PES filter membrane into small pieces for DNA extraction.

[0014] Preferably, the air sample collection is performed using a medium-flow particulate matter sampler and a total suspended particulate matter sampler.

[0015] Preferably, the sampling duration is 24 hours.

[0016] Preferably, the sampling filter membrane is transported and preserved using dry ice.

[0017] Preferably, the centrifugation at 3-5℃ and 100-300 g for 2-4 hours is equivalent to centrifugation at 4℃ and 200 g for 3 hours.

[0018] Preferably, the pore size of the PES filter membrane is 0.22 μm.

[0019] Preferably, the small piece has an area of ​​0.2-0.5 cm². 2 .

[0020] Preferably, the small piece is 0.5*0.4 cm.

[0021] Preferably, the specific steps are as follows:

[0022] a. Sampling filter membrane preparation: Use sterile glass fiber filter membranes and store them in sterile foil bags or petri dishes before sampling;

[0023] b. Air sample collection: Samples were collected using a medium-flow particulate matter sampler and a total suspended particulate matter sampling head;

[0024] c. Sampling filter membrane transportation and preservation: After sampling, the samples are preserved and transported using dry ice;

[0025] d. Pretreatment of sampling filter membrane: Under aseptic conditions, transfer the glass fiber filter membrane containing the collected eDNA into a centrifuge tube, and add 1×PBS buffer until the filter membrane is completely submerged;

[0026] e. Elution and collection: Tighten the centrifuge tube cap to prevent leakage, centrifuge at 200 g for 3 hours at 4°C to elute the precipitated particulate matter;

[0027] f. Filtration and enrichment: Gently shake the centrifuge tube and pour the resuspended solution into a vacuum filtration flask for filtration. Use a 0.22 μm PES filter membrane to enrich the precipitated particulate matter.

[0028] g. Rinse the centrifuge tube with 50 mL of sterile water and filter again (repeat step f) to improve elution and enrichment efficiency;

[0029] h. Cut the PES filter membrane into 0.5*0.4 cm pieces in a sterile petri dish and transfer them to a 2 mL extraction tube;

[0030] i. Add 1000 µL of extraction solution to the extraction tube and vortex to mix. Add proteinase K, shake to mix for 5 s, and incubate at 37°C for 30 minutes on a constant temperature shaker at 180 rpm.

[0031] j. Add 400 µL of preheated 20% sodium dodecyl sulfate, mix gently, and incubate in a 65°C water bath for 2 hours, inverting and mixing once every 30 minutes during the process;

[0032] k. Centrifuge the water-baked centrifuge tubes at 12,000 rpm for 3 minutes, and transfer the supernatant to a new centrifuge tube;

[0033] 1. Add an equal volume of chloroform-isoamyl alcohol solution and mix by inverting for 2 minutes. Centrifuge at 12,000 rpm for 5 minutes. Transfer the supernatant to a new centrifuge tube and repeat this step once.

[0034] m. Transfer the supernatant to a new centrifuge tube, add 600 µL of isoamyl alcohol and mix well. After standing at room temperature for 1 hour, centrifuge at 12000 rpm for 10 minutes at 4°C and discard the supernatant.

[0035] n. Add 1 mL of 70% ethanol to the centrifuge tube to wash the DNA precipitate, discard the supernatant, and repeat this step once;

[0036] o. Allow the container to air dry at room temperature until the alcohol has completely evaporated, then add 50 µL of sterile water to dissolve the DNA.

[0037] A second objective of this invention is to provide the application of the above-described method in the extraction of airborne eDNA.

[0038] Compared with the prior art, the present invention has the following technical effects:

[0039] This invention provides an experimental analysis method for collecting and extracting eDNA from the atmospheric environment, which has the following advantages compared with existing technologies:

[0040] 1. This invention uses glass fiber filter membranes, resulting in low cost;

[0041] 2. This invention improves the collection efficiency of airborne eDNA by using a medium-flow-rate sampler and long-term sampling.

[0042] 3. This invention reduces DNA degradation during transportation by preserving the transport sampling filter membrane at low temperatures;

[0043] 4. This invention employs an elution enrichment method, which improves the enrichment efficiency of particulate matter and DNA on large-size filter membranes or adsorbent materials.

[0044] 5. Use ultrapure 1×PBS buffer (Phosphate Buffered Saline) to elute large or multiple filter membranes to increase the concentration of subsequent DNA extraction. This invention uses low temperature and low speed centrifugation, which reduces the degradation rate of DNA fragments during elution and enrichment, thereby maintaining the integrity of the extracted DNA fragments.

[0045] 6. This invention uses a 0.22 μm PES (polyethersulfone) membrane to filter and enrich particulate matter in the eluent, thereby improving the quality of DNA extraction; the PES membrane is cut into small pieces and proteinase K is added to improve the efficiency and quality of subsequent DNA extraction.

[0046] 7. This invention significantly improves the quality of DNA extraction by optimizing the sampling and extraction methods for eDNA;

[0047] 8. This invention standardizes the sampling and extraction process of airborne eDNA, avoids differences in sample DNA quality caused by non-standard methods, improves the enrichment of airborne eDNA and the efficiency and quality of subsequent DNA extraction, ensures the accuracy of subsequent experimental analysis results, and simplifies the analysis work of experimental personnel.

[0048] 9. This invention has a wide range of applications and is well-suited for various atmospheric environments. The environmental DNA extracted using the method of this invention has a high concentration and good integrity. Furthermore, the DNA extracted using this method can be amplified using universal primers for different species, resulting in excellent amplification results and facilitating subsequent species diversity analysis using eDNA macrobarcoding technology. Attached Figure Description

[0049] Figure 1 It represents the DNA concentration collected by filter membranes of different materials.

[0050] Figure 2 These represent the DNA concentrations obtained at different sampling times.

[0051] Figure 3 These are the DNA concentrations under different transport and storage conditions.

[0052] Figure 4 This refers to the DNA concentration under different elution buffer types.

[0053] Figure 5 It is the DNA concentration collected by the filter membrane under different elution methods.

[0054] Figure 6 It refers to the DNA concentration under different treatments of the filter membrane.

[0055] Figure 7 It refers to the DNA concentration at different types of filter membranes.

[0056] Figure 8 These are the dominant animal and plant species detected in actual case applications. Detailed Implementation

[0057] The basic experiments of this invention are as follows (Examples 1-8 are all improvements on the basic experiments):

[0058] 1. Air sample collection

[0059] The sampling points were located in Yuexiu District, Guangzhou, mainly concentrated near Guangzhou Zoo (Xianlie Middle Road). This area has a rich variety of flora and fauna, and the airborne eDNA contains information on various plants and animals. At the main entrance of Guangzhou Zoo, three sampling points (numbered H1, H2, and H3, with an adjacent sampling point spacing of approximately 100 m) were evenly selected along both sides of Xianlie Middle Road for sampling. Three samples were collected from each sampling point, for a total of nine air samples. The sampling flow rate of the medium-flow particulate matter sampler (Laoying 2050 air / intelligent TSP integrated sampler) was checked using a flow calibrator. If the flow rate error exceeded ±2% of the sampler's set flow rate (100 L / min), the sampling flow rate should be calibrated. The sterilized air collection filter membrane (the filter membrane used in Examples 2-7 was a glass fiber filter membrane) was placed on the filter screen in the sterilized sampling clip, with the rough side of the filter membrane facing the air inlet direction. The filter membrane was then firmly pressed to ensure no air leakage. Install the sampling head (Total Suspended Particulates Sampler, TSP), set the sampling time according to the sampler's instructions, and start sampling (temperature 26.3℃-36.9℃, humidity 53%-67%, atmospheric pressure 100.7-101.6 kPa). The sampling duration is 24 hours (the sampling duration for Examples 1 and 3-7 is 24 hours). For the transportation of the airborne eDNA filter membrane, dry ice is used for preservation and transportation (dry ice is used for preservation in Examples 1-2 and 4-7). During transportation, it must not be squeezed, inverted, or subjected to significant vibration.

[0060] 2. Specific steps for DNA extraction from the filter membrane.

[0061] A. Using tweezers, roll up a 90 mm diameter air collection filter membrane on a sterile workbench, and then insert the rolled-up filter membrane into a 50 mL sterile centrifuge tube.

[0062] B. Add 50 mL of 1× ultrapure PBS buffer (PBS buffer was used in Examples 1-3 and 5-7) to the test tube, tighten the cap, and then centrifuge at 4°C and 200 g for 3 hours to precipitate particulate matter. Keep the supernatant for later use.

[0063] C. Using sterilized tweezers, remove the 0.22 μm PES filter membrane (PES filter membrane used in Examples 1-6) from the sterile packaging and place it on the gauze of a sterilized filter membrane funnel (for support and replacement - to avoid cross-contamination). Then screw the funnel onto a 1-liter glass bottle and connect the funnel to a vacuum pump. Gently shake the test tube after centrifugation in step B to resuspend the precipitate at the bottom of the tube and pour it into the funnel, while not disturbing the collecting filter membrane. Then begin suction filtration until all liquid is completely filtered.

[0064] D. After filtration, use sterilized forceps to place the PES filter membrane (in Examples 1 and 5, the filter membrane is kept intact; in Examples 2-4 and 7, it is cut into 0.5*0.4 cm pieces) into a 2 mL extraction tube.

[0065] E: Add 1000 µL of extraction buffer (S0 extraction buffer: 1) 6.8 mL of 1M NaH2PO4 + 93.2 mL of 1M Na2HPO4 6.8 mL, mix and adjust the pH to 8.0 with NaOH; 2) Add 200 mL of 0.5M EDTA (pH=8.0), 100 mL of 1M Tris-HCl and 300 mL of 3M NaCl to the mixture; 3) Use ddH2O to make up to 1 L, sterilize and set aside) into a 2 mL extraction tube and vortex to mix, add proteinase K (20 mg / mL), shake to mix for 5 s, and incubate at 37℃ on a constant temperature shaker at 180 rpm for 30 minutes.

[0066] F: Add 400 µL of 20% sodium dodecyl sulfonate (SDS, w / v) preheated at 65°C, mix gently, and incubate in a 65°C water bath for 2 hours, inverting and mixing once every 30 minutes.

[0067] G: Centrifuge the centrifuge tubes after water bath at 12,000 rpm for 3 minutes, and transfer the supernatant to a new centrifuge tube.

[0068] H: Add an equal volume of chloroform-isoamyl alcohol (24:1, v / v), mix by inverting for 2 minutes, centrifuge at 12000 rpm for 5 minutes, transfer the supernatant to a new centrifuge tube, and repeat this step once.

[0069] I: Transfer the supernatant to a new centrifuge tube, add 600 µL of isoamyl alcohol and mix well. After standing at room temperature for 1 hour, centrifuge at 12000 rpm for 10 minutes at 4°C and discard the supernatant.

[0070] J: Add 1 mL of 70% ethanol aqueous solution (v / v) to the centrifuge tube to wash the DNA precipitate, centrifuge at 12000 rpm for 1 minute, discard the supernatant and repeat this step once.

[0071] K: Open the centrifuge tube and dry it at room temperature for 3-5 minutes to allow the alcohol residue to evaporate completely. Add 50 µL of sterile water to dissolve the DNA and store it at -20°C.

[0072] 3. Determine the concentration of DNA extracted from air samples.

[0073] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0074] Example 1:

[0075] The sampling point selection and air collection filter membrane in step 1 are as follows, and the remaining steps are the same as the basic experiment described above.

[0076] 1. Sampling Site Selection: The sampling sites were located in Yuexiu District, Guangzhou, mainly concentrated near Guangzhou Zoo (Xianlie Middle Road). This area has a large number of plant and animal species, and airborne eDNA contains various biological information. Sampling was conducted near Xianlie Middle Road, the main entrance of Guangzhou Zoo. Airborne eDNA was collected using both glass fiber and quartz fiber filters (three replicates for each filter), resulting in a total of six air samples. The sampling instruments, methods, and duration were consistent with the basic experiments to assess the subsequent DNA content.

[0077] 2. DNA was extracted from the air samples using a DNA extraction method, with strictly consistent operating procedures. The concentration of the extracted product was determined using Nanodrop. Results are as follows: Figure 1 There was no significant difference in the DNA content collected by glass fiber membrane and quartz fiber membrane, while the cost of glass fiber membrane was less than one-tenth that of quartz fiber membrane.

[0078] Example 2:

[0079] The sampling point selection and sampling duration in step 1 are as follows, and the remaining steps are the same as the basic experiment described above.

[0080] 1. Sampling Site Selection: The sampling sites were located in Yuexiu District, Guangzhou, mainly concentrated near Guangzhou Zoo (Xianlie Middle Road). Sampling was conducted near Xianlie Middle Road at Guangzhou Huanghuagang Zoo, with samples collected at 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h (three replicates for each sampling time), for a total of 21 air samples. The sampling instruments and filter membranes used were consistent to assess the subsequent DNA content.

[0081] 2. DNA was extracted from the air samples using a DNA extraction method, with strictly consistent operating procedures. The concentration of the extracted product was determined using Nanodrop. Results are as follows: Figure 2 The longer the sampling time, the higher the DNA content. The highest DNA content was obtained when the sampling time was 24 hours.

[0082] Example 3:

[0083] The sampling point selection, sample preservation method, and preservation time in step 1 are as follows; the remaining steps are the same as the basic experiment described above.

[0084] 1. Sampling Site Selection: The sampling sites were located in Yuexiu District, Guangzhou, mainly concentrated near Guangzhou Zoo (Xianlie Middle Road). Sampling was conducted near Xianlie Middle Road in Guangzhou Zoo, using the same sampling instruments, sampling filters, and sampling duration. A total of 21 air samples were collected. After sampling, the samples were preserved using the following methods: room temperature storage for 12 h and 24 h, ice pack refrigeration (0~4℃) for 12 h, 24 h, and 48 h, and dry ice storage for 24 h and 48 h (three replicates per week) to assess the subsequent DNA content.

[0085] 2. DNA was extracted from the air samples using a DNA extraction method, with strictly consistent operating procedures. The concentration of the extracted product was determined using Nanodrop. Results are as follows: Figure 3 Using dry ice for transportation and preservation of filter membranes yields the best results.

[0086] Example 4:

[0087] The sampling point selection and sample processing methods in step 2-B are as follows, and the remaining steps are the same as the basic experiment described above.

[0088] 1. Sampling Site Selection: The sampling sites were located in Yuexiu District, Guangzhou, mainly concentrated near Guangzhou Zoo (Xianlie Middle Road). Samples were collected near the entrance of Guangzhou Zoo, totaling 9 air samples. All samples used the same collection method, equipment, sampling time, and adsorption filter membrane to ensure consistency in subsequent DNA content assessment.

[0089] 2. Sample preparation: Add 50 mL of sterile water, 75% alcohol solution (v / v) and 1× ultrapure PBS buffer (Phosphate Buffered Saline) to the test tubes in step A for elution (3 replicates for each treatment).

[0090] DNA was extracted from air samples using the same DNA extraction method, with strictly consistent operational procedures. The concentration of the extracted products was detected using a nucleic acid detector (Nanodrop). Results are as follows: Figure 4 The results showed that the DNA concentration extracted with PBS buffer was the highest.

[0091] Example 5:

[0092] The sampling point selection and the elution method for the sampling filter membrane in step 2-B are as follows, and the remaining steps are the same as the basic experiment described above.

[0093] 1. Sampling Site Selection: The sampling sites were located in Yuexiu District, Guangzhou, mainly concentrated near Guangzhou Zoo (Xianlie Middle Road). Nine air samples were collected near Xianlie Middle Road at Guangzhou Zoo. The sampling methods, equipment, sampling time, and adsorption filter were all identical, used to assess subsequent DNA content.

[0094] 2. Three elution methods were used on the sampling filter membrane: Treatment 1, oscillation at 6 m / s for 30 s using a nucleic acid homogenizer; Treatment 2, centrifugation at 12000 r / min for 20 min at 4℃ using a low-temperature centrifuge; Treatment 3, centrifugation at 200 g for 3 hours at 4℃ (three replicates for each treatment).

[0095] DNA was extracted from air samples using the same DNA extraction method, with strictly consistent operational procedures. The concentration of the extracted products was determined using Nanodrop. Results are as follows: Figure 5 The highest concentration of DNA was extracted from treatment 3.

[0096] Example 6:

[0097] The sampling point selection and the selection and processing methods for the filter membrane material in step 2-D are as follows, and the remaining steps are the same as the basic experiment described above.

[0098] 1. Sampling Site Selection: The sampling sites were located in Yuexiu District, Guangzhou, mainly concentrated near Guangzhou Zoo (Xianlie Middle Road). Nine air samples were collected near Xianlie Middle Road at Guangzhou Zoo. The sampling methods, equipment, sampling time, and adsorption filter membranes were all identical, used to assess subsequent DNA content.

[0099] 2. Using a PES filter membrane, perform three treatments on the filter membrane: Treatment 1, keep the filter membrane intact; Treatment 2, cut it into pieces of about 1*1 cm; Treatment 3, cut it into pieces of about 0.5*0.4 cm (each treatment is repeated 3 times).

[0100] DNA was extracted from air samples using the same DNA extraction method, with strictly consistent operational procedures. The concentration of the extracted products was determined using Nanodrop. Results are as follows: Figure 6 The highest DNA concentration was extracted from samples cut into 0.5*0.4cm pieces after treatment 3.

[0101] Example 7:

[0102] The selection of sampling points and the selection of filter membrane material in step 2-C are as follows, and the remaining steps are the same as the basic experiment described above.

[0103] 1. Sampling Site Selection: The sampling sites for this study were located in Yuexiu District, Guangzhou City, mainly concentrated near Guangzhou Zoo (Xianlie Middle Road). A total of 6 air samples were collected near Xianlie Middle Road at Guangzhou Zoo. The sampling methods, equipment, sampling time, and adsorption filter membranes were all identical, and these samples were used to assess the subsequent DNA content.

[0104] 2. To compare the extraction efficiency of different filter membrane materials, filter membranes made of mixed cellulose (50 mm in size and 0.25 µm in pore size) and PES (polyethersulfone) were used respectively.

[0105] DNA was extracted from air samples using the same DNA extraction method, with strictly consistent operational procedures. The concentration of the extracted products was determined using Nanodrop. Results are as follows: Figure 7 The highest DNA concentration was extracted using PES (polyethersulfone) filtration membranes.

[0106] Example 8:

[0107] 1. Air sample collection

[0108] The sampling sites were located in Yuexiu District, Guangzhou, mainly concentrated near Guangzhou Zoo (Xianlie Middle Road). This area boasts a rich variety of flora and fauna, and the airborne eDNA contains diverse information on these species. At the main entrance of Guangzhou Zoo, three sampling points (numbered H1, H2, and H3, with an approximate spacing of 100m) were evenly selected along both sides of Xianlie Middle Road. Three samples were collected from each point, for a total of nine air samples. A flow calibrator was used to check the sampling flow rate of the medium-flow particulate matter sampler (Laoying 2050 air / intelligent TSP integrated sampler). If the flow rate error exceeded ±2% of the sampler's set flow rate (100 L / min), the sampling flow rate should be calibrated. The sterilized air collection filter membrane (glass fiber filter membrane, 90 mm in diameter, 0.3 μm pores) was placed on the filter screen inside the sterilized sampling clip, with the rough side of the filter membrane facing the air intake direction. The filter membrane was then firmly pressed together to ensure no leakage. Install the sampling head (total suspended particulate matter sampling head), set the sampling time according to the sampler's instruction manual, and start sampling (temperature 26.3℃-36.9℃, humidity 53%-67%, atmospheric pressure 100.7-101.6 kPa). The sampling duration is 24 hours. For the transportation of air eDNA filter membranes, use dry ice for storage and transport. Do not squeeze, invert, or subject to significant vibration during transportation.

[0109] 2. Specific steps for DNA extraction from the filter membrane.

[0110] A. Using tweezers, roll up a 90 mm diameter air collection filter membrane on a sterile workbench, and then insert the rolled-up filter membrane into a 50 mL sterile centrifuge tube.

[0111] B. Add 50 mL of ultrapure 1×PBS buffer to the test tube, tighten the cap, and then centrifuge at 200 g for 3 hours at 4°C to precipitate particulate matter. Keep the supernatant for later use.

[0112] C. Using sterilized tweezers, remove the 0.22 μm PES membrane filter from the sterile packaging and place it on the gauze of a sterilized filter funnel. Then, screw the funnel onto a 1-liter glass bottle and connect the funnel to a vacuum pump. Gently shake the test tube after centrifugation in step B to resuspend the precipitate at the bottom of the tube and pour it into the funnel. Do not disturb the collection filter membrane. Then, begin suction filtration until all the liquid has passed through the PES filter membrane.

[0113] D. After filtration, use sterilized forceps to transfer the PES filter membrane to a sterilized petri dish, and then use sterilized scissors to cut the filter membrane into small pieces (approximately 0.20 cm each). 2 (0.5*0.4 cm), and put it into a 2 mL extraction tube.

[0114] E: Add 1000 µL of extraction buffer (S0 extraction buffer) to a 2 mL extraction tube and vortex to mix. Add proteinase K (20 mg / mL), vortex to mix for 5 s, and incubate at 37°C for 30 minutes on a constant temperature shaker at 180 rpm.

[0115] F: Add 400 µL of 20% sodium dodecyl sulfonate (SDS, w / v) preheated at 65°C, mix gently, and incubate in a 65°C water bath for 2 hours, inverting and mixing once every 30 minutes.

[0116] G: Centrifuge the centrifuge tubes after water bath at 12,000 rpm for 3 minutes, and transfer the supernatant to a new centrifuge tube.

[0117] H: Add an equal volume of chloroform-isoamyl alcohol (24:1, v / v), mix by inverting for 2 minutes, centrifuge at 12000 rpm for 5 minutes, transfer the supernatant to a new centrifuge tube, and repeat this step once.

[0118] I: Transfer the supernatant to a new centrifuge tube, add 600 µL of isoamyl alcohol and mix well. After standing at room temperature for 1 hour, centrifuge at 12000 rpm for 10 minutes at 4°C and discard the supernatant.

[0119] J: Add 1 mL of 70% ethanol aqueous solution (v / v) to the centrifuge tube to wash the DNA precipitate. Centrifuge at 12000 rpm for 1 minute and remove the supernatant. Repeat this step once for the tube.

[0120] K: Open the centrifuge tube and dry it at room temperature for 3-5 minutes to allow the alcohol residue to evaporate completely. Add 50 µL of sterile water to dissolve the DNA and store it at -20°C.

[0121] 3. DNA concentration and flora and fauna diversity in air samples

[0122] DNA extracted from air samples was amplified by PCR, and the products were subjected to next-generation sequencing. High-quality rRNA gene amplicon sequences were analyzed using the open-source microbial ecology quantitative analysis software (QIIME2). Noise reduction was performed using DATA2 sequence reads in the QIIME2 system. The noise-reduced sequences were grouped into one class of ASVs with 100% similarity. Finally, SILVA132 was used for phylogenetic classification of the ASV sequences (http: / / www.mothur.org / wiki / Taxonnomy_outline), species annotation was performed, and species abundance information was obtained. The results are detailed in Table 1 and... Figure 8 .

[0123] The results showed that the concentrations of airborne DNA collected and extracted in this invention were 10.6, 8.2, and 11.5 ng / μL, respectively. PCR amplification quality was good, and the method successfully amplified both animal and plant DNA using universal primers. After effective sequence splicing, the number of reads for both animals and plants was approximately 280,000. After species annotation, 59, 30, and 52 animal species, and 121, 124, and 128 higher plant species were detected in the three samples, respectively. The environmental DNA extracted using this invention has a high concentration and good integrity. Furthermore, the DNA extracted using this method, amplified with universal primers for different species, yields good amplification results, facilitating subsequent species diversity analysis using eDNA macrobarcoding technology.

[0124] Table 1. DNA concentration and number of plant and animal species extracted from air samples. .

Claims

1. A method for efficient collection and extraction of airborne eDNA, characterized in that, Includes the following steps: a. Sampling filter membrane preparation: Use sterile glass fiber filter membrane; b. Air sample collection; c. Sampling filter membrane transportation and storage: Store and transport at -80℃ to -70℃; d. Pretreatment of sampling filter membrane: Immerse the sampling filter membrane in PBS buffer; e. Elution and collection: Centrifuge the mixture of PBS buffer and sampling filter membrane at 3-5℃ and 100-300 g for 2-4 hours to elute the precipitated particulate matter; f. Filtration and enrichment: After resuspending the solution from step e, filter it under vacuum and enrich the precipitated particulate matter using a PES filter membrane; g. Divide the PES filter membrane into small pieces for DNA extraction.

2. The method according to claim 1, characterized in that, The air samples were collected using a medium-flow particulate matter sampler and a total suspended particulate matter sampler.

3. The method according to claim 2, characterized in that, The sampling duration is 24 hours.

4. The method according to claim 1, characterized in that, The sampling filter membrane is transported and preserved using dry ice.

5. The method according to claim 1, characterized in that, The centrifugation at 3-5℃ and 100-300 g for 2-4 hours is equivalent to centrifugation at 4℃ and 200 g for 3 hours.

6. The method according to claim 1, characterized in that, The PES filter membrane has a pore size of 0.22 μm.

7. The method according to claim 1, characterized in that, The small piece has an area of ​​0.2-0.5 cm². 2 .

8. The method according to claim 6, characterized in that, The small piece measures 0.5 x 0.4 cm.

9. The method according to claim 1, characterized in that, The specific steps are as follows: a. Sampling filter membrane preparation: Use sterile glass fiber filter membranes and store them in sterile foil bags or petri dishes before sampling; b. Air sample collection: Samples were collected using a medium-flow particulate matter sampler and a total suspended particulate matter sampling head; c. Sampling filter membrane transportation and preservation: After sampling, the samples are preserved and transported using dry ice; d. Pretreatment of sampling filter membrane: Under aseptic conditions, transfer the glass fiber filter membrane containing the collected eDNA into a centrifuge tube, and add 1×PBS buffer until the filter membrane is completely submerged; e. Elution and collection: Tighten the centrifuge tube cap to prevent leakage, centrifuge at 200 g for 3 hours at 4°C to elute the precipitated particulate matter; f. Filtration and enrichment: Gently shake the centrifuge tube and pour the resuspended solution into a vacuum filtration flask for filtration. Use a 0.22 μm PES filter membrane to enrich the precipitated particulate matter. g. Rinse the centrifuge tubes with 50 mL of sterile water and filter again to improve elution and enrichment efficiency; h. Cut the PES filter membrane into 0.5*0.4 cm pieces in a sterile petri dish and transfer them to a 2 mL extraction tube; i. Add 1000 µL of extraction solution to the extraction tube and vortex to mix. Add proteinase K, shake to mix for 5 s, and incubate at 37°C for 30 minutes on a constant temperature shaker at 180 rpm. j. Add 400 µL of preheated 20% sodium dodecyl sulfate, mix gently, and incubate in a 65°C water bath for 2 hours, inverting and mixing once every 30 minutes during the process; k. Centrifuge the water-baked centrifuge tubes at 12,000 rpm for 3 minutes, and transfer the supernatant to a new centrifuge tube; 1. Add an equal volume of chloroform-isoamyl alcohol solution and mix by inverting for 2 minutes. Centrifuge at 12,000 rpm for 5 minutes. Transfer the supernatant to a new centrifuge tube and repeat this step once. m. Transfer the supernatant to a new centrifuge tube, add 600 µL of isoamyl alcohol and mix well. After standing at room temperature for 1 hour, centrifuge at 12000 rpm for 10 minutes at 4°C and discard the supernatant. n. Add 1 mL of 70% ethanol to the centrifuge tube to wash the DNA precipitate, discard the supernatant, and repeat this step once; o. Allow the container to air dry at room temperature until the alcohol has completely evaporated, then add 50 µL of sterile water to dissolve the DNA.

10. The use of the method according to any one of claims 1-9 in the extraction of airborne eDNA.

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