CN105368949A - Detection method of city wastewater treatment plant/station microbial aerosol - Google Patents

Abstract

The invention discloses a method for detecting microbial aerosols in urban sewage treatment plants/processing stations, comprising the following steps: 1) using a medium-flow portable atmospheric sampler to collect microbial aerosols at test points in urban sewage treatment plants/processing stations, collecting Sampling the meteorological parameters at the time, 2) eluting the particulate matter on the sampling membrane and extracting its total DNA, 3) amplification and high-throughput sequencing of the target fragment, 4) sequence analysis, identifying the community structure of microbial aerosols, 5) microbial Aerosol community structure diversity and similarity analysis, 6) Microbial aerosol dissipation characteristics and source apportionment. The invention provides a feasible method for extracting trace DNA in microbial aerosols of urban sewage treatment plants/processing stations, and provides the most fundamental premise and basis for analyzing the escape characteristics and diversity of microbial aerosols.

Description

A detection method for microbial aerosols in urban sewage treatment plants/processing stations

technical field

The invention relates to the technical field of environmental microbial analysis, in particular to a method for detecting and analyzing all microorganisms in microbial aerosols in urban sewage treatment plants/processing stations.

Background technique

Bioaerosols refer to living aerosol particles (including microbial particles such as bacteria, fungi, and viruses) and active particles (pollen, spores, etc.) and various plasmids released into the air by living organisms. A variety of microorganisms exist in sewage. In the process of sewage treatment, aeration, mechanical stirring and sludge dehydration can release them into the surrounding air in the form of aerosols. Studies have shown that microbial aerosols emitted from urban sewage treatment plants/treatment stations contain a large number of pathogenic bacteria or opportunistic pathogens, which have great potential for the health of workers in sewage treatment plants/sewage treatment stations, nearby residents and the environment. harm. Therefore, to find out the community structure of microbial aerosols in urban sewage treatment plants and sewage treatment stations, and to analyze the succession and escape characteristics of the microbial flora of bioaerosols in the process of escaping, will have a great impact on the control and development of microbial aerosols in sewage treatment plants. Personnel health protection has important research and guiding significance.

At present, the collection methods of microbial aerosols emitted from urban sewage treatment plants/stations (WWTP/WWTS for short) mainly include cultivable methods and non-culturable methods.

1) Agar medium-Andersen solid impact sampling method is an international standard cultivable microbial aerosol collection method, which can study the characteristics of microbial aerosol community structure, concentration and particle size distribution. This method is a traditional microbial culture method, which is cumbersome to operate and requires a lot of manpower and material resources; and studies have also shown that the cultureable microorganisms only account for 1%-10% of environmental microorganisms, therefore, this method cannot efficiently and effectively reflect urban environmental pollution. Distribution and composition characteristics of microbial aerosols in sewage treatment plants.

2) The SKC-Sampler liquid impact sampling method is an international standard non-culturable collection method for microbial aerosols. As a standard method, it is widely used to collect all microorganisms in aerosols, and can reflect the information of microbial communities more comprehensively. Obtaining the DNA of microbial aerosols is the prerequisite for the subsequent analysis of microbial characteristics. However, the number of microorganisms in the air is much lower than that of other habitats (soil, water), and it has been a long-standing practice to directly extract DNA from microorganisms in the air without culturing. problem.

The SKC-Sampler liquid sampler has a small flow rate (the rated flow rate is 12.5L air/min), and it takes a long time to collect samples to obtain sufficient sample volume to extract DNA; the microbial aerosol collection solution (PBS buffer) in this method A large amount of volatilization will occur during the collection process, and the collection liquid needs to be added repeatedly to ensure its normal operation; at the same time, due to the fact that the collection liquid will freeze under low temperature conditions (<0°C), this method cannot complete the sample collection task at any time .

Refer to the common large-flow air sampler (flow > 100L/min) and the air sampler is generally installed at a fixed sampling point, but for the investigation arrangement between different sewage treatment plants/processing stations, and between multiple sampling points frequently Requests for replacements were not met.

The above shortcomings make the two international standard bioaerosol collection methods unable to achieve the purpose of on-site sampling of all microorganisms in the microbial aerosol of urban sewage treatment plants, as well as the subsequent analysis of microbial community structure and escape characteristics.

Contents of the invention

The purpose of the present invention is to provide a method for efficient collection and analysis of all microorganisms in microbial aerosols of urban sewage treatment plants/treatment stations, which can overcome the application of nutrient agar-Andersen solid impact sampling method and SKC-Sampler liquid impact sampling method Disadvantages in the collection of microbial aerosols in urban sewage treatment plants.

In order to achieve the above object, the technical scheme adopted in the present invention is as follows:

A method for detecting microbial aerosols in urban sewage treatment plants/processing stations, comprising the following steps:

1) Preparation of the sampler and its accessories, preparation of the sampling film and its collection device, and high temperature and high pressure sterilization;

2) On-site sampling of microbial aerosols in urban sewage treatment plants, run the air sampling pump, filter the air onto the sterile glass fiber filter membrane, trap air microorganisms on the sampling membrane, and record the meteorological parameters during the sampling process;

3) Collection and storage of the sampling film. Use sterile tweezers to transfer the sampling film that traps airborne microorganisms to sterile aluminum foil and seal it. If the sample cannot be processed immediately, store it in a freezer at -20°C or -80°C. Store in the refrigerator;

4) DNA extraction, extracting the microbial DNA on the glass fiber membrane;

5) Target fragment amplification, bacterial 16SrRNA and fungal 18S gene PCR amplification;

6) High-throughput sequencing, using high-throughput sequencing technology to sequence the target sequences of bacteria and fungi;

7) Sequence analysis, filter the original data of the DNA sequence of the target fragment off the machine to obtain the optimized sequence, remove the chimera sequence and perform OTU cluster analysis, compare and analyze the representative sequence of the OTU in the Silva database, and identify Classification of bacteria and fungi in microbial aerosols;

8) Community structure and diversity analysis, according to the classification information of identified bacteria and fungi, analyze the community structure and diversity of microbial aerosols;

Can also include:

9) Source apportionment and evaluation of escape law, compare the similarity between microbial aerosol samples in different sewage treatment sections, and at the same time conduct source apportionment for microbial aerosol in urban sewage treatment plants to comprehensively evaluate the escape characteristics of microbial aerosol;

10) The construction of the characteristic sequence database, establishing the characteristic sequence information database of bacteria and fungi in the microbial aerosol of urban sewage treatment plants/treatment stations, especially the sequence information of pathogenic bacteria.

The detection method can be applied to the detection of microbial aerosols in sewage treatment plants/sewage treatment stations under different operating processes and different meteorological conditions, that is, both indoor and outdoor atmospheric environments can be realized.

In order to realize the object of the present invention better, further technical scheme is as follows:

The sampler in the step 1) is a medium-flow total suspended particle sampler, equipped with a total suspended particulate matter (Total Suspended Particulate, referred to as TSP) particle cutter, the sampling membrane is a hydrophilic glass fiber membrane, and the sampling membrane collection device includes Bacteria tweezers, aluminum foil paper, ziplock bags, glass plates with a diameter of 90mm or sterile disposable plastic petri dishes. Since the use of electricity in urban sewage treatment plants/processing stations is strictly controlled, it is necessary to prepare additional power output coils (Determined by site conditions), after the line connection is completed, install the sterilized TSP cutter and filter membrane on the sampling pump to complete the collection of microbial aerosol samples at the sampling point.

The installation height of the sampling pump in the step 2) is the height of human breathing, that is, the vertical height of 1.5m from the ground; for the biological treatment unit of the sewage treatment plant, it is at the water surface, 1.5m from the vertical height of the ground, and 1.5m from the vertical height of the ground. Three sampling points are set at 4m. The sampling flow rate is 100L/min, and the glass fiber membrane is a circular sampling membrane with a diameter of 90mm and a retention rate of >99.95% for 0.3μm standard particles. The sampling time is set to 1h, 2h, 4h, 8h, 12h and 24h. Record the weather conditions at that time.

The sterile tweezers and sterile aluminum foil in step 3) are wrapped in kraft paper or a high-temperature-resistant and high-pressure container in a high-temperature and high-pressure sterilizer in advance, and sterilized at 121° C. for 15 minutes.

The total DNA extraction of microorganisms attached to the particles of the glass fiber membrane in the step 4) includes the following instruments, utensils and reagents: low-temperature low-speed centrifuge, vacuum diaphragm pump, glass filtration system, scissors, tweezers, 50mL serum bottle, 1× PBS buffer, polyethersulfone filter membrane (PES) with a pore size of 0.22 μm and a diameter of 25 mm, DNA extraction kit, except for the centrifuge and suction pump, all others need to be sterilized at 121 ° C for 15 min; total DNA extraction The method is as follows: put the glass fiber membrane sample frozen in the refrigerator at -20°C or -80°C in the ultra-clean bench, use sterile tweezers to pick up the sampling membrane, cut it into pieces with sterile scissors and place it in a sterile In a 50mL serum bottle, take 40mL of sterile 1×PBS buffer to wash the cut sampling membrane and immerse it, cap it and turn the serum bottle upside down several times to wash as much particles from the sampling membrane into the buffer as possible , put it in a low-temperature and low-speed centrifuge, centrifuge at 200g for 2 hours, install a vacuum filtration system, pour the above-mentioned centrifuged buffer solution (avoiding the shredded filter membrane) into the filter cup, concentrate the particles on the PES membrane, and use Pick up the filter membrane with sterile tweezers, and use a DNA extraction kit to extract the total DNA from the particles concentrated in the filter membrane. Since the total DNA content of microorganisms in the air is low, in the last step of total DNA extraction, use 50 μL solution to collect the total DNA. DNA to increase the final concentration of total DNA.

For the amplification of the target fragment in the step 5), primer 338F/806R (5'-ACTCCTACGGGAGGCAGCA-3'/5'-GGACTACHVGGGTWTCTAAT-3') is used for bacterial 16S rRNA amplification, and primer 0817F/1196R is used for fungal 18S gene amplification (5'-TTAGCATGGAATAATRRAATAGGA-3'/5'-TCTGGACCTGGTGAGTTTCC-3').

In the step 6) high-throughput sequencing, the IluuminaMiseq sequencing platform is used, the PE250 sequencing library is used for bacteria, and the PE300 sequencing library is used for fungi.

In the step 9), the source analysis and the evaluation of the escape law of the microbial aerosol are compared with the main sewage treatment section of the urban sewage plant/treatment station (coarse grid, fine grid, primary sedimentation tank, aeration tank, secondary sedimentation tank, Oxidation ditch, etc.) and the background control point (upwind direction, downwind direction of the sewage plant/station, etc.) Evaluate its escape law.

Beneficial effect:

A kind of detection method of microbial aerosol of urban sewage treatment plant/treatment station that the present invention proposes, through portable air sampler, adopts membrane sampling method to air TSP collection to complete to different sewage treatment plants/treatment stations, all of different sites For the collection of air microorganisms, on the premise of collecting a sufficient volume of air samples, the optimized DNA extraction method overcomes the problem of low microbial content in air samples and the difficulty of obtaining total DNA from samples, and can provide high-concentration and high-purity total DNA samples for Subsequent high-throughput sequencing provides the basis.

The commonly used Andersen solid impact sampling method based on culture counting method, SKC liquid impact sampling method (sampling time 24h) and the method of the present invention (sampling time 4h) are respectively used to collect bacterial aerosol samples in the same sewage station, sequence analysis The results of the distribution of microbial phyla in the samples are shown in Figure 6. It can be seen from the figure that the Andersen sampling method only obtained bacteria belonging to the two phyla of Firmicutes (62.97%) and Proteobacteria (37.03%), and the SKC sampling method with a sampling time of 24 hours obtained Firmicutes (46.62%), Proteobacteria (41.25%) ) and Actinobacteria (12.13%) three bacterial phyla, and adopt the method provided by the invention, only need 4h to obtain seven kinds of bacteria including Cyanobacteria, Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, Deinococcus-Thermus, Chloroflexi Door of bacteria. That is, the present invention provides a detection method for microbial aerosols in urban sewage treatment plants/processing stations. Under the premise of overcoming the disadvantages of the Andersen cultivable sampling method and the SKC sampling method, the use time is greatly shortened, the operation is simple and not limited by meteorological conditions, Better completion of the collection and characterization of microbial aerosols.

Description of drawings

Fig. 1 is the flow chart of the analysis method of microbial aerosol in urban sewage treatment plant;

Figure 2 is a distribution map of microbial aerosol sampling points in sewage treatment stations;

Fig. 3 is the PCR gel electrophoresis figure of bacteria and fungi in the embodiment, wherein 3 (a) is the electrophoresis figure of bacteria, and 3 (b) is the electrophoresis figure of fungi;

Fig. 4 is the histogram of the community structure of bacteria based on the phylum distribution in the embodiment;

Fig. 5 is the histogram of the community structure of the fungi based on the phylum distribution in the embodiment;

Fig. 6 is a histogram of the community structure distribution of bacterial aerosols in the same sewage station based on the Andersen, SKC sampling method (sampling time 24h) and the method of the present invention (sampling time 4h).

detailed description

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be described in further detail below with reference to the accompanying drawings in conjunction with specific embodiments. Obviously, the specific embodiments and accompanying drawings described here are illustrative rather than limiting Rather, the scope of the present invention should not be limited by the exemplary implementation, but should only be limited by the claims and their equivalents. Based on the implementation cases in the present invention, those skilled in the art should not make creative efforts Below, all other examples obtained belong to the protection scope of the present invention.

Example 1

As shown in Figure 1, a kind of detection method of urban sewage treatment station (WWTS) microbial aerosol, comprises steps as follows:

1) Preparation of the sampler and its accessories, preparation of the sampling film and its collection device, and high temperature and high pressure sterilization;

2) On-site collection of microbial aerosols in urban sewage treatment plants, arrangement of sampling points, operation of air sampling pumps, suction and filtration of air onto sterile glass fiber filter membranes, and retention of airborne microorganisms on the sampling membranes;

3) Collection and storage of the sampling film. Use sterile tweezers to transfer the sampling film that traps airborne microorganisms to sterile aluminum foil and seal it. If the sample cannot be processed immediately, store it in a freezer at -20°C or -80°C. Store in the refrigerator;

4) DNA extraction, extracting the microbial DNA on the glass fiber membrane;

5) Target fragment amplification, bacterial 16SrRNA and fungal 18S gene PCR amplification;

6) High-throughput sequencing, using high-throughput sequencing technology to sequence the target sequences of bacteria and fungi;

7) Sequence analysis, filter the original data of the DNA sequence of the target fragment off the machine, obtain the optimized sequence, remove the chimera sequence and perform OTU cluster analysis, compare and analyze the representative sequence of OTU in the Sliva database, and identify Classification of bacteria and fungi in microbial aerosols;

8) Community structure and diversity analysis, according to the classification information of identified bacteria and fungi, analyze the community structure and diversity of microbial aerosols;

9) Source apportionment and evaluation of escape law, compare the similarity between microbial aerosol samples in different sewage treatment sections, and at the same time conduct source apportionment for microbial aerosol in urban sewage treatment plants to comprehensively evaluate the escape characteristics of microbial aerosol;

10) The construction of the characteristic sequence database, establishing the characteristic sequence information database of bacteria and fungi in the microbial aerosol of urban sewage treatment plants/treatment stations, especially the sequence information of pathogenic bacteria.

The step 2) is: select a site with a floor area of 193m ³ , a daily sewage treatment scale of 100m ³ /d, the main source of sewage is domestic sewage from office areas and family areas, and the treatment process is a three-dimensional circulation integrated oxidation ditch. In the urban sewage treatment station, the sampler used is Wuhan Tianhong Instrument Co., Ltd. TH-150C intelligent medium-flow total suspended particle sampler, equipped with a TSP particle cutter, with a flow rate of 100L/min, as shown in Figure 2. In the sewage station (indoor ) Set up the sampling equipment at a horizontal distance of 1m from the three-dimensional circulation integrated oxidation ditch rotary brush, and the vertical height is at the height of human breathing (1.5m), and set up the sampling equipment at the entrance of the sewage station (outdoor) at a horizontal distance of 1m from the entrance of the sewage station 2m, and the vertical height is at the height of human breathing (1.5m). In advance, the various parts of the TSP cutter are first scrubbed with ultrapure water, and then sterilized with 75% medical alcohol. Circular glass fiber sampling membrane with retention rate >99.95%, sampling membrane collection equipment including sterile tweezers, aluminum foil, ziplock bag, glass petri dish with a diameter of 90mm or sterile disposable plastic petri dish adopts high temperature and high pressure at 121 ℃ Sterilize for 15 minutes, install the sterilized TSP cutter and glass fiber filter membrane on the sampling pump, arrange 3 samplers at each sampling point to collect microbial aerosol samples as parallel samples, and set the sampling time of indoor sampling points to 1h. 2h, 4h, 8h, 12h, 24h, indoor sampling is completed in 4 days, at the same time, water samples in the three-dimensional circulation integrated oxidation ditch are taken, and the sampling time of the outdoor microbial aerosol sampling point is set to 24h, and the weather at that time is recorded while sampling Conditions, including temperature, humidity, relative humidity, light intensity, wind speed, etc. After sampling, record the air pressure, average temperature, and gas volume under standard conditions displayed on the sampler during the sampling process.

The step 4) involves the following instruments, utensils and reagents: low-temperature low-speed centrifuge, vacuum diaphragm pump (Tianjin Jinteng GM-0.33A type), glass filter (50mL filter cup, glass filter head with a diameter of 20mm in the frosted part, 1L Erlenmeyer flask), scissors, tweezers, 50mL serum bottle, 1×PBS buffer solution (pH7.4, containing 0.27gKH2PO4, 1.42gNa2HPO4, 8gNaCl, 0.2gKCl in each liter of solution), a pore diameter of 0.22μm, a diameter of 25mm Ethersulfone membrane (PES), DNA extraction kit (MO-BIO DNAIsolationKitComponents) except for the centrifuge and the filtration pump, all others need to be sterilized at 121°C for 15 minutes. Place different glass fiber membrane TSP samples in the ultra-clean bench, use sterile tweezers to pick up the sampling membrane, and use sterile scissors to clean it. Cut it into pieces and place it in a sterile 50mL serum bottle, take 40mL of sterile 1×PBS buffer to wash the cut sampling membrane and immerse it, cover it and turn the serum bottle upside down several times, wash the particles in the sampling membrane to the buffer solution, put it in a low-temperature and low-speed centrifuge, centrifuge at 200g for 2h, install a vacuum filtration system, pour the above-mentioned centrifuged buffer solution (avoid the shredded filter membrane) into the filter cup, and concentrate the particles to a pore size of 0.22 On a polyethersulfone filter membrane (PES) membrane with a diameter of 25 mm and a diameter of 25 mm, use sterile tweezers to clamp the filter membrane, and follow the instructions of the DNA extraction kit to extract the total DNA from the particles concentrated by the filter membrane. The total DNA content of microorganisms in the medium is low. In the last step of total DNA extraction, 50 μL solution was used to replace the 100 μL solution in the instructions to collect the total DNA to increase the final concentration of total DNA. The DNA extraction results showed that the sampling time was 1h and 2h. Indoor microbial aerosol samples were not successfully extracted to obtain DNA because the collected gas was too small, and DNA was successfully extracted from the remaining samples.

The step 5) is: bacterial 16SrRNA amplification uses primer 338F/806R (5'-ACTCCTACGGGAGGCAGCA-3'/5'-GGACTACHVGGGTWTCTAAT-3'), fungal 18S gene amplification primer uses primer 0817F/1196R (5'-TTAGCATGGAATAATRRAATAGGA -3'/5'-TCTGGACCTGGTGAGTTTCC-3'), PCR experiment using TransGenAP221-02: TransStartFastpfuDNAPolymerase, 20μL PCR reaction system is as follows: 5×FastPfuBuffer 4μL, 2.5mMdNTPs 2μL, ForwardPrimer (5μM) 0.8μL, ReversePrimer (5μM) 0.8μL, FastPfuPolymerase0.4μL, 10ngTemplateDNA, add ddH ₂ 0 to 20μL, PCR amplification program is as follows: a=1×(3minutesat95℃), b=27×(30secondsat95℃; 30secondsat55℃; 45secondsat72℃), c=10minutesat72℃, 10 ℃untilhaltedbyuser, bacterial and fungal PCR results are shown in Figure 3 (Note to Figure 3: Indoor aerosol samples are represented by IBA, and samples of different sampling times of 4h, 8h, 12h, and 24h are represented by IBA-4, IBA-8, and IBA-12 respectively , IBA-24, outdoor samples are represented by OBA, outdoor samples with a sampling time of 24 hours are represented by OBA-24, water samples are represented by W, Figures (a) and (b) are the PCR results of bacteria and fungi of all samples, respectively. gel electrophoresis).

In the step 6) high-throughput sequencing, the IluuminaMiseq sequencing platform is used, the PE250 sequencing library is used for bacteria, and the PE300 sequencing library is used for fungi.

The step 8) is to analyze the community structure and diversity of microbial aerosols between the sewage station and the sewage station gate, the sewage station and the water sample according to the identified bacteria and fungal classification information, as shown in Figure 4 and Figure 5 respectively It is the Treebar diagram of bacteria and fungi when the classification level is phylum. The left side of the picture is the hierarchical clustering analysis (bray-curtis algorithm) based on the community composition between samples, and the right side is the histogram of the community structure of the sample. It can be seen from the results that for the microbial aerosol samples in the sewage station, the sample with a sampling time of 4h has already covered the bacteria and fungi with a longer sampling time (8h, 12h, 24h), and extending the sampling time is important for reflecting the microbial gas in the sewage station. The contribution of the community structure and diversity of the sol is small, and at the same time, the community structure similarity between the microbial aerosols in the sewage station and outdoor, and the community structure in the sewage station and water samples is relatively high.

The above results show that the microbial aerosol detection method proposed by the present invention is feasible for the analysis of microbial aerosols in sewage treatment stations.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention within.

Claims (10)

1. a detection method of urban sewage treatment plant/processing station microbial aerosol, is characterized in that, comprises the following steps: 1) Preparation of the sampler and its accessories, preparation of the sampling film and its collection device, and high temperature and high pressure sterilization; 2) On-site sampling of microbial aerosols in urban sewage treatment plants, run the air sampling pump, filter the air onto the sterile glass fiber filter membrane, trap air microorganisms on the sampling membrane, and record the meteorological parameters during the sampling process; 3) Collection and storage of the sampling film. Use sterile tweezers to transfer the sampling film that traps airborne microorganisms to sterile aluminum foil and seal it. If the sample cannot be processed immediately, store it in a freezer at -20°C or -80°C. Store in the refrigerator; 4) DNA extraction, extracting the microbial DNA on the glass fiber membrane; 5) Target fragment amplification, bacterial 16SrRNA and fungal 18S gene PCR amplification; 6) High-throughput sequencing, using high-throughput sequencing technology to sequence the target sequences of bacteria and fungi; 7) Sequence analysis, filter the original data of the DNA sequence of the target fragment off the machine to obtain the optimized sequence, remove the chimera sequence and perform OTU cluster analysis, compare and analyze the representative sequence of the OTU in the Silva database, and identify Classification of bacteria and fungi in microbial aerosols; 8) Community structure and diversity analysis, according to the classification information of identified bacteria and fungi, the community structure and diversity of microbial aerosols are analyzed. 2. detection method as claimed in claim 1, it is characterized in that, in described step 1), sampler adopts medium-flow total suspended particle portable sampler, is equipped with the particulate matter cutter of total suspended particle, and sampling membrane is hydrophilic Sampling membrane collection devices include sterile tweezers, aluminum foil, ziplock bags, glass plates with a diameter of 90mm or sterile disposable plastic petri dishes. 3. detection method as claimed in claim 1, it is characterized in that, described step 2) in sampling pump installation height is human breathing height, promptly apart from ground vertical height 1.5m place; For the biological treatment unit place of sewage treatment plant, respectively Set three sampling points at the water surface, at a vertical height of 1.5m from the ground, and at a vertical height of 4m from the ground; the sampling flow rate is 100L/min, the glass fiber membrane is 90mm in diameter, and the interception rate of 0.3μm standard particles is >99.95% Circular sampling film, the sampling time is set to 1h, 2h, 4h, 8h, 12h and 24h, and the meteorological conditions at that time are recorded while sampling. 4. detection method as claimed in claim 1, is characterized in that, described step 4) in the particle matter of glass fiber membrane, the total DNA extraction of the microorganism that adheres to comprises following instrument, utensil and reagent: low-temperature low-speed centrifuge, vacuum diaphragm Pump, glass filtration system, water-based filter membrane matching the size of the glass filtration system, scissors, tweezers, 50mL serum bottle, 1×PBS buffer solution, DNA extraction kit, except for the centrifuge and suction pump, everything else needs to be in the Sterilize at 121°C for 15 minutes. 5. detection method as claimed in claim 1 or 4, is characterized in that, the extraction method of total DNA in described step 4) is as follows: the glass fiber membrane sample that freezes in the refrigerator of-20 ℃ or-80 ℃ Place it in an ultra-clean bench, use sterile tweezers to pick up the sampling membrane, cut it into pieces with sterile scissors and place it in a sterile 50mL serum bottle, take an appropriate amount of sterile 1×PBS buffer solution to wash the cut glass fiber Submerge the membrane, cap it and turn the serum bottle upside down several times, rinse the particles of the sampling membrane into the buffer solution, place it in a low-temperature and low-speed centrifuge, centrifuge at 200g for 2 hours, install a vacuum filtration system, and put the above centrifuged Avoid the shredded filter membrane and pour the buffer solution into the filter cup, concentrate the particles onto the water-based membrane, pick up the filter membrane with sterile tweezers, and perform total DNA analysis on the particles concentrated by the filter membrane according to the instructions of the DNA extraction kit. Due to the low total DNA content of microorganisms in the air, in the final step of total DNA extraction, 50 μL solution was used to collect the total DNA to increase the final concentration of total DNA. 6. The detection method according to claim 1, characterized in that, for the amplification of the target fragment in the step 5), primer 338F/806R is used for amplification of bacterial 16SrRNA, and primer 0817F/1196R is used for amplification of fungal 18S gene. 7. The detection method according to claim 1, wherein the high-throughput sequencing in step 6) uses the Iluumina Miseq sequencing platform, the PE250 sequencing library is used for bacteria, and the PE300 sequencing library is used for fungi. 8. The detection method according to any one of claims 1-7, characterized in that, the detection method also includes: source analysis of microbial aerosols and evaluation of the law of escape, comparing microbial aerosol samples in different sewage treatment sections At the same time, the source apportionment of microbial aerosols in urban sewage treatment plants was carried out to comprehensively evaluate the escape characteristics of microbial aerosols. 9. detection method as claimed in claim 8, it is characterized in that, described detection method also comprises: the construction of characteristic sequence database, set up the bacterium and fungal sequence information database of characteristic in urban sewage treatment plant/processing station microbial aerosol, Especially the sequence information of pathogenic bacteria. 10. The detection method according to claim 8, characterized in that, the source analysis and the evaluation of the escape law of the microbial aerosol are compared with the coarse grid, the fine grid, and the primary sewage treatment section of the urban sewage plant/treatment station. The similarity between microbial species in sedimentation tanks, aeration tanks, secondary sedimentation tanks or oxidation ditches and background control point sewage plants/stations in the upwind or downwind microbial aerosols, in order to analyze the characteristic microorganisms from the process of urban sewage species, and combined with meteorological parameters to evaluate its dissipation law.