CN105368949A - Detection method of city wastewater treatment plant/station microbial aerosol - Google Patents
Detection method of city wastewater treatment plant/station microbial aerosol Download PDFInfo
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Abstract
The invention discloses a detection method of city wastewater treatment plant/station microbial aerosol. The detection method includes the steps of firstly, using a medium-flow portable atmosphere sampler to collect the microbial aerosol of city wastewater treatment plant/station test points, and collecting and sampling the meteorological parameters at the moment; secondly, eluting particles on a sampling membrane, and extracting the total DNA of the particles; thirdly, performing target fragment amplification and high-throughput sequencing; fourthly, performing sequence analysis to identify the community structure of the microbial aerosol; fifthly, analyzing the diversity and similarity of the community structure of the microbial aerosol; sixthly, analyzing the dissipation features and sources of the microbial aerosol. The method is a feasible method for extracting trace DNA in the city wastewater treatment plant/station microbial aerosol, and a fundamental premise and foundation is provided for the analysis of the dissipation features and diversity of the microbial aerosol.
Description
Technical field
The present invention relates to environmental microorganism analysis technical field, be specifically related to the determination method of whole microorganism in municipal sewage plant/treatment station microbial aerosol.
Background technology
Bioaerosol refer to the lived aerosol particles of tool (comprising the microorganism particles such as bacterium, fungi, virus) and active particle (pollen, spore etc.) and by have the body of vital activity be discharged into various plasmids in air.The multiple-microorganism existed in sewage, in sewage treatment process, the links such as aeration, mechanical stirring and sludge dewatering can make it discharge in the form of an aerosol in the air of periphery.Research shows, containing a large amount of pathogenic bacterium or conditioned pathogen in the microbial aerosol of municipal sewage plant/treatment station loss, has larger potential hazard to sewage work/sewage works staff, neighbouring residents ' health and environment.Thus, verify the structure of community of municipal sewage plant, sewage works microbial aerosol, resolve the succession of herbaceous loss feature of microorganism species in loss process of bioaerosol, for the control of sewage work's microbial aerosol and personnel health's protection, all there is important research and directive significance.
At present, mainly comprise can cultural method and can not cultural method two kinds for the microbial aerosol acquisition method of municipal sewage plant/treatment station (wastewatertreatmentplant/station, be called for short WWTP/WWTS) loss.
1) nutrient agar-Andersen solid impact formula sampling method is the Bacterial diversity aerosol acquisition method of international standard, can study features such as the structure of community of microbial aerosol, concentration and size distribution.The method is traditional microbial culture method, and complex operation needs a large amount of manpower and materials; And research also shows, educable microorganism only accounts for the 1%-10% of environmental microorganism, and thus, the method can not reflect distribution and the composition characteristic of municipal sewage plant's microbial aerosol efficiently, effectively.
2) SKC-Sampler liquid knockout formula sampling method is that the microbial aerosol of international standard can not cultivate acquisition method, is widely used in the collection of whole microorganism in aerosol, can more comprehensively reflects for microflora's information as standard method.The DNA obtaining microbial aerosol is the prerequisite of follow-up microorganism feature analysis, but, microbe population in air is much lower relative to other habitats (soil, water body), and directly extracting DNA to the microorganism in air without culturing process is a difficult problem all the time.
SKC-Sampler Liquid sampler flow little (rated flow is 12.5L air/min), obtain enough sample size extraction DNA needs long sample collection; Microbial aerosol in the method is collected liquid (PBS damping fluid) and can be volatilized in a large number in sample collecting process, needs repeatedly to add and collects liquid to ensure that it normally runs; Meanwhile, be limited by its collection liquid and can freeze under cold condition (< 0 DEG C), the method can not complete the sample collecting task of a time in office.
Air sampler with reference to common mass flow atmospheric sampling thief (flow > 100L/min) is generally installed on certain fixed sample point, and can not meet for the requirement changed frequent between the investigation arrangement between different sewage treatment plant/treatment station and multiple sampling point.
Above shortcoming, makes the bioaerosol acquisition method of two kinds of international standards, all can not realize the spot sampling object to microorganism whole in municipal sewage plant's microbial aerosol, and the parsing of follow-up biological community structure and loss feature.
Summary of the invention
The object of the present invention is to provide the method for whole microorganism highly effective gathering and analysis in a kind of municipal sewage plant/treatment station microbial aerosol, namely can overcome nutrient agar medium-Andersen solid impact formula sampling method and SKC-Sampler liquid knockout formula sampling method and be applied to drawback in municipal sewage plant's microbial aerosol collection process.
To achieve these goals, the technical solution used in the present invention is as follows:
A detection method for municipal sewage plant/treatment station microbial aerosol, comprises the following steps:
1) sampling thief and accessory thereof prepare, and sampling film and sampling film thereof collect preparation and the autoclave sterilization of utensil;
2) municipal sewage plant's microbial aerosol spot sampling, runs air sampling pump, and suction filtration air is on aseptic glass fiber filter, and entrapped air microorganism, on sampling film, records the meteorologic parameter in sampling process;
3) sampling film collection and deposit, with aseptic nipper the sampling film of entrapped air microorganism is transferred on sterile aluminum foil paper and seals, as subsequent disposal can not be carried out to sample immediately, by stand-by in the refrigerator of its frozen-20 DEG C or-80 DEG C;
4) DNA extraction, extracts the microbial DNA on glass fibre membrane;
5) object fragment amplification, bacterial 16 S rRNA and the amplification of fungi 18S gene PCR;
6) high-flux sequence, utilizes the aim sequence of high throughput sequencing technologies to bacterium and fungi to check order;
7) sequential analysis, the object fragment DNA sequence dna of lower machine is carried out filtration treatment to raw data, be optimized sequence, OTU cluster analysis is carried out after removing mosaic sequence, the representative sequence of OTU is compared in Silva database, the classification of bacterium and fungi in Identifying micro-organisms aerosol;
8) structure of community and diversity analysis, according to bacterium and the classified information of fungi of qualification, analyzes the structure of community of microbial aerosol and diversity;
Can also comprise:
9) source resolution and the assessment of loss rule, compare the similarity between different sewage process workshop section microbial aerosol sample, carry out source resolution simultaneously to municipal sewage plant's microbial aerosol, with the loss feature of comprehensive assessment microbial aerosol;
10) structure of characteristic sequence database, sets up bacterium and the fungal sequence information database of feature in municipal sewage plant/treatment station microbial aerosol, the especially sequence information of pathogenic bacterium.
This detection method can be applied to the detection of the sewage work/sewage works microbial aerosol under different operation process and DIFFERENT METEOROLOGICAL CONDITIONS, and namely indoor and outdoors atmospheric environment all can realize.
In order to realize the object of the invention better, further technical scheme is as follows:
Described step 1) in sampling thief be middle flow total suspended particle sampler, be equipped with overall suspended pellet (TotalSuspendedParticulate, be called for short TSP) cutter for particles, sampling film is hydrophilic glass fibre membrane, sampling film is collected utensil and is comprised aseptic tweezers, aluminium-foil paper, valve bag, diameter is the glass plate of 90mm or aseptic disposable plastic culture dish, because in municipal sewage plant/treatment station, the use of electric power is subject to strict supervision, so need additionally to provide power input lines circle some (because field condition determines) for oneself, connection is complete, the TSP cutting unit of sterilizing and filter membrane are installed on sampling pump, the collection of the microbial aerosol sample of sampling point can be completed.
Described step 2) middle sampling pump mounting height behaviour breathing height, namely apart from vertical height 1.5m place, ground; For the biological processing unit place of sewage work, respectively three sampling points are set at water surface place, apart from vertical height 1.5m place, ground, apart from vertical height 4m place, ground.Sampling flow is 100L/min, and glass fibre membrane is diameter 90mm, circular sampling film to the rejection >99.95% of 0.3 μm of standard particle, and the sampling time is set to 1h, 2h, 4h, 8h, 12h and 24h, record meteorological conditions at that time while sampling.
Described step 3) in aseptic nipper and sterile aluminum foil paper, in advance in high temperature, high-pressure sterilizing pot with kraft paper or high temperature resistant, high pressure vessel parcel, 121 DEG C of sterilizing 15min.
Described step 4) in glass fibre membrane particulate matter on the total DNA extraction of microorganism that adheres to comprise following instrument, utensil and reagent: low temperature low speed centrifuge, vacuum diaphragm pump, glass filtration system, scissors, tweezers, 50mL serum bottle, 1 × PBS damping fluid, the polyethersulfone membranes (PES) that aperture is 0.22 μm, diameter is 25mm, DNA extraction kit, except whizzer and suction filter pump, other all need at 121 DEG C of sterilizing 15min, the extracting method of STb gene is as follows: frozen glass fibre membrane sample in the refrigerator of-20 DEG C or-80 DEG C is placed in super clean bench, with aseptic tweezers gripping sampling film, shredded by sterile scissors and be placed in aseptic 50mL serum bottle, get sampling film that the aseptic 1 × PBS wash buffer of 40mL shreds and by its submergence, the serum bottle that turns upside down after capping for several times, rinse the particulate matter of sampling film as much as possible in damping fluid, be placed in low temperature low speed centrifuge, the centrifugal 2h of 200g, vacuum filtering system is installed, by above-mentioned centrifugal after damping fluid (avoiding the filter membrane shredded) pour in filter bowl, concentrated granular thing is on PES film, with this filter membrane of aseptic nipper gripping, by DNA extraction kit, the particulate matter that filter membrane concentrates is carried out to the extraction of STb gene, because the STb gene content of microbes in air is lower, in the final step of total DNA extraction, with 50 μ L solution collection STb gene, to improve the ultimate density of STb gene.
Described step 5) in the amplification of object fragment, bacterial 16 S rRNA increases and adopts primer 338F/806R (5'-ACTCCTACGGGAGGCAGCA-3'/5'-GGACTACHVGGGTWTCTAAT-3'), and fungi 18S gene amplification primer adopts primer 0817F/1196R (5'-TTAGCATGGAATAATRRAATAGGA-3'/5'-TCTGGACCTGGTGAGTTTCC-3').
Described step 6) middle high-flux sequence, adopt IluuminaMiseq order-checking platform, bacterium adopts PE250 sequencing library, and fungi adopts PE300 sequencing library.
Described step 9) in the source resolution of microbial aerosol and the assessment of loss rule, similarity between contrast municipal wastewater treatment plant/treatment station main body sewage treatment plant (coarse rack, fine fack, preliminary sedimentation tank, aeration tank, second pond, oxidation ditch etc.) and the microbe species of background control spots (Sewage Plant/upwind of standing, lower wind direction etc.) microbial aerosol, to resolve the feature microbe species derived from municipal effluent process, and in conjunction with meteorologic parameter, assessment is made to its loss rule.
Beneficial effect:
The detection method of a kind of municipal sewage plant/treatment station microbial aerosol that the present invention proposes, by portable air sampler, film sampling method is adopted to collect air TSP with the collection completing the whole air microbes to different sewage treatment plant/treatment station, different website, under the prerequisite of collecting enough volumes of air samples, it is low that the DNA extraction method optimized overcomes content of microorganisms in air sample, be difficult to the difficult problem obtaining sample total DNA, the STb gene sample that concentration is high, purity is high can be provided, provide basis for subsequent high pass measures sequence.
Adopt the Andersen solid impact actuated sampling method based on cultivation counting process conventional at present, SKC liquid knockout formula sampling method (sampling time 24h) and the inventive method (sampling time 4h) to gather bacterial aerosol sample in same sewage plant respectively, in sequencing analysis sample, the result of microbial bacteria door distribution is as accompanying drawing 6.As seen from the figure, Andersen sampling method only obtains the bacterium being under the jurisdiction of Firmicutes (62.97%) and Proteobacteria (37.03%) two bacterium door, sampling time is that the SKC sampling method of 24h obtains Firmicutes (46.62%), the bacterium of Proteobacteria (41.25%) and Actinobacteria (12.13%) three bacterium door, and adopt method provided by the invention, only need 4h to obtain and comprise Cyanobacteria, Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, Deinococcus-Thermus, Chloroflexi is on the bacterium of interior seven kinds of bacterium doors.Namely the detection method that the invention provides a kind of municipal sewage plant/treatment station microbial aerosol is under overcoming Andersen and can cultivating the prerequisite of sampling method and SKC sampling method drawback, the employing time shortens greatly, easy and simple to handle and do not limit by meteorological conditions, better complete the collection to microbial aerosol and sign.
Accompanying drawing explanation
Fig. 1 is the analytical procedure schema of municipal sewage plant's microbial aerosol;
Fig. 2 is sewage works microbial aerosol sampling point distributions figure;
Fig. 3 is the PCR gel electrophoresis figure of bacterium and fungi in embodiment, and wherein 3 (a) is bacterium electrophorogram, and 3 (b) is fungi electrophorogram;
Fig. 4 is the structure of community histogram that in embodiment, bacterium distributes based on class;
Fig. 5 is the structure of community histogram that in embodiment, fungi distributes based on class;
Fig. 6 is the structure of community histogram that Andersen, SKC sampling method (sampling time 24h) distributes based on class to bacterial aerosol in same sewage plant with the inventive method (sampling time 4h).
Embodiment
For making object of the present invention, technical scheme and advantage are clearly understood, referring to accompanying drawing also in conjunction with specific embodiments, the present invention is described in further detail, obviously, specific embodiment described herein and accompanying drawing are exemplary and nonrestrictive, scope of the present invention should not limited by exemplary enforcement, and should only be limited by claims and equivalency range thereof, based on the case study on implementation in the present invention, those of ordinary skill in the art are not making under creative work prerequisite, other embodiments all obtained, all belong to the scope of protection of the invention.
Embodiment 1
As shown in Figure 1, a kind of detection method of municipal sewage treatment station (WWTS) microbial aerosol, comprises step as follows:
1) sampling thief and accessory thereof prepare, and sampling film and sampling film thereof collect preparation and the autoclave sterilization of utensil;
2) municipal sewage plant's microbial aerosol collection in worksite, arranges sampling point, runs air sampling pump, and suction filtration air is on aseptic glass fiber filter, and entrapped air microorganism is on sampling film;
3) sampling film collection and deposit, with aseptic nipper the sampling film of entrapped air microorganism is transferred on sterile aluminum foil paper and seals, as subsequent disposal can not be carried out to sample immediately, by stand-by in the refrigerator of its frozen-20 DEG C or-80 DEG C;
4) DNA extraction, extracts the microbial DNA on glass fibre membrane;
5) object fragment amplification, bacterial 16 S rRNA and the amplification of fungi 18S gene PCR;
6) high-flux sequence, utilizes the aim sequence of high throughput sequencing technologies to bacterium and fungi to check order;
7) sequential analysis, the object fragment DNA sequence dna of lower machine is carried out filtration treatment to raw data, be optimized sequence, OTU cluster analysis is carried out after removing mosaic sequence, the representative sequence of OTU is compared in Sliva database, the classification of bacterium and fungi in Identifying micro-organisms aerosol;
8) structure of community and diversity analysis, according to bacterium and the classified information of fungi of qualification, analyzes the structure of community of microbial aerosol and diversity;
9) source resolution and the assessment of loss rule, compare the similarity between different sewage process workshop section microbial aerosol sample, carry out source resolution simultaneously to municipal sewage plant's microbial aerosol, with the loss feature of comprehensive assessment microbial aerosol;
10) structure of characteristic sequence database, sets up bacterium and the fungal sequence information database of feature in municipal sewage plant/treatment station microbial aerosol, the especially sequence information of pathogenic bacterium.
Described step 2) be: select place's floor space to be 193m
3, sewage day treatment scale be 100m
3/ d, sewage source is mainly the sanitary sewage of Office Area and dependents' district, treatment process is the municipal sewage treatment station of stereoscopic circulation integrated oxidation ditch, sampling thief used is flow total suspended particle sampler in the rainbow finite instrument leading company TH-150C intelligence of Wuhan, be equipped with TSP cutter for particles, flow is 100L/min, as shown in Figure 2, sample devices is set up in distance stereoscopic circulation integrated oxidation ditch to turn brush horizontal throw is 1m by (indoor) in sewage plant, vertical height is behaved and is breathed At The Height (1.5m), it is 2m that sample devices is set up in distance sewage plant doorway horizontal throw by sewage plant doorway place (outdoor), vertical height is behaved and is breathed At The Height (1.5m), in advance first all parts of TSP cutting unit is cleaned with ultrapure water, then degerming sterilization is carried out with the medical alcohol of 75%, diameter is 90mm, to the circular glass fiber sampling film of the rejection >99.95% of 0.3 μm of standard particle, sampling film is collected utensil and is comprised aseptic tweezers, aluminium-foil paper, valve bag, diameter is that the glass culture dish of 90mm or aseptic disposable plastic culture dish adopt 121 DEG C of high temperature, autoclaving 15min, the TSP cutting unit of sterilizing and glass fiber filter are installed on sampling pump, often place's sampling point is laid 3 sampling thiefs and is collected microbial aerosol sample as Duplicate Samples simultaneously, the indoor sampling point sampling time is set to 1h, 2h, 4h, 8h, 12h, 24h, indoor sampling divides and completes for 4 days, simultaneously, get the water sample in stereoscopic circulation integrated oxidation ditch, the outdoor microbial aerosol sampling point sampling time is set to 24h, record meteorological conditions at that time while sampling, comprise temperature, humidity, relative humidity, intensity of illumination, wind speed etc., sampling terminates, air pressure in the sampling process that record sampling thief shows, medial temperature, gas volume etc. under the normal conditions.
Described step 4) relate to following instrument, utensil and reagent: low temperature low speed centrifuge, vacuum diaphragm pump (Tian Jinjin rises GM-0.33A type), glass filter (50mL filter bowl, frosted portion diameter are glass filter, the 1L triangular flask of 20mm), scissors, tweezers, 50mL serum bottle, 1 × PBS damping fluid (pH7.4, containing 0.27gKH2PO4 in often liter of solution, 1.42gNa2HPO4,8gNaCl, 0.2gKCl), the polyethersulfone membranes (PES) that aperture is 0.22 μm, diameter is 25mm, DNA extraction kit (MO-BIO
dNAIsolationKitComponents) except whizzer and suction filter pump, other all need at 121 DEG C of sterilizing 15min, different glass tunica fibrosa TSP sample is placed in super clean bench, with aseptic tweezers gripping sampling film, shredded by sterile scissors and be placed in aseptic 50mL serum bottle, get sampling film that the aseptic 1 × PBS wash buffer of 40mL shreds and by its submergence, the serum bottle that turns upside down after capping for several times, rinse the particulate matter of sampling film in damping fluid, be placed in low temperature low speed centrifuge, the centrifugal 2h of 200g, vacuum filtering system is installed, by above-mentioned centrifugal after damping fluid (avoiding the filter membrane shredded) pour in filter bowl, concentrated granular thing to aperture is 0.22 μm, diameter is on polyethersulfone membranes (PES) film of 25mm, with this filter membrane of aseptic nipper gripping, according to the operation instruction of DNA extraction kit, the particulate matter concentrated filter membrane carries out the extraction of STb gene, because the STb gene content of microbes in air is lower, in the final step of total DNA extraction, 100 μ L solution collection STb gene in specification sheets are replaced with 50 μ L solution, to improve the ultimate density of STb gene, DNA extraction result shows, sampling time is that the indoor microbial aerosol sample of 1h and 2h is too little because collecting gas, unsuccessful extraction obtains DNA, all the other samples all extract DNA success.
Described step 5) be: bacterial 16 S rRNA increases and adopts primer 338F/806R (5'-ACTCCTACGGGAGGCAGCA-3'/5'-GGACTACHVGGGTWTCTAAT-3'), fungi 18S gene amplification primer adopts primer 0817F/1196R (5'-TTAGCATGGAATAATRRAATAGGA-3'/5'-TCTGGACCTGGTGAGTTTCC-3'), PCR experiment adopts TransGenAP221-02:TransStartFastpfuDNAPolymerase, the PCR reaction system of 20 μ L is as follows: 5 × FastPfuBuffer4 μ L, 2.5mMdNTPs2 μ L, ForwardPrimer (5 μMs) 0.8 μ L, ReversePrimer (5 μMs) 0.8 μ L, FastPfuPolymerase0.4 μ L, 10ngTemplateDNA, add ddH
20 to 20 μ L, pcr amplification program is as follows: a=1 × (3minutesat95 DEG C), b=27 × (30secondsat95 DEG C, 30secondsat55 DEG C, 45secondsat72 DEG C), c=10minutesat72 DEG C, 10 DEG C of untilhaltedbyuser, bacterium and fungi PCR the results are shown in accompanying drawing 3, and (Fig. 3 annotates: indoor aerosols sample IBA represents, different sampling stages 4h, 8h, the sample of 12h, 24h uses IBA-4 respectively, IBA-8, IBA-12, IBA-24 represents, outdoor sample OBA represents, the sampling time is that the outdoor sample OBA-24 of 24h represents, water sample W represents, figure (a) and (b) are respectively bacterium and the fungi PCR result gel electrophoresis figure of all samples).
Described step 6) middle high-flux sequence, adopt IluuminaMiseq order-checking platform, bacterium adopts PE250 sequencing library, and fungi adopts PE300 sequencing library.
Described step 8) be, according to bacterium and the classification of fungi information of qualification, to in sewage plant and sewage plant doorway, structure of community and the diversity of the microbial aerosol in sewage plant and between water sample are analyzed, Fig. 4 and Fig. 5 is when categorization levels is door (phylum) respectively, the Treebar figure of bacterium and fungi, the left side of picture is the Hierarchical clustering analysis (bray-curtis algorithm) based on group's composition between sample, the right is the structure of community histogram of sample, as can be seen from the results, for the microbial aerosol sample in sewage plant, sampling time is that the sample of 4h has covered longer sampling time (8h, 12h, bacterium 24h) and fungi class, extend the sampling time little for the structure of community and multifarious contribution reflecting microbial aerosol in sewage plant, simultaneously, with outdoor microbial aerosol in sewage plant, structure of community similarity in sewage plant and between water sample is higher.
Above result shows, the analysis of microbial aerosol detection method to sewage works microbial aerosol that the present invention proposes is feasible.
The foregoing is only better embodiment of the present invention, be not limited to the present invention, all do within the spirit and principles in the present invention any amendment, equivalent to replace and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a detection method for municipal sewage plant/treatment station microbial aerosol, is characterized in that, comprise the following steps:
1) sampling thief and accessory thereof prepare, and sampling film and sampling film thereof collect preparation and the autoclave sterilization of utensil;
2) municipal sewage plant's microbial aerosol spot sampling, runs air sampling pump, and suction filtration air is on aseptic glass fiber filter, and entrapped air microorganism, on sampling film, records the meteorologic parameter in sampling process;
3) sampling film collection and deposit, with aseptic nipper the sampling film of entrapped air microorganism is transferred on sterile aluminum foil paper and seals, as subsequent disposal can not be carried out to sample immediately, by stand-by in the refrigerator of its frozen-20 DEG C or-80 DEG C;
4) DNA extraction, extracts the microbial DNA on glass fibre membrane;
5) object fragment amplification, bacterial 16 S rRNA and the amplification of fungi 18S gene PCR;
6) high-flux sequence, utilizes the aim sequence of high throughput sequencing technologies to bacterium and fungi to check order;
7) sequential analysis, the object fragment DNA sequence dna of lower machine is carried out filtration treatment to raw data, be optimized sequence, OTU cluster analysis is carried out after removing mosaic sequence, the representative sequence of OTU is compared in Silva database, the classification of bacterium and fungi in Identifying micro-organisms aerosol;
8) structure of community and diversity analysis, according to bacterium and the classified information of fungi of qualification, analyzes the structure of community of microbial aerosol and diversity.
2. detection method as claimed in claim 1, it is characterized in that, described step 1) in sampling thief adopt in flow total suspended particles Portable sampling device, be equipped with the cutter for particles of overall suspended pellet, sampling film is hydrophilic glass fibre membrane, and sampling film collects that utensil comprises aseptic tweezers, aluminium-foil paper, valve bag, diameter are the glass plate of 90mm or aseptic disposable plastic culture dish.
3. detection method as claimed in claim 1, is characterized in that, described step 2) middle sampling pump mounting height behaviour breathing height, namely apart from vertical height 1.5m place, ground; For the biological processing unit place of sewage work, respectively three sampling points are set at water surface place, apart from vertical height 1.5m place, ground, apart from vertical height 4m place, ground; Sampling flow is 100L/min, and glass fibre membrane is diameter 90mm, circular sampling film to the rejection >99.95% of 0.3 μm of standard particle, and the sampling time is set to 1h, 2h, 4h, 8h, 12h and 24h, record meteorological conditions at that time while sampling.
4. detection method as claimed in claim 1, it is characterized in that, described step 4) in glass fibre membrane particulate matter on the total DNA extraction of microorganism that adheres to comprise following instrument, utensil and reagent: low temperature low speed centrifuge, vacuum diaphragm pump, glass filtration system, water system filter membrane, scissors, tweezers, 50mL serum bottle, the 1 × PBS damping fluid supporting with glass filtration system dimension, DNA extraction kit, except whizzer and suction filter pump, other all need at 121 DEG C of sterilizing 15min.
5. the detection method as described in claim 1 or 4, it is characterized in that, described step 4) in the extracting method of STb gene as follows: frozen glass fibre membrane sample in the refrigerator of-20 DEG C or-80 DEG C is placed in super clean bench, with aseptic tweezers gripping sampling film, shredded by sterile scissors and be placed in aseptic 50mL serum bottle, get glass fibre membrane that 1 × PBS wash buffer aseptic in right amount shreds and by its submergence, the serum bottle that turns upside down after capping for several times, rinse the particulate matter of sampling film in damping fluid, be placed in low temperature low speed centrifuge, the centrifugal 2h of 200g, vacuum filtering system is installed, by above-mentioned centrifugal after the damping fluid filter membrane avoiding shredding pour in filter bowl, concentrated granular thing is on water system film, with this filter membrane of aseptic nipper gripping, according to the operation instruction of DNA extraction kit, the particulate matter concentrated filter membrane carries out the extraction of STb gene, because the STb gene content of microbes in air is lower, in the final step of total DNA extraction, with 50 μ L solution collection STb gene, to improve the ultimate density of STb gene.
6. detection method as claimed in claim 1, is characterized in that, described step 5) in the amplification of object fragment, bacterial 16 S rRNA increases and adopts primer 338F/806R, and fungi 18S gene amplification primer adopts primer 0817F/1196R.
7. detection method as claimed in claim 1, is characterized in that, described step 6) middle high-flux sequence, adopt IluuminaMiseq order-checking platform, bacterium adopts PE250 sequencing library, and fungi adopts PE300 sequencing library.
8. the detection method as described in as arbitrary in claim 1-7, it is characterized in that, described detection method also comprises: the source resolution of microbial aerosol and the assessment of loss rule, similarity relatively between different sewage process workshop section microbial aerosol sample, source resolution is carried out to municipal sewage plant's microbial aerosol, with the loss feature of comprehensive assessment microbial aerosol simultaneously.
9. detection method as claimed in claim 8, it is characterized in that, described detection method also comprises: the structure of characteristic sequence database, sets up bacterium and the fungal sequence information database of feature in municipal sewage plant/treatment station microbial aerosol, the especially sequence information of pathogenic bacterium.
10. detection method as claimed in claim 8, it is characterized in that, the source resolution of described microbial aerosol and the assessment of loss rule, similarity between the upwind of contrast municipal wastewater treatment plant/treatment station main body sewage treatment plant coarse rack, fine fack, preliminary sedimentation tank, aeration tank, second pond or oxidation ditch and background control spots Sewage Plant/stand or the microbe species of lower wind direction microbial aerosol, to resolve the feature microbe species derived from municipal effluent process, and in conjunction with meteorologic parameter, assessment is made to its loss rule.
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Cited By (4)
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| CN107227339A (en) * | 2017-06-29 | 2017-10-03 | 西南科技大学 | Environment microbial aerosol monitoring method in closed type domestic animal house |
| CN111454941A (en) * | 2020-04-13 | 2020-07-28 | 武汉轻工大学 | Sampling liquid and sampling method of DNA viruses in aerosol |
| CN113881665A (en) * | 2021-11-19 | 2022-01-04 | 北京工业大学 | Microbial aerosol DNA extraction method |
| CN116223138A (en) * | 2022-12-07 | 2023-06-06 | 哈尔滨工业大学 | Pretreatment method for community diversity detection of microbial aerosol |
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