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

Detection method of city wastewater treatment plant/station microbial aerosol Download PDF

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CN105368949A
CN105368949A CN201510866928.2A CN201510866928A CN105368949A CN 105368949 A CN105368949 A CN 105368949A CN 201510866928 A CN201510866928 A CN 201510866928A CN 105368949 A CN105368949 A CN 105368949A
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韩云平
许光素
刘俊新
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Research Center for Eco Environmental Sciences of CAS
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Abstract

本发明公开了一种城市污水处理厂/处理站微生物气溶胶的检测方法,包括以下步骤:1)采用中流量便携式大气采样器对城市污水处理厂/处理站收集测试点的微生物气溶胶,收集采样当时的气象参数,2)洗脱采样膜上的颗粒物并提取其总DNA,3)目的片段的扩增和高通量测序,4)序列分析,鉴定微生物气溶胶的群落结构,5)微生物气溶胶群落结构多样性及相似性分析,6)微生物气溶胶逸散特征及源解析。本发明提供了一种提取城市污水处理厂/处理站微生物气溶胶中微量DNA可行的方法,为解析微生物气溶胶的逸散特征和多样性提供了最根本的前提和基础。

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

一种城市污水处理厂/处理站微生物气溶胶的检测方法A detection method for microbial aerosols in urban sewage treatment plants/processing stations

技术领域technical field

本发明涉及环境微生物分析技术领域,具体涉及城市污水处理厂/处理站微生物气溶胶中全部微生物的检测分析方法。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.

背景技术Background technique

生物气溶胶是指具有生命的气溶胶粒子(包括细菌、真菌、病毒等微生物粒子)和活性粒子(花粉、孢子等)以及由有生命活性的机体所释放到空气中的各种质粒。污水中存在的多种微生物,在污水处理过程中,曝气、机械搅拌及污泥脱水等环节可使其以气溶胶的形式释放周边的空气中。研究表明,城市污水处理厂/处理站逸散的微生物气溶胶中含有大量的致病菌或条件致病菌,对污水处理厂/污水处理站工作人员、附近居民健康及环境具有较大的潜在危害。因而,探明城市污水处理厂、污水处理站微生物气溶胶的群落结构,解析生物气溶胶的微生物菌群在逸散过程中的演替规律逸散特征,对于污水处理厂微生物气溶胶的控制及人员健康防护都具有重要的研究和指导意义。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.

目前,城市污水处理厂/处理站(wastewatertreatmentplant/station,简称WWTP/WWTS)逸散的微生物气溶胶采集方法主要包括可培养方法和不可培养方法两种。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)琼脂培养基-Andersen固体撞击式采样法是国际标准的可培养微生物气溶胶采集方法,可对微生物气溶胶的群落结构、浓度以及粒径分布等特征进行研究。该方法为传统的微生物培养方法,操作繁琐,需要大量的人力物力;而研究也表明,可培养的微生物仅占环境微生物的1%-10%,因而,该方法并不能高效、有效地反映城市污水处理厂微生物气溶胶的分布及组成特征。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)SKC-Sampler液体撞击式采样法是国际标准的微生物气溶胶不可培养采集方法,作为标准方法广泛应用于气溶胶中全部微生物的收集,对于微生物群落信息能够较全面的反映。获取微生物气溶胶的DNA是后续微生物特征解析的前提,但是,空气中的微生物数量相对于其他生境(土壤、水体)要低很多,不经培养过程直接对空气中的微生物提取DNA是一直以来的难题。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.

SKC-Sampler液体采样器流量小(额定流量为12.5L空气/min),要获取足够的样本量提取DNA需要长时间的样品收集;该方法中的微生物气溶胶收集液(PBS缓冲液)在样品采集过程中会大量挥发,需要反复添加收集液以保证其正常运行;同时,受制于其收集液在低温条件(<0℃)下会结冰,该方法并不能完成在任一时间的样品采集任务。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 .

参考常见的大流量大气采样器(流量>100L/min)的大气采样器一般安装于某固定采样点,而对于不同污水处理厂/处理站之间的调研安排、以及多个采样点之间频繁更换的要求并不能满足。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.

以上缺点,使得两种国际标准的生物气溶胶采集方法,都不能实现对城市污水处理厂微生物气溶胶中全部微生物的现场采样目的,以及后续的微生物群落结构及逸散特征的解析。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.

发明内容Contents of the invention

本发明的目的在于提供一种城市污水处理厂/处理站微生物气溶胶中全部微生物高效采集和分析的方法,即能够克服营养琼脂-Andersen固体撞击式采样法和SKC-Sampler液体撞击式采样法应用于城市污水处理厂微生物气溶胶收集过程中的弊端。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.

为了实现上述目的,本发明采用的技术方案如下:In order to achieve the above object, the technical scheme adopted in the present invention is as follows:

一种城市污水处理厂/处理站微生物气溶胶的检测方法,包括以下步骤:A method for detecting microbial aerosols in urban sewage treatment plants/processing stations, comprising the following steps:

1)采样器及其配件准备,采样膜及其采样膜收集器具的准备及高温高压灭菌;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)城市污水处理厂微生物气溶胶现场采样,运行空气采样泵,抽滤空气至无菌的玻璃纤维滤膜上,截留空气微生物至采样膜上,记录采样过程中的气象参数;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)采样膜的收集及存放,用无菌镊子将截留空气微生物的采样膜转移至无菌铝箔纸上密封,如不能立即对样品进行后续处理,将其冻存-20℃或者-80℃的冰箱内待用;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提取,提取玻璃纤维膜上的微生物DNA;4) DNA extraction, extracting the microbial DNA on the glass fiber membrane;

5)目的片段扩增,细菌16SrRNA和真菌18S基因PCR扩增;5) Target fragment amplification, bacterial 16SrRNA and fungal 18S gene PCR amplification;

6)高通量测序,利用高通量测序技术对细菌和真菌的目的序列进行测序;6) High-throughput sequencing, using high-throughput sequencing technology to sequence the target sequences of bacteria and fungi;

7)序列分析,将下机的目的片段DNA序列对原始数据进行过滤处理,得到优化序列,去除嵌合体序列后进行OTU聚类分析,对OTU的代表序列在Silva数据库中进行比对分析,鉴定微生物气溶胶中细菌和真菌的分类;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)群落结构及多样性分析,根据鉴定的细菌和真菌的分类信息,对微生物气溶胶的群落结构和多样性进行分析;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;

还可以包括:Can also include:

9)源解析及逸散规律评估,比较不同污水处理工段微生物气溶胶样品之间的相似性,同时对城市污水处理厂微生物气溶胶进行源解析,以综合评估微生物气溶胶的逸散特征;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)特征序列数据库的构建,建立城市污水处理厂/处理站微生物气溶胶中特征的细菌和真菌序列信息数据库,尤其是致病菌的序列信息。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.

该检测方法可以应用于不同运行工艺及不同气象条件下的污水处理厂/污水处理站微生物气溶胶的检测,即室内和室外大气环境均可实现。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.

为了更好地实现本发明目的,进一步的技术方案如下:In order to realize the object of the present invention better, further technical scheme is as follows:

所述步骤1)中采样器为中流量总悬浮微粒采样器,配以总悬浮颗粒物(TotalSuspendedParticulate,简称TSP)的颗粒物切割器,采样膜为亲水性的玻璃纤维膜,采样膜收集器具包括无菌的镊子、铝箔纸、自封袋、直径为90mm的玻璃平板或者无菌一次性塑料培养皿,由于在城市污水处理厂/处理站内电力的使用受到严格管制,所以需要额外自备电力输出线圈若干(因现场情况决定),线路连接完毕,将灭菌的TSP切割器和滤膜安装于采样泵上,即可完成采样点的微生物气溶胶样品的采集。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.

所述步骤2)中采样泵安装高度为人呼吸高度,即距地面垂直高度1.5m处;对于污水处理厂的生物处理单元处,分别在水面处、距地面垂直高度1.5m处、距地面垂直高度4m处设置三个采样点。采样流量为100L/min,玻璃纤维膜为直径90mm、对0.3μm标准粒子的截留率>99.95%的圆形采样膜,采样时间设置为1h,2h,4h,8h,12h和24h,采样的同时记录当时的气象条件。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.

所述步骤3)中无菌镊子和无菌铝箔纸,事先在高温、高压灭菌锅中用牛皮纸或者耐高温、高压容器包裹,121℃灭菌15min。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.

所述步骤4)中玻璃纤维膜的颗粒物上附着的微生物的总DNA提取包括以下仪器、器具和试剂:低温低速离心机、真空隔膜泵、玻璃过滤系统、剪刀、镊子、50mL血清瓶、1×PBS缓冲液,孔径为0.22μm、直径为25mm的聚醚砜滤膜(PES),DNA提取试剂盒,除离心机和抽滤泵外,其他均需在121℃灭菌15min;总DNA的提取方法如下:将冻存于-20℃或者-80℃的冰箱内的玻璃纤维膜样品置于超净台中,用无菌的镊子夹取采样膜,用无菌剪刀将其剪碎置于无菌的50mL血清瓶中,取40mL无菌的1×PBS缓冲液冲洗剪碎的采样膜并将其浸没,封盖后上下颠倒血清瓶数次,尽可能多地冲洗采样膜的颗粒物至缓冲液中,将其置于低温低速离心机中,200g离心2h,安装真空过滤系统,将上述离心后的缓冲液(避开剪碎的滤膜)倒入滤杯中,浓缩颗粒物至PES膜上,用无菌镊子夹取该滤膜,用DNA提取试剂盒对滤膜浓缩的颗粒物进行总DNA的提取,由于空气中微生物的总DNA含量较低,在总DNA提取的最后一步,用50μL溶液收集总DNA,以提高总DNA的最终浓度。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.

所述步骤5)中目的片段的扩增,细菌16SrRNA扩增采用引物338F/806R(5'-ACTCCTACGGGAGGCAGCA-3'/5'-GGACTACHVGGGTWTCTAAT-3'),真菌18S基因扩增引物采用引物0817F/1196R(5'-TTAGCATGGAATAATRRAATAGGA-3'/5'-TCTGGACCTGGTGAGTTTCC-3')。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').

所述步骤6)中高通量测序,采用IluuminaMiseq测序平台,细菌采用PE250测序文库,真菌采用PE300测序文库。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.

所述步骤9)中微生物气溶胶的源解析和逸散规律评估,对比城市污水厂/处理站主体污水处理工段(粗格栅、细格栅、初沉池、曝气池、二沉池、氧化沟等)与背景对照点(污水厂/站的上风向、下风向等)微生物气溶胶的微生物种类之间的相似性,以解析来源于城市污水过程中的特征微生物种类,并结合气象参数对其逸散规律做出评估。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.

有益效果:Beneficial effect:

本发明提出的一种城市污水处理厂/处理站微生物气溶胶的检测方法,通过便携式的空气采样器,采用膜采样法对空气TSP收集以完成对不同污水处理厂/处理站、不同站点的全部空气微生物的收集,在收集足够体积空气样品的前提下,优化的DNA提取方法克服了空气样品中微生物含量低,难以获取样品总DNA的难题,可提供浓度高、纯度高的总DNA样品,为后续高通量测序提供基础。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.

采用目前常用的基于培养计数法的Andersen固体冲击式采样法、SKC液体撞击式采样法(采样时间24h)与本发明方法(采样时间4h)分别对同一污水站内细菌气溶胶样品进行采集,测序分析样品中微生物菌门分布的结果如附图6。由图可见,Andersen采样法仅获取了隶属于Firmicutes(62.97%)和Proteobacteria(37.03%)两个菌门的细菌,采样时间为24h的SKC采样法获取了Firmicutes(46.62%)、Proteobacteria(41.25%)和Actinobacteria(12.13%)三个菌门的细菌,而采用本发明提供的方法,仅需4h即可获得包括Cyanobacteria,Proteobacteria,Actinobacteria,Firmicutes,Bacteroidetes,Deinococcus-Thermus,Chloroflexi在内的七种菌门的细菌。即本发明提供一种城市污水处理厂/处理站微生物气溶胶的检测方法在克服了Andersen可培养采样法和SKC采样法弊端的前提下,采用时间大大缩短、操作简便且不受气象条件限制,更好的完成了对微生物气溶胶的采集和表征。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.

附图说明Description of drawings

图1为城市污水处理厂微生物气溶胶的分析方法流程图;Fig. 1 is the flow chart of the analysis method of microbial aerosol in urban sewage treatment plant;

图2为污水处理站微生物气溶胶采样点分布图;Figure 2 is a distribution map of microbial aerosol sampling points in sewage treatment stations;

图3为实施例中细菌和真菌的PCR凝胶电泳图,其中3(a)为细菌电泳图,3(b)为真菌电泳图;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;

图4为实施例中细菌基于门类分布的群落结构柱状图;Fig. 4 is the histogram of the community structure of bacteria based on the phylum distribution in the embodiment;

图5为实施例中真菌基于门类分布的群落结构柱状图;Fig. 5 is the histogram of the community structure of the fungi based on the phylum distribution in the embodiment;

图6为Andersen、SKC采样法(采样时间24h)与本发明方法(采样时间4h)对同一污水站内细菌气溶胶基于门类分布的群落结构柱状图。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).

具体实施方式detailed description

为使本发明的目的、技术方案和优点更加清楚明白,以下参照附图并结合具体实施例,对本发明作进一步详细说明,显然,此处所描述的具体实施例及附图为示例性而非限制性的,本发明的范围不应当由示例性实施所限制,而应当仅由权利要求书及其等同范围所限制,基于本发明中的实施案例,本领域普通技术人员在没有做出创造性劳动前提下,所获得的所有其它实施例,均属于本发明保护的范围。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.

实施例1Example 1

如图1所示,一种城市污水处理站(WWTS)微生物气溶胶的检测方法,包括步骤如下:As shown in Figure 1, a kind of detection method of urban sewage treatment station (WWTS) microbial aerosol, comprises steps as follows:

1)采样器及其配件准备,采样膜及其采样膜收集器具的准备及高温高压灭菌,;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)城市污水处理厂微生物气溶胶现场采集,布置采样点,运行空气采样泵,抽滤空气至无菌的玻璃纤维滤膜上,截留空气微生物至采样膜上;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)采样膜的收集及存放,用无菌镊子将截留空气微生物的采样膜转移至无菌铝箔纸上密封,如不能立即对样品进行后续处理,将其冻存-20℃或者-80℃的冰箱内待用;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提取,提取玻璃纤维膜上的微生物DNA;4) DNA extraction, extracting the microbial DNA on the glass fiber membrane;

5)目的片段扩增,细菌16SrRNA和真菌18S基因PCR扩增;5) Target fragment amplification, bacterial 16SrRNA and fungal 18S gene PCR amplification;

6)高通量测序,利用高通量测序技术对细菌和真菌的目的序列进行测序;6) High-throughput sequencing, using high-throughput sequencing technology to sequence the target sequences of bacteria and fungi;

7)序列分析,将下机的目的片段DNA序列对原始数据进行过滤处理,得到优化序列,去除嵌合体序列后进行OTU聚类分析,对OTU的代表序列在Sliva数据库中进行比对分析,鉴定微生物气溶胶中细菌和真菌的分类;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)群落结构及多样性分析,根据鉴定的细菌和真菌的分类信息,对微生物气溶胶的群落结构和多样性进行分析;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)源解析及逸散规律评估,比较不同污水处理工段微生物气溶胶样品之间的相似性,同时对城市污水处理厂微生物气溶胶进行源解析,以综合评估微生物气溶胶的逸散特征;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)特征序列数据库的构建,建立城市污水处理厂/处理站微生物气溶胶中特征的细菌和真菌序列信息数据库,尤其是致病菌的序列信息。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.

所述步骤2)为:选择一处占地面积为193m3、污水日处理规模为100m3/d、污水来源主要为办公区及家属区的生活污水、处理工艺为立体循环一体化氧化沟的城市污水处理站,所用采样器为武汉天虹仪表有限责任公司TH-150C智能中流量总悬浮微粒采样器,配以TSP颗粒物切割器,流量为100L/min,如图2所示,污水站内(室内)将采样设备架设于距立体循环一体化氧化沟转刷水平距离为1m、垂直高度为人呼吸高度处(1.5m),污水站门口处(室外)将采样设备架设于距污水站门口水平距离为2m、垂直高度为人呼吸高度处(1.5m),事先对TSP切割器的各个部件首先用超纯水擦洗,然后用75%的医用酒精进行除菌消毒,直径为90mm、对0.3μm标准粒子的截留率>99.95%的圆形玻璃纤维采样膜、采样膜收集器具包括无菌的镊子、铝箔纸、自封袋、直径为90mm的玻璃培养皿或者无菌一次性塑料培养皿采用121℃高温、高压灭菌15min,将灭菌的TSP切割器和玻璃纤维滤膜安装于采样泵上,每处采样点布设3台采样器同时收集微生物气溶胶样品作为平行样,室内采样点采样时间设置为1h,2h,4h,8h,12h,24h,室内采样分4天完成,同时,取立体循环一体化氧化沟内的水样,室外微生物气溶胶采样点采样时间设置为24h,采样的同时记录当时的气象条件,包括温度、湿度、相对湿度、光照强度、风速等,采样结束,记录采样器上显示的采样过程中的气压、平均温度、标准状况下的气体体积等。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.

所述步骤4)涉及以下仪器、器具和试剂:低温低速离心机、真空隔膜泵(天津津腾GM-0.33A型)、玻璃过滤器(50mL滤杯、磨砂部分直径为20mm的玻璃滤头、1L三角瓶)、剪刀、镊子、50mL血清瓶、1×PBS缓冲液(pH7.4,每升溶液中含0.27gKH2PO4,1.42gNa2HPO4,8gNaCl,0.2gKCl),孔径为0.22μm、直径为25mm的聚醚砜滤膜(PES),DNA提取试剂盒(MO-BIODNAIsolationKitComponents)除离心机和抽滤泵外,其他均需在121℃灭菌15min,将不同玻璃纤维膜TSP样品置于超净台中,用无菌的镊子夹取采样膜,用无菌剪刀将其剪碎置于无菌的50mL血清瓶中,取40mL无菌的1×PBS缓冲液冲洗剪碎的采样膜并将其浸没,封盖后上下颠倒血清瓶数次,冲洗采样膜的颗粒物至缓冲液中,将其置于低温低速离心机中,200g离心2h,安装真空过滤系统,将上述离心后的缓冲液(避开剪碎的滤膜)倒入滤杯中,浓缩颗粒物至孔径为0.22μm、直径为25mm的聚醚砜滤膜(PES)膜上,用无菌镊子夹取该滤膜,按照DNA提取试剂盒的使用说明,对滤膜浓缩的颗粒物进行总DNA的提取,由于空气中微生物的总DNA含量较低,在总DNA提取的最后一步,用50μL溶液取代说明书中的100μL溶液收集总DNA,以提高总DNA的最终浓度,DNA提取结果显示,采样时间为1h和2h的室内微生物气溶胶样品因收集气体太小,未成功提取获得DNA,其余样品均提取DNA成功。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.

所述步骤5)为:细菌16SrRNA扩增采用引物338F/806R(5'-ACTCCTACGGGAGGCAGCA-3'/5'-GGACTACHVGGGTWTCTAAT-3'),真菌18S基因扩增引物采用引物0817F/1196R(5'-TTAGCATGGAATAATRRAATAGGA-3'/5'-TCTGGACCTGGTGAGTTTCC-3'),PCR实验采用TransGenAP221-02:TransStartFastpfuDNAPolymerase,20μL的PCR反应体系如下:5×FastPfuBuffer4μL,2.5mMdNTPs2μL,ForwardPrimer(5μM)0.8μL,ReversePrimer(5μM)0.8μL,FastPfuPolymerase0.4μL,10ngTemplateDNA,补加ddH20至20μL,PCR扩增程序如下:a=1×(3minutesat95℃),b=27×(30secondsat95℃;30secondsat55℃;45secondsat72℃),c=10minutesat72℃,10℃untilhaltedbyuser,细菌和真菌PCR结果见附图3(图3注释:室内气溶胶样品用IBA表示,不同采样时间4h,8h,12h,24h的样品分别用IBA-4,IBA-8,IBA-12,IBA-24表示,室外样品用OBA表示,采样时间为24h的室外样品用OBA-24表示,水样用W表示,图(a)和(b)分别为所有样品的细菌和真菌PCR结果凝胶电泳图)。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).

所述步骤6)中高通量测序,采用IluuminaMiseq测序平台,细菌采用PE250测序文库,真菌采用PE300测序文库。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.

所述步骤8)为,根据鉴定的细菌和真菌分类信息,对污水站内与污水站门口、污水站内与水样之间的微生物气溶胶的群落结构和多样性进行分析,图4和图5分别是在分类水平为门(phylum)的情况下,细菌和真菌的Treebar图,图片的左边是样本间基于群落组成的层次聚类分析(bray-curtis算法),右边是样本的群落结构柱状图,从结果可以看出,对于污水站内的微生物气溶胶样品,采样时间为4h的样品已经覆盖了更长采样时间(8h,12h,24h)的细菌和真菌门类,延长采样时间对于反映污水站内微生物气溶胶的群落结构和多样性的贡献小,同时,污水站内与室外的微生物气溶胶、污水站内与水样之间的群落结构相似性较高。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.

以上结果表明,本发明提出的微生物气溶胶检测方法对污水处理站微生物气溶胶的分析是可行的。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.

以上所述仅为本发明的较佳实施方案,并不用于限制本发明,凡在本发明的精神和原则之内作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。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)

1.一种城市污水处理厂/处理站微生物气溶胶的检测方法,其特征在于,包括以下步骤:1. a detection method of urban sewage treatment plant/processing station microbial aerosol, is characterized in that, comprises the following steps: 1)采样器及其配件准备,采样膜及其采样膜收集器具的准备及高温高压灭菌;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)城市污水处理厂微生物气溶胶现场采样,运行空气采样泵,抽滤空气至无菌的玻璃纤维滤膜上,截留空气微生物至采样膜上,记录采样过程中的气象参数;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)采样膜的收集及存放,用无菌镊子将截留空气微生物的采样膜转移至无菌铝箔纸上密封,如不能立即对样品进行后续处理,将其冻存-20℃或者-80℃的冰箱内待用;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提取,提取玻璃纤维膜上的微生物DNA;4) DNA extraction, extracting the microbial DNA on the glass fiber membrane; 5)目的片段扩增,细菌16SrRNA和真菌18S基因PCR扩增;5) Target fragment amplification, bacterial 16SrRNA and fungal 18S gene PCR amplification; 6)高通量测序,利用高通量测序技术对细菌和真菌的目的序列进行测序;6) High-throughput sequencing, using high-throughput sequencing technology to sequence the target sequences of bacteria and fungi; 7)序列分析,将下机的目的片段DNA序列对原始数据进行过滤处理,得到优化序列,去除嵌合体序列后进行OTU聚类分析,对OTU的代表序列在Silva数据库中进行比对分析,鉴定微生物气溶胶中细菌和真菌的分类;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)群落结构及多样性分析,根据鉴定的细菌和真菌的分类信息,对微生物气溶胶的群落结构和多样性进行分析。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.如权利要求1所述的检测方法,其特征在于,所述步骤1)中采样器采用中流量总悬浮微粒便携式采样器,配以总悬浮颗粒物的颗粒物切割器,采样膜为亲水性的玻璃纤维膜,采样膜收集器具包括无菌的镊子、铝箔纸、自封袋、直径为90mm的玻璃平板或者无菌一次性塑料培养皿。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.如权利要求1所述的检测方法,其特征在于,所述步骤2)中采样泵安装高度为人呼吸高度,即距地面垂直高度1.5m处;对于污水处理厂的生物处理单元处,分别在水面处、距地面垂直高度1.5m处、距地面垂直高度4m处设置三个采样点;采样流量为100L/min,玻璃纤维膜为直径90mm、对0.3μm标准粒子的截留率>99.95%的圆形采样膜,采样时间设置为1h,2h,4h,8h,12h和24h,采样的同时记录当时的气象条件。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.如权利要求1所述的检测方法,其特征在于,所述步骤4)中玻璃纤维膜的颗粒物上附着的微生物的总DNA提取包括以下仪器、器具和试剂:低温低速离心机、真空隔膜泵、玻璃过滤系统、与玻璃过滤系统尺寸配套的水系滤膜、剪刀、镊子、50mL血清瓶、1×PBS缓冲液,,DNA提取试剂盒,除离心机和抽滤泵外,其他均需在121℃灭菌15min。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.如权利要求1或4所述的检测方法,其特征在于,所述步骤4)中总DNA的提取方法如下:将冻存于-20℃或者-80℃的冰箱内的玻璃纤维膜样品置于超净台中,用无菌的镊子夹取采样膜,用无菌剪刀将其剪碎置于无菌的50mL血清瓶中,取适量无菌的1×PBS缓冲液冲洗剪碎的玻璃纤维膜并将其浸没,封盖后上下颠倒血清瓶数次,冲洗采样膜的颗粒物至缓冲液中,将其置于低温低速离心机中,200g离心2h,安装真空过滤系统,将上述离心后的缓冲液避开剪碎的滤膜倒入滤杯中,浓缩颗粒物至水系膜上,用无菌镊子夹取该滤膜,按照DNA提取试剂盒的使用说明,对滤膜浓缩的颗粒物进行总DNA的提取,由于空气中微生物的总DNA含量较低,在总DNA提取的最后一步,用50μL溶液收集总DNA,以提高总DNA的最终浓度。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.如权利要求1所述的检测方法,其特征在于,所述步骤5)中目的片段的扩增,细菌16SrRNA扩增采用引物338F/806R,真菌18S基因扩增引物采用引物0817F/1196R。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.如权利要求1所述的检测方法,其特征在于,所述步骤6)中高通量测序,采用IluuminaMiseq测序平台,细菌采用PE250测序文库,真菌采用PE300测序文库。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.如权利要求1-7任一所述的检测方法,其特征在于,所述检测方法还包括:微生物气溶胶的源解析及逸散规律评估,比较不同污水处理工段微生物气溶胶样品之间的相似性,同时对城市污水处理厂微生物气溶胶进行源解析,以综合评估微生物气溶胶的逸散特征。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.如权利要求8所述的检测方法,其特征在于,所述检测方法还包括:特征序列数据库的构建,建立城市污水处理厂/处理站微生物气溶胶中特征的细菌和真菌序列信息数据库,尤其是致病菌的序列信息。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.如权利要求8所述的检测方法,其特征在于,所述微生物气溶胶的源解析和逸散规律评估,对比城市污水厂/处理站主体污水处理工段粗格栅、细格栅、初沉池、曝气池、二沉池或氧化沟与背景对照点污水厂/站的上风向或下风向微生物气溶胶的微生物种类之间的相似性,以解析来源于城市污水过程中的特征微生物种类,并结合气象参数对其逸散规律做出评估。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.
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