CN108841942B - PM2.5 bacterial community composition source rapid analysis and risk assessment method - Google Patents
PM2.5 bacterial community composition source rapid analysis and risk assessment method Download PDFInfo
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Abstract
The invention discloses a PM2.5 bacterial community composition source rapid analysis and risk assessment method, which belongs to the technical field of microbial analysis and mainly comprises sampling of a PM2.5 urban functional area, extraction of microbial genome DNA, PCR amplification of extracted DNA products, purification of PCR products, fluorescence quantification, high-throughput sequencing, bacterial diversity and abundance analysis and risk prediction. Compared with the traditional culture method, the method reflects the flora structure of a research sample more carefully and comprehensively, realizes the rapid and accurate analysis of the bacterial diversity of the particle sample, and is an effective method for evaluating the urban air environment quality and predicting the disease risk. In a word, the invention has the advantages of wide coverage, high detection accuracy and sensitivity, more scientific and reliable prediction result and the like.
Description
Technical Field
The invention belongs to the technical field of microbiological analysis, and particularly relates to a rapid analysis and risk assessment method for PM2.5 bacterial community composition sources.
Background
Researches in recent years show that the particulate matters become the primary factors causing serious air pollution in most urban environments in China, and the pollutants are inhalable particulate matters (main components of haze) in most days. PM2.5 has a smaller particle size relative to PM10, longer residence time in the atmosphere, longer transport distance, and is a carrier for many toxic and hazardous substances due to its larger specific surface area. The biological aerosol can transmit allergen and toxin to cause respiratory diseases of human bodies, so that the understanding of the composition of air microorganisms and the influence of atmospheric environment is of great significance.
In recent years, molecular biology technology has been developed into a mature and effective technology in the aspect of researching microbial community structures of water bodies, soil and sediments, but only a few reports are reported on the research of air samples, and few researches are carried out on the source analysis based on the structure of the particle bacterial community. The community composition and diversity characteristics of the bacterial population can be obtained at the gene level based on 16S rRNA gene sequencing analysis. The high-throughput sequencing technology is a revolutionary change of the traditional sequencing technology, can comprehensively reflect the distribution characteristics of the bacterial community while rapidly and accurately analyzing multiple samples, and is an effective technology for analyzing the bacterial community in the atmospheric particulates.
Because some environments such as animal wastes, sewage systems, plants and the like are often rich in or accompanied by pathogenic bacteria and allergic substances, the generated bacterial aerosol can be potentially harmful to human health. In the prior art, the source of the PM2.5 bacterial community is analyzed, but the possibility of infectivity of diseases caused by bacteria is ignored, and outbreak of the diseases is likely to grow, so that the analysis of the source of the PM2.5 bacterial community is not enough.
Disclosure of Invention
Aiming at the technical problems, the invention provides a rapid analysis and risk assessment method for PM2.5 bacterial community composition sources.
The technical scheme of the invention is as follows: a PM2.5 bacterial community composition source rapid analysis and risk assessment method comprises the following steps:
(1) PM2.5 sample collection: sterilizing sampling tool, arranging sterilized quartz filter membrane (20.3cm × 25.4cm) at sampling port of air sampler, and setting flow rate at 1050L min-1Sampling at a height of 10-12m from the ground, and storing the sample film loaded with the PM2.5 sample at-20 ℃ after sampling is finished;
(2) extracting sample DNA: shearing the sample membrane into uniform fragments on an ultra-clean bench by using sterilized scissors and tweezers, scraping a sample on the sample membrane by using a sterile toothbrush stick, ensuring the completeness of the sampling, reducing the doping of a filter membrane as much as possible, after the scraping of the sample is finished, shearing the sample by using the scissors, dividing the sample into four steps, adding the four steps into a grinding bead sleeve for oscillation, extracting DNA in the sample after carrying out water bath treatment at 65 ℃ for 10min, measuring the concentration and purity of the DNA by using a NanoDrop2000 ultramicro protein nucleic acid analyzer, and then storing the sample at-20 ℃;
(3) PCR amplification and product purification: performing primary PCR amplification on the DNA extracted in the step (2) by using a Primer of a 16S V4 area to obtain a primary PCR product, wherein a PCR amplification reaction system of 16S rDNA is 50 mu L, the primary PCR product comprises 10 mu L of DNA template (about 50-100 ng of DNA in each 50 mu L amplification system) and 1.5 mu L of Forward Primer (10 mu mol. L)-1) 1.5. mu.L of Reverse Primer (10. mu. mol. L)-1) 1 μ L of KAPA HiFi Hot start (1U μ L)-1) 1.5. mu.L of dNTPs (10 mmol. multidot.L)-1) 10 μ L of KAPA HiFi Fidelity Buffer (5X), 24.5 μ L of PCR-grade water. Carrying out primary purification treatment on the primary PCR product to obtain a primary purified product; connecting the Index and the joint by using the primary purified product as a template, and performing secondary PCR amplification to obtain a secondary PCR product, wherein the PCR amplification reaction system is 50 mu L, the secondary PCR product comprises 5 mu L of DNA template (50-100 ng of DNA in each 50ul of amplification system) and 1.5 mu L of Forward Primer (10 mu mol. L)-1) 1.5. mu.L of Reverse Primer (10. mu. mol. L)-1) 1 μ L of KAPA HiFi Hot start (1U μ L)-1) 1.5. mu.L of dNTPs (10 mmol. multidot.L)-1) 10 μ L of KAPA HiFi Fidelity Buffer (5X), 19.5 μ L of PCR-grade water. Carrying out secondary purification treatment on the secondary PCR product to obtain a secondary purified product;
(4) fluorescence quantification and high throughput sequencing: performing fluorescent quantitative detection on the secondary purified product by using the Qubit, then performing library construction on a library construction kit by using TruSeq, evaluating the quality of a sequencing library by using an Agilent Bioanalyzer 2100 system, and finally performing high-throughput sequencing on an Illumina HiSeq 2500 platform; obtaining an effective sequence and correcting the direction of the effective sequence;
(5) and (3) data analysis: selecting 75,000 sequences from each sample data, intercepting 240bp of each reads, removing chimeras, selecting OTU (with the similarity of 97%) and comparing the OTU with a Greenene database, removing archaea sequences in the OTU and obtaining sequence information; obtaining a PM2.5 bacterial community composition structure, analyzing the diversity of the flora, the relative abundance of each PM2.5 bacteria and the abundance of the dominant bacterial community, and further researching and analyzing the PM2.5 source environment;
(6) risk prediction: an equation model is established by adopting a Logistic regression method, the pathogenic coefficient of the PM2.5 bacterial community is calculated according to the relative abundance of each PM2.5 bacterium in the step (5), then the risk prediction of the disease is carried out on the unknown sample according to the general propagation rule of the disease, and the prediction result is obtained;
the equation model is as follows:
F(t)＝A[PNs(t)Δt-μNh(t)Δt] (3)
s(t)+h(t)＝1 (4)
h(t)＝(1-P)Δt (5)
wherein, P is the pathogenic coefficient of PM2.5 bacterial community; x is the number ofiRelative abundance of each PM2.5 bacterium; beta is aiParameters of a Logistic regression model; alpha is alphaiMutation coefficients are for each bacterial population; Δ miThe number of mutations for each bacterial population; Δ niThe number of generations of division of each bacterium; f (t) is the risk prediction score for the disease; a is a risk correction coefficient; n is the total number of people in the monitoring area; s (t) is the proportion of healthy people in the monitoring area at a certain moment; h (t) is the ratio of the number of pathogenic people caused by PM2.5 bacterial communities in a monitoring area at a certain moment; mu is the daily cure rate of the population with disease due to the PM2.5 bacterial community.
Further, the sterilization treatment mode of the quartz filter membrane in the step (1) is to bake the quartz filter membrane in a muffle furnace at 450 ℃ for 2 hours, and the baking mode can be used for performing virus killing and sterilization treatment on the inside and the outside of the quartz filter membrane.
Further, the V4 region specific primer sequence forward primer in step (3): AYTGGGYDTAAAGNG, reverse primer: TACNVGGGTATCTAATCC are provided.
Further, the reaction procedure of the primary PCR amplification and the secondary PCR amplification in the step (3) is as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, 25 cycles, and final extension at 72 ℃ for 5 min.
Further, the method of the primary purification treatment in the step (3) comprises the following steps: and detecting the primary PCR product by using 2% agarose gel electrophoresis, and purifying by using 1X AMPure XP Beads.
Further, the method of the secondary purification treatment in the step (3) comprises the following steps: and removing small fragments of primer dimers from the secondary PCR product by adopting 1X AMPure XP Beads, purifying and screening target fragments by adopting 0.7X +0.15X AMPure XP Beads, and carrying out purity detection by using 1.5% agarose gel electrophoresis.
Further, the extraction of DNA from the sample in step (2) is carried out by using the standard method of the PowerSoil DNA Isolation Kit.
Further, the prediction result in the step (6) is: risk of disease development of the PM2.5 bacterial community, disease spread rate, and disease control rate. And corresponding prevention and control measures can be taken in time according to the risk prediction result.
Compared with the prior art, the invention has the beneficial effects that:
(1) according to the method, the high-throughput sequencing technology is utilized to rapidly analyze the composition and diversity of the bacterial community in the PM2.5, the relative abundance of the PM2.5 bacterial community is calculated, and the potential bacterial source of the PM2.5 is analyzed based on the composition of the bacterial community, so that the method can be used as an effective means for evaluating the urban air environment quality.
(2) The invention utilizes two rounds of PCR amplification, improves the sensitivity and specificity of PCR, and overcomes the problems of low sensitivity, poor specificity and the like of common PCR detection;
(3) the invention also establishes a risk prediction method, an equation model is established by adopting a Logistic regression method, the pathogenic coefficient of the PM2.5 bacterial community is calculated according to the relative abundance of each PM2.5 bacterium, then the risk prediction of the disease is carried out on an unknown sample according to the general propagation rule of the disease, and the prediction results of the pathogenic risk of the PM2.5 bacterial community, the diffusion rate of the disease, the prevention and control rate and the like are obtained;
in a word, the invention has the advantages of wide coverage, high detection accuracy and sensitivity, more scientific and reliable prediction result and the like.
Drawings
FIG. 1 is a flow chart of a method of the present invention;
FIG. 2 is a histogram of the taxonomic horizontal bacterial community of each sample gate of the present invention;
FIG. 3 is a heat map of the dominant bacteria clustering at the taxonomic level of each sample genus of the present invention.
Detailed Description
The invention will be described in more detail below with reference to the accompanying figures 1-3 and specific embodiments.
As shown in fig. 1, a method for rapid analysis of PM2.5 bacterial community composition source and risk assessment, based on the PM2.5 bacterial community in the atmospheric environment within 2017 months 2-5 in the city of february, includes the following steps:
(1) PM2.5 sample collection: the sampling tool is firstly sterilized, a sterilized quartz filter membrane (20.3cm multiplied by 25.4cm) is arranged at a sampling port of the air sampler, the sterilization treatment mode of the quartz filter membrane is to bake the quartz filter membrane in a muffle furnace at 450 ℃ for 2 hours, and the baking mode can sterilize and sterilize the inside and outside of the quartz filter membrane. The flow rate of the air sampler is set to 1050L-min-1Sampling at a height of 10-12m from the ground, and storing the sample film loaded with the PM2.5 sample at-20 ℃ after sampling is finished;
(2) extracting sample DNA: cutting the sample membrane into uniform fragments on an ultra-clean bench by using sterilized scissors and tweezers, scraping a sample on the sample membrane by using a sterile toothbrush stick, ensuring the completeness of the sampling, reducing the doping of a filter membrane as much as possible, after the scraping of the sample is finished, cutting the sample by using the scissors, dividing the sample into four steps, adding the four steps into a grinding bead sleeve for oscillation, extracting DNA in the sample by using a PowerSoil DNA Isolation Kit operation standard method after carrying out water bath treatment at 65 ℃ for 10min, measuring the concentration and the purity of the DNA by using a NanoDrop2000 ultramicro protein nucleic acid analyzer, and storing the DNA at-20 ℃;
(3) PCR amplification and product purification: performing primary PCR amplification on the DNA extracted in the step (2) by using a primer of a 16S V4 region to obtain a primary PCR product, wherein the sequence of the V4 region specific primer is as follows: AYTGGGYDTAAAGNG, reverse primer: TACNVGGGTATCTAATCC are provided. The PCR amplification reaction system of 16S rDNA is 50 mu L, comprises 10 mu L of DNA template (50-100 ng of DNA in each 50ul of amplification system) and 1.5 mu L of Forward Primer (10 mu mol. L)-1) 1.5. mu.L of Reverse Primer (10. mu. mol. L)-1) 1 μ L of KAPA HiFi Hot start (1U μ L)-1) 1.5. mu.L of dNTPs (10 mmol. multidot.L)-1) 10 μ L of KAPA HiFi Fidelity Buffer (5X), 24.5 μ L of PCR-grade water. The reaction procedure is as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, 25 cycles, and final extension at 72 ℃ for 5 min. Detecting the primary PCR product by using 2% agarose gel electrophoresis, and purifying by using 1X AMPure XP Beads to obtain a primary purified product; connecting the Index and the joint by using the primary purified product as a template, and performing secondary PCR amplification to obtain a secondary PCR product, wherein the PCR amplification reaction system is 50 mu L, the secondary PCR product comprises 5 mu L of DNA template (50-100 ng of DNA in each 50ul of amplification system) and 1.5 mu L of Forward Primer (10 mu mol. L)-1) 1.5. mu.L of Reverse Primer (10. mu. mol. L)-1) 1 μ L of KAPA HiFi Hot start (1U μ L)-1) 1.5. mu.L of dNTPs (10 mmol. multidot.L)-1) 10 μ L of KAPA HiFi Fidelity Buffer (5X), 19.5 μ L of PCR-grade water. The reaction procedure is as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, 25 cycles, and final extension at 72 ℃ for 5 min. Removing small fragments of primer dimers from the secondary PCR product by using 1X AMPure XP Beads, purifying and screening target fragments by using 0.7X +0.15X AMPure XP Beads, and performing purity detection by using 1.5% agarose gel electrophoresis to obtain a secondary purified product;
(4) fluorescence quantification and high throughput sequencing: performing fluorescent quantitative detection on the secondary purified product by using the Qubit, then performing library construction on a library construction kit by using TruSeq, evaluating the quality of a sequencing library by using an Agilent Bioanalyzer 2100 system, and finally performing high-throughput sequencing on an Illumina HiSeq 2500 platform; obtaining an effective sequence and correcting the direction of the effective sequence;
(5) and (3) data analysis: selecting 75,000 sequences from each sample data, intercepting 240bp of each reads, removing chimeras, selecting OTU (with the similarity of 97%) and comparing the OTU with a Greenene database, removing archaea sequences in the OTU and obtaining sequence information; obtaining a PM2.5 bacterial community composition structure, analyzing the diversity of the flora, the relative abundance of various types of PM2.5 bacteria and the abundance of the dominant bacterial community, and further researching and analyzing the PM2.5 source environment;
(6) risk prediction: and (3) establishing an equation model by using a Logistic regression method, calculating the pathogenic coefficient of the PM2.5 bacterial community according to the relative abundance of each PM2.5 bacterium in the step (5), predicting the risk of the disease of an unknown sample according to the general propagation rule of the disease, and obtaining the prediction results of the pathogenic risk of the PM2.5 bacterial community, the diffusion rate of the disease, the prevention and control rate of the disease and the like. And corresponding prevention and control measures can be taken in time according to the risk prediction result.
The equation model is as follows:
F(t)＝A[PNs(t)Δt-μNh(t)Δt] (3)
s(t)+h(t)＝1 (4)
h(t)＝(1-P)Δt (5)
wherein, P is the pathogenic coefficient of PM2.5 bacterial community; x is the number ofiRelative abundance of each PM2.5 bacterium; beta is aiParameters of a Logistic regression model; alpha is alphaiMutating for each bacterial populationA coefficient; Δ miThe number of mutations for each bacterial population; Δ niThe number of generations of division of each bacterium; f (t) is the risk prediction score for the disease; a is a risk correction coefficient; n is the total number of people in the monitoring area; s (t) is the proportion of healthy people in the monitoring area at a certain moment; h (t) is the ratio of the number of pathogenic people caused by PM2.5 bacterial communities in a monitoring area at a certain moment; mu is the daily cure rate of the population with disease due to the PM2.5 bacterial community.
Analysis of Experimental results
To study the phylogenetic relationship of different OTUs, as well as the difference of dominant species in different samples, multiple sequence alignments were performed using the Muscle software (Version 3.8.31). Based on species annotation results for OTUs representative sequences, taxonomic information was obtained and separately at each taxonomic level: and (3) counting community compositions of various samples, and analyzing the diversity and the abundance of dominant groups of the bacterial communities.
Species annotation results indicated that a total of 20 phyla of bacteria were detected in the samples, and as shown in FIG. 2, the highest relative abundance was Proteobacteria (Proteobacteria, 63.87%), and the proportion of Proteobacteria in each sample was F > J > G > K > I > A > B > C > D > E > H. Cyanobacteria (11.21%) and Firmicutes (5.80%) are the dominant groups ranked 2 nd and 3 rd in average abundance, with average relative abundances of 19.82% and 9.31%, respectively. In addition, the phyla of bacteria with high relative abundance were Bacteroidetes (3.27%), actinomycetemcomita (actinobacillia, 2.90%), Deinococcus-Thermus (Deinococcus-Thermus, 2.05%), Verrucomicrobia (1.75%), and phytophthora (plancomycetes, 1.51%) and acidobacter (acidobactiria, 1.12%) in this order, and the group of bacteria with low occupation ratio was classified as the category of otherkind.
And (3) knotting: at the phylogenetic classification level, Proteobacteria, Cyanobactria, Firmicutes and Bacteroides account for 84.15% of the total gene abundance, while Proteobacteria, Cyanobactria, Firmicutes and Bacteroides are mainly the predominant bacteria in freshwater environments, indicating that freshwater is likely to be the PM functional region in the Changzhou city2.5The most predominant source of middle-living bacteria.
Genus taxonomic level dominant bacterial clustering heatmaps are shown in FIG. 3, mainly for the dominant bacteria genus with average relative abundance > 1%, among which Pseudomonas (Pseudomonas) and Acinetobacter (Acinetobacter). Rubellimicobium, Sphingomonas, Methylobacterium and Acinetobacter are distributed in soil environment; pseudomonas, Sphingomonas and Methylobacterium are also associated with plants; indicating that soil and plants are also PM2.5Is the source of the bacterium.
And (3) knotting: by comparison, spring PM of Changzhou city2.5The diversity of the medium bacterial community is higher, and the Proteobacteria, the cyanobacter and the Firmicutes are the dominant bacterial community ranked at the top 3. The horizontally dominant bacteria of genus are mainly genus Chroococcoides (6.03%), Rubellicinobium (5.95%), Microcystis (4.86%) and Sphingomonas (3.16%). Changzhou spring city functional area PM2.5The main source environment of the mesobacteria is fresh water, and then soil and plants.
And (4) conclusion: the experimental result shows that the PM can be rapidly analyzed by utilizing a high-throughput sequencing technology2.5The composition and diversity of the medium bacterial community are effective technologies for analyzing the bacterial community in the atmospheric particulates. Because some environments such as animal wastes, sewage systems, plants and the like are often rich in or accompanied by pathogenic bacteria and allergic substances, the generated bacterial aerosol can be potentially harmful to human health. PM analysis based on bacterial community composition2.5Potential environmental sources can be used as an effective means for evaluating the environmental quality of urban air.
According to the relative abundance of the phylum of bacteria, the equation model is applied to obtain that the pathogenic factor is 0.547, the score value after one week of risk prediction is 3.6, the score is 2-4, the score is low, the score is 4-6, the score is medium, the score is 6-8, the score is high, and the score is high, so that the pathogenic factor of the PM2.5 bacterial community in the Changzhou city is high, the risk is controllable and the like.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (3)
1. A PM2.5 bacterial community composition source rapid analysis and risk assessment method is characterized by comprising the following steps:
(1) PM2.5 sample collection: sterilizing a sampling tool, arranging a sterilized quartz filter membrane at a sampling port of an air sampler, sampling at a position 10-12m away from the ground, and storing a sample membrane loaded with a PM2.5 sample at-20 ℃ after sampling is finished;
(2) extracting sample DNA: shearing the sample membrane into uniform fragments on a super clean bench by using sterilized scissors and tweezers, scraping the sample on the sample membrane by using a sterile toothbrush, shearing the sample by using the scissors, adding the sample into a grinding bead sleeve for oscillation in four steps, extracting DNA in the sample after treating the sample in water bath at 65 ℃ for 10min, measuring the concentration and purity of the DNA by using a NanoDrop2000 ultramicro protein nucleic acid analyzer, and storing the sample at-20 ℃;
(3) PCR amplification and product purification: performing primary PCR amplification on the DNA extracted in the step (2) by using a primer in a 16S V4 area to obtain a primary PCR product, and performing primary purification treatment on the primary PCR product to obtain a primary purified product; connecting the Index and the joint by taking the primary purified product as a template, performing secondary PCR amplification to obtain a secondary PCR product, and performing secondary purification treatment on the secondary PCR product to obtain a secondary purified product;
(4) fluorescence quantification and high throughput sequencing: performing fluorescent quantitative detection on the secondary purified product by using the Qubit, then performing library construction on a library construction kit by using TruSeq, evaluating the quality of a sequencing library by using an Agilent Bioanalyzer 2100 system, and finally performing high-throughput sequencing on an Illumina HiSeq 2500 platform; obtaining an effective sequence and correcting the direction of the effective sequence;
(5) and (3) data analysis: selecting 75,000 sequences from each sample data, intercepting 240bp of each reads, removing chimeras, selecting an OTU, comparing the OTU with a Greenene database, removing archaea sequences in the OTU, and obtaining sequence information; obtaining a PM2.5 bacterial community composition structure, analyzing the diversity of the flora, the relative abundance of various types of PM2.5 bacteria and the abundance of the dominant bacterial community, and further researching and analyzing the PM2.5 source environment;
(6) risk prediction: an equation model is established by adopting a Logistic regression method, the pathogenic coefficient of the PM2.5 bacterial community is calculated according to the relative abundance of various PM2.5 bacteria in the step (5), then the risk prediction of the disease is carried out on an unknown sample according to the general propagation rule of the disease, and the prediction result is obtained;
the equation model is as follows:
F(t)＝A[PNs(t)Δt-μNh(t)Δt] (3)
s(t)+h(t)＝1 (4)
h(t)＝(1-P)Δt (5)
wherein, P is the pathogenic coefficient of PM2.5 bacterial community; x is the number ofiRelative abundance of each PM2.5 bacterium; beta is aiParameters of a Logistic regression model; alpha is alphaiMutation coefficients are for each bacterial population; Δ miThe number of mutations for each bacterial population; Δ niThe number of generations of division of each bacterium; f (t) is the risk prediction score for the disease; a is a risk correction coefficient; n is the total number of people in the monitoring area; s (t) is the proportion of healthy people in the monitoring area at a certain moment; h (t) is the ratio of the number of pathogenic people caused by PM2.5 bacterial communities in a monitoring area at a certain moment; mu is the daily cure rate of the population suffering from the disease due to the PM2.5 bacterial community;
the V4 region specific primer sequence forward primer in the step (3): AYTGGGYDTAAAGNG, reverse primer: TACNVGGGTATCTAATCC, respectively;
the V4 region specific primer sequence reverse primer in the step (3): TACNVGGGTATCTAATCC, forward primer: AYTGGGYDTAAAGNG, respectively;
the reaction procedures of the primary PCR amplification and the secondary PCR amplification in the step (3) are as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, 25 cycles, and extension at 72 ℃ for 5 min;
the method for the primary purification treatment in the step (3) comprises the following steps: detecting the primary PCR product by using 2% agarose gel electrophoresis, and purifying by using 1X AMPure XP Beads;
the secondary purification treatment method in the step (3) comprises the following steps: and removing small fragments of primer dimers from the secondary PCR product by adopting 1X AMPure XP Beads, purifying and screening target fragments by adopting 0.7X +0.15X AMPure XP Beads, and carrying out purity detection by using 1.5% agarose gel electrophoresis.
2. The method for rapid analysis and risk assessment of the bacterial community composition origin of PM2.5 according to claim 1, wherein the sterilization treatment of said quartz filter membrane in step (1) is muffle furnace baking at 450 ℃ for 2 h.
3. The method for rapid analysis and risk assessment of bacterial community composition origin of PM2.5 according to claim 1, wherein the DNA extraction in step (2) is performed by using the standard method of the PowerSoil DNA Isolation Kit.
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