CN108841942A - A kind of PM2.5 bacterial community composition source is quickly analyzed and methods of risk assessment - Google Patents
A kind of PM2.5 bacterial community composition source is quickly analyzed and methods of risk assessment Download PDFInfo
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Abstract
The invention discloses a kind of PM2.5 bacterial community composition sources quickly to analyze and methods of risk assessment, belong to microbiological analysis technical field, this method mainly includes the sampling of urban function region PM2.5, microbe genome DNA extracts, PCR amplification is carried out to the DNA product extracted, PCR product purifies, fluorescent quantitation, high-flux sequence simultaneously carries out Phylogenetic diversity of bacteria and enrichment analysis and risk profile.Relative to culture-based method, the Bacterial community for reflecting study sample of the more careful overall picture of this method, the Phylogenetic diversity of bacteria for fast and accurately analyzing aerosol sample is not only realized, while being the effective ways for evaluating urban air environmental quality and predictive disease risk.In short, the present invention has many advantages, such as that broad covered area, accuracy in detection and high sensitivity, prediction result are more scientific reliable.
Description
Technical field
The invention belongs to microbiological analysis technical fields, and in particular to a kind of PM2.5 bacterial community composition source is quickly divided
Analysis and methods of risk assessment.
Background technique
Recent study show particulate matter have become cause China's majority urban environment air pollution it is serious it is primary because
Element, and pollutant is pellet (haze main constituents) in most number of days.PM2.5 relative to PM10 and
Say that partial size is smaller, the residence time in an atmosphere is longer, transmission range is farther, and since its specific surface area is bigger, becomes perhaps
The carrier of more poisonous and harmful substances.Bioaerosol can propagate anaphylactogen and toxin, and human body is caused to suffer from respiratory disease, so
The influence for understanding air microbe composition and atmospheric environment is of great significance.
In recent years, Protocols in Molecular Biology has evolved into terms of research water body, soil, deposit biological community structure
For a kind of effective technology of maturation, but to the only a small amount of report of the research of air sample, and rare research is based on particulate matter bacterium
Structure of community carries out source analysis.It can obtain the group of bacterial groups at the genetic level based on the analysis of 16S rRNA gene sequencing
Fall composition and Biodiversity Characteristics.High throughput sequencing technologies are the changes to conventional sequencing technology revolution, quick, accurate
It can comprehensively reflect bacterial community distribution characteristics while analyzing Multi-example, be to analyze bacterial groups in Atmospheric particulates to have
Effect technology.
Since certain environment such as animal wastes, sewerage and plant etc. are often rich in or association pathogenic bacteria and anaphylaxis object
Matter, therefore its bacterial aerosol generated may have potential hazard to human health.In the prior art, mostly for right
The source of PM2.5 bacterial community is analyzed, and ignores disease caused by bacterium there may be infectiousness, it is possible that
Therefore the explosive growth of disease only analyze to the source of PM2.5 bacterial community or inadequate.
Summary of the invention
For the technical problem present on, the present invention provide a kind of PM2.5 bacterial community composition source quickly analyze and
Methods of risk assessment.
The technical scheme is that：A kind of PM2.5 bacterial community composition source is quickly analyzed and methods of risk assessment, packet
Include following steps：
(1) PM2.5 sample acquires：First sampling device is carried out disinfection sterilization treatment, is set in the thief hatch of air sampler
Quartz filter (20.3cm × 25.4cm) after setting sterilization treatment, flow set 1050Lmin-1, apart from ground 10-12m
It is sampled at height, load is had to the sample film of PM2.5 sample after the completion of to be sampled, is saved at -20 DEG C；
(2) sample DNA extracts：Using the scissors of sterilization treatment, tweezers super-clean bench by the sample film be cut into uniformly it is broken
Piece reduces the incorporation of filter membrane to the greatest extent on the basis of guaranteeing that sampling is complete using the sample on sterile toothbrush label scraping sample film,
Sample scraping finish, recycle scissors the sample is carried out to shred processing, and be divided into four steps be added in grinding bead casing into
Row oscillation extracts the DNA in sample, then with NanoDrop2000 ultramicron protein nucleic acid after 65 DEG C of water bath processing 10min
After DNA concentration described in analysis-e/or determining and purity, it is placed in -20 DEG C and saves；
(3) PCR amplification and product purification：The DNA extracted in step (2) is subjected to a PCR using 16S V4 area's primer
Amplification, obtains a PCR product, and the pcr amplification reaction system of 16S rDNA is 50 μ L, DNA profiling (every 50ul comprising 10 μ L
In amplification system, DNA about 50~100ng), Forward Primer (10 μm of olL of 1.5 μ L-1), the Reverse of 1.5 μ L
Primer(10μmol·L-1), KAPA HiFi Hot start (the 1U μ L of 1 μ L-1), the dNTPs (10mmolL of 1.5 μ L-1), the KAPA HiFi Fidelity Buffer (5X) of 10 μ L, the PCR-grade water of 24.5 μ L.To a PCR
Product carries out a purification process, obtains a purified product；Index is connected as template using a purified product and is connect
Head carries out secondary PCR amplification, obtains secondary PCR product, and pcr amplification reaction system is 50 μ L, and the DNA profiling comprising 5 μ L is (every
In 50ul amplification system, DNA about 50~100ng), Forward Primer (10 μm of olL of 1.5 μ L-1), 1.5 μ L's
Reverse Primer(10μmol·L-1), KAPA HiFi Hot start (the 1U μ L of 1 μ L-1), the dNTPs of 1.5 μ L
(10mmol·L-1), the KAPA HiFi Fidelity Buffer (5X) of 10 μ L, the PCR-grade water of 19.5 μ L.It is right again
The secondary PCR product carries out secondarily purified processing, obtains secondarily purified product；
(4) fluorescent quantitation and high-flux sequence：Fluorogenic quantitative detection is carried out to the secondarily purified product using Qubit,
Then library kit progress library construction is being built using TruSeq, 2100 system evaluation of Agilent Bioanalyzer is used to survey
Sequence Library Quality finally carries out high-flux sequence on 2500 platform of Illumina HiSeq；Ordered sequence is obtained, and is corrected
Ordered sequence direction；
(5) data are analyzed：75,000 sequence is chosen from each sample data, and every reads is intercepted into 240bp, and
Chimera is removed, OTU (similitude 97%) is then chosen and is compared with Greengene database, archaeal sequence therein is removed,
Obtain sequence information；It obtains PM2.5 bacterial community composed structure, and analyzes the relative abundance of bacterial diversity, each PM2.5 bacterium
And predominant bacteria monoid abundance, further research and analyse the source PM2.5 environment；
(6) risk profile：Equation model is established using the Logistic Return Law, it is thin according to each PM2.5 described in step (5)
The relative abundance of bacterium calculates the pathogenic coefficient of PM2.5 bacterial community, further according to disease general propagation law to unknown sample
The risk profile of disease is carried out, and obtains prediction result；
The equation model is as follows：
F (t)=A [PNs (t) Δ t- μ Nh (t) Δ t] (3)
+ h (t)=1 s (t) (4)
H (t)=(1-P) Δ t (5)
Wherein, P is the pathogenic coefficient of PM2.5 bacterial community；xiFor the relative abundance of each PM2.5 bacterium；βiFor Logistic
Parameters in Regression Model；αiFor each bacterial population change coefficient；ΔmiFor the mutation quantity of each bacterial population；ΔniFor each bacterium point
The generation number split；F (t) is the risk profile score value of disease；A is risk correction factor；N is the total number of persons for monitoring region；s
(t) the healthy number ratio in region is monitored for a certain moment；H (t) is to monitor region internal cause PM2.5 bacterial community at a certain moment
Pathogenic number ratio；μ is the day cure rate of the pathogenic crowd because of PM2.5 bacterial community.
Further, the sterilization treatment mode of quartz filter described in step (1) is to toast 2h in 450 DEG C of Muffle furnaces, is dried
Roasting mode can be to progress exterminating bacterium processing inside and outside quartz filter.
Further, the area V4 specific primer sequence forward primer described in step (3)：AYTGGGYDTAAAGNG, reversely
Primer：TACNVGGGTATCTAATCC.
Further, a PCR amplification described in step (3) and the response procedures of secondary PCR amplification are：95 DEG C of initial denaturations
3min, 95 DEG C of denaturation 30s react 30s, 72 DEG C of extension 30s at 55 DEG C of annealing temperature, and totally 25 circulations, finally extend at 72 DEG C
5min。
Further, the method for a purification process described in step (3) is：By 2% agar of a PCR product
Sugared detected through gel electrophoresis is purified using 1X AMPure XP Beads.
Further, the method for secondarily purified processing described in step (3) is：The secondary PCR product is used into 1X
AMPure XP Beads removes primer dimer small fragment, purifies screening mesh using 0.7X+0.15X AMPure XP Beads
Segment, carry out purity detecting with 1.5% agarose gel electrophoresis.
Further, it is PowerSoil DNA Isolation that the DNA in extraction sample described in step (2), which is used,
Kit kit operation standard method.
Further, prediction result described in step (6) is：The diffusion of the pathogenic risk, disease of PM2.5 bacterial community
The prevention and control rate of rate and disease.Corresponding prevention and control measure can be taken in time according to risk profile result.
Compared with prior art, beneficial effects of the present invention are：
(1) present invention quickly analyzes bacterial community composition and diversity in PM2.5 using high throughput sequencing technologies, calculates
The relative abundance of PM2.5 bacterial community out is based on the potential bacterial origin of bacterial community composition analysis PM2.5, can be used as and comment
The effective means of valence urban air environmental quality.
(2) present invention utilizes two-wheeled PCR amplification, improves the sensibility and specificity of PCR, overcomes regular-PCR detection
The problems such as sensitivity is low, poor specificity；
(3) present invention has also set up Risk Forecast Method, equation model is established using the Logistic Return Law, according to each
The relative abundance of PM2.5 bacterium calculates the pathogenic coefficient of PM2.5 bacterial community, further according to disease general propagation law to not
Know that sample carries out the risk profile of disease, and the pathogenic risk, the diffusivity of disease and prevention and control rate etc. for obtaining PM2.5 bacterial community
Prediction result；
In short, the present invention has broad covered area, accuracy in detection and a high sensitivity, prediction result is more scientific reliable etc. excellent
Point.
Detailed description of the invention
Fig. 1 is flow chart of the method for the present invention；
Fig. 2 is each sample door categorization levels bacterial community column accumulation graph of the present invention；
Fig. 3 is each sample category categorization levels predominant bacteria cluster thermal map of the present invention.
Specific embodiment
1-3 and specific embodiment to carry out the present invention further detailed description with reference to the accompanying drawing
As shown in Figure 1, a kind of PM2.5 bacterial community composition source is quickly analyzed and methods of risk assessment, with Changzhou
PM2.5 bacterial community is basic data in the 2-5 month in 2017 in one's duty atmospheric environment, is included the following steps：
(1) PM2.5 sample acquires：First sampling device is carried out disinfection sterilization treatment, is set in the thief hatch of air sampler
Quartz filter (20.3cm × 25.4cm) after setting sterilization treatment, the sterilization treatment mode of the quartz filter are in 450 DEG C of horses
Not baking oven bakes 2h, and roasting mode can be to progress exterminating bacterium processing inside and outside quartz filter.By the flow set of air sampler
For 1050Lmin-1, sampled at the 10-12m height of ground, will load after the completion of to be sampled has PM2.5 sample
Sample film saves at -20 DEG C；
(2) sample DNA extracts：Using the scissors of sterilization treatment, tweezers super-clean bench by the sample film be cut into uniformly it is broken
Piece reduces the incorporation of filter membrane to the greatest extent on the basis of guaranteeing that sampling is complete using the sample on sterile toothbrush label scraping sample film,
Sample scraping finish, recycle scissors the sample is carried out to shred processing, and be divided into four steps be added in grinding bead casing into
Row oscillation, after 65 DEG C of water bath processing 10min, with PowerSoil DNA Isolation Kit kit operation standard method
Extract the DNA in sample, then the DNA concentration and purity described with the measurement of NanoDrop2000 ultramicron protein nucleic acids instrument
Afterwards, it is placed in -20 DEG C and saves；
(3) PCR amplification and product purification：The DNA extracted in step (2) is subjected to a PCR using 16S V4 area's primer
Amplification, obtains a PCR product, wherein the area the V4 specific primer sequence forward primer：AYTGGGYDTAAAGNG, reversely
Primer：TACNVGGGTATCTAATCC.The pcr amplification reaction system of 16S rDNA is 50 μ L, and the DNA profiling comprising 10 μ L is (every
In 50ul amplification system, DNA about 50~100ng), Forward Primer (10 μm of olL of 1.5 μ L-1), 1.5 μ L's
Reverse Primer(10μmol·L-1), KAPA HiFi Hot start (the 1U μ L of 1 μ L-1), the dNTPs of 1.5 μ L
(10mmol·L-1), the KAPA HiFi Fidelity Buffer (5X) of 10 μ L, the PCR-grade water of 24.5 μ L.Reaction
Program is：95 DEG C of initial denaturation 3min, 95 DEG C of denaturation 30s react 30s at 55 DEG C of annealing temperature, and 72 DEG C of extension 30s, totally 25 are followed
Ring, finally in 72 DEG C of extension 5min.2% agarose gel electrophoresis of PCR product is detected, 1X AMPure is used
XP Beads is purified, and a purified product is obtained；Index and connector are connected by template of a purified product, into
The amplification of row secondary PCR obtains secondary PCR product, and pcr amplification reaction system is 50 μ L, and (every 50ul expands the DNA profiling comprising 5 μ L
In increasing system, DNA about 50~100ng), Forward Primer (10 μm of olL of 1.5 μ L-1), the Reverse of 1.5 μ L
Primer(10μmol·L-1), KAPA HiFi Hot start (the 1U μ L of 1 μ L-1), the dNTPs (10mmolL of 1.5 μ L-1), the KAPA HiFi Fidelity Buffer (5X) of 10 μ L, the PCR-grade water of 19.5 μ L.Response procedures are：95
DEG C initial denaturation 3min, 95 DEG C of denaturation 30s react 30s, 72 DEG C of extension 30s at 55 DEG C of annealing temperature, and totally 25 circulations, finally exist
72 DEG C of extension 5min.Primer dimer small fragment is removed using 1X AMPure XP Beads to by the secondary PCR product again,
Screening target fragment is purified using 0.7X+0.15X AMPure XP Beads, carries out purity with 1.5% agarose gel electrophoresis
Detection, obtains secondarily purified product；
(4) fluorescent quantitation and high-flux sequence：Fluorogenic quantitative detection is carried out to the secondarily purified product using Qubit,
Then library kit progress library construction is being built using TruSeq, 2100 system evaluation of Agilent Bioanalyzer is used to survey
Sequence Library Quality finally carries out high-flux sequence on 2500 platform of Illumina HiSeq；Ordered sequence is obtained, and is corrected
Ordered sequence direction；
(5) data are analyzed：75,000 sequence is chosen from each sample data, and every reads is intercepted into 240bp, and
Chimera is removed, OTU (similitude 97%) is then chosen and is compared with Greengene database, archaeal sequence therein is removed,
Obtain sequence information；Obtain PM2.5 bacterial community composed structure, and analyze bacterial diversity, all kinds of PM2.5 bacteriums it is relatively rich
Degree and predominant bacteria monoid abundance, further research and analyse the source PM2.5 environment；
(6) risk profile：Equation model is established using the Logistic Return Law, it is thin according to each PM2.5 described in step (5)
The relative abundance of bacterium calculates the pathogenic coefficient of PM2.5 bacterial community, further according to disease general propagation law to unknown sample
The risk profile of disease is carried out, and obtains the prevention and control of the pathogenic risk, the diffusivity of disease and disease of PM2.5 bacterial community
The prediction results such as rate.Corresponding prevention and control measure can be taken in time according to risk profile result.
The equation model is as follows：
F (t)=A [PNs (t) Δ t- μ Nh (t) Δ t] (3)
+ h (t)=1 s (t) (4)
H (t)=(1-P) Δ t (5)
Wherein, P is the pathogenic coefficient of PM2.5 bacterial community；xiFor the relative abundance of each PM2.5 bacterium；βiFor Logistic
Parameters in Regression Model；αiFor each bacterial population change coefficient；ΔmiFor the mutation quantity of each bacterial population；ΔniFor each bacterium point
The generation number split；F (t) is the risk profile score value of disease；A is risk correction factor；N is the total number of persons for monitoring region；s
(t) the healthy number ratio in region is monitored for a certain moment；H (t) is to monitor region internal cause PM2.5 bacterial community at a certain moment
Pathogenic number ratio；μ is the day cure rate of the pathogenic crowd because of PM2.5 bacterial community.
Analysis of experimental results
It is soft using Muscle in order to study the difference of sociales in the Phylogenetic Relationships of different OTU, and different samples
Part (Version 3.8.31) carries out Multiple Sequence Alignment.The species annotation of sequence is represented based on OTUs as a result, obtaining taxology letter
It ceases and respectively in each categorization levels：Boundary, door, guiding principle, mesh, section, category and kind count group's composition of each sample, analyze bacterial flora
Fall diversity and dominant groups abundance.
Species annotation the result shows that detect 20 Bacteriophytas, as shown in Fig. 2, it is deformation that relative abundance is highest altogether in sample
Bacterium door (Proteobacteria, 63.87%), Proteobacteria accounting size is F > J > G > K > I > A in each sample
> B > C > D > E > H.Cyanobacteria door (Cyanobacteria, 11.21%) and Firmicutes (Firmicutes), 5.80%)
It is the dominant groups of average abundance ranking the 2nd and the 3rd, average relative abundance is respectively 19.82% and 9.31%.In addition, opposite
The higher Bacteriophyta of abundance be followed successively by Bacteroidetes (Bacteroidetes, 3.27%), actinomyces door (Actinobacteria,
2.90%), abnormal cocci-Thermus door (Deinococcus-Thermus, 2.05%), wart germ door
(Verrucomicrobia, 1.75%) and floating mould door (Planctomycetes, 1.51%) and acidfast bacilli door
The lower bacterial groups of accounting are classified as Others class by (Acidobacteria, 1.12%).
Brief summary：From the point of view of door categorization levels, Proteobacteria, Cyanobacteria, Firmicutes and
Bacteroidetes occupies the 84.15% of total gene abundance, and Proteobacteria, Cyanobacteria,
Firmicutes and Bacteroidetes is mainly the predominant bacteria in fresh water environment, shows that fresh water is likely to be Chang Zhoucheng
City functional areas PM2.5The main source of middle bacterium.
Belong to categorization levels predominant bacteria cluster thermal map as shown in figure 3, being mainly the predominant bacteria of averagely relative abundance > 1%
Belong to, wherein pseudomonas (Pseudomonas) and Acinetobacter (Acinetobacter).Rubellimicrobium,
Sphingomonas (Sphingomonas), Methylobacter (Methylobacterium) and more points of Acinetobacter
It is distributed in soil environment；Pseudomonas, Sphingomonas and Methylobacterium are also related to plant；Show soil
Earth and plant are also PM2.5The source of middle bacterium.
Brief summary：Through comparing, Changzhou spring PM2.5The diversity of middle bacterial community is higher, Proteobacteria,
Cyanobacteria and Firmicutes is before ranking 3 predominant bacteria monoid.The horizontal predominant bacteria of category mainly has quasi- first chromosphere
Trentepohlia (Chroococcidiopsis, 6.03%), Rubellimicrobium (5.95%), microcystis kutz (Microcystis,
And Sphingomonas (3.16%) 4.86%).Changzhou spring urban function region PM2.5The main source environment of middle bacterium is
Secondly fresh water is soil and plant.
Conclusion：The experimental results showed that can quickly analyze PM using high throughput sequencing technologies2.5Middle bacterial community composition and
Diversity is the effective technology for analyzing bacterial groups in Atmospheric particulates.Due to certain environment such as animal wastes, sewerage and
Plant etc. is often rich in or association pathogenic bacteria and anaphylaxis substance, therefore its bacterial aerosol generated may have human health
Potential hazard.Based on bacterial community composition analysis PM2.5Potential environmental sources can be used as evaluation urban air environmental quality
Effective means.
According to the relative abundance of above-mentioned Bacteriophyta, above-mentioned equation model is applied, show that pathogenic coefficient is 0.547, risk is pre-
Surveying score value after a week is 3.6 points, according to standards of grading 2-4 point be low-risk, 4-6 points be medium risk, 6-8 divides is high wind
Danger, 8-10 divide for high risk it is found that this time the pathogenic coefficient of Changzhou PM2.5 bacterial community is higher, and controllable risk is medium.
Finally it should be noted that：The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
Present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that：It still may be used
To modify to technical solution documented by previous embodiment or equivalent replacement of some of the technical features；And
These are modified or replaceed, the spirit and model of technical solution of the embodiment of the present invention that it does not separate the essence of the corresponding technical solution
It encloses.
Claims (8)
1. a kind of PM2.5 bacterial community composition source is quickly analyzed and methods of risk assessment, which is characterized in that including following step
Suddenly：
(1) PM2.5 sample acquires：First sampling device is carried out disinfection sterilization treatment, is gone out in the thief hatch setting of air sampler
Bacterium treated quartz filter, is sampled at the 10-12m height of ground, and load had PM2.5 sample after the completion of to be sampled
The sample film of product saves at -20 DEG C；
(2) sample DNA extracts：The sample film is cut into uniform fragment in super-clean bench using the scissors of sterilization treatment, tweezers, benefit
With the sample on sterile toothbrush label scraping sample film, scissors is recycled to carry out the sample to shred processing, and be divided into four steps and add
Enter into grinding bead casing and vibrated, after 65 DEG C of water bath processing 10min, extracts the DNA in sample, then use
After the measurement of NanoDrop2000 ultramicron protein the nucleic acids instrument DNA concentration and purity, it is placed in -20 DEG C and saves；
(3) PCR amplification and product purification：The DNA extracted in step (2) is subjected to a PCR amplification using 16S V4 area's primer,
A PCR product is obtained, a purification process is carried out to a PCR product, obtains a purified product；With described primary
Purified product is that template connects Index and connector, carries out secondary PCR amplification, obtains secondary PCR product, then to the secondary PCR
Product carries out secondarily purified processing, obtains secondarily purified product；
(4) fluorescent quantitation and high-flux sequence：Fluorogenic quantitative detection is carried out to the secondarily purified product using Qubit, then
Library kit progress library construction is being built using TruSeq, is using 2100 system evaluation of Agilent Bioanalyzer that text is sequenced
Library quality finally carries out high-flux sequence on 2500 platform of Illumina HiSeq；Ordered sequence is obtained, and is corrected effective
Sequence direction；
(5) data are analyzed：75,000 sequence is chosen from each sample data, every reads is intercepted into 240bp, and remove
Then chimera is chosen OTU and is compared with Greengene database, removes archaeal sequence therein, obtain sequence information；?
PM2.5 bacterial community composed structure out, and analyze the relative abundance and predominant bacteria class of bacterial diversity, all kinds of PM2.5 bacteriums
Group's abundance, further researchs and analyses the source PM2.5 environment；
(6) risk profile：Equation model is established using the Logistic Return Law, all kinds of PM2.5 bacteriums according to step (5)
Relative abundance calculate the pathogenic coefficient of PM2.5 bacterial community, further according to disease general propagation law to unknown sample into
The risk profile of row disease, and obtain prediction result；
The equation model is as follows：
F (t)=A [PNs (t) Δ t- μ Nh (t) Δ t] (3)
+ h (t)=1 s (t) (4)
H (t)=(1-P) Δ t (5)
Wherein, P is the pathogenic coefficient of PM2.5 bacterial community；xiFor the relative abundance of each PM2.5 bacterium；βiFor Logistic recurrence
Model parameter；αiFor each bacterial population change coefficient；ΔmiFor the mutation quantity of each bacterial population；ΔniFor the division of each bacterium
Generation number；F (t) is the risk profile score value of disease；A is risk correction factor；N is the total number of persons for monitoring region；S (t) is
Healthy number ratio in a certain moment monitoring region；H (t) is the cause for monitoring region internal cause PM2.5 bacterial community at a certain moment
Patient's number ratio；μ is the day cure rate of the pathogenic crowd because of PM2.5 bacterial community.
2. a kind of PM2.5 bacterial community composition source as described in right 1 is quickly analyzed and methods of risk assessment, feature exist
In the sterilization treatment mode of quartz filter described in step (1) is to toast 2h in 450 DEG C of Muffle furnaces.
3. a kind of PM2.5 bacterial community composition source as described in right 1 is quickly analyzed and methods of risk assessment, feature exist
In the area V4 specific primer sequence forward primer described in step (3)：AYTGGGYDTAAAGNG, reverse primer：
TACNVGGGTATCTAATCC。
4. a kind of PM2.5 bacterial community composition source as described in right 1 is quickly analyzed and methods of risk assessment, feature exist
In the area V4 specific primer sequence reverse primer described in step (3)：TACNVGGGTATCTAATCC, forward primer：
AYTGGGYDTAAAGNG。
5. a kind of PM2.5 bacterial community composition source as described in right 1 is quickly analyzed and methods of risk assessment, feature exist
In the response procedures of a PCR amplification described in step (3) and secondary PCR amplification are：95 DEG C of initial denaturation 3min, 95 DEG C of denaturation
30s reacts 30s, 72 DEG C of extension 30s at 55 DEG C of annealing temperature, totally 25 circulations, finally in 72 DEG C of extension 5min.
6. a kind of PM2.5 bacterial community composition source as described in right 1 is quickly analyzed and methods of risk assessment, feature exist
In the method for a purification process described in step (3) is：2% agarose gel electrophoresis of PCR product is examined
It surveys, is purified using 1X AMPure XP Beads.
7. a kind of PM2.5 bacterial community composition source as described in right 1 is quickly analyzed and methods of risk assessment, feature exist
In the method for secondarily purified processing described in step (3) is：The secondary PCR product is used into 1X AMPure XP Beads
Primer dimer small fragment is removed, screening target fragment is purified using 0.7X+0.15X AMPure XP Beads, with 1.5% fine jade
Sepharose electrophoresis carries out purity detecting.
8. a kind of PM2.5 bacterial community composition source as described in right 1 is quickly analyzed and methods of risk assessment, feature exist
In it is the operation of PowerSoil DNA Isolation Kit kit that the DNA in extraction sample described in step (2), which is used,
Standard method.
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| CN110176275A (en) * | 2019-05-22 | 2019-08-27 | 中国药科大学 | The macro genomic data analysis method in oral cavity based on high-flux sequence |
| CN112735530A (en) * | 2021-01-22 | 2021-04-30 | 中国科学院北京基因组研究所(国家生物信息中心) | Method for tracing sample based on flora structure |
| CN114334003A (en) * | 2021-12-22 | 2022-04-12 | 中国水产科学研究院南海水产研究所 | Fermented golden pomfret deep learning quality discrimination method and system based on single molecule sequencing |
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| CN104568680A (en) * | 2015-01-14 | 2015-04-29 | 浙江大学 | Community monitoring method of microorganism carried by air particulate matters with multiple particle sizes |
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| CN106548439A (en) * | 2016-10-21 | 2017-03-29 | 常州市环境监测中心 | A kind of system for residential quarters Ecology Environment Evaluation |
| CN106929578A (en) * | 2017-03-06 | 2017-07-07 | 常州市环境监测中心 | The evaluation method of planktonic bacteria group in a kind of Taihu Lake water body |
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| CN110176275A (en) * | 2019-05-22 | 2019-08-27 | 中国药科大学 | The macro genomic data analysis method in oral cavity based on high-flux sequence |
| CN112735530A (en) * | 2021-01-22 | 2021-04-30 | 中国科学院北京基因组研究所(国家生物信息中心) | Method for tracing sample based on flora structure |
| CN114334003A (en) * | 2021-12-22 | 2022-04-12 | 中国水产科学研究院南海水产研究所 | Fermented golden pomfret deep learning quality discrimination method and system based on single molecule sequencing |
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