CN105462956A - Sample pretreatment method for extracting microorganism total DNA in biological aerosol - Google Patents
Sample pretreatment method for extracting microorganism total DNA in biological aerosol Download PDFInfo
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- CN105462956A CN105462956A CN201410498133.6A CN201410498133A CN105462956A CN 105462956 A CN105462956 A CN 105462956A CN 201410498133 A CN201410498133 A CN 201410498133A CN 105462956 A CN105462956 A CN 105462956A
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Abstract
The invention relates to a sample pretreatment method for extracting microorganism total DNA in biological aerosol. The method includes the steps that a filter membrane is adopted to collect air microorganisms, 0.8% normal saline is used as a buffer solution, the microorganisms are fully separated under the oscillation action of a shaking table supplemented by glass beads, and membrane stripping liquid is filtered through a 25-mm filter membrane, so that the microorganisms are concentrated on the surface of a membrane with the area of 1.96 cm<2> and oscillated, walls of the microorganisms, with thick cell walls, on the surface of the membrane are broken preliminarily, and follow-up DNA extraction is facilitated. The sample pretreatment method is easy to operate, harmless to the human body and capable of effectively increasing the DNA extraction amount.
Description
Technical field
The present invention relates to microbe research field in air aerosol, specifically, relate to a kind of sample-pretreating method extracting microorganism total DNA in bioaerosol.
Background technology
Bioaerosol be the lived aerosol particles of tool (comprising the microorganism particles such as bacterium, fungi, virus) and active particle (pollen, spore etc.) and by have the body of vital activity be discharged into plasmid etc. in air.Because bio-aerosol concentration is low in air, first needs to collect biomone in air to its research, make air microbe be able to enrichment.Current collection method mainly contains three kinds: (1) utilizes impact sampler to make bioaerosol strike selective medium surface, for subsequent analysis after target gas microorganism growth; (2) utilize sampling thief to absorb bioaerosol in air in damping fluid, use membrane filtration damping fluid, make microorganism adsorption in filter membrane surface, for subsequent analysis; (3) instrument is utilized by the bioaerosol direct filtration in air to filter membrane surface.(1) kind method is mainly used in Bacterial diversity in collection of biological aerosol, due to Bacterial diversity in air only account for whole microorganism less than 10, so most investigator adopts latter two sample collecting mode to gather the whole microorganisms in air at present, and utilize molecular biology method to analyze the air microbe that filter membrane is collected, thus more comprehensively react the situation of actual air microorganism.
At present, the bottleneck of microbe research whole in air is to effective extraction of bioaerosol sample total DNA.Utilize filter membrane to collect air microbe in prior art, the pre-treating process of DNA extraction has following several usually: a kind of is directly using adsorbing the filter membrane of bioaerosol as sample, carries out the operation of extracting DNA to whole film; Another kind is first separated by the air microbe on film using damping fluid as medium, is filtered on the less film of area, and long-pending less film carries out the operation of extracting DNA over there.But first method, because filter membrane area is comparatively large, in the process of DNA extraction, causes DNA loss amount larger; Although second method is lost less in leaching process, the damping fluid used in de-filming process, as 0.1%TritonX-100 damping fluid or the phosphate buffered saline buffer containing 0.03% tween 20, organism containing harmful HUMAN HEALTH, and damping fluid layoutprocedure is complicated, complex steps.Therefore, a kind of pre-treating process of the DNA extraction harmless and simple to operate to human non-toxic is badly in need of.
Summary of the invention
The object of the present invention is to provide a kind of simple to operate, harmful to human non-toxic, and effectively can improve the sample-pretreating method of microorganism total DNA in the bioaerosol of DNA extraction amount.
A kind of sample-pretreating method extracting microorganism total DNA in bioaerosol provided by the invention, comprises the following steps:
(1) sample collection: the microorganism in air ambient in bioaerosol collected by sampling thief, is filtered on fibrous filter membrane;
(2) sample demoulding process: be laid in sterile chamber by smooth for the fibrous filter membrane of absorption bioaerosol, particulate absorbent, facing to above container, adds sterile glass beads and stroke-physiological saline solution, oscillation treatment on constant-temperature table;
(3) film process crossed by sample: the elutriant after step (2) being processed, adopt micropore cellulose acetate membrane filtration, again micropore cellulose acetate filter membrane is put into sterile chamber, oscillation treatment on the oscillator, the solids after process is used for the extraction of STb gene.
In method of the present invention, in step (1), sampling thief is TSP air total particle sampling thief.
Sampling time is 24-48h.The preferred sampling time is 24h.
Wherein, the fibrous filter membrane of step (1) is silica fiber filter membrane or glass fiber filter, and diameter is 48-90mm.Preferred silica fiber filter membrane, preferred diameter is 90mm.
After step (1) sample collecting terminates, the filter membrane of bioaerosol can be kept at-80 DEG C of refrigerators and also can be directly used in subsequent operations.
In the inventive method, the sterile glass beads diameter that step (2) adds is 4-6mm, and quantity is between 40-60.
Wherein, the stroke-physiological saline solution concentration that step (2) adds is 0.8%-0.9%, and add-on is with complete wetting fibrous filter membrane and have residue to be as the criterion.
Wherein, step (2) constant-temperature table working conditions is 100-120rpm process 10-12h.
In the inventive method, step (3) micropore cellulose acetate filter membrane diameter is 10-25mm, and aperture is 0.22-0.45 μm.Preferred microporous cellulose acetate filter membrane diameter is 25mm, and aperture is 0.22 μm.
Wherein, the oscillation treatment condition of step (3) is 6.0-8.0m/s, process 40-60s.
The present invention is on the basis to existing pre-treating process, improve: utilize filter membrane to collect air microbe, complicated buffered soln and organic reagent is instead of as damping fluid using physiological saline, under shaking table oscillation action, and granulated glass sphere collision in addition, microorganism is separated from filter membrane fully, improves demoulding efficiency, simplify operation steps, improve the security of experiment.Liquid parting is being filtered on the less film of area, diameter is selected to be the membrane filtration of 25mm in one embodiment of the present of invention, microbial concentration is made to be convenient to follow-up oscillation treatment in comparatively small area film surface (1.96cm2), contribute to the abundant broken wall of microorganism that film surface attachment cell walls is thicker, as gram-positive microorganism, improve the efficiency of follow-up DNA extraction.The inventive method is not only simple to operate, harmful to human non-toxic, and can effectively improve DNA extraction amount.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
Sample collecting: gather air bioaerosol sample in Beijing City Agriculture and Forestry Institute.Sampling instrument is air total particulate collector (Qingdao Lao answers Environmental Protection Technology Co., Ltd), and the sampling time is 48h.By sampling after microbe filter to diameter be on the silica fiber filter membrane of 90mm, then silica fiber filter membrane is divided into 3 parts.
1st part, according to the method for the application, be laid in smooth for the fibrous filter membrane of absorption bioaerosol in 50ml sterile centrifugation tube along tube wall, the side filter membrane not adsorbing microorganism againsts centrifugal tube wall, particulate absorbent faces up, and guarantees that granulated glass sphere directly contacts absorption surface.Adding diameter is the sterile glass beads 40 of 5mm and the stroke-physiological saline solution of 0.8%, and the add-on of stroke-physiological saline solution is with complete wetting fibrous filter membrane and have residue to be as the criterion.Centrifuge tube is fixed on constant-temperature table, 100rpm process 12h.Need in operation to guarantee fully to contact with air microbe adsorption plane in the process that granulated glass sphere rotates at shaking table.Oscillation treatment on constant-temperature table; Adopt the filter membrane (diameter is 25mm, and aperture is the micropore cellulose acetate filter membrane of 0.22 μm) that area is less, the elutriant after above-mentioned process is filtered.Then by above-mentioned millipore filtration, the centrifuge tube of 1.5ml is put into, on the oscillator with the speed of 6.0m/s, process 50s.Solids after process is adopted DNA extraction kit (MO-BIO
dNAIsolationKit) DNA extraction is carried out.
2nd part, without process, directly DNA extraction operation is carried out to the filter membrane of absorption bioaerosol;
3rd part, the method reported is adopted to carry out pre-treatment.Be laid in the sterilized petri dishes of 90mm by smooth for the fibrous filter membrane of absorption bioaerosol, add demoulding damping fluid, use 0.1%TritonX-100.Be fixed on by plate on constant-temperature table, by the elutriant after above-mentioned process after process, employing diameter is 25mm, and aperture is the micropore cellulose acetate membrane filtration of 0.22 μm.Directly DNA extraction operation is carried out to the filter membrane of absorption bioaerosol.
(ThermoNANODROP2000 after testing, USA), through the filter membrane of pre-treatment of the present invention, namely the 1st part of DNA concentration extracted is 40.5ng/ μ l, without the filter membrane of pre-treatment, the DNA concentration of the 2nd part and the 3rd part extraction is difference 18.5ng/ μ l and 30.7ng/ μ l.
Embodiment 2
Sample collecting: in poulty house, Beijing, gathers air bioaerosol sample in hen house.Sampling instrument is air total particulate collector (Qingdao Lao answers Environmental Protection Technology Co., Ltd), and the sampling time is 24h.
1st part, according to the method for the application, be laid in 50ml sterile centrifugation tube along tube wall is smooth by the silica fiber filter membrane of absorption bioaerosol, the side filter membrane not adsorbing microorganism againsts centrifugal tube wall, particulate absorbent faces up, and guarantees that granulated glass sphere directly contacts absorption surface., adding diameter is the sterile glass beads 50 of 5mm and the stroke-physiological saline solution of 0.8%, and the add-on of stroke-physiological saline solution is with complete wetting silica fiber filter membrane and have residue to be as the criterion.Centrifuge tube is fixed on constant-temperature table, 100rpm process 12h.Need in operation to guarantee fully to contact with air microbe adsorption plane in the process that granulated glass sphere rotates at shaking table.Oscillation treatment on constant-temperature table; Adopt the filter membrane (diameter is 25mm, and aperture is the micropore cellulose acetate filter membrane of 0.22 μm) that area is less, the elutriant after above-mentioned process is filtered.Then by above-mentioned millipore filtration, the centrifuge tube of 1.5ml is put into, on the oscillator with the speed of 6.0m/s, process 45s.Solids after process is adopted DNA extraction kit (MO-BIO
dNAIsolationKit) DNA extraction is carried out.
2nd part, without process, directly DNA extraction operation is carried out to the silica fiber filter membrane of absorption bioaerosol;
3rd part, the method reported is adopted to carry out pre-treatment.Be laid in the sterilized petri dishes of 90mm by smooth for the fibrous filter membrane of absorption bioaerosol, add demoulding damping fluid, use 0.1%TritonX-100.Be fixed on by plate on constant-temperature table, by the elutriant after above-mentioned process after process, employing diameter is 25mm, and aperture is the micropore cellulose acetate membrane filtration of 0.22 μm.Finally, directly DNA extraction operation is carried out to the filter membrane of absorption bioaerosol.
After testing (ThermoNANODROP2000, USA).1st part, the DNA concentration of the 2nd part and the 3rd part film extraction is respectively 65.7ng/ μ l, 40.6ng/ μ l and 49.2ng/ μ l.
Interpretation of result
The concentration of the above-mentioned bioaerosol microbial DNA adopting different pre-treating processs to extract in two kinds of environment of comparative analysis, result display is through the fibrous filter membrane of pre-treating process process of the present invention, and the concentration of DNA extraction is higher than the fibrous filter membrane without pre-treatment; Compare with an organic solvent as the treatment process of liquid parting, the concentration using the inventive method to extract DNA higher than in prior art with an organic solvent as the concentration of liquid parting treatment process, and liquid parting of the present invention configuration is simple, use safety; Meanwhile, the inventive method goes for the pre-treatment of the filter membrane of the absorption bioaerosol that varying environment gathers.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. extract a sample-pretreating method for microorganism total DNA in bioaerosol, it is characterized in that, comprise the following steps:
(1) sample collection: the microorganism in air ambient in bioaerosol collected by sampling thief, is filtered on fibrous filter membrane;
(2) sample demoulding process: be laid in sterile chamber by smooth for the fibrous filter membrane of absorption bioaerosol, particulate absorbent, facing to above container, adds sterile glass beads and stroke-physiological saline solution, oscillation treatment on constant-temperature table;
(3) film process crossed by sample: the elutriant after step (2) being processed, adopt micropore cellulose acetate membrane filtration, again micropore cellulose acetate filter membrane is put into sterile chamber, oscillation treatment on the oscillator, the solids after process is used for the extraction of STb gene.
2. sample-pretreating method as claimed in claim 1, is characterized in that, in step (1), sampling thief is air total particle sampling thief.
3. sample-pretreating method as claimed in claim 1, it is characterized in that, the fibrous filter membrane of step (1) is silica fiber filter membrane or glass fiber filter, and diameter is 48-90mm.
4. sample-pretreating method as claimed in claim 1, it is characterized in that, step (1) sampling time is 24-48h.
5. sample-pretreating method as claimed in claim 1, is characterized in that, after step (1) sample collecting terminates, the filter membrane of bioaerosol can be kept at-80 DEG C of refrigerators.
6. sample-pretreating method as claimed in claim 1, it is characterized in that, the sterile glass beads diameter that step (2) adds is 4-6mm, and quantity is between 40-60.
7. sample-pretreating method as claimed in claim 1, it is characterized in that, the stroke-physiological saline solution concentration that step (2) adds is 0.8%-0.9%, and add-on is with complete wetting fibrous filter membrane and have residue to be as the criterion.
8. sample-pretreating method as claimed in claim 1, it is characterized in that, step (2) constant-temperature table working conditions is 100-120rpm process 10-12h.
9. sample-pretreating method as claimed in claim 1, it is characterized in that, step (3) micropore cellulose acetate filter membrane diameter is 10-25mm, and aperture is 0.22-0.45 μm.
10. sample-pretreating method as claimed in claim 1, it is characterized in that, the oscillation treatment condition of step (3) is 6.0-8.0m/s, process 40-60s.
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| CN105200039A (en) * | 2015-10-21 | 2015-12-30 | 中国科学院生态环境研究中心 | Extraction method of atmospheric particulate matter genome DNA |
| CN107384756A (en) * | 2017-08-01 | 2017-11-24 | 云南中烟工业有限责任公司 | A kind of collection device and method of essence spice for cigarette contaminating microorganisms |
| CN113005027A (en) * | 2020-11-25 | 2021-06-22 | 中国矿业大学 | Device and method for measuring distribution of microbial particles in air |
| CN113817608A (en) * | 2021-10-18 | 2021-12-21 | 福建中烟工业有限责任公司 | Separation and enrichment method of microorganisms on surface of alcoholized tobacco leaf |
| CN113881665A (en) * | 2021-11-19 | 2022-01-04 | 北京工业大学 | Microbial aerosol DNA extraction method |
| CN116223138A (en) * | 2022-12-07 | 2023-06-06 | 哈尔滨工业大学 | Pretreatment method for community diversity detection of microbial aerosol |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105200039A (en) * | 2015-10-21 | 2015-12-30 | 中国科学院生态环境研究中心 | Extraction method of atmospheric particulate matter genome DNA |
| CN105200039B (en) * | 2015-10-21 | 2019-01-25 | 中国科学院生态环境研究中心 | The extracting method of Fine Particles genomic DNA |
| CN107384756A (en) * | 2017-08-01 | 2017-11-24 | 云南中烟工业有限责任公司 | A kind of collection device and method of essence spice for cigarette contaminating microorganisms |
| CN107384756B (en) * | 2017-08-01 | 2023-09-15 | 云南中烟工业有限责任公司 | Device and method for collecting tobacco essence and spice polluted microorganisms |
| CN113005027A (en) * | 2020-11-25 | 2021-06-22 | 中国矿业大学 | Device and method for measuring distribution of microbial particles in air |
| CN113817608A (en) * | 2021-10-18 | 2021-12-21 | 福建中烟工业有限责任公司 | Separation and enrichment method of microorganisms on surface of alcoholized tobacco leaf |
| CN113881665A (en) * | 2021-11-19 | 2022-01-04 | 北京工业大学 | Microbial aerosol DNA extraction method |
| CN116223138A (en) * | 2022-12-07 | 2023-06-06 | 哈尔滨工业大学 | Pretreatment method for community diversity detection of microbial aerosol |
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