CN104962472A - Method for effectively enriching air medium microorganisms on basis of magnetic bead method - Google Patents
Method for effectively enriching air medium microorganisms on basis of magnetic bead method Download PDFInfo
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- CN104962472A CN104962472A CN201510395441.0A CN201510395441A CN104962472A CN 104962472 A CN104962472 A CN 104962472A CN 201510395441 A CN201510395441 A CN 201510395441A CN 104962472 A CN104962472 A CN 104962472A
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Abstract
The invention provides a method for effectively enriching air medium microorganisms on the basis of a magnetic bead method. The method comprises the following steps: placing a filter membrane in a to-be-detected environment, performing the sampling in an adsorption manner, and placing the filter membrane after the sampling into an extraction container; adding an acid buffer solution with pH value of 2.0 to 2.5, then oscillating for 5 to 10 minutes, and then carrying out the ultrasonic dispersion for 10 to 20 minutes; adding ferroferric oxide magnetic beads into the extraction container, and continuously ultrasonically dispersing for 10 to 30 seconds; taking another separation container, charging the acid buffer solution into the separation container, pouring the ultrasonically-dispersed solution into the separation container, oscillating, uniformly mixing, standing for 1 to 10 minutes, and then separating magnetic beads to obtain separated magnetic beads. By adopting the technical solution, the air medium microorganisms with high enrichment amount, high concentration and convenience in detection can be obtained on the premise of low cost, so that the problem of difficulty in tracking, enriching and quantifying in the monitoring and analysis process of air medium microorganisms can be effectively solved.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of based on paramagnetic particle method efficiently concentrating air dielectric method of microorganism.
Background technology
Since nineteen forty-three, the U.S. implemented " aerosol infection " plan, just start extensive concern microbial aerosol both at home and abroad.Bacterium in microbial aerosol comprises nonpathogenic bacteria, pathogenic bacterium and conditionality pathogenic bacterium, and they all cause human infection by airborne transmission to a certain extent, and even the pathogenic bacterium of minute quantity just directly can cause the infection of respiratory tract.Therefore, reinforcement is most important to controlling Human Health Risk with the biological study of the mankind's closely-related microfluidic aerosol.
At present, air microbe is commonly used detection method and can be divided into culture method and non-culture method two class.Culture-based method utilizes selective medium to carry out microorganism separation and Culture, and because air microbe is of a great variety, and air microbe Culturability is very low, less than 1%, makes air microbe information research be subject to great restriction.Based on the microbe genome DNA research of non-culture method in environmental sample, wherein, DGGE, TGGE, qPCR, T-RFLP and high throughput sequencing technologies are based on molecular biological conventional analytical techniques, by pcr amplification and molecular fingerprint technology, fast biological community structure compared and detect.But, research shows compared to the microbe genome DNA content in soil, ight soil and water, in air, content is lower, more be difficult to enrichment, the efficiency gathered is low, and the requirement of detection technique to microorganism is higher, gatherer process, treating processes and testing process is all needed to do strict control and just can obtain effective analytical results.
Summary of the invention
The technical problem of enrichment is difficult to for air dielectric microorganism, the invention provides a kind of based on paramagnetic particle method efficiently concentrating air dielectric method of microorganism, high enriching quantity, high density can be obtained under low cost prerequisite, be convenient to the air dielectric microorganism that detects, effectively solve in air dielectric microorganism monitoring and analytic process the problem that difficult trackings, difficult enrichment, difficulty are quantitative.
To this, the technical solution used in the present invention is:
A kind of based on paramagnetic particle method efficiently concentrating air dielectric method of microorganism, comprise the following steps:
Steps A: be placed in environment to be measured by filter membrane and carry out adsorption sampling, puts in extraction vessel by the filter membrane after sampling;
Step B: acidic buffer solution is joined in the extraction vessel of steps A, then vibrate 5 ~ 10min,
Step C: extraction vessel is put in ultrasonic disperse equipment and carries out ultrasonic disperse 10 ~ 20min;
Step D: add Z 250 magnetic bead in extraction vessel, continues ultrasonic disperse 10 ~ 30s;
Step e: separately get a separation container, add described acidic buffer solution, then the liquid in the extraction vessel after step D ultrasonic disperse is poured into wherein, vibration mixes rear standing 1 ~ 10min, then carries out Beads enrichment, obtains the magnetic bead be separated;
Wherein, the pH value of described acidic buffer solution is 2.0 ~ 2.5.
Contriver is by creationary experimental study, it is unexpected that discovery sour environment be more conducive to the enrichment of air dielectric microorganism, adopt this technical scheme, be conducive to the air dielectric microorganism obtaining high density, be convenient to detection, effectively solve difficult tracking, difficult enrichment, difficult quantitative problem in air dielectric microorganism monitoring and analytic process.
As a further improvement on the present invention, described acidic buffer solution is any one in citrate-phosphate disodium hydrogen damping fluid or PBS phosphate buffered saline buffer.
As a further improvement on the present invention, in step D, the particle diameter of described Z 250 magnetic bead is 100 ~ 400nm.
As a further improvement on the present invention, in step e, described Beads enrichment comprises: lived by magnet package with Parafilm, be positioned in above-mentioned beaker solution carry out stirring to adsorb magnetic bead as much as possible with tweezers after clamping; Separately get 50mL centrifuge tube sealed membrane is torn to put into centrifuge tube, add 30mL stroke-physiological saline solution, after sonic oscillation 40 ~ 60s, sealed membrane is pressed from both sides out, by centrifugal for residual solution 12min, rotating speed 5000r/min, after outwelling supernatant liquor, magnetic bead in pipe is transferred in clean centrifuge tube for subsequent use, obtain the magnetic bead be separated.
As a further improvement on the present invention, the concentration of described stroke-physiological saline solution is 0.9% physiological saline, and at 121 DEG C sterilizing 20min.
As a further improvement on the present invention, in step e, the method for described Beads enrichment is: be placed on magnetic frame by described separation container, carries out a Beads enrichment, sucks liquid, retains magnetic bead, obtains the magnetic bead be separated.
As a further improvement on the present invention, in step B, the rotating speed of the equipment of described vibration is 2800 ~ 3000r/min.
As a further improvement on the present invention, in step C, extraction vessel is placed on and is equipped with in the beaker of mixture of ice and water, ultrasonic 4min, pause 3min, repetitive operation three times.
As a further improvement on the present invention, the amount of the acidic buffer solution added in step e is not less than the amount of the acidic buffer solution added in step B.
Preferred a kind of based on paramagnetic particle method efficiently concentrating air dielectric method of microorganism further, comprise the steps:
(1) shred sampling membrane and be placed in beaker, be positioned in 50mL centrifuge tube with tweezers careful tear-off filter membrane front adsorption layer;
(2) acidic buffer solution 40mL is added;
(3) centrifuge tube is positioned over vibrator, regulates vibrator rotating speed to 2800 ~ 3000r/min, vibration 5 ~ 10min;
(4) above-mentioned centrifuge tube is positioned over is equipped with in the 250mL beaker of mixture of ice and water, ultrasonic 4min, pause 3min, operate continuously three times;
(5) in centrifuge tube, add the Z 250 of 25mg particle diameter 200nm, ultrasonic 20s makes it be uniformly dispersed;
(6) separately get 500mL beaker and add the identical acidic buffer solution of 50mL, after the liquid in centrifuge tube is poured in beaker, vibration mix rear standing 3min;
(7) with Parafilm, magnet package is lived, be positioned over after clamping with tweezers in above-mentioned beaker solution and carry out stirring until adsorb maximum magnetic bead;
(8) separately get 50mL centrifuge tube sealed membrane is torn to put into centrifuge tube, add 30mL stroke-physiological saline solution, after sonic oscillation 40 ~ 60s, sealed membrane is pressed from both sides out;
(9) by centrifugal for residual solution 12min, rotating speed 5000r/min, after outwelling supernatant liquor, magnetic bead in pipe is transferred in clean centrifuge tube for subsequent use.
Wherein, the pH value of described acidic buffer solution is 2.0 ~ 2.5, and described acidic buffer solution is any one in citrate-phosphate disodium hydrogen damping fluid or PBS phosphate buffered saline buffer.
Present invention also offers a kind of method extracting air dielectric microbe genome DNA based on paramagnetic particle method, adopt and obtain based on paramagnetic particle method efficiently concentrating air dielectric method of microorganism collection microorganism the magnetic bead having adsorbed microorganism as above, then the microorganism of magnetic bead absorption is carried out to the extraction of genomic dna.
Compared with prior art, adopt technical scheme of the present invention, in acid condition can efficiently concentrating air dielectric microorganism, can obtain under low cost prerequisite high-recovery, high density, be convenient to detect air dielectric microorganism, greatly improve the detectability of air dielectric microorganism, effectively solve difficult tracking, difficult enrichment, difficult quantitative problem in air dielectric microorganism monitoring and analytic process; Be applicable to accurately, research and application air dielectric microorganism properties study quickly and easily.
Employing the present invention is based on magnetic bead technology, in acid condition can efficiently concentrating air dielectric microorganism, can be used for the detection paper etc. of real-time PCR, fluorescent bacteria dyeing, the separation of feature bacterium and enlarged culturing, feature bacterium.Wherein important application is for extracting high density, high-quality air dielectric microbe genome DNA, multianalysis air dielectric microbial species group character, and for analyzing air dielectric microorganism characterizing gene, target fragment etc., all provide strong substrate guarantee based on DNA Study on product, the research border of air dielectric microorganism is extended to trace by the enrichment effect of the method, substantially increases the detectability of air dielectric microorganism.
Accompanying drawing explanation
Fig. 1 is the process schematic of an embodiment of the present invention based on paramagnetic particle method efficiently concentrating air dielectric method of microorganism;
Fig. 2 is the spectrophotofluorimetry canonical plotting of an embodiment of the present invention;
Fig. 3 is the quantitative verification smog bin device schematic diagram of the another kind of embodiment of the present invention;
Fig. 4 is the fluorescent dye counting diagram of the another kind of embodiment of the present invention.
Embodiment
Below in conjunction with accompanying drawing, preferably embodiment of the present invention is described in further detail.
If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.
Embodiment 1:
As shown in Figure 1, a kind of based on paramagnetic particle method efficiently concentrating air dielectric method of microorganism, specifically comprise the following steps:
A: air dielectric microorganism gathers
(1) sample devices: large discharge TSP (PM10) sampling thief that Qingdao LaoShan Application Technology Research Institute produces, gathers air sample and collects on 230mm × 180mm glass fibre membrane filter membrane;
(2) sample duration: 10h, and maximum collection flow is 1.05m
3/ h;
(3) sampling position: chicken raising factory.
B: adopt paramagnetic particle method enriched air medium microorganism
(1) shred sampling membrane and be placed in beaker, get 1/4 size filter membrane and be positioned in 3 50mL centrifuge tubes respectively with tweezers careful tear-off filter membrane front adsorption layer;
(2) in 3 centrifuge tubes, add citrate-phosphate disodium hydrogen buffered soln, adjust ph is 2.2,7.2,10.0 respectively, is acidity, neutrality, alkalescence;
(3) centrifuge tube is positioned over vibrator, regulates vibrator speed to 3000r/min, vibration 10min;
(4) above-mentioned centrifuge tube is positioned over is equipped with in the 250mL beaker of mixture of ice and water, ultrasonic 4min, pause 3min, operate continuously three times;
(5) in centrifuge tube, adding 25mg particle diameter respectively is 200nm nano ferriferrous oxide, and ultrasonic 20s makes it be uniformly dispersed;
(6) separately get the citrate-phosphate disodium hydrogen buffered soln that 500mL beaker adds 50mL same pH, after the liquid in centrifuge tube is poured in corresponding beaker, vibration mix rear standing 3min;
(7) with Parafilm, magnet package is lived, be positioned over after clamping with tweezers in above-mentioned beaker solution and carry out stirring until adsorb maximum magnetic bead;
(8) separately get 50mL centrifuge tube sealed membrane is torn to put into centrifuge tube, add 30mL stroke-physiological saline solution, after sonic oscillation 40 ~ 60s, sealed membrane is pressed from both sides out;
(9) centrifugal 12min, rotating speed 5000r/min, transfer in clean centrifuge tube for subsequent use by magnetic bead in pipe after outwelling supernatant liquor;
(10) the Lysing Matix E pipe centrifugal middle magnetic bead being transferred to MP FastDNA Spin Kit for Soil carries out DNA extraction.
C: interpretation of result
(1) air dielectric microorganism organic efficiency
The air dielectric microbe genome DNA of paramagnetic particle method enrichment is carried out fluorescent dyeing, and adopt fluorescent spectrophotometer assay, can obtain typical curve as shown in Figure 2, DNA concentration is as shown in table 1, and wherein control group extracts DNA for not adopting technical solution of the present invention.
The air dielectric microbe genome DNA concentration of table 1 paramagnetic particle method enrichment
Result shows: under acidic solution condition, adopts paramagnetic particle method enriched air medium microorganism to have the advantage of high-recovery, high density.
(2) extracting method quality evaluation
Adopt molecular biology software high-flux sequence result to be corrected errors in printing, after the comparison of SILVA database, obtain different sample operation separating unit (OTU), and then obtain the similarity angle of cut under different population rank between sample, as shown in table 2.
Table 2 different sample room similarity angle
Note: sample number into spectrum respective operations is: 1-contrasts, and 2-is acid, and 3-is neutral, 4-alkalescence; About 42 °, the chance sample angle of cut.
Result shows: under acidic solution condition, adopt paramagnetic particle method enriched air medium microorganism all to have similarity angle minimum under distance threshold is 0.03 or 0.06 level and largely on be less than 42 °, the chance sample angle of cut, illustrate that this method can zero deflection ground enriched air medium microorganism.
D. conclusion
Adopt a kind of based on paramagnetic particle method efficiently concentrating air dielectric method of microorganism in the present embodiment, from test result analysis, the present invention can efficiently concentrating air dielectric microorganism in acid condition, improve the detectability of air dielectric microorganism, low cost obtains the air dielectric microorganism of high density, is therefore applicable to accurately, research and application air dielectric microorganism properties study quickly and easily.
Embodiment 2:
A kind of based on paramagnetic particle method efficiently concentrating air dielectric method of microorganism, specifically comprise the following steps:
One, air dielectric microorganism gathers
(1) sample devices: large discharge TSP (PM10) sampling thief that Qingdao LaoShan Application Technology Research Institute produces, gathers air sample and collects on 230mm × 180mm glass fibre membrane filter membrane; The quantitative verification smog bin device schematic diagram adopted as shown in Figure 3;
(2) sample duration: 10h, and maximum collection flow is 1.05m
3/ h;
(3) sampling position: 10m
3controllable ration verifying smog chamber, setting bacterial concentration is 10
6cell/m
3;
(4) enriching method: by paramagnetic particle method and damping fluid dispersion method, sampling film is processed respectively.
Two, paramagnetic particle method enriched air medium microorganism
(1) shred sampling membrane and be placed in beaker, get 1/4 size filter membrane and be positioned in 50mL centrifuge tube with tweezers careful tear-off filter membrane front adsorption layer;
(2) in centrifuge tube, add citrate-phosphate disodium hydrogen buffered soln, adjust ph is 2.2, is acidic conditions;
(3) centrifuge tube is positioned over vibrator, regulates vibrator speed to 3000r/min, vibration 10min;
(4) above-mentioned centrifuge tube is positioned over is equipped with in the 250mL beaker of mixture of ice and water, ultrasonic 4min, pause 3min, operate continuously three times;
(5) in centrifuge tube, adding 25mg particle diameter is 200nm nano ferriferrous oxide, and ultrasonic 20s makes it be uniformly dispersed;
(6) separately get the citrate-phosphate disodium hydrogen buffered soln that 500mL beaker adds 50mL same pH, after the liquid in centrifuge tube is poured in corresponding beaker, vibration mix rear standing 3min;
(7) with Parafilm, magnet package is lived, be positioned over after clamping with tweezers in above-mentioned beaker solution and carry out stirring until adsorb maximum magnetic bead;
(8) separately get 50mL centrifuge tube sealed membrane is torn to put into centrifuge tube, add 30mL stroke-physiological saline solution, after sonic oscillation 40 ~ 60s, sealed membrane is pressed from both sides out;
(9) centrifugal 12min, rotating speed 5000r/min, transfer in clean centrifuge tube for subsequent use by magnetic bead in pipe after outwelling supernatant liquor;
(10) extraction sample is carried out DAPI (4,6-diamidino-2-phenylindone) photofluorometer total count, result as shown in Figure 4.
Three, interpretation of result
Respectively to adopting the sample of paramagnetic particle method and the enrichment of damping fluid dispersion method to carry out DAPI fluorescence counting, the bacterial number obtained in different methods acquisition air dielectric is as shown in table 3:
Table 3 air dielectric bacterial concentration DAPI coloration result
| Paramagnetic particle method | Damping fluid dispersion method | Theoretical value | |
| Concentration (Cell/L) | 790 | 462 | 1000 |
Result shows: under acidic solution condition, adopts paramagnetic particle method enriched air medium microbe population much larger than damping fluid dispersion method, and relatively theoretical value.Therefore, adopt a kind of based on paramagnetic particle method efficiently concentrating air dielectric method of microorganism in the present embodiment, can in acid condition can efficiently concentrating air dielectric microorganism.
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple deduction or replace can also be made, all should be considered as belonging to protection scope of the present invention.
Claims (10)
1., based on a paramagnetic particle method efficiently concentrating air dielectric method of microorganism, it is characterized in that, comprise the following steps:
Steps A: be placed in environment to be measured by filter membrane and carry out adsorption sampling, puts in extraction vessel by the filter membrane after sampling;
Step B: acidic buffer solution is joined in the extraction vessel of steps A, then vibrate 5 ~ 10min,
Step C: extraction vessel is put in ultrasonic disperse equipment and carries out ultrasonic disperse 10 ~ 20min;
Step D: add Z 250 magnetic bead in extraction vessel, continues ultrasonic disperse 10 ~ 30s;
Step e: separately get a separation container, add described acidic buffer solution, then the liquid in the extraction vessel after step D ultrasonic disperse is poured into wherein, vibration mixes rear standing 1 ~ 10min, then carries out Beads enrichment, obtains the magnetic bead be separated;
Wherein, the pH value of described acidic buffer solution is 2.0 ~ 2.5.
2. according to claim 1 based on paramagnetic particle method efficiently concentrating air dielectric method of microorganism, it is characterized in that: described acidic buffer solution is any one in citrate-phosphate disodium hydrogen damping fluid or PBS phosphate buffered saline buffer.
3. according to claim 1ly it is characterized in that: in step D based on paramagnetic particle method efficiently concentrating air dielectric method of microorganism, the particle diameter of described Z 250 magnetic bead is 100 ~ 400nm.
4. according to claim 1 based on paramagnetic particle method efficiently concentrating air dielectric method of microorganism, it is characterized in that: in step e, described Beads enrichment comprises: lived by magnet package with Parafilm, be positioned in above-mentioned beaker solution carry out stirring to adsorb magnetic bead as much as possible with tweezers after clamping; Separately get 50mL centrifuge tube sealed membrane is torn to put into centrifuge tube, add 30mL stroke-physiological saline solution, after sonic oscillation 40 ~ 60s, sealed membrane is pressed from both sides out, by centrifugal for residual solution 12min, rotating speed 5000r/min, after outwelling supernatant liquor, magnetic bead in pipe is transferred in clean centrifuge tube for subsequent use, obtain the magnetic bead be separated.
5. according to claim 4 based on paramagnetic particle method efficiently concentrating air dielectric method of microorganism, it is characterized in that: the concentration of described stroke-physiological saline solution is 0.9% physiological saline, and at 121 DEG C sterilizing 20min.
6. according to claim 1 ~ 3 any one based on paramagnetic particle method efficiently concentrating air dielectric method of microorganism, it is characterized in that: in step e, the method of described Beads enrichment is: be placed on magnetic frame by described separation container, carry out a Beads enrichment, suck liquid, retain magnetic bead, obtain the magnetic bead be separated.
7. according to claim 1 ~ 5 any one based on paramagnetic particle method efficiently concentrating air dielectric method of microorganism, it is characterized in that: in step B, the rotating speed of described oscillator device is 2800 ~ 3000r/min.
8. according to claim 1 ~ 5 any one based on paramagnetic particle method efficiently concentrating air dielectric method of microorganism, it is characterized in that: in step C, extraction vessel be placed on and be equipped with in the beaker of mixture of ice and water, ultrasonic 4min, pause 3min, repetitive operation three times.
9. according to claim 1 ~ 5 any one based on paramagnetic particle method efficiently concentrating air dielectric method of microorganism, it is characterized in that: the amount of the acidic buffer solution added in step e is not less than the amount of the acidic buffer solution added in step B.
10. one kind is extracted the method for air dielectric microbe genome DNA based on paramagnetic particle method, it is characterized in that: adopt the magnetic bead obtaining having adsorbed microorganism based on paramagnetic particle method efficiently concentrating air dielectric method of microorganism collection microorganism as described in claim 1 ~ 9 any one, then the microorganism of magnetic bead absorption is carried out to the extraction of genomic dna.
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| CN105784441A (en) * | 2016-03-15 | 2016-07-20 | 华东理工大学 | Magnetic membrane enrichment and separation device and using method thereof in solid phase spectrum detection |
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| CN114032173B (en) * | 2022-01-11 | 2022-03-15 | 至美时代生物智能科技(北京)有限公司 | Closed air sampling bottle |
| CN115845806A (en) * | 2022-11-23 | 2023-03-28 | 杭州富集生物科技有限公司 | Adsorption particle product for enriching liquid phase/gas phase sample biological particles and preparation method thereof |
| CN115845806B (en) * | 2022-11-23 | 2023-09-29 | 杭州富集生物科技有限公司 | Adsorption particle product for enriching liquid phase/gas phase sample biological particles and preparation method thereof |
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