CN107810280A - Utilize impingement method while collection and the method for detection bacterium and virus - Google Patents
Utilize impingement method while collection and the method for detection bacterium and virus Download PDFInfo
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- CN107810280A CN107810280A CN201680034726.4A CN201680034726A CN107810280A CN 107810280 A CN107810280 A CN 107810280A CN 201680034726 A CN201680034726 A CN 201680034726A CN 107810280 A CN107810280 A CN 107810280A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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Abstract
The present invention relates to it is a kind of using impingement method simultaneously collect and the method for detection bacterium and virus, its object is to provide it is a kind of using impingement method and meanwhile collect and the method for detection bacterium and virus, it can quickly and easily collect bacterium and virus in air quickly.The present invention as being collected using laboratory and detection bacterium and viral method simultaneously, including：Injecting step, the air sampler TSA culture mediums for being coated with Host Strains being arranged in laboratory, and it is ejected into viral simultaneously in laboratory with another bacterium again, virus will be applied to the Host Strains of the TSA culture mediums as host, another bacterium is not the viral host again, and it forms bacterium colony in the TSA culture mediums for smearing virulent Host Strains and can distinguished；Microorganism-collecting step, the bacterium sprayed and virus are collected using air sampler, and generate sample；Incubation step, cultivate the sample using air sampler generation and so that bacterium colony is formed；Calculation procedure, after incubation step, calculate and bacterial multiplication is suppressed the quantity of plaque (plaque) that is formed by virus infection in the sample, and calculate in the sample by the propagation of collected bacterium and formed injection bacterium bacterium colony quantity, for collecting bacterium and virus in TSA culture mediums, bacterium and virus are collected in TSA culture mediums using air sampler.
Description
Technical field
Collected and the method for detection bacterium and virus, be related to a kind of using hitting simultaneously using impingement method the present invention relates to a kind of
Striking is collected simultaneously and the method for detection bacterium and virus, and it can be utilized in a kind of collection simultaneously of culture medium and detection air
Bacterium and virus.
Background technology
Recently, as atmosphere pollution becomes serious, various such as virus, Escherichia coli or moulds one are contained in room air
The pathogen of sample, and also uprised as the various content of organics of smell root.
Generally, for virus, plant virus, animal virus and bacterial virus (phagocytosis are divided into according to the host of parasitism
Body), and the virus moved in an atmosphere be 0.02~0.2 μm, size is very small, research get up it is highly difficult, and not only into
For damaged with variform to humans and animals disease the reason for, and also can economically be caused according to different situations
Huge infringement.
In the prior art, realized by the following method to collect virus as described above：So that virus infect in
Cell monolayer (cell monolayer) simultaneously manufactures viral solution, and the viral solution is put on into the solid by sterilization treatment
After on culture medium, the plaque number for being mixed in host's (host) fluid nutrient medium and being generated after rocking is calculated, so as to collection virus
Operation is realized by more subjobs, so having in terms of collection virus needs plenty of time and operation to become complicated and ask
Topic.
In addition, there are impingement method, ablution and filtration method etc. in the microbe sampling method used now, with regard to the shock
It is following method for the situation of method：Andersen sampler (Anderson Sieve Sampler) is by the pancreas containing 3% gelatin
Peptone phosphate nutrient solution is put into petri diss, then collects the virus being present in the particle contained by air, still,
Collected viral limited amount system and be not certain, the problem of so as to quantitative analysis method is not used as.
In addition, for the situation of ablution, there are as below methods：Viral concentration material as polyethylene glycol is put into
In impinger (impinger), air is aspirated with certain flow velocity, so as to collect the virus of the aerosol in air, still,
The virus being collected into viral concentration material is put into by the pipe of sterilizing by methods described, and after being filtered with filter paper, is put into
Formalin, then virus is fixed and collected, the problem of more subjobs is performed untill collection so having.
In addition, for the situation of filtration method so that gelatin filter dissolves at once on culture medium top, counts after incubation
Calculate formed bacterium colony (colony) and collect bacterium, and for virus so that gelatin filter is melted in physiology salt
Water or 0.1% peptone solution, using stirring after magnet makes it fully dissolve, by with the solution of membrane filter and host one
Rise and be applied to culture medium top, calculate the plaque to be formed (plaque) quantity after incubation and collect virus, held so as to have
The problem of row more subjobs.
In addition, at present, for wanting in an indoor collection virus and the situation of bacterium, with the following method：First,
A kind of bacterium (or virus) is ejected into interior, after the bacterium (or virus) of injection is collected, chamber is cleaned and (killed
Bacterium), another viral (or bacterium) interior will be ejected into again again, and described viral (or bacterium) is collected, so as to have
Following problem：In order in an indoor collection virus and bacterium, identical operation twice should at least be tested repeatedly, so needing very
Long acquisition time, and the washing and cleaning operation of chamber should be carried out individually, so needing the overall operation time grown very much.
In addition, in order to grasp degree of danger and exposure limit according to microbe species, it is necessary to indoors to logical under environment
The bacterium and virus for crossing air sampler are collected, but up to the present the method for collecting virus and bacterium simultaneously fails to
Find.
Look-ahead technique document Prior Art
(patent document 1) publication publication publication number spy 2003-0085829 (2003.110.07)
(patent document 2) publication publication publication number 10-2010-00584442 (2010.05.25)
The content of the invention
It is an object of the invention to provide it is a kind of using impingement method simultaneously collect and the method for detection bacterium and virus, its energy
It is enough quickly and easily to collect and detect quickly the bacterium in air and virus.
The present invention as being collected using laboratory and detection bacterium and viral method simultaneously, including：Injecting step,
TSA (the Tryptic Soy Agar of Host Strains will be coated with：Tryptose soya agar) culture medium be arranged on laboratory in
Air sampler, it is ejected into viral simultaneously in laboratory with another bacterium again, virus will be applied to the TSA culture mediums
Host Strains are as host, and another bacterium is not the viral host, and it is smearing the TSA cultures of virulent Host Strains
Bacterium colony is formed in base and can be distinguished；Microorganism-collecting step, the bacterium sprayed and virus are collected using air sampler, and
Generate sample；Incubation step, cultivate the sample using air sampler generation and so that bacterium colony is formed；Calculation procedure,
After incubation step, calculate and bacterial multiplication suppressed the quantity of plaque (plaque) that is formed by virus infection in the sample,
And calculate in the sample by the propagation of collected bacterium and formed injection bacterium bacterium colony (colony) quantity, just
For TSA culture mediums collect bacterium and virus, bacterium and virus are collected in TSA culture mediums using air sampler.
The present invention passes through one using being coated with the culture medium of host (host) by impingement method while collecting bacterium and virus
Individual test specimen can calculate the quantity and colony counts of plaque, and plaque formation has the virus being in contact with the bacterium colony of host, bacterium
Fall formed with the bacterium being in contact with culture medium.
The present invention can collect the bacterium being present in air and virus simultaneously with simple method, so as to rapidly grasp
Pathogenic microbes situation in air and movement is predicted, and then the counte-rplan for this can be made.
The present invention collects bacterium and virus using impingement method, thus with the existing microorganism using ablution and filtration method
Collection is compared, and it is simple to collect process.
The present invention is carried out in the laboratory based on actual inhabitation or living space, so can carry out relative to actual life
The accurate microorganism detection (collection) of environment living.
The present invention carries out microorganism-collecting in the laboratory based on actual inhabitation or living space, it is possible to is applied to
The benchmark test of air purifier.
Brief description of the drawings
Fig. 1 is the frame diagrammatic illustration for representing bacterium and collection virus process according to the present invention
Fig. 2 is the diagrammatic illustration for the concept for showing the air sampler according to the present invention
Fig. 3 is the photo diagrammatic illustration for showing result according to an embodiment of the invention
Fig. 4 is Fig. 3 " A " enlarged photograph diagrammatic illustration
Embodiment
Fig. 1 is to show the frame diagrammatic illustration according to the virus of the present invention and the collection process of bacterium, and Fig. 2 is shown according to this hair
The diagrammatic illustration of the concept of bright air sampler.
The present invention includes：Injecting step, air sampling of the germy TSA culture mediums in laboratory will be smeared
Device, and be ejected into viral simultaneously in laboratory with another bacterium again, virus will be applied to the bacterium of the TSA culture mediums
As host, and another bacterium is not the viral host, its shape in the TSA culture mediums for smearing virulent Host Strains
Into bacterium colony and can distinguish；
Microorganism-collecting step, the bacterium sprayed and virus are collected using air sampler, and generate sample；
Incubation step, cultivate the sample using air sampler generation and so that bacterium colony is formed；
Calculation procedure, after incubation step, calculate and bacterial multiplication is suppressed to be formed by virus infection in the sample
The quantity of plaque (plaque), and calculate the bacterium colony of the injection bacterium formed in the sample by the propagation of collected bacterium
(colony) quantity,
For collecting bacterium and virus from TSA culture mediums, collected using air sampler in TSA culture mediums bacterium and
Virus.
The injecting step sprays in the step in a laboratory simultaneously as by the virus of desired collection and bacterium, will
The TSA culture mediums for being coated with Host Strains are installed on air sampler in laboratory, are sprayed virus and bacterium using sprayer
Into laboratory.
The laboratory is provided with the 30m based on actual inhabitation or living space3To 120m3Closing space, spray it is micro-
Before biology, using air cleaning unit come cleaner assay room air, air cleaning unit is provided with what is carried out using uviol lamp
Sterilization and HEPA (High Efficiency Particle Air：Highly effective air) filter etc., and ceiling fan is operated, to examination
Test while room air is stirred and spray microorganism.
Now, with regard to sprayed virus, for bacterium, respectively in advance to every mL viruses (PFU, plaque forming
unit：Plaque forming unit) quantity and per mL bacterial numbers (CFU, colony forming unit：CFU) enter
Row is quantitative, then sprays, fluid nutrient medium now is distilled water or PBS (phosphate buffer saline：Phosphoric acid delays
Rush salt) solution.In addition, be set as that the amount sprayed in the case of the volume difference of room is also different, but with air sampler
The form that the plaque/clump count collected virus/bacterium and cultivated is no more than per culture medium 300CFU (PFU) is sprayed.It means that
Preferably, experimenter is provided to collect concentration in advance, and calculated concentration is sprayed in the form of being consistent with collecting amount.
Do not limit the manufacture for the TSA culture mediums for being coated with Host Strains for being used in the injecting step especially, but
It is that the TSA culture mediums for being coated with Host Strains can manufacture in the following way：Host Strains are incubated at liquid training in advance
After supporting base, take about 1mL and 5mL 0.7% agar (agar) to mix and be coated on 25mL TSA tops.The Host Strains
Clean work station progress is applied to, clean work station completely cuts off the possibility of external contamination using air curtain function.
Now, the feelings collected in the amount of the TSA culture mediums for being coated with Host Strains for more than 20mL and using impingement method
Under condition, it is preferable that make the measurer of TSA culture mediums for 25mL.When the amount of the culture medium is less than 20mL, according to the size of microorganism
And difference is shown on inertia force, so the relative step-down of collection efficiency meeting, therefore as virus, size hour, in order to carry
High collection efficiency, the amount of culture medium should be increased.Accordingly, most preferably, the measurer of the culture medium is for 25mL.
The virus and host bacteria refer to bacterial virus and belong to the bacterium of the host of the bacterial virus, not to it
Species is especially limited.For example, the bacterium can use staphylococcus aureus, Escherichia coli etc., and by institute
The virus that bacterium is stated as host can use SA11, T3, PHI-X174 etc..
Refer to the other bacteriums for the host for being not belonging to bacterial virus with the bacterium sprayed together with the virus, and not
Its species is especially limited.For example, the bacterium can use MRSE (Staphylococcus epi
Dermidis), serratia marcescens (Serratia Marcescens) etc..
The PHI-X174 is as being host (host) with Escherichia coli (Escherichia coli) and bred
The bacterial virus of 0.027 μm of size, it is the DNA bacteriophages for the coat protein (capsid) being configured to around nucleic acid.It is described big
The one kind of enterobacteria as the enteric bacteria for belonging to bacillus, substantially nonpathogenic bacteria, and inhabit human body and various animals
Enteral, be distributed widely in also by excreta in nature.It is negative in Gram's staining, and size is 2~4 μ
m。
The serratia marcescens (Serratia Marcescens) is led to as shaft-like gram negative enteric bacteria
Often there is pathogenicity to the respiratory apparatus of adult, the urinary tract and the stomach and intestine of children, and be galore present in environment, and wet
The bathroom propagation of gas weight, forms pink or Zhu Huangse film.Serratia marcescens is classified as BL1 (Biosafety
level：Bio-safety grade) grade a diameter of 0.4 μm of bacterium.
Now, the jet particle size of the sprayer and performance are illustrated in following [table 1], PHI-X174, large intestine bar
Bacterium (Escherichia coli), serratia marcescens (Serratia Marcescens) are such as shown in [table 2].
[table 1]
[table 2]
The microorganism-collecting step be used as in germy TSA culture mediums are smeared collect simultaneously using the bacterium as
The step of virus of host and other bacteriums sprayed together, after spraying microorganism (virus and bacterium) by injecting step,
So that ceiling fan stops, in order to realize room air and spray homogenizing for microorganism, after 10~30 minutes, being adopted using air
The impingement method that sample device is carried out, i.e. so that the virus and bacterium that are ejected into laboratory impinge upon the TSA cultures for being coated with Host Strains
Base, so as to generate sample.
In the injecting step and collection virus step, it is 23 ± 3 DEG C of temperature that the temperature and humidity conditions in laboratory, which perform,
And relative humidity 50 ± 5%.(but relative humidity suggestion is the humidity that bacteriophage will not be caused to inactivate.)
The incubation step is cultivated as the test specimen for causing the collection step by virus and bacterium to collect
Step, cultivated in the thermostat of 35 DEG C of aerobic conditions, after the culture, the sample inner virus that are cultured and thin
The plaque and colony counts of bacterium are embodied as falling into the scope of every 50~200PFU of culture medium (CFU), and with petri diss
The plaque and the form of the bacterium colony formed by bacterium that generation is formed by virus simultaneously are cultivated.
For the calculation procedure, in by plaque and bacterium colony of the culture to be present in petri diss, differentiation is bitten
The colony counts of spot and bacterium and calculating, plaque suppress the propagation of Host Strains to generate by virus, and the bacterium colony of bacterium enters
The hole (hole) of ram and it is collected and is bred on TSA culture mediums.
In other words, collected in germy TSA culture mediums are smeared using the bacterium as during the virus of host, according to
Whether virus breeds to hinder the propagation of the bacterium as host, and generates hole in the bacterium colony of bacterium, so calculating as above institute
State the plaque to be formed (plaque) quantity.
Increased in addition, calculate the bacterium (bacterium for being not belonging to the host of virus) sprayed with virus simultaneously by culture
The colony counts grown and formed.
While the present invention formed as described above collect and the method for detection bacterium and virus for, be coated with place
In the TSA of main bacterium, collected simultaneously using the bacterium as the virus of host and other bacteriums, so can be simple by the use of impingement method
And the collection of the two kinds of microorganisms separately performed in the prior art is performed quickly.
Hereinafter, following more specific detail is carried out to the present invention.
Embodiment 1
- experiment Host Strains：Escherichia coli (Escherichia coli)
- it is coated with the TSA culture mediums of Host Strains：25mL
- experiment virus：PHI-X174
- experiment bacterium：Serratia marcescens (Sarratia Marcescens)
Air sampler：MAS-100NT (Merck Products, nominal flow rate 100L/min, sampling volume:50L, head are straight
Footpath:10cm)
Laboratory：Volume 60m3, 23 ± 3 DEG C of temperature, relative humidity 50 ± 5%
Condition：For the manufacture of the culture medium for collection, power supply is operated more than 10 minutes, so as to from fully completely cutting off
Take out preprepared 1mL E. coli broths in the clean work station of the condition of external contamination, and with the 0.7% of 5mL
Agar mixes, and is coated on 25mL TSA culture mediums top, then make its be dried to it is sufficiently hard untill, be ready to.Will
Ready culture medium is installed on the air sampler backed off after random laboratory in laboratory.Before injection bacterium and virus, operating
Uviol lamp is sterilized, and (is filtered using HEPA filter units using air cleaning unit, utilize the apparatus of air conditioning
Carry out temperature and humidity regulation) realize cleaning.Afterwards so that ceiling fan operates about 10 minutes, while utilizes sprayer injection PHI-X174
With serratia marcescens (Sarratia Marcescens), and homogenized, then, turn off ceiling fan, 30 points are stagnated making its
Zhong Hou, it is collected using air sampler.
It is small that the test specimen being collected into as described above is carried out in thermostat at a temperature of 35 DEG C to 24 with aerobic condition
When cultivate, and observe according to PHI-X174 whether breed whether hinder the propagation of Escherichia coli so as to formed plaque, whether
The bacterium colony of pink is formed by the propagation of serratia marcescens (Sarratia Marcescens).
Fig. 3 is the photo diagrammatic illustration for representing the result for the embodiment of the present invention 1, and Fig. 4 is Fig. 3 " A " enlarged photograph example
Diagram, as phage-infect suppresses in host the propagation of host, it is observed that in the Escherichia coli for the yellow smeared
Layer generation plaque.Furthermore, it is possible to it was observed that, using impingement method culture medium upper collection to bacterium bred, and according to
The circular pink bacterium colony of hole (hole) shape generation.
The present invention is not limited to the specific preferred embodiment, certainly, the institute in without departing from claims
It is required that idea of the invention in the state of, if there is the technical staff of general knowledge in the technical field that the present invention belongs to,
Then anyone can carry out various deformation implementation, belong to the change of this identical in the scope described in claims.
Claims (3)
- Collected simultaneously using impingement method and the method for detection bacterium and virus 1. a kind of, it is collects using a laboratory simultaneously And the method for detection bacterium and virus, it is described that using impingement method, the feature of collection and the method for detection bacterium and virus exists simultaneously In, including：Injecting step, will be coated with Host Strains TSA culture mediums be arranged on laboratory in air sampler, and by virus and Another bacterium is ejected into laboratory simultaneously again, and virus will be applied to the Host Strains of the TSA culture mediums as host, and separately A kind of bacterium is not the viral host, and it forms bacterium colony in the TSA culture mediums for smearing virulent Host Strains and can Distinguish；Microorganism-collecting step, the bacterium sprayed and virus are collected using air sampler, and generate sample；Incubation step, cultivate the sample using air sampler generation and so that bacterium colony is formed；Calculation procedure, after incubation step, calculate and bacterial multiplication is suppressed the plaque that is formed by virus infection in the sample Quantity, and calculate in the sample by the propagation of collected bacterium and formed injection bacterium bacterium colony quantity,For collecting bacterium and virus in TSA culture mediums, bacterium and disease are collected in TSA culture mediums using air sampler Poison.
- 2. it is according to claim 1 using impingement method simultaneously collect and the method for detection bacterium and virus, it is characterised in thatThe TSA culture mediums for being coated with the Host Strains are to cultivate Host Strains in liquid medium within to take 1mL and 5mL afterwards 0.7% agar mixes and is coated on 25mLTSA tops and manufactures.
- 3. according to claim 1 or 2, using impingement method, collection and the method for detection bacterium and virus, its feature exist simultaneously In,The Host Strains are staphylococcus aureus or Escherichia coli,It is SA11 either T3 or PHI-X174 using the Host Strains as the virus of host,It is MRSE or serratia marcescens with the bacterium sprayed together with virus.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150085039A KR101694895B1 (en) | 2015-06-16 | 2015-06-16 | Method for simultaneous sampling and quantification of bacteria and host-based virus |
| KR10-2015-0085039 | 2015-06-16 | ||
| PCT/KR2016/006329 WO2016204500A1 (en) | 2015-06-16 | 2016-06-15 | Method for collecting and detecting bacteria and viruses simultaneously by means of impaction technique |
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| Publication Number | Publication Date |
|---|---|
| CN107810280A true CN107810280A (en) | 2018-03-16 |
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| CN201680034726.4A Pending CN107810280A (en) | 2015-06-16 | 2016-06-15 | Utilize impingement method while collection and the method for detection bacterium and virus |
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| Country | Link |
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| KR (1) | KR101694895B1 (en) |
| CN (1) | CN107810280A (en) |
| WO (1) | WO2016204500A1 (en) |
Cited By (1)
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|---|---|---|---|---|
| CN110734951A (en) * | 2019-09-05 | 2020-01-31 | 武汉东湖科创中试基地科技有限公司 | Detection method of virus removal rate of air purifier |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113029883B (en) * | 2021-03-24 | 2022-04-12 | 江苏瑞城检测技术有限公司 | Detection equipment for air disinfection effect |
| FR3132437A1 (en) * | 2022-02-08 | 2023-08-11 | Jean Angelidis | Test box for air and surface contamination by microorganisms, in particular bacteria and/or contaminating moulds/yeasts; Associated method of operation. |
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| KR20030085829A (en) | 2002-05-02 | 2003-11-07 | 조병철 | Method for Collecting Aerosol Viruses by Using Impinger |
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- 2015-06-16 KR KR1020150085039A patent/KR101694895B1/en active Active
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- 2016-06-15 WO PCT/KR2016/006329 patent/WO2016204500A1/en active Application Filing
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| CN2689216Y (en) * | 2003-06-19 | 2005-03-30 | 广东省职业病防治院 | Biological granular sampling and enriching systems |
| JP2012021829A (en) * | 2010-07-13 | 2012-02-02 | Ihi Corp | Virus detection unit and virus detection device |
| JP2013172665A (en) * | 2012-02-24 | 2013-09-05 | Azbil Corp | Method for grasping attaching characteristics of microorganism, and method for evaluating test environment of microorganisms |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110734951A (en) * | 2019-09-05 | 2020-01-31 | 武汉东湖科创中试基地科技有限公司 | Detection method of virus removal rate of air purifier |
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| Publication number | Publication date |
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| KR101694895B1 (en) | 2017-01-11 |
| KR20160148770A (en) | 2016-12-27 |
| WO2016204500A1 (en) | 2016-12-22 |
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