CN105087797A - Method for determining microorganism varieties and pathogenic microorganism pollution conditions in air - Google Patents
Method for determining microorganism varieties and pathogenic microorganism pollution conditions in air Download PDFInfo
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- CN105087797A CN105087797A CN201510514716.8A CN201510514716A CN105087797A CN 105087797 A CN105087797 A CN 105087797A CN 201510514716 A CN201510514716 A CN 201510514716A CN 105087797 A CN105087797 A CN 105087797A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
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Abstract
The invention discloses a method for determining microorganism varieties and pathogenic microorganism pollution conditions in air. The method for determining microorganism varieties in air comprises the following steps: 1) acquiring microorganisms in air to be detected by using a sampling filter membrane to obtain microorganisms in the air to be detected on the sampling filter membrane; 2) extracting DNAs (deoxyribonucleic acids) in the air to be detected on the sampling filter membrane to obtain unpurified microorganism DNAs; purifying the unpurified microorganism DNAs with magnetic beads to obtain microorganism DNAs in the air; and 3) sequencing the microorganism DNAs in the air, and determining the microorganism varieties in the air to be detected according to the sequences of the microorganism DNAs in the air. The experiment proves that the method for determining microorganism varieties in air can be used for determining the microorganism varieties in the air, and the method for determining pathogenic microorganism pollution conditions in air can be used for determining the existence of pathogenic microorganism pollution in the air.
Description
Technical field
The present invention relates in biological technical field the method determining microbes in air kind and pathogenic microorganism pollution situation.
Background technology
The particle size scope suspended in air is usually between 10nm to 100 μm.Irregular due to the shape of Atmospheric particulates, therefore the size of aerodynamically diameter, Atmospheric particulates are divided into TSP (Totalsuspendedparticulate usually, aerodynamic diameter≤100 μm), PM10 (coarseparticulate, aerodynamic diameter≤10 μm), PM2.5 (fine particle, aerodynamic diameter≤2.5 μm) and PM0.1 (superfine particulate matter, aerodynamic diameter≤0.1 μm) etc.International community is to the extensive concern of the particulate matter of these two kinds of particle size range of PM10 and PM2.5, mainly come from the reason of two aspects: one, from the angle of the particle size distribution range of suspended particulate, the particulate matter of about 10 μm particle size range is in the peak value of coarseparticulate relative abundance distribution, is the important composition of coarseparticulate; Simultaneously the particulate matter of particle diameter about 2.5 μm be generally in slightly, peak valley between fine particle two mode, be the part that in Atmospheric particulates, particle diameter is less, and be also the particle size interval that the pollutent discharged by mankind's activity mainly exists.Two, using health effect as considerations, due to the reason of human physiological structure, the particulate matter of PM10 and PM2.5 size is not filtered and obstruction ability completely: the particulate matter that general aerodynamic diameter is less than 10 μm can enter the upper respiratory tract of people, the particulate matter being less than 2.5 μm can enter segmental bronchus or bronchial end, particulate matter below 1 μm can reach alveolar, and even can penetrate alveolar when being less than 0.1 μm and enter blood circulation of human body.Therefore PM10 is also called as " pellet ", and PM2.5 is also called as " entering lung particulate matter ".PM2.5 due to its particle diameter little, more easily enter human body, and specific surface area is larger, various toxic heavy metal, acidic cpd, organic pollutant and the microorganism such as bacterium and virus in easy enriched air, the potential health hazard produced human body is larger, the impact of PM2.5 on HUMAN HEALTH mainly comprises: the infringement on respiratory system, the impact on cardiovascular systems, on neural impact, on immune impact, simultaneously also have certain relation with cancer and inborn defect.
Play an important role as in Atmospheric particulates forming process, and accounting can reach the microbe composition of 4%-80% in pellet, rarely have in former research and relate to, fast or technology maturation, this directly causes people very limited for the cognition of microbial species in pellet in the analysis that this qualification mainly coming from biological components does not have a chemical composition.But clearly, because the size of bacterium is generally between 0.2 μm-10 μm, mostly should be gathered in theory in the particle size range of pellet, simultaneously because its there is biological activity and part to have potential infection pathogenic, the composition distribution understanding them is very important on understanding the impact of pellet on human health.In the U.S., annual nosocomial infection has 1,300,000, and therefore 9.9 ten thousand people can get killed.And in China, study report on a large scale for current several and point out, the ward infection of similar tumour hospital is about 2%-3%, and fails to report about 2%.In addition, other specific environments such as household, hospital, food factory, pharmaceutical factory, large-scale public life, public place of entertainment also easily infects.Microbe species in good ambient air microorganism detection means such as specific environment air, the detection of environmental situation are most important, but present stage, this type of research still had to be strengthened.
Current air microbe species identification method mainly comprises cultivates and DNA sequence dna comparison.The species differentiated in particulate matter by the method for cultivating generally are not subject to the impact of grain diameter, but because environmental microorganism major part all can not be cultivated in laboratory conditions, the quantity of species very limited (comprising limited bacterium and fungi) therefore can be identified; And calculate that microorganism concn actual in air is inaccurate by the unit colony number of turning out.Mainly passed through the 16SrRNA sequence (or 18SrRNA sequence of fungi) of amplification bacterium by the method for DNA sequence dna Identifying micro-organisms kind, obtain based composition information through micro-array chip or order-checking and compare with the sequence of Known Species in database.But because the particle diameter of a pellet little order of magnitude compared with TSP, biological components wherein or extractible DNA measure relatively less, so the research of current most air microbe is still carried out based on TSP.
But 16SrRNA amplification technique makes the discriminating of bacterial species can only be confined to " genus " or " genus " above level, the pathogenecity due to bacterium differs all very large, therefore abundant not to the understanding of pathogenic bacterium between " kind " or " strain "; And the error that exponential amplification brings makes the calculating of flora relative abundance be inaccurate.Simultaneously, the sequence information of bacterium can only be obtained to the primary treatment (such as only doing 16SrRNA amplification) of sample, composition as understood fungi also needs again to carry out 18SrRNA sequence amplification, and secondary operation makes the relative content error calculating bacterium and fungi strengthen.In addition, the amplimer of general use is 16S universal primer, but in fact universal primer designs based on the known array of limited quantity, might not to all species all " general ", may in the amplification of some species sequence efficiency lower, bring amplification error equally.Consider the healthy effect of airborne microorganism to people, virus is also the pathogenic agent of can not ignore, but also can ignore by 16SrRNA augmentation detection.
Along with the development of high throughput sequencing technologies and the raising of computing power, metagenomics (metagenomics) is more and more applied in environmental microorganism Identification of Species, and the microorganism comprising the complex environments such as soil, ocean, oral cavity, enteron aisle forms all to have and relates to.The advantage of metagenomics is to have abandoned to the dependence of cultivating and the pre-amplification to specific DNA fragments, the genomic dna of extracting directly sample, " kind " level can be accurate to by sequence alignment, and the time of day of microbe survival in environment can be reflected accurately.But with the environmental facies ratio carried out metagenomics and study, Atmospheric particulates, especially the biotic component of pellet, or DNA content is relatively less, use traditional method to be difficult to obtain enough DNA amount to carry out order-checking and build storehouse, be therefore a technical barrier with the microorganism in metagenomics technique study air suspended particulated always.
Summary of the invention
Technical problem to be solved by this invention how to determine the pollution situation of microbe species in air and air.
For solving the problems of the technologies described above, the present invention provide firstly the method determining microbes in air kind.
The method determining microbes in air kind provided by the present invention, comprises following 1)-3):
1) gather microbes in air to be measured with sampling membrane, obtain the microbes in air to be measured be positioned on described sampling membrane;
2) be positioned at the DNA of the microbes in air to be measured on described sampling membrane described in extraction, obtain unpurified microbial DNA; With magnetic bead, purifying is carried out to described unpurified microbial DNA, obtain microbes in air DNA;
3) described microbes in air DNA is checked order, determine described microbes in air kind to be measured according to the sequence of described microbes in air DNA.
Above-mentionedly determine in the method for microbes in air kind, described microbes in air DNA directly checks order without amplification (amplification in vitro).Described method does not comprise the step increased to described microbes in air DNA.
Above-mentionedly determine that, in the method for microbes in air kind, described magnetic bead specifically can be Beckman Coulter Inc. (BeckmanCoulter, Inc.) product, model is AMPureXPcat.no.A63881.
Above-mentionedly determine that, in the method for microbes in air kind, described air can be specific environment air.Described specific environment air specifically can be specific ambient air to be measured.Described specific environment air can be the air at atmospheric arbitrary place of earth surface, can for there being the air of the environment of human living, the air of the environment also can lived for nobody's class.The described air having the air of the environment of human living specifically to can be the indoor or outdoors of the various playground of the mankind, as the air of densely populated public place, household, hospital ward or hospital outpatient place, food factory, pharmaceutical factory, large-scale public life, public place of entertainment.In one embodiment of the invention, described ambient air to be measured is that hospital is outdoor and hospital is indoor.
Above-mentionedly determine in the method for microbes in air kind, described 2) following 21 can be comprised) and 22):
21) be positioned at microbes in air water to be measured on described sampling membrane or PBS elutes from described sampling membrane by described, obtain the liquid containing microbes in air to be measured; With the liquid containing microbes in air to be measured described in filtration membrane filtration, obtain being positioned at the microbes in air to be measured on described PES film;
22) extract the microbes in air DNA to be measured be positioned on described PES film, obtain described unpurified microbial DNA; With described magnetic bead, purifying is carried out to described unpurified microbial DNA, obtain microbes in air DNA.
Above-mentionedly determine in the method for microbes in air kind, the aperture of described filtration film can be 0.01-0.2 micron.
Above-mentionedly determine in the method for microbes in air kind, described filtration film can be PES film.Described PES film specifically can be quite your life science (PallLifeSciences) product, and model is 0.2 μm of Supor200PESmembranediscfilter, cat.no.66234.
Above-mentionedly determine in the method for microbes in air kind, the described extraction microbes in air DNA to be measured be positioned on described PES film also can comprise and shredding with on the described PES film of described microbes in air to be measured.
Above-mentionedly determine in the method for microbes in air kind, described 22) can be: extract test kit with soil DNA and extract described microbes in air DNA to be measured, obtain described unpurified microbial DNA; With described magnetic bead, purifying is carried out to described unpurified microbial DNA, obtain microbes in air DNA.
When extracting test kit with described soil DNA and extracting described microbes in air DNA to be measured, described soil DNA only need be utilized to extract test kit the DNA of described microbes in air to be measured is discharged from microbes in air described to be measured, do not need to extract test kit with described soil DNA and purifying is carried out to the DNA discharged, now obtain described unpurified microbial DNA.With described magnetic bead, purifying is carried out to described unpurified microbial DNA and obtain described microbes in air DNA.
In one embodiment of the invention, described soil DNA extracts the pillar that test kit contains purify DNA, after the DNA of described microbes in air to be measured being discharged from microbes in air described to be measured with described soil DNA extraction test kit, with the pillar that described magnetic bead is replaced in described soil DNA extraction test kit, purifying is carried out to described unpurified microbial DNA, obtain described microbes in air DNA.
Above-mentionedly determine that, in the method for microbes in air kind, described magnetic bead also can replace with the test kit utilizing magnetic beads for purifying DNA, as AgencourtAMPureXP.
Above-mentionedly determine in the method for microbes in air kind, it is that the name of Mobio company is called that described soil DNA extracts test kit
the test kit of DNAIsolationKit or Qiagen company
dNAStoolMiniKit.
Above-mentionedly determine in the method for microbes in air kind, the aperture of described sampling membrane can be 0.01-0.2 micron, as 0.2 micron.
Described sampling membrane gathers microbes in air to be measured and can be and gather microbes in air to be measured with the detachable filter that described sampling membrane is housed.Described detachable filter can be loaded in air sampling pump.
Described air sampling pump body can be SKC air sampling pump.Described detachable filter specifically can be the detachable filter of Pall company.Described sampling membrane specifically can be PTEE filter membrane.
Above-mentionedly determine in the method for microbes in air kind, when described sampling membrane gathers microbes in air to be measured, can be 4L/min by the air capacity of described sampling membrane.The time of described collection can be 23 hours.Described sampling membrane gathers microbes in air to be measured and can be and gather described microbes in air to be measured incessantly.
Above-mentionedly determine in the method for microbes in air kind, 3) the available high-flux sequence platform of described order-checking carries out.Described high-flux sequence platform specifically can be Hiseq, Miseq, IonTorrent, Proton, SOLiD, 454 or single-molecule sequencing device.
Above-mentionedly determine in the method for microbes in air kind, before described microbes in air DNA is checked order, also can comprise and whole genome amplification is carried out to described microbes in air DNA.
Above-mentionedly determine in the method for microbes in air kind, the described sequence according to described microbes in air DNA is determined that described microbes in air kind to be measured can be the microbial DNA in the sequence of described microbes in air DNA and reference database to compare and is determined described microbes in air kind to be measured.
Described reference database can be common biological data storehouse.Described reference database specifically can be at least one of the common ward infection bacterial genomes database of the full-length genome composition of all nosocomial infection bacteriums in M5NR database, Nr database, Silva database, Greengenes database, RDP database, CARD database and NCBI.
Above-mentionedly determine in the method for microbes in air kind, also can comprise before the microbial DNA in the sequence of described microbes in air DNA and reference database is compared, remove the sequence that the low quality data in the sequencing result of described microbes in air DNA, joint pollution and the mankind pollute.
The sequence that the described removal mankind pollute realizes to the sequence in human genome reference sequences Hg19 by removing comparison in described sequencing result.Described comparison can utilize SOAP, Bwa and/or Blat software to carry out.
Based on the result of described comparison, biological analysis software can be utilized to carry out species taxonomy, to determine described microbes in air kind.Described biological analysis software can be Metaphlan, Megan, Mother, MG-RAST and/or Galaxy.
For solving the problems of the technologies described above, present invention also offers the system extracting microbes in air DNA.
The system of extraction microbes in air DNA provided by the present invention, is made up of following A 1 and A2:
A1, the reagent extracting microbes in air DNA and/or test kit;
A2, for purify DNA magnetic bead or utilize the test kit of magnetic beads for purifying DNA.
In the system of said extracted microbes in air DNA, reagent described in A1 and/or test kit can only for slightly the carrying of DNA and/or DNA of microorganism in release air.The reagent of described extraction microbes in air DNA and/or test kit can be soil DNA described in the above-mentioned method determining microbes in air kind and extract test kit.The described magnetic bead for purify DNA can be magnetic bead described in the above-mentioned method determining microbes in air kind.The described test kit of magnetic beads for purifying DNA that utilizes can be the test kit utilizing magnetic beads for purifying DNA described in the above-mentioned method determining microbes in air kind.
For solving the problems of the technologies described above, present invention also offers the system determining microbes in air kind.
The system determining microbes in air kind provided by the present invention, is made up of at least one in the system of described extraction microbes in air DNA and following B1, B2 and B3 these three kinds:
B1, the instrument gathering microbes in air and/or instrument;
B2, high-flux sequence platform;
B3, carry out the biological software of sequence alignment and/or microorganism classification.
Above-mentionedly determine in the system of microbes in air kind, the instrument of described collection microbes in air and/or instrument can be above-mentionedly determines sampling membrane described in the method for microbes in air kind, described detachable filter and/or described air sampling pump.
Described high-flux sequence platform can be business-like high-flux sequence platform, as Hiseq, Miseq, IonTorrent, Proton, SOLiD, 454 and/or single-molecule sequencing device.Described Hiseq specifically can be HiSeq2000 or HiSeq2500 of Illumina Products.
Described biological software of carrying out sequence alignment and/or microorganism classification specifically can be SOAP, Bwa, Blat, Metaphlan, Megan, Mother, MG-RAST and/or Galaxy software.
For solving the problems of the technologies described above, present invention also offers and describedly determine the application of the method for microbes in air kind in monitor air pollution situation.
Whether described detection air pollution carries out containing pathogenic microorganism by detecting in air to be measured, and containing pathogenic microorganism in described air to be measured, described air to be measured is polluted by pathogenic microorganism or candidate is polluted by pathogenic microorganism; Not containing pathogenic microorganism in described air to be measured, described air to be measured is not polluted by pathogenic microorganism or candidate is not polluted by pathogenic microorganism.Described detection air pollution specifically can comprise: utilize and describedly determine that the method for microbes in air kind determines the species number of pathogenic microorganism in air to be measured, utilize and describedly determine that the method for microbes in air kind determines the species number of all microorganisms in described air to be measured, air pollution is detected: described ratio is 0, described free of air pollution to be measured according to the ratio of the species number of all microorganisms in the species number of pathogenic microorganism in described air to be measured and described air to be measured; Described ratio is greater than 0, and described air to be measured is contaminated, and described ratio is larger, and described air to be measured is contaminated more serious.
Described abovely determine that in the application of the method for microbes in air kind in monitor air pollution situation, described pollution specifically can be the pollution that pathogenic microorganism causes.
For solving the problems of the technologies described above, present invention also offers the system of described extraction microbes in air DNA or describedly determining that the system of microbes in air kind is in the application determining microbes in air kind or detect in air pollution.
In above-mentioned application, described pollution specifically can be the pollution that pathogenic microorganism causes.
Current indoor and outdoor microbiological analysis method, mainly contains three kinds: air jet flow, qPCR and grand genome method.Wherein, air jet flow method, cheap, but the low and most microorganism of sensitivity cannot be cultivated; QPCR method, fast, but can only detect several microorganism, and there is the problem that various microorganism disturbs mutually; Grand genome method, can be qualitative, quantitative, but price is higher, speed is slower.
The present invention is intended at least to solve one of technical problem existed in prior art.For this reason, one object of the present invention be to propose a kind of can the means of particular environment detection microbes in air kind quickly and easily.
According to an aspect of the present invention, the invention provides a kind of method determining specific environment microbes in air kind.According to embodiments of the invention, the method comprises the following steps: for described specific environment, utilizes air sampling pump to carry out air sampling, to obtain the testing sample comprising air microbe, wherein said air sampling pump comprises detachable filter and filter membrane; DNA extraction is carried out, to obtain mixing microorganisms DNA to the described testing sample comprising air microbe; Described mixing microorganisms DNA is checked order, to obtain sequencing result; Described sequencing result and reference database are compared, to obtain comparison result; And based on described comparison result, determine the microbe species in described specific environment air.
Wherein, described specific environment air specifically can be specific ambient air to be measured (i.e. any environment to be measured).Described specific environment air can be the air at atmospheric arbitrary place of earth surface, can for there being the air of the environment of human living, the air of the environment also can lived for nobody's class.The described air having the air of the environment of human living specifically to can be the indoor or outdoors of the various playground of the mankind, as the air of densely populated public place, household, hospital ward or hospital outpatient place, food factory, pharmaceutical factory, large-scale public life, public place of entertainment.In one embodiment of the invention, described ambient air to be measured is that hospital is outdoor and hospital is indoor.
Contriver is surprised to find, and utilizes method of the present invention, can effectively detect the microbe species determined in specific environment air, and then based on the microbe species in specific environment air, can determine the pollution situation of this specific environment.In addition, the method cost is low, efficiency is high, fast and easy, all common microbiological that can be contained in disposable detection testing sample, and can quantitative and qualitative analysis simultaneously, and highly sensitive, result accurately and reliably.
In addition, the method determining specific environment microbes in air kind according to the above embodiment of the present invention, can also have following additional technical characteristic:
According to embodiments of the invention, described air sampling pump is SKC air sampling pump, and described detachable filter is the detachable filter of Pall company, and described filter membrane is PTEE filter membrane.Thus, during sampling, steady air current, air input is constant, and sample effect is good.Further, adopt the detachable filter of Pall company, then acquisition system is reusable, and repeating utilization factor is high, and each sample effect otherness is little, and homogeneity is good; Adopt PTEE filter membrane, more effectively can collect the microorganism in air, and convenient and swift.
According to embodiments of the invention, the diameter of described PTEE filter membrane coordinates with described detachable filter, and aperture is 0.2 micron.Thus, sample effect is good, is convenient to subsequent analysis.
According to embodiments of the invention, in specific environment, with the air input of at least 4L/min, within 23 hours, carry out described air sampling incessantly.Thereby, it is possible to the lung exposure of simulation normal people is to the situation of microorganism, sample effect is good, is conducive to subsequent analysis, and significantly can provide the accuracy of result.Wherein, the half respiratory capacity (staying the airshed that can touch lung surface in lung) that air-flow adopts simulation normal people to breathe, the namely air input of 4L/min, and can air input be strengthened, shorten inlet period.The uninterruptedly sampling that adopts 23 hours, be simulation people in specific environment (i.e. environment to be measured, as indoor or outdoors) one day the amount of all microorganisms of breathing, thus make method of the present invention science, rigorous more.
The extraction of air microbe is an industry difficult problem always.According to embodiments of the invention, utilize Mobio company
dNAIsolationKit or Qiagen company
dNAStoolMiniKit carries out described DNA extraction.Thereby, it is possible to effectively extract air microbe DNA from testing sample.According to preferred embodiments more of the present invention, utilize Mobio company
dNAIsolationKit, by following steps, carries out described DNA extraction: take out filter membrane, and after carrying out break process, utilizes SolutionC1, aqua sterilisa or PBS to shake mixing and dissolve half an hour, to obtain the testing sample comprising air microbe; In described testing sample, add SolutionC1, carry out washing after mixing successively and grind and the first centrifugal treating, to obtain the first centrifugal rear liquid; Get the supernatant in described first centrifugal rear liquid, and add SolutionC2, carry out first successively after vortex mixing and hatch and the second centrifugal treating, to obtain the second centrifugal rear liquid; Get the supernatant in described second centrifugal rear liquid, and add SolutionC3, carry out second successively after vortex mixing and hatch and the 3rd centrifugal treating, to obtain the 3rd centrifugal rear liquid; And the supernatant got in described 3rd centrifugal rear liquid, and add SolutionC4, obtain mixing microorganisms DNA after vortex mixing.Thus, air microbe DNA extraction is remarkably productive, is conducive to the carrying out of subsequent step.
According to embodiments of the invention, carry out described concussion mixing and dissolve 30min.Thus, the collection of testing sample is conducive to.
According to embodiments of the invention, utilize vortex instrument, under 1400rpm rotating speed, carry out described washing grind process 30min.Thus, it is good that washing grinds treatment effect, is conducive to the carrying out of subsequent step.
According to embodiments of the invention, under room temperature, 10000g condition, carry out described first centrifugal 30s.Thus, centrifugal effect is good, is conducive to the carrying out of subsequent step.
According to embodiments of the invention, at 4 DEG C, carry out described first hatch 5min.Thus, hatch effective, be conducive to the carrying out of subsequent step.
According to embodiments of the invention, under room temperature, 10000g condition, carry out described second centrifugal 1min.Thus, centrifugal effect is good, is conducive to the carrying out of subsequent step.
According to embodiments of the invention, at 4 DEG C, carry out described second hatch 5min.Thus, hatch effective, be conducive to the carrying out of subsequent step.
According to embodiments of the invention, under room temperature, 10000g condition, carry out described 3rd centrifugal 1min.Thus, centrifugal effect is good, is conducive to the carrying out of subsequent step.
According to embodiments of the invention, before carrying out described order-checking, comprise the step of described mixing microorganisms DNA being carried out to purifying further.Thus, the carrying out of follow-up order-checking, analytical procedure is conducive to.
According to some embodiments of the present invention, the magnetic bead of Jin Maige company or AgencourtAMPureXP purification process is utilized to carry out described purifying.Thus, DNA purification effect is good.
According to embodiments of the invention, before carrying out described order-checking, comprise the step of described mixing microorganisms DNA being carried out to whole genome amplification further.Thereby, it is possible to realize the effective amplification to trace dna in mixing microorganisms DNA, be conducive to the carrying out of follow-up order-checking, analytical procedure.
According to some embodiments of the present invention, whole genome amplification test kit is utilized to carry out described whole genome amplification.Thus, good to the amplification homogeneity of mixing microorganisms DNA, various microorganism all can effectively be increased, remarkably productive.
According to embodiments of the invention, utilize be selected from Hiseq, Miseq, IonTorrent, Proton, SOLiD, 454 and at least one of single-molecule sequencing device carry out described order-checking.Thereby, it is possible to effectively determine the sequence of mixing microorganisms DNA, and result is accurate, reliable, is conducive to the carrying out of subsequent step.
According to embodiments of the invention, carry out the order-checking of two end 200bp.Thus, check order effective, efficiency is high, and obtain sequencing result be conducive to follow-up compare of analysis.
According to embodiments of the invention, described reference database is at least one being selected from common microbiological database, common drug resistant gene database and common ward infection bacterial genomes database.Thus, according to the microbe species paid close attention in specific environment, corresponding reference database can be selected, and then for after compare of analysis, effectively can improve efficiency and the effect of compare of analysis.
According to some embodiments of the present invention, described reference database is at least one being selected from M5NR database, Nr database, Silva database, Greengenes database, RDP database and CARD database.Thus, compare of analysis efficiency is high, effective.
According to embodiments of the invention, described common ward infection bacterial genomes database is made up of the full-length genome of ward infection bacteriums all in NCBI.Thus, the carrying out of follow-up compare of analysis step is conducive to.
According to embodiments of the invention, before carrying out described compare of analysis, comprise further, remove the sequence that the low quality data in described sequencing result, joint pollution and the mankind pollute.Thus, be conducive to the carrying out of compare of analysis, the accuracy of compare of analysis result can be significantly improved.
According to embodiments of the invention, the sequence removing mankind's pollution realizes to the sequence in human genome reference sequences Hg19 by removing comparison in described sequencing result.
According to embodiments of the invention, utilize be selected from SOAP, Bwa and Blat software one of at least carry out described compare of analysis.Thus, the efficiency of compare of analysis is high, and result accurately, reliably.
According to embodiments of the invention, based on described comparison result, biological analysis software is utilized to carry out species taxonomy, to determine the microbe species in described specific environment air.According to preferred embodiments more of the present invention, described biological analysis software is at least one being selected from Metaphlan, Megan, Mother, MG-RAST and Galaxy.Thereby, it is possible to effectively realize the species taxonomy of air microbe in testing sample.
According to a further aspect in the invention, present invention also offers a kind of method determining specific environment pollution situation.According to embodiments of the invention, the method comprises the following steps: utilize the foregoing method determining specific environment microbes in air kind, determines the microbe species in described specific environment air; Based on the common bacterial classification of air microbe, the microbiota in described specific environment air is divided into common air microorganism and specificity air microbe; Pathogenic microorganism is screened, to determine the pathogenic microorganism kind in described specific environment air from described specificity air microbe; Based on the pathogenic microorganism kind in described specific environment air, determine the dustiness of described specific environment, wherein,
All microbe species in pathogenic microorganism kind/specific environment air in the dustiness=specific environment air of specific environment; And based on the dustiness of described specific environment, determine the pollution situation of described specific environment.
Contriver is surprised to find, and utilizes the method effectively can determine the pollution situation of specific environment.In addition, the method determining specific environment pollution situation of the present invention, cost is low, efficiency is high, convenient and swift, and highly sensitive, and result accurately and reliably, can provide strong scientific basis for researchs such as environmental monitorings.
In addition, the method determining specific environment pollution situation according to the above embodiment of the present invention, can also have following additional technical characteristic:
According to embodiments of the invention, the common bacterial classification of described air microbe is following 48 kinds: hidden geodermatophilus obscurus (Geodermatophilusobscurus), Modestobactermarinus, occupy rock tooth coccus (Blastococcussaxobsidens), addicted to root Kocuria kristinae ad (Kocuriarhizophila), micrococcus luteus (Micrococcusluteus), candiyeast (CandidatusNitrospiradefluvii), radiation hardness pseudomonas (Methylobacteriumradiotolerans), hot bifid (Thermbifidafusca), Nocardia bacteria (Nocardioidessp.), propionibacterium acnes (Propionibacteriumacnes), the short shape bacillus (Brachybacteriumfaecium) of excrement, Cellvibrio (Cellvibriogilvus), Arthrobacter (Arthrobacterphenanthrenivorans), Da Songweier nocardia (Nocardiopsisdassonvillei), meat bacillus (Carnobacteriumsp.), tiny bacterium (Microbacteriumtestaceum), Paracoccus denitrificans (Paracoccusdenitrificans), motionless lid coccus (Kytococcussedentarius), Coriolis blood bacillus (Sanguibacterkeddieii), the dynamic coccus (Kineococcusradiotolerans) of radiation, pineapple multi-source bacterium (Pantoeaananatis), Yue Hanxunshi Bacterium lacticum (Lactobacillusjohnsonii), fertilizer cellulomonas cartae (Cellulomonasfimi), lactobacillus crispatus (Lactobacilluscrispatus), the unusual coccus in Gobi desert (Deinococcusgobiensis), to make pottery Salmonella in distress (Thauerasp.), pantoea agglomerans (Pantoeavagans), clostridium perfringens (Clostridiumperfringens), lactobacillus reuteri (Lactobacillusreuteri), Pseudomonas stutzeri (Pseudomonasstutzeri), South Pole bacillus (Psychrobactercryohalolentis), lactobacillus salivarius (Lactobacillussalivarius), bacillus megaterium (Bacillusmegaterium), A Shi Arthrobacter (Arthrobacterarilaitensis), Ramlibactertataouinensis, middle village Salmonella (Nakamurellamultipartita), baby suis (Streptococcusinfantarius), pale yellow mycobacterium (Mycobacteriumgilvum), chlorophenol Arthrobacter (Arthrobacterchlorophenolicus), outer chain Methylobacterium (Methylobacteriumextorquens), Methylobacterium (Methylobacteriumchloromethanicum), coryneform bacteria (Corynebacteriumefficiens), addicted to maltose Stenotrophomonas (Stenotrophomonasmaltophilia), intend promise Ka Shi Alba (Nocardiopsisalba), South Pole bacterium (Psychrobacterarcticus), green thermophilic actinomycete (Saccharomonosporaviridis), streptomyces coelicolor (Streptomycescoelicolor) and leukonid (Leuconostocmesenteroides).
Wherein, common air microorganism is air common bacteria, substantially no matter takes these bacterial classifications of what sample to be certain existence; And the specificity air microbe special bacterial classification that to be specific sample have, namely can have special representativeness illustrate that air sample has in particular circumstances mutually should bacterial classification.
According to embodiments of the invention, based on the pathogenic microorganism kind in described specific environment air, before determining the dustiness of described specific environment, comprise the step of pathogenic microorganism kind the selection result being carried out to certificate authenticity further.Thus, ensure that the environmental situation result finally determined is accurate, reliable.
According to embodiments of the invention, by calculating relative abundance and the order-checking saturation ratio of the pathogenic microorganism in described specific environment air, determine the reliability of pathogenic microorganism kind the selection result, the saturation ratio that wherein checks order is that each sample sequencing data amount reaches 5 ~ 7Gb base number, and the relative abundance of each microbe species that each sample is detected all is not less than 1%, is that described pathogenic microorganism kind the selection result indicates reliably.Wherein, it should be noted that, " relative abundance " number of species number in ecological Shang Zhi group, and in this article pointer to each sample, the ratio of the genome stdn summation of the genome standardized value of its each microbe species be detected and microbe species that all the other are detected, wherein " genome standardized value " refers to the ratio of the Genome Size that unique sequencing data amount of this microbe species is corresponding to this microbe species in comparison.
According to embodiments of the invention, utilize relative abundance and the order-checking saturation ratio of the pathogenic microorganism in specific environment air described in biological analysis computed in software, described biological analysis software is at least one being selected from Metaphlan, Megan, Mother, MG-RAST and Galaxy.Thereby, it is possible to effectively calculate the relative abundance and order-checking saturation ratio that obtain target pathogenic microorganism, be conducive to the carrying out of certificate authenticity.
According to embodiments of the invention, based on the dustiness of described specific environment, determine the pollution situation of described specific environment, comprise further: when dustiness≤0.3%, described specific environment is pollution-free; When dustiness is 0.3%-0.6%, described specific environment slight pollution; When dustiness is 0.6%-1%, described specific environment intermediate pollution; As dustiness >1%, described specific environment serious pollution.Thereby, it is possible to effectively determine the pollution situation of specific environment.
Experiment proves, of the present inventionly determine the microbe species that the method for microbes in air kind can be used for determining in air, the present invention is when detecting sample at the air choosing hospital's indoor and outdoors, determine the content of microorganisms in the air sample of hospital's indoor and outdoors, and further determined that hospital indoor altogether containing 5 kinds of pathogenic microorganisms, outdoor is not containing pathogenic microorganism.The microbes in air DNA determining to obtain in the method for microbes in air kind of the present invention can directly be used for checking order, decrease in traditional method the step that the microbes in air DNA directly obtained increases, microbial DNA information in the erroneous air that can avoid increases causes, further enhancing the reliability of result.In addition, the present invention also establishes the method determining air pathogenic microorganism pollution situation further on the basis determining microbes in air kind, the method determines hospital indoor altogether containing 5 kinds of pathogenic microorganisms, outdoor is not containing pathogenic microorganism, show, there is the pollution of pathogenic microorganism the hospital indoor chosen, and hospital outdoor there is no the pollution of pathogenic microorganism.The present invention also according to relative abundance and the order-checking saturation ratio of the pathogenic microorganism obtained, determines that the pathogenic microorganism kind the selection result obtained is reliable.
Experiment proves, of the present inventionly determines the microbe species that the method for microbes in air kind can be used for determining in air, of the present inventionly determines the method for air pathogenic microorganism pollution situation can be used for determining whether to have in air the pollution of pathogenic microorganism.
Accompanying drawing explanation
Fig. 1 is the ratio of common microbiological and special microbes in air in indoor air in hospital.Wherein, " having " represents common microbiological in indoor air in hospital, and " peculiar " represents the indoor special microbes in air of hospital.
Fig. 2 is the ratio of common microbiological and special microbes in air in hospital's outside air.Wherein, " having " represents common microbiological in hospital's outside air, and " peculiar " represents the outdoor special microbes in air of hospital.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Below for certain hospital's outside air and room air, concrete defining method of setting forth microbes in air kind and air pollution.
Specific environment in following embodiment all refers to corresponding environment to be measured, i.e. the indoor (as Outpatient Hall) of certain hospital or the outdoor of certain hospital, and the outdoor of certain hospital refers to the environment away from hospital outpatient place and the intensive place of patient herein.
PES film in following embodiment is quite your life science (PallLifeSciences) product, and model is 0.2 μm of Supor200PESmembranediscfilter, cat.no.66234.。
Magnetic bead (AmpureBeads) in following embodiment is Beckman Coulter Inc. (BeckmanCoulter, Inc.) product, and model is AMPureXPcat.no.A63881.
High-flux sequence platform Hiseq in following embodiment is the HiSeq2000 of Illumina Products.
The determination of embodiment 1, microbes in air kind and air pollution
One, the determination of microbes in air kind
One) determination of microbe species in room air
When determining microbe species in certain indoor air in hospital, have chosen the indoor place of 5 different hospitals (Outpatient Hall, two different wards, pharmacy, cash desk) altogether and carry out the collection of sample and the determination of indoor microbe species, concrete grammar is as follows:
1, sample
Equipment: SKC air sampling pump; The detachable filter of Pall company and PTEE filter membrane, the diameter of PTEE filter membrane coordinates with this detachable filter, and aperture is 0.2 micron.
Method: SKC air sampling pump is placed in certain hospital indoor, the intake velocity of SKC air sampling pump is adjusted to 4L/min; The detachable filter of the Pall company that PTEE filter membrane is housed correctly is inserted continuous sampling after in SKC air sampling pump after 23 hours, PTEE filter membrane to be disassembled, obtain the sampling membrane gathering microbes in air.
2, microbial DNA extracts
The sampling membrane of microbes in air is had to roll the collection of step 1, there is the one side of microbes in air inside, one side without microbes in air is outside, then put it in 50mL centrifuge tube, in this centrifuge tube, add PBS the air particle on sampling membrane is eluted, abandon sampling membrane, obtain the suspension containing microbes in air; By the PES membrane filtration of the suspension containing microbes in air, obtain the PES film collecting microorganism.
Mobio company
dNAIsolationKit extracts above-mentioned collection to be had the microbial DNA on the PES film of microorganism and carries out purifying with magnetic bead (AmpureBeads) to microbial DNA, and concrete grammar is as follows:
1) clamp the PES film collected and have microorganism with sterilized tweezers, have the PES film of microorganism to shred collection by sterile scissors, obtain PES film fragment, PES film fragment is placed in 15ml centrifuge tube (PowerBeadTubes);
2) to step 1) be equipped with in the centrifuge tube of PES film fragment and add 60 μ lSolutionC1 and (detect SolutionC1 in advance, if there is precipitation in SolutionC1, SolutionC1 is placed in 60 DEG C of water-baths to precipitating CL), turn upside down and mix for several times, then this centrifuge tube is fixed on vortex instrument adapter, 30min is processed under maximum speed of revolution 1400rpm, to the suspension as much as possible of the microorganism on PES film in the solution, centrifugal 30s under room temperature 10000g, abandon precipitation, obtain supernatant liquor 1;
3) by 400-500 μ l step 2) supernatant liquor 1 be placed in a clean 2mlCollectionTube (test kit provides), and 250 μ lSolutionC2 are added in supernatant liquor 1, vortex mixing 5s, hatch 5min for 4 DEG C, centrifugal 1min under room temperature 10000g, abandon precipitation, obtain supernatant liquor 2;
4) by 600 μ l steps 3) supernatant liquor 2 be placed in a new EP pipe, in supernatant liquor 2, add 200 μ lSolutionC3, vortex mixes, and hatch 5min for 4 DEG C, under room temperature 10000g, centrifugal 1min, abandons precipitation, obtains supernatant liquor 3;
5) by 750 μ l steps 4) supernatant liquor 3 be placed in a new 2mlEP pipe, in supernatant liquor 3, add 1200 μ lSolutionC4 (SolutionC4 first shakes up before using), vortex mixing 5s, obtains unpurified microbial DNA solution;
6) adding in the unpurified microbial DNA solution of step 5 is that the magnetic bead (AmpureBeads) of unpurified microbial DNA solution 1.5 times of volumes carries out purifying to microbial DNA, concrete operation step carries out according to the operation instruction of magnetic bead (AmpureBeads), obtains microbial DNA in room air after operation terminates.
3, whole genome amplification
Microbial DNA in the room air of step 2 is dissolved in 20 μ lEB, quantitatively, use whole genome amplification test kit QIAGENREPLI-g to carry out whole genome amplification to microbial DNA in the room air of step 2, obtain microbial DNA in the room air of whole genome amplification.
4, check order
Use the standard Library development flow of Illumina to complete and build storehouse, adopt the strategy of two end 200bp, with high-flux sequence platform Hiseq, microbial DNA in the room air of step 2 is checked order, obtain microbial DNA sequence in room air.
Use the standard Library development flow of Illumina to complete and build storehouse, adopt the strategy of two end 200bp, with high-flux sequence platform Hiseq, microbial DNA in the room air of the whole genome amplification of step 3 is checked order, obtain microbial DNA sequence in the room air of indoor whole genome amplification.
Found that, in the room air of step 2 whole genome amplification of microbial DNA and step 3 room air in microbial DNA all reach the requirement of order-checking, no matter that in the room air to step 2, microbial DNA checks order, or microbial DNA in the room air of the whole genome amplification of step 3 is checked order, all can complete order-checking.
5, compare of analysis
In the room air that step 4 is obtained microbial DNA sequence and whole genome amplification room air in microbial DNA sequence carry out following process and sequence alignment respectively: remove the low quality data in sequencing result, joint pollutes and the mankind pollute the sequence of (namely comparison is to the sequence in human genome reference sequences Hg19), then SOAP software and reference database is utilized to compare, to obtain comparison result.Wherein, reference database comprises M5NR database, Nr database, Silva database, Greengenes database, RDP database and CARD database, and the common ward infection bacterial genomes database that in NCBI, the full-length genome of all nosocomial infection bacteriums forms.
6, microbe species is determined
Based on the comparison result of microbial DNA sequence in step 5 pair room air, the microorganism of the biological analysis softwares such as Metaphlan, Megan, Mother, MG-RAST and Galaxy to hospital indoor is utilized to carry out species taxonomy, to determine the microbe species in specific environment (hospital namely to be measured is indoor) air.
Based on the comparison result of microbial DNA sequence in the room air of step 5 pair whole genome amplification, the microorganism of the biological analysis softwares such as Metaphlan, Megan, Mother, MG-RAST and Galaxy to hospital indoor is utilized to carry out species taxonomy, to determine the microbe species in specific environment (hospital namely to be measured is indoor) air.
Result shows, the microbe species determined with microbial DNA in the room air directly obtained is consistent with the microbe species determined with microbial DNA in the room air after whole genome amplification, showing can with according to step one) in 1 and 2 room airs directly obtained microbial DNA directly carry out checking order and carrying out next step analysis, and need not whole genome amplification be carried out.
As a result, in the air sample of the indoor different location of hospital, comprise altogether 308 kinds of total microorganisms (being classified to the level of genus).
Two) determination of microbe species in outside air
When determining microbe species in certain hospital's outside air, have chosen 5 different hospital's outdoor site altogether and carry out the collection of sample and the determination of outdoor microbe species, concrete grammar is as follows:
According to step one) in step 1 and 2 method, certain hospital indoor is replaced with certain hospital outdoor, other steps are all constant, obtain microbial DNA in outside air.
According to step one) in the method for step 3, whole genome amplification is carried out to microbial DNA in outside air, obtains microbial DNA in the outside air of whole genome amplification.
According to step one) in the sequence measurement of step 4, microbial DNA in room air is replaced with respectively microbial DNA in the outside air of microbial DNA and whole genome amplification in above-mentioned outside air, other steps are all constant, obtain microbial DNA sequence in the outside air of microbial DNA sequence and whole genome amplification in outside air.
To microbial DNA sequence in the outside air of microbial DNA sequence in outside air and whole genome amplification according to step one) in the compare of analysis method of step 5 process and carry out sequence alignment, then utilize the biological analysis softwares such as Metaphlan, Megan, Mother, MG-RAST and Galaxy to carry out species taxonomy to the microorganism of hospital outdoor based on this sequence alignment result.
Found that, the microbe species determined microbial DNA sequence in outside air and microbial DNA sequence analysis in the outside air of whole genome amplification is consistent, showing can with according to step one) in 1 and 2 outside airs directly obtained microbial DNA directly carry out checking order and carrying out next step analysis, and need not whole genome amplification be carried out.In the air sample of the outdoor different location of hospital, comprise altogether 452 kinds of total microorganisms (being classified to the level of genus).
Two, the determination of air pollution
1, microbe species is distinguished
Based on common microbiological in air, the microbiota in hospital indoor and hospital's outside air is divided into microorganism and special microbes in air in common air.In air, common microbiological is following 48 kinds: hidden geodermatophilus obscurus (Geodermatophilusobscurus), Modestobactermarinus, occupy rock tooth coccus (Blastococcussaxobsidens), addicted to root Kocuria kristinae ad (Kocuriarhizophila), micrococcus luteus (Micrococcusluteus), candiyeast (CandidatusNitrospiradefluvii), radiation hardness pseudomonas (Methylobacteriumradiotolerans), hot bifid (Thermbifidafusca), Nocardia bacteria (Nocardioidessp.), propionibacterium acnes (Propionibacteriumacnes), the short shape bacillus (Brachybacteriumfaecium) of excrement, Cellvibrio (Cellvibriogilvus), Arthrobacter (Arthrobacterphenanthrenivorans), Da Songweier nocardia (Nocardiopsisdassonvillei), meat bacillus (Carnobacteriumsp.), tiny bacterium (Microbacteriumtestaceum), Paracoccus denitrificans (Paracoccusdenitrificans), motionless lid coccus (Kytococcussedentarius), Coriolis blood bacillus (Sanguibacterkeddieii), the dynamic coccus (Kineococcusradiotolerans) of radiation, pineapple multi-source bacterium Pantoeaananatis, Yue Hanxunshi Bacterium lacticum (Lactobacillusjohnsonii), fertilizer cellulomonas cartae (Cellulomonasfimi), lactobacillus crispatus (Lactobacilluscrispatus), the unusual coccus in Gobi desert (Deinococcusgobiensis), to make pottery Salmonella in distress (Thauerasp.), pantoea agglomerans (Pantoeavagans), clostridium perfringens (Clostridiumperfringens), lactobacillus reuteri (Lactobacillusreuteri), Pseudomonas stutzeri (Pseudomonasstutzeri), South Pole bacillus (Psychrobactercryohalolentis), lactobacillus salivarius (Lactobacillussalivarius), bacillus megaterium (Bacillusmegaterium), A Shi Arthrobacter (Arthrobacterarilaitensis), Ramlibactertataouinensis, middle village Salmonella (Nakamurellamultipartita), baby suis (Streptococcusinfantarius), pale yellow mycobacterium (Mycobacteriumgilvum), chlorophenol Arthrobacter (Arthrobacterchlorophenolicus), outer chain Methylobacterium (Methylobacteriumextorquens), Methylobacterium (Methylobacteriumchloromethanicum), coryneform bacteria (Corynebacteriumefficiens), addicted to maltose Stenotrophomonas (Stenotrophomonasmaltophilia), intend promise Ka Shi Alba (Nocardiopsisalba), South Pole bacterium (Psychrobacterarcticus), green thermophilic actinomycete (Saccharomonosporaviridis), streptomyces coelicolor (Streptomycescoelicolor) and leukonid (Leuconostocmesenteroides).
In indoor air in hospital, comprise common microbiological (see table 1) and 5 kinds of indoor special microbes in airs (see table 2) of hospital in 36 kinds of air, in air, common microbiological accounts for the ratio of microorganism total amount in indoor air in hospital is 87.8%, the ratio that the indoor special microbes in air of hospital accounts for microorganism total amount in indoor air in hospital is 12.2%, as shown in Figure 1.
Common microbiological in 36 kinds of air in table 1, indoor air in hospital
5 species specific microbes in airs in table 2, indoor air in hospital
In air, common microbiological accounts for the ratio of microorganism total amount in hospital's outside air is 80%, and the ratio that the outdoor special microbes in air of hospital accounts for microorganism total amount in hospital's outside air is 20%, as shown in Figure 2.
2, pathogenic microorganism kind is determined
Pathogenic microorganism is screened, to determine the pathogenic microorganism kind in indoor air in hospital in microbes in air special from the indoor special microbes in air of 5 kinds of hospitals that step 1 obtains and hospital's outside air.Wherein, the pathogenic microorganism information in the definition base ncbi database of pathogenic microorganism is carried out.
Found that, the indoor special microbes in air of 5 kinds of hospitals that step 1 obtains is pathogenic microorganism, and these 5 kinds of pathogenic microorganisms are respectively as Acinetobacter bauamnnii (Acinetobacterbaumannii), meningitis Neisser formula bacterium (Neisseriameningitidis), Acinetobacter lwoffii (Acinetobacterlwoffii), streptococcus pneumoniae (Streptococcuspneumoniae) and mycobacterium tuberculosis (Mycobacteriumparascrofulaceum).
3, certificate authenticity is carried out to pathogenic microorganism kind the selection result
Utilize Metaphlan to calculate relative abundance and the order-checking saturation ratio of the pathogenic microorganism in hospital indoor and hospital's outside air, determine the reliability of pathogenic microorganism kind the selection result.Wherein, when each sample sequencing data amount reaches 5-7Gb base number (saturation ratio that namely checks order is 5-7Gb base number), and the relative abundance of pathogenic microorganism that is detected of each sample is when being all not less than 1%, show that this pathogenic microorganism kind the selection result is reliable.Wherein, " relative abundance " number of species number in ecological Shang Zhi group, and in this article pointer to each sample, the ratio of the genome stdn summation of all the other each microorganisms be detected in the genome standardized value of its pathogenic microorganism be detected and this sample, wherein " genome standardized value " refers to the ratio of the sequencing data amount of this microorganism and the Genome Size of this microorganism.
Result shows, and in room air, in microbial DNA and outside air, the sequencing data amount of microbial DNA all reaches 5-7Gb base number, meets the requirement of order-checking saturation ratio.In 5 kinds of indoor pathogenic microorganisms of hospital, the relative abundance of Acinetobacter bauamnnii (Acinetobacterbaumannii) is 3.92%, the relative abundance of meningitis Neisser formula bacterium (Neisseriameningitidis) is 2.74%, the relative abundance of Acinetobacter lwoffii (Acinetobacterlwoffii) is 2.93%, the relative abundance of streptococcus pneumoniae (Streptococcuspneumoniae) is 1.75%, the relative abundance of mycobacterium tuberculosis (Mycobacteriumparascrofulaceum) is 1.87%, thus, can determine that the selection result of the indoor pathogenic microorganism of hospital is all reliable.The relative abundance of the pathogenic microorganism of hospital outdoor all below 0.3% (much smaller than 1%), therefore thinks hospital's pathogenic microorganism of not having of outdoor.
4, environmental pathogenic microorganism dustiness is determined
Based on the pathogenic microorganism of hospital indoor, determine the environmental pollution degree of hospital indoor, wherein, all microbe species numbers in the pathogenic microorganism species number/specific environment air in specific environment pathogenic microorganism dustiness=specific environment air.
Thus, calculate: the environmental pathogenic microorganism dustiness of hospital indoor is 0.32%, and the environmental pathogenic microorganism dustiness of hospital outdoor is 0.
Show, the environment of hospital indoor has the pollution of pathogenic microorganism, and the environment of hospital outdoor is without the pollution of pathogenic microorganism.
Claims (24)
1. determine the method for microbes in air kind, comprise following 1)-3):
1) gather microbes in air to be measured with sampling membrane, obtain the microbes in air to be measured be positioned on described sampling membrane;
2) be positioned at the DNA of the microbes in air to be measured on described sampling membrane described in extraction, obtain unpurified microbial DNA; With magnetic bead, purifying is carried out to described unpurified microbial DNA, obtain microbes in air DNA;
3) described microbes in air DNA is checked order, determine described microbes in air kind to be measured according to the sequence of described microbes in air DNA.
2. method according to claim 1, is characterized in that: described microbes in air DNA directly checks order without amplification.
3. method according to claim 1 and 2, is characterized in that: described 2) comprise following 21) and 22):
21) be positioned at microbes in air water to be measured on described sampling membrane or PBS elutes from described sampling membrane by described, obtain the liquid containing microbes in air to be measured; With the liquid containing microbes in air to be measured described in filtration membrane filtration, obtain being positioned at the microbes in air to be measured on described PES film;
22) extract the microbes in air DNA to be measured be positioned on described PES film, obtain described unpurified microbial DNA; With described magnetic bead, purifying is carried out to described unpurified microbial DNA, obtain microbes in air DNA.
4. method according to claim 3, is characterized in that: described 22) extracting described microbes in air DNA to be measured for extracting test kit with soil DNA, obtaining described unpurified microbial DNA; With described magnetic bead, purifying is carried out to described unpurified microbial DNA, obtain microbes in air DNA.
5. method according to claim 4, is characterized in that: described soil DNA extraction test kit is that the name of Mobio company is called
the test kit of DNAIsolationKit or Qiagen company
dNAStoolMiniKit.
6., according to described method arbitrary in claim 1-5, it is characterized in that: the aperture of described sampling membrane is 0.01-0.2 micron.
7., according to described method arbitrary in claim 1-6, it is characterized in that: 3) described order-checking high-flux sequence platform carries out.
8. the system of following I or II:
I, extract the system of microbes in air DNA, be made up of following A 1 and A2:
A1, the reagent extracting microbes in air DNA and/or test kit;
A2, for purify DNA magnetic bead or utilize the test kit of magnetic beads for purifying DNA;
II, determine the system of microbes in air kind, be made up of at least one in the system of described extraction microbes in air DNA and following B1, B2 and B3 these three kinds:
B1, the instrument gathering microbes in air and/or instrument;
B2, high-flux sequence platform;
B3, carry out the biological software of sequence alignment and/or microorganism classification.
9. arbitraryly in claim 1-7 describedly determine the application of the method for microbes in air kind in monitor air pollution situation.
10. system described in claim 8 is in the application determining microbes in air kind or detect in air pollution.
11. 1 kinds of methods determining specific environment microbes in air kind, is characterized in that, comprise the following steps:
For described specific environment, utilize air sampling pump to carry out air sampling, to obtain the testing sample comprising air microbe, wherein said air sampling pump comprises detachable filter and filter membrane;
DNA extraction is carried out, to obtain mixing microorganisms DNA to the described testing sample comprising air microbe;
Described mixing microorganisms DNA is checked order, to obtain sequencing result;
Described sequencing result and reference database are compared, to obtain comparison result; And
Based on described comparison result, determine the microbe species in described specific environment air.
12. methods according to claim 11, is characterized in that: described air sampling pump is SKC air sampling pump, and described detachable filter is the detachable filter of Pall company, and described filter membrane is PTEE filter membrane,
Optionally, the diameter of described PTEE filter membrane coordinates with described detachable filter, and aperture is 0.2 micron.
13. methods according to claim 12, is characterized in that: in specific environment, with the air input of at least 4L/min, within 23 hours, carry out described air sampling incessantly.
14. methods according to claim 11, is characterized in that: utilize Mobio company
dNAIsolationKit or Qiagen company
dNAStoolMiniKit carries out described DNA extraction.
15. methods according to claim 14, is characterized in that: utilize Mobio company
dNAIsolationKit, by following steps, carries out described DNA extraction:
Take out filter membrane, and after carrying out break process, utilize SolutionC1, aqua sterilisa or PBS to shake mixing and dissolve half an hour, to obtain the testing sample comprising air microbe;
In described testing sample, add SolutionC1, carry out washing after mixing successively and grind and the first centrifugal treating, to obtain the first centrifugal rear liquid;
Get the supernatant in described first centrifugal rear liquid, and add SolutionC2, carry out first successively after vortex mixing and hatch and the second centrifugal treating, to obtain the second centrifugal rear liquid;
Get the supernatant in described second centrifugal rear liquid, and add SolutionC3, carry out second successively after vortex mixing and hatch and the 3rd centrifugal treating, to obtain the 3rd centrifugal rear liquid; And
Get the supernatant in described 3rd centrifugal rear liquid, and add SolutionC4, after vortex mixing, obtain mixing microorganisms DNA,
Optionally, carry out described concussion mixing and dissolve 30min,
Optionally, utilize vortex instrument, under 1400rpm rotating speed, carry out described washing grind process 30min,
Optionally, under room temperature, 10000g condition, carry out described first centrifugal 30s,
Optionally, at 4 DEG C, carry out described first hatch 5min,
Optionally, under room temperature, 10000g condition, carry out described second centrifugal 1min,
Optionally, at 4 DEG C, carry out described second hatch 5min,
Optionally, under room temperature, 10000g condition, carry out described 3rd centrifugal 1min.
16. methods according to claim 11, is characterized in that: before carrying out described order-checking, comprise the step of described mixing microorganisms DNA being carried out to purifying further,
Optionally, the magnetic bead of Jin Maige company or AgencourtAMPureXP purification process is utilized to carry out described purifying,
Optionally, before carrying out described order-checking, comprise the step of described mixing microorganisms DNA being carried out to whole genome amplification further,
Optionally, whole genome amplification test kit is utilized to carry out described whole genome amplification.
17. methods according to claim 11, is characterized in that: utilize be selected from Hiseq, Miseq, IonTorrent, Proton, SOLiD, 454 and at least one of single-molecule sequencing device carry out described order-checking,
Optionally, the order-checking of two end 200bp is carried out.
18. methods according to claim 11, is characterized in that: described reference database is at least one being selected from common microbiological database, common drug resistant gene database and common ward infection bacterial genomes database,
Optionally, described reference database is at least one being selected from M5NR database, Nr database, Silva database, Greengenes database, RDP database and CARD database,
Optionally, described common ward infection bacterial genomes database is made up of the full-length genome of ward infection bacteriums all in NCBI.
19. methods according to claim 11, is characterized in that: before carrying out described compare of analysis, comprise further, remove the sequence that the low quality data in described sequencing result, joint pollution and the mankind pollute,
Optionally, the sequence removing mankind's pollution realizes to the sequence in human genome reference sequences Hg19 by removing comparison in described sequencing result,
Optionally, utilize be selected from SOAP, Bwa and Blat software one of at least carry out described compare of analysis,
Optionally, based on described comparison result, biological analysis software is utilized to carry out species taxonomy, to determine the microbe species in described specific environment air,
Optionally, described biological analysis software is at least one being selected from Metaphlan, Megan, Mother, MG-RAST and Galaxy.
20. 1 kinds of methods determining specific environment pollution situation, is characterized in that, comprise the following steps:
Utilize the method described in any one of claim 11-19, determine the microbe species in described specific environment air;
Based on the common bacterial classification of air microbe, the microbiota in described specific environment air is divided into common air microorganism and specificity air microbe;
Pathogenic microorganism is screened, to determine the pathogenic microorganism kind in described specific environment air from described specificity air microbe;
Based on the pathogenic microorganism kind in described specific environment air, determine the dustiness of described specific environment, wherein,
All microbe species in pathogenic microorganism kind/specific environment air in the dustiness=specific environment air of specific environment; And
Based on the dustiness of described specific environment, determine the pollution situation of described specific environment.
21. methods according to claim 20, is characterized in that, the common bacterial classification of described air microbe is following 48 kinds:
Hidden geodermatophilus obscurus, Modestobactermarinus, occupy rock tooth coccus, addicted to root Kocuria kristinae ad, micrococcus luteus, candiyeast, radiation hardness pseudomonas, hot bifid, Nocardia bacteria, propionibacterium acnes, the short shape bacillus of excrement, Cellvibrio, Arthrobacter, Da Songweier nocardia, meat bacillus, tiny bacterium, Paracoccus denitrificans, motionless lid coccus, Coriolis blood bacillus, the dynamic coccus of radiation, pineapple multi-source bacterium, Yue Hanxunshi Bacterium lacticum, fertilizer cellulomonas cartae, lactobacillus crispatus, the unusual coccus in Gobi desert, to make pottery Salmonella in distress, pantoea agglomerans, clostridium perfringens, lactobacillus reuteri, Pseudomonas stutzeri, South Pole bacillus, lactobacillus salivarius, bacillus megaterium, A Shi Arthrobacter, Ramlibactertataouinensis, middle village Salmonella, baby suis, pale yellow mycobacterium, chlorophenol Arthrobacter, outer chain Methylobacterium, Methylobacterium, coryneform bacteria, addicted to maltose Stenotrophomonas, intend promise Ka Shi Alba, South Pole bacterium, green thermophilic actinomycete, streptomyces coelicolor and leukonid.
22. methods according to claim 20, it is characterized in that, based on the pathogenic microorganism kind in described specific environment air, before determining the dustiness of described specific environment, comprise the step of pathogenic microorganism kind the selection result being carried out to certificate authenticity further.
23. methods according to claim 22, it is characterized in that, by calculating relative abundance and the order-checking saturation ratio of the pathogenic microorganism in described specific environment air, determine the reliability of pathogenic microorganism kind the selection result, the saturation ratio that wherein checks order is that each sample sequencing data amount reaches 5-7Gb base number, and the relative abundance of each microbe species that each sample is detected all is not less than 1%, is that described pathogenic microorganism kind the selection result indicates reliably
Optionally, utilize relative abundance and the order-checking saturation ratio of the pathogenic microorganism in specific environment air described in biological analysis computed in software, described biological analysis software is at least one being selected from Metaphlan, Megan, Mother, MG-RAST and Galaxy.
24. methods according to claim 20, is characterized in that, based on the dustiness of described specific environment, determine the pollution situation of described specific environment, comprise further:
When dustiness≤0.3%, described specific environment is pollution-free;
When dustiness is 0.3%-0.6%, described specific environment slight pollution;
When dustiness is 0.6%-1%, described specific environment intermediate pollution;
As dustiness >1%, described specific environment serious pollution.
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| CN114164260A (en) * | 2020-09-10 | 2022-03-11 | 中国科学院青岛生物能源与过程研究所 | Indoor micro-ecological environment livability detection method based on human body conditioned pathogens |
| WO2022051975A1 (en) * | 2020-09-10 | 2022-03-17 | The Procter & Gamble Company | Systems and methods of determining hygiene condition of interior space |
| CN114045217A (en) * | 2022-01-11 | 2022-02-15 | 至美时代生物智能科技(北京)有限公司 | Air microorganism sampling device and air microorganism sampling and detecting integrated device |
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