CN107190055A - Soil bacteria high flux absolute quantification method - Google Patents
Soil bacteria high flux absolute quantification method Download PDFInfo
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Abstract
The invention discloses a kind of soil bacteria high flux absolute quantification method, comprise the following steps：1), by E.coli HTAQ GFP thalline, vortex is resuspended in sterilized water, and regulation bacteria suspension is in ultraviolet specrophotometer OD600nm=1.0；2) absolute quantitation, is carried out to internal standard bacterial strain HTAQ GFP, internal standard bacterial strain E.coli HTAQ GFP absolute contents in bacterium solution are obtained；3), internal standard bacterial strain HTAQ GFP bacterium solutions are to soil sample and stir and evenly mix for addition；4) soil DNA, is extracted, 16S rRNA gene amplicon high-flux sequences are carried out, bacterial quorum sensing classification composition information and correspondence relative abundance is obtained；5) absolute content for obtaining soil native bacterium total amount and each taxon, is calculated according to Escherichia relative abundances in internal standard bacterial strain HTAQ GFP absolute contents and high-flux sequence result.
Description
Technical field
The invention belongs to edaphon field, it is related to and a kind of integrates high-flux sequence platform and bacterium internal standard method
Soil bacteria high flux absolute quantitation technology.
Background technology
Soil Microorganism substantial amounts, species is various, in cycle of matter in the earth, industrial and agricultural production or even medical domain etc.
Indispensable effect is played.Study biological community structure succession process in specific ecological environment, obtain its species and
Number change information, for further explaination with there is weight using microbial biochemical mechanism, protection microbial resources and diversity
Want meaning.
Observed from Leeuwenhoek first passage microscope after the presence of microorganism, it is a variety of to be used to probe into the microcosmic generation of microorganism
The method on boundary is arisen at the historic moment.Prior art has soil bacteria sizing technique：Dyeing-fluorescence microscope counting method, flat board culture are counted
Method, chloroform fumigate biomass C n2 method, quantitative real-time PCR etc., and soil bacterial community research method：Phosphatide
Aliphatic acid (PLFA) analytical technology, high throughput sequencing technologies etc..Wherein, flat board culture counting method or it can meet while obtaining micro- life
The requirement of species and quantity information, but only 1%-3% microorganism can be turned out using the technology in environment；High pass is measured
Sequence technology can meet a large amount of requirements for obtaining biological community structure classification information, but be confined to contain with relative abundance sign species
Amount.In Same Community structure, what relative abundance was characterized is advantage degree of the species in the structure of community, but can not be with
This speculates growth and reduction of the species (across sample) in time scale and space scale.
By technology restriction, for a long time, scholars are only capable of grinding to carry out with the increase and decrease that relative abundance substitutes sign microorganism
Study carefully, be the number change for more precisely representing microorganism, microbiologic population's classification information can be obtained again by being badly in need of exploitation one kind
The technology of bacterial community absolute content composition can be obtained, the defect of existing method is made up, it is ecological to further microorganisms
Tool is of great significance.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of soil bacteria high flux absolute quantification method；The present invention is solved
The deficiencies in the prior art, there is provided a kind of suitable for soil bacteria high flux absolute quantitation by taking bacterium domain in microorganism as an example
Technology, is conducive to improving the ecological research of number of bacteria, so that further research and utilization soil bacteria.
In order to solve the above-mentioned technical problem, a kind of soil bacteria high flux absolute quantification method of present invention offer, including with
Lower step：
1), internal standard bacterial strain is obtained：
By E.coli HTAQ-GFP thalline (centrifugation obtains incubated overnight E.coli HTAQ-GFP thalline), vortex is resuspended in
In sterilized water, regulation bacteria suspension is in ultraviolet specrophotometer OD600nm=1.0 (now cell concentration is about 5 × 108CFU mL-1)；
2), internal standard bacterial strain absolute quantitation：
Absolute quantitation is carried out to internal standard bacterial strain HTAQ-GFP using fluorescence microscope or colony counting method, obtained in bacterium solution
Mark bacterial strain E.coli HTAQ-GFP absolute contents；
3), to soil sample, (addition concentration is 10 to addition internal standard bacterial strain HTAQ-GFP bacterium solutions6cells g-1Left and right) and stir mixed
It is even, soil container is placed on ice bath during stirring；
4) soil DNA, is extracted, 16S rRNA gene amplicon high-flux sequences are carried out, bacterial quorum sensing classification is obtained
Constitute information and correspondence relative abundance (door, guiding principle, mesh, section, category)；
5), according to Escherichia relative abundances in internal standard bacterial strain HTAQ-GFP absolute contents and high-flux sequence result
Calculate the absolute content for obtaining soil native bacterium total amount and each taxon (door, guiding principle, mesh, section, category).
It is used as the improvement of the soil bacteria high flux absolute quantification method of the present invention：
The step 2) in microscope to internal standard bacterial strain HTAQ-GFP absolute quantitations operation be：First by clean cover glass
(22mm × 26mm) is placed on slide, and (being slowly added) 8 μ L steps 1 are added at cover glass and slide slot edge) institute
The bacteria suspension obtained, randomly selects 20 visuals field and photographs to record, fluorescence signal is counted, and is hanged according to visual field size correspondence bacterium
Liquid product calculates internal standard bacterial strain HTAQ-GFP concentration in bacterium solution.
It is used as the further improvement of the soil bacteria high flux absolute quantification method of the present invention：
The step 3) in bacteria suspension addition be：No more than 10% (that is, the 10% of≤dry ground weight) of dry ground weight,
5 ± 1min is stirred and evenly mixed, for bacterial growth influence quantitative result in preventing and treating whipping process, the process is carried out on ice bath.
It is used as the further improvement of the soil bacteria high flux absolute quantification method of the present invention：The step 5) in soil soil
Writing bacteria total amount (T) calculation formula isRi represents Escherichia phases in addition internal standard bacterial strain treatment group
To abundance；Rc represents to be not added with Escherichia relative abundances in internal standard strain control group, and such as soil is not used muck and can not set
Put control group, Rc=0；Ii is internal standard bacterial strain HTAQ-GFP absolute contents (microscopic counting result or plate count result)；
Obtain after soil bacteria total amount, each taxon absolute content is equal to (T+Ii) and the taxon relative abundance
Product.
In summary, the invention provides a kind of the complete of the soil bacteria absolute quantitation based on platform by high-flux sequence
Adjusting technique, including：(1) optimization two kinds of operating process absolute quantitation internal standard bacterial strains of microscope count method and colony counting method are provided to add
Dosage.(2) so that bacterial cell is internal standard substance and combines high throughput sequencing technologies, bacterium high flux Absolute quantification is realized.(3)
The relative abundance and absolute content information of bacterial community composition are obtained simultaneously.High flux bacterium absolute quantitation constructed by the present invention
Technical method is simple, can disclose bacterium exact amount under environment succession and change, make to compare more accurately and reliably across sample, can enter one
Step is applied to the ecological association area research of number of bacteria.
The invention process of the present invention is specific as follows：
Microorganism can be divided into ancient bacterium, bacterium, three domains of fungi according to rRNA homologys.The present invention is led to by taking bacterium domain as an example
Cross bacterium specific primer and enter performing PCR amplification enrichment to a bacterial 16 S rRNA fragment, then carry out high-flux sequence point
Analysis.Under normal circumstances, sequencing can be carried out to 50,000-10 ten thousand DNA moleculars in a sample, measure sequence pair is obtained with this
The division bacteria information (door, guiding principle, mesh, section, category) answered, and the total sequence bar number relative abundance of sample DNA is accounted for DNA sequence dna bar number
(%) characterizes the content of variety classes bacterium, to reach the purpose to a sample bacterium structure of community research.
Research shows that sequencing depth is deeper, more can reflect the truth of biological community structure, but be sequenced into accordingly
This can also increase.For balance both sides relation, the sequencing depth of one sample of in the market is generally in 50,000 sequences or so.It is relatively rich
Degree can characterize species advantage degree as a content ratio in a sample, and when comparing for different sample rooms, with
Percent abundance is biased as more just losing.For example in two pedotheques, A and B samples it is containing bacterial number respectively
107cells g-1With 109cells g-1, certain bacterium category percent abundance in two samples is all 1%, characterizes this with percentage thin
Content of the Pseudomonas in two samples just indifference, and actually its absolute content in A and B samples is respectively 105cells
g-1With 107cells g-1, there are magnitude differences.So when comparing across sample, the sign of relative abundance may draw
Rise and mislead, really change so as to cover structure of community, and the result that absolute content is characterized is more accurate.
To realize the absolute quantitation of bacterium high-flux sequence, the present invention adds absolute content known to one plant in pedotheque
Bacterium E.coli HTAQ-GFP somatic cells belong to Escherichia coli as internal standard substance, the bacterium, major survival in enteron aisle,
Content in the soil of muck is not applied can be neglected.It is allowed to obtain group by high-flux sequence jointly with bacterium in sample
Fall the relative abundance of composition, for example, add internal standard bacterial strain to 106cells g-1, it is assumed that its place in last high-flux sequence data
The ratio of category is 1%, then soil bacteria total amount is 106÷ 1%=108cells g-1, then it is multiplied by with total amount other classification
Percentage is to obtain corresponding absolute content.
There is researcher to attempt to extract soil DNA again to reach the mesh of absolute quantitation after adding DNA of bacteria in pedotheque
, and added in the present invention be microorganism cell to soil, it is close with soil native bacterium cellular attributes, undergo it is similar
Lose (soil DNA extraction step), so as to the absolute content of preferably estimation soil background bacterium；And can be directly to bacterium
Body cell is quantified, as a result more accurate, and these two aspects is all an advantage over DNA as internal standard substance.
Absolute quantitation is carried out to internal standard bacterial strain the invention provides two methods：Flat board culture counting method and fluorescence microscope
Counting method.Flat board culture counting method is consistent with traditional operation；Microscope count method is improved in loading step, and standard operation is
Slide is first placed, drop is added dropwise, then is capped cover glass, the problem of existing is can not to know the follow-up meter of dropwise addition droplet size influence
Calculate, and the easily observation of generation aeration and calculating.Be in the present invention after cover glass is placed on slide, from cover glass with
8 μ L liquid are added in gap between slide, it is ensured that given volume is convenient to be calculated, and it also avoid the generation of bubble.
The present invention compared with prior art, by definitely containing known to the simple addition in original high-flux sequence flow
The internal standard bacterial strain E.coli HTAQ-GFP of amount can obtain each point of bacterium simultaneously so as to realize the absolute quantitation of soil bacterial community
Bacterium real change in the relative abundance and absolute content of class unit, reaction environment During Succession, makes to compare across sample and more may be used
Lean on, advance the ecological research of bacterial number.
Brief description of the drawings
The embodiment to the present invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 is workflow schematic diagram of the invention.
Fig. 2 is internal standard bacterial strain HTAQ-GFP microscopic countings result and flat board culture count results dependency relation figure.
Fig. 3 is fluorescence microscope sample-adding method (a) and traditional sample-adding method (b) comparison diagram in this method.
Fig. 4 is principal component analysis figure.
Fig. 5 is the horizontal absolute content (a) of door and relative abundance (b) comparison diagram in sodium azide processing soil.
Fig. 6 changes (b) trend comparison diagram to belong to horizontal absolute content (a) in sodium azide processing soil with relative abundance.
Embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This.
(1) preparation method of the E.coli HTAQ-GFP bacterium in the present invention is：By E.coli O157:H7EDL933 is made
Competent cell, heat shock imports green fluorescent protein (GFP) plasmid (Cloning vector pGFPuv), can be according to《Molecule gram
Grand experiment guide (third edition)》Middle calcium chloride prepares competence Escherichia coli method and carried out, and is specially：
1) one single bacterium colony of picking from 37 DEG C of culture 14-18h flat board, goes to one containing 100mL LB culture mediums
In 500mL triangular flasks, in 37 DEG C of 250rpm cultures 3h.
2) bacterium is gone in a sterile, disposable, ice-cold 50mL PA tube, placed on ice
10min, makes culture be cooled to 0 DEG C.
3) 4000rpm centrifuges 10min and obtains cell under the conditions of 4 DEG C.
4) nutrient solution is poured out, pipe is inverted 1min so that last trace nutrient solution flows to end.
5) per the 0.1mol L of 50mL initial incubations liquid 30mL precoolings-1CaCl2-MgCl2(80mmol L-1MgCl2,
20mmol L-1CaCl2) every part of cell precipitation is resuspended.
6) 4000rpm centrifuges 10min and reclaims cell under the conditions of 4 DEG C.
7) nutrient solution is poured out, pipe is inverted 1min so that last trace of liquid is flow to end.
8) per the 50mL initial incubations thing ice-cold 0.1mol L of 2mL-1CaCl2Every part of cell precipitation is resuspended.
9) now, subsequent transformation experiment can directly be carried out or by cell cryopreservation in -80 DEG C.
10) 50 μ L competent cell suspensions are drawn with the sterile pipette tip of cooling to be transferred in 1.5mL sterile centrifugation tubes, added
The μ L of GFP plasmids 1, gently rotate to mix content, 30min are placed in ice.
11) 1.5mL centrifuge tubes are put into 42 DEG C of water-baths, stand and place 90s.
12) quickly 1.5mL centrifuge tubes are transferred in ice bath, cell is cooled down 1-2min.
13) 250 μ L are added to balance to the LB culture mediums of room temperature, 1h is cultivated under the conditions of 37 DEG C of 250rpm.
14) 200 μ L bacterium solutions are taken equably to be coated in LB flat boards, LB flat boards contain antibiotic Ampicillin concentration for 100 μ g
mL-1, incubated overnight under the conditions of being upside down in 37 DEG C after liquid is absorbed, picking has the bacterium colony of green fluorescence under uviol lamp.
15) in 20mL LB liquid mediums, (concentration of Ampicillin containing antibiotic is 100 μ under the conditions of 37 DEG C, 250rpm
g mL-1) in incubated overnight 16h.
16) it is stand-by in -80 DEG C of preservations by fungi preservation in 25% glycerine.
(2) preparation method of the internal standard bacterial strain HTAQ-GFP bacteria suspension mother liquors in the present invention is：
1) the bacterial strain HTAQ-GFP for drawing the preservation of the glycerine of 200 μ L 25% is inoculated in 20mL LB liquid (containing antibiotic
Ampicillin concentration is 100 μ g mL-1) in culture medium.
2) 14-18h is cultivated under the conditions of 37 DEG C, 250rpm.
3) incubated overnight bacterium solution is transferred in 50mL centrifuge tubes, supernatant is given up in centrifugation under the conditions of 4 DEG C of 5000rpm, obtains
Thalline.
4) bacteria suspension is centrifuged again after thalline being resuspended with 40mL aqua sterilisas, gives up supernatant and obtains thalline.Repeat the step
Once.
5) it will finally obtain thalline to be resuspended in aqua sterilisa bacteria suspension mother liquor is made, with ultraviolet specrophotometer by bacteria suspension
OD600nm1.0 are adjusted to, now bacterial strain HTAQ-GFP concentration ≈ 5 × 108CFU mL-1。
Embodiment 1, using fluorescence microscope counting method and flat board culture counting method internal standard bacterial strain HTAQ-GFP is carried out it is exhausted
To quantitative, following steps are carried out successively：
1) prepared by 2 times of gradient dilution bacteria suspensions：
The above-mentioned internal standard bacterial strain HTAQ-GFP bacteria suspensions mother liquors of 500 μ L are drawn in 1.5mL sterile centrifugation tubes, 500 μ L are added
Blow and beat and mix after aqua sterilisa, 2 times of dilution bacteria suspensions are made.500 μ L are drawn from 2 times of dilution bacteria suspensions to centrifuge in 1.5mL sterilizings
Guan Zhong, adds and mixing is blown and beaten after 500 μ L aqua sterilisas, and 4 times of dilution bacteria suspensions are made；By that analogy, 2 times of gradient dilution bacterium are made to hang
Liquid (mother liquor is diluted 2,4,8,16,32,64,128 times).
2) fluorescence microscope counting method：
First clean cover glass (22mm × 26mm) is placed on slide.The above-mentioned internal standard bacterial strain HTAQ- of 8 μ L are drawn respectively
2 times of gradient dilution bacteria suspensions of GFP, in cover glass with being slowly added at slide slot edge.It is placed under fluorescence microscope, with
20 visuals field of machine selection are taken pictures to be counted to fluorescence signal, and bacterial strain in bacteria suspension is calculated according to visual field correspondence bacteria suspension volume
HTAQ-GFP concentration (cells mL-1)。
3) flat board culture counting method：
By 2 times of gradient dilution bacteria suspensions of internal standard bacterial strain HTAQ-GFP (mother liquor is diluted 2,4,8,16,32,64,128 times)
10 times of gradient dilutions are distinguished to 102CFU mL-1, draw 100 μ L and be laid on LB flat boards that (concentration of Ampicillin containing antibiotic is
100μg mL-1).Incubated overnight 14-18h, records clump count and calculates bacterial strain HTAQ-GFP concentration (CFU mL in bacteria suspension-1)。
4) fluorescence microscope count results/flat board culture count results standard curve is drawn：
Obtain after two kinds of count method results (as shown in table 1), using microscopic counting result as abscissa (X), flat board
Culture count results are ordinate (Y), draw internal standard 2 times of bacterial strain, two kinds of gradient dilution bacteria suspension method of counting standard curve, such as
Shown in Fig. 2.
The microscope count method of table 1 and flat board culture count method result
By examples detailed above, we can obtain drawing a conclusion：This method to fluorescence microscope loading methods by changing
Enter, reduce and the bubble that bacteria suspension is produced during cover glass is covered on slide again is first added dropwise in the past so that count results
It is more accurate.Fluorescence microscope visual field Green fluorescence signal is as shown in Figure 3 a, more convenient without GFP labeled strains compared to tradition
Count, and fluorescence microscope count results include can not cultivate the thalline HTAQ-GFP of state, it is true closer to internal standard bacterial strain
Add value so that follow-up result of calculation is more accurate.As shown in Fig. 2 fluorescence microscope counting method result is counted with flat board culture
Method result has preferable dependency relation (R2=0.998), show that fluorescence microscope counting method result is stable, available for internal standard bacterial strain
Absolute quantitation.In addition, flat board culture counting method also can carry out absolute quantitation to it, the scope of application of the technology is expanded, is fitted
For different condition laboratory.
Embodiment 2, addition internal standard bacterial strain HTAQ-GFP, obtaining soil bacteria absolute content, (total amount and each categorization levels contain
Amount)
1) internal standard bacterial strain is added to sample：
200 μ L, 10 times of dilution bacterial strain HTAQ-GFP bacteria suspensions mother liquors are drawn in the beaker for filling 10g soil samples (dry ground),
In stirring and evenly mixing 5min under condition of ice bath, bacterial strain HTAQ-GFP final concentration of 10 is obtained6cells g-1The soil sample of left and right.To add
The soil sample of isometric sterilized water is used as control treatment.
2) soil DNA is extracted：
Weigh 0.25g steps 1) gained soil extract soil DNA, this example uses MoBioDNA is carried
Kit is taken, operating procedure is such as according to kit standard flow.
3) high-flux sequence：
Soil sample DNA is subjected to bacterial 16 S rRNA gene amplicons high-flux sequence (Illumina Miseq platforms), obtained
Obtain the relative abundance of each categorization levels of pedotheque bacterial community (door, guiding principle, mesh, section, category).It need to further illustrate, high pass
Measure in sequencing result classification annotation, microorganism classification can identify " door detailed outline section category " five different classifications levels, " category " water
Flat to belong to technology and reach most accurate classification, internal standard bacterial strain HTAQ-GFP is categorized as Escherichia
(Escherichia)。
4) result is calculated：
Internal standard bacterial strain HTAQ-GFP absolute contents and relative abundance (bacterial strain HTAQ-GFP are obtained from high-flux sequence result
It is categorized as Escherichia, i.e. Escherichia relative abundances) after, according to formulaCalculate soil original inhabitants
Bacteria total amount, Ri represents Escherichia relative abundances in addition internal standard bacterial strain treatment group；Rc represents to be not added with internal standard bacterial strain pair
According to Escherichia relative abundances in group, such as muck, which is not used, in soil can be not provided with control group, Rc=0；Ii is internal standard bacterial strain
HTAQ-GFP absolute contents (fluorescence microscope count results).Obtain after soil bacteria total amount, each taxon absolute content etc.
In (T+Ii) and the product of the taxon relative abundance.
It is (high with relative abundance obtaining internal standard bacterial strain absolute content (fluorescence microscope count results) by examples detailed above
Flux sequencing result) after, samples-soil native bacterium total amount can be calculated according to formula.Internal standard bacterial strain HTAQ-GFP adds in treatment group
Plus concentration is 1.23 × 106cells g-1(Ii)；In high-flux sequence data, Escherichia relative abundances be 0.0006 ±
Escherichia relative abundances are 0.0000230 ± 0.0000100 (Rc) in 0.0002 (Ri), control group, therefore, bring public affairs into
FormulaCalculate, soil native bacterium total amount T=2.31 × 109±8.92×108cells g-1。
After soil native bacterium total amount is obtained, you can according to the taxon phase in (T+Ii) × high flux data result
The absolute content of the taxon is drawn to abundance.For example, in treatment group, Acidobacteria bacterium door is relative in door level
Abundance is that 0.241 ± 0.018, Acidobacteria bacterium door absolute content is [(2.31 × 109±8.92×108)+(1.23
×106cells g-1)] × (0.241 ± 0.018)=9.59 × 108+1.42×108cells g-1.It is tested soil native bacterium
Horizontal absolute content (the cells g of door-1) constitute as shown in Fig. 5 (a).In category level Gp16 belong to relative abundance be 0.0052 ±
0.012, Gp16 category absolute content is [(2.31 × 109±8.92×108)+(1.23×106cells g-1)]×(0.0052
± 0.012)=2.10 × 108+6.14×107cells g-1.In this way, just can obtain different classifications level in treatment group (door,
Guiding principle, mesh, section, category) on absolute content composition.
Embodiment 3, soil bacteria high flux absolute quantification method are applied to sodium azide and handle soil sample, carry out successively as follows
Step：
1) in soil addition 0.1% sodium azide (w/w), under the conditions of 28 DEG C cultivate 14 days, take respectively culture 0 day
And the soil sample 10g of 14 days is in case follow-up addition internal standard bacterial strain HTAQ-GFP.
2) internal standard bacterial strain is added to sample：
200 μ L, 10 times of dilution bacterial strain HTAQ-GFP bacteria suspensions mother liquors are drawn in the beaker for filling 10g soil samples (dry ground),
In stirring and evenly mixing 5min under condition of ice bath, bacterial strain HTAQ-GFP final concentration of 10 is obtained6cells g-1The soil sample of left and right.
3) soil DNA is extracted：
Weigh 0.25g steps 2) gained soil extract soil DNA, this example uses MoBioDNA is carried
Kit is taken, operating procedure is such as according to kit standard flow.
4) high-flux sequence：
Soil sample DNA is subjected to bacterial 16 S rRNA gene amplicons high-flux sequence (Illumina Miseq platforms), obtained
Obtain the relative abundance of each categorization levels of pedotheque bacterial community (door, guiding principle, mesh, section, category).
5) according to calculation in embodiment 2, the absolute total amount of native bacterium and difference in sodium azide processing soil are obtained
The absolute content of bacterium in categorization levels.
By examples detailed above, it can be learnt according to soil bacteria high flux absolute quantification method, soil sample is by sodium azide
Reason is after 14 days, and native bacterium total amount is by 3.86 × 108±5.27×107cells g-1Significantly reduce to 1.27 × 108±7.09×
106cells g-1.In door level, sodium azide has preferable inhibition to most bacterium door, shows as absolute content and shows
Write and decline, such as Acidobacteria, Actinobacteria, Candidatus Saccharibacteria,
Gemmatimonadetes etc..In category level, 97.36% bacterium belongs to absolute content and shows downward trend, wherein
Conexibacter, Phenylobacterium, Porphyobacter, Gp16 are significantly reduced, specific delta data such as Fig. 6 a institutes
Show.
From above example as can be seen that the present invention can pass through simple internal standard bacterial strain on the basis of high-flux sequence
HTAQ-GFP addition reaches the purpose to soil bacterial community high flux absolute quantitation, and method is simple, is widely used, can be many
Promote the use of in field.
Contrast experiment 1, the microscope count method in embodiment 1 made into as described in routine techniques, i.e. first place and carry glass
Piece, is added dropwise drop, then be capped cover glass；Remaining is equal to embodiment 1.
Acquired results are：As shown in Figure 3 b, using traditional loading methods, bubble is easily produced in loading liquid, so that
Obtain and fill incomplete between cover glass and slide so that area of visual field correspondence liquid volume is less than normal, so that internal standard bacterial strain
Absolute quantitation result is less than normal.
Contrast experiment 2, make the addition concentration of the internal standard bacterial strain HTAQ-GFP in embodiment 2 into 1.23 × 1010cells
g-1(T10)、1.23×109cells g-1(T9)、1.23×108cells g-1(T8)、1.23×107cells g-1(T7)、
1.23×105cells g-1(T5)。
Acquired results are：The absolute total amount of soil native bacterium is calculated according to formula in embodiment 2, as shown in table 2, as a result
Show, addition internal standard bacterial strain HTAQ-GFP concentration is 1.23 × 105、1.23×107、1.23×108、1.23×109、1.23×
1010cells g-1The calculated absolute total amount of soil native bacterium is 1.23 × 10 with addition concentration6cells g-1Without significance difference
It is different.But, the horizontal bacterium absolute content composition principal component analysis of category shows, various concentrations internal standard bacterial strain HTAQ-GFP addition meeting
Certain influence is produced on bacterial community result.As shown in figure 4, T10-T8 bacterial communities belong to horizontal absolute content composition distance collation
Farther out, T7-T5 distance collations are nearer.Therefore, it is 10 in internal standard bacterial strain HTAQ-GFP additions8-1010cells g-1In the range of
When, the addition of external source bacterial strain is larger on the indigenous structure of community influence of soil, and addition is 105、107cells g-1With 106cells
g-1Additive effect is close, and influences smaller to background structure of community.
Table 2, the absolute total amount of soil native bacterium
Contrast experiment 3, Setup Experiments and embodiment 3 are consistent, but without internal standard bacterial strain HTAQ-GFP, remaining is equal
In embodiment 3.
Acquired results are：Because without internal standard bacterial strain HTAQ-GFP additions, the absolute of soil bacteria can not be calculated
Content, can only be represented with the result of high-flux sequence relative abundance.Relative abundance result shows that soil is handled by sodium azide
After 14 days, Bacteriophyta such as Acidobacteria, Actinobacteria, Candidatus Saccharibacteria,
Gemmatimonadetes is remarkably decreased with actual absolute content and is not inconsistent (Fig. 5 b) without significant changes.In category level,
Conexibacter, Phenylobacterium, Porphyobacter, Gp16 significantly reduce (Fig. 6 b), definitely contain with actual
Amount, which is remarkably decreased, completely contradicts.The characterization result of relative abundance can cause misleading conclusion, it is impossible to reflect the true of bacterial content
Consolidation.
Finally, in addition it is also necessary to it is noted that listed above is only several specific embodiments of the invention, it is clear that be not limited to
Above example, can also there is many deformations.One of ordinary skill in the art can directly export from present disclosure
Or all deformations associated, it is considered as protection scope of the present invention.
Claims (4)
1. soil bacteria high flux absolute quantification method, it is characterized in that comprising the following steps：
1), internal standard bacterial strain is obtained：
By E.coli HTAQ-GFP thalline, vortex is resuspended in sterilized water, and regulation bacteria suspension is in ultraviolet specrophotometer OD600nm
=1.0；
2), internal standard bacterial strain absolute quantitation：
Absolute quantitation is carried out to internal standard bacterial strain HTAQ-GFP using fluorescence microscope or colony counting method, internal standard bacterium in bacterium solution is obtained
Strain E.coli HTAQ-GFP absolute contents；
3), internal standard bacterial strain HTAQ-GFP bacterium solutions are to soil sample and stir and evenly mix for addition, and soil container is placed on ice bath during stirring；
4) soil DNA, is extracted, 16S rRNA gene amplicon high-flux sequences are carried out, bacterial quorum sensing classification composition is obtained
Information and correspondence relative abundance；
5), calculated according to Escherichia relative abundances in internal standard bacterial strain HTAQ-GFP absolute contents and high-flux sequence result
Obtain the absolute content of soil native bacterium total amount and each taxon.
2. soil bacteria high flux absolute quantification method according to claim 1, it is characterized in that：
The step 2) in microscope to internal standard bacterial strain HTAQ-GFP absolute quantitations operation be：Clean cover glass is first placed in load glass
On piece, from cover glass and slide slot edge at add 8 μ L steps 1) obtained by bacteria suspension, randomly select 20 visuals field and take pictures
Record, is counted to fluorescence signal, and calculates internal standard bacterial strain HTAQ- in bacterium solution according to visual field size correspondence bacteria suspension volume
GFP concentration.
3. soil bacteria high flux absolute quantification method according to claim 2, it is characterized in that：
The step 3) in bacteria suspension addition be：No more than the 10% of dry ground weight, 5 ± 1min is stirred and evenly mixed, is stirred for preventing and treating
Bacterial growth influences quantitative result during mixing, and the process is carried out on ice bath.
4. soil bacteria high flux absolute quantification method according to claim 3, it is characterized in that：
The step 5) in soil native bacterium total amount (T) calculation formula beRi represents to add internal standard bacterium
Escherichia relative abundances in strain treatment group；It is relatively rich that Rc represents to be not added with Escherichia in internal standard strain control group
Muck, which is not used, in degree, such as soil can be not provided with control group, Rc=0；Ii is internal standard bacterial strain HTAQ-GFP absolute contents；
Obtain after soil bacteria total amount, each taxon absolute content is equal to multiplying for (T+Ii) and the taxon relative abundance
Product.
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