CN105331702B - The method of quantitative determination liquor fermentation process microbiologic population composition - Google Patents
The method of quantitative determination liquor fermentation process microbiologic population composition Download PDFInfo
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Abstract
The present invention relates to the methods that microbiologic population during a kind of quick measurement liquor fermentation forms, it is characterized in that the specific amplified of the specific microorganism species of some during by reaching liquor fermentation using door specific primer in PCR reaction systems, and quantitative amplification is carried out using qPCR systems, obtain quantitative judge.The present invention is according to known microorganisms class, Bacteroidetes, Proteobacteria, Firmicutes are separately designed, the primer of mutual bacteria door and actinomyces door, the quantitative amplification based on door specific primer the macrostructure of microbiologic population can change during quickly investigating liquor fermentation in the level of door.
Description
Technical field
It is to be based on door specific primer the present invention relates to microbiologic population's operating method in biotechnology
(phylum-specific primer, PSP) measures the quick PSP- of microbiologic population's composed structure during liquor fermentation
QPCR quantitative approach.
Background technology
Natural complexity microbiologic population is generally by tens, hundreds of even more microorganism groups not of the same race at and the composition
Dynamic change can occur under different environmental conditions.The major part of strain included by almost all of natural microbial group
The mankind know about it is very few, so to the natural microbial group during liquor fermentation drop into row scene quickly structure composition quantitatively also
It is extremely difficult.Common technology includes at present：
First, classical culture protocols；For example most of microorganism belongs to and does not cultivate bacterium in pit mud, is trained using conventional medium
Foster cellar mud microorganisms speed is slow, and appraisal amount is very big；
Second is that pyrophosphoric acid high-flux sequence method；This is a kind of structure of community quantitative determination side of domestic and international prevalence at present
Method is generally completed by biotech company's Delegation Server, expensive, and the sequencing number that can be returned is up to hundreds of thousands
Item, generally can be with qualitative, quantitative to category；
Third, FISH technology；FISH, that is, fluorescence in situ hybridization technologies are mutual based on base
The principle of benefit, by the nucleic acid sequence of a bit of (usual 15-30 base, such as 16S rRNA segments) fluorescent marker as spy
Needle hybridizes with the target sample on glass slide, and with the single-minded combination of target sequence, particular core is shown by detecting hybridization site fluorescence
The presence of nucleotide sequence and quantity；
Fourth, DGGE/TGGE technologies；DGGE, that is, denatured gradient gel electrophoresis technologies are
It is proposed at first for detecting DNA mutation within 1979 by Fischer and Lerman.Myers etc. uses " GC in 1985 in DGGE
Clamping plate " and heteroduplex technology.Muzyer etc. confirms this technology in microorganisms group genetic diversity and kind for 1993
It is highly effective in terms of constellation variance.Temperature gradient gel electrophoresis (the temperature gradient of chemical denaturant were replaced later
Gel electrophoresis, TGGE) it is deriving technology.DGGE/TGGE is the difference that behavior is melted according to double-stranded DNA segment,
Detach similar length but the different DNA marker segment (rRNA or rDNA) of sequence in PCR product.One on DGGE/TGGE figures
Band can represent a microbe groups, and categorization levels can conveniently judge dominant bacteria or function bacterium up to kind；
Fifth, T-RFL technologies；T-RFLP, that is, Terminal-Restriction fragment length poly-
Morphism technologies are developed by RFLP.T-RFLP technical principles are by mono- primer mark fluorescence of PCR, by PCR amplification
Product digestion with restriction enzyme generates the restriction fragment of different length, and a portion contains fluorescence, forms T-RFLP
Collection of illustrative plates discloses the information such as type, quantity of microorganism in sample；
Sixth, RAPD technologies；RAPD is a kind of polymorphic monitoring technology of DNA, and by PCR amplification, amplified production is through agarose
After gel electrophoresis separation, ethidium bromide staining, to detect DNA fragmentation polymorphism.It is a kind of genome to entire unknown nucleotide sequence
Carry out the molecular engineering of polymorphism analysis.RAPD technologies are unique in that：1) mould pull DNA requirement it is few, purity requirement is low, adopts
Use random primer；2) it is easy to operate quickly, be not required to molecule hybridization and etc., directly progress DNA polymorphism analysis；3) primer needed for
Short, the binding site in whole gene group is more, and coverage rate is big, and primer can be used with, a variety of primer pair genomes can be used to carry out
The Polymorphism Analysis of large area；4) recall rate is high.But RAPD result poor reproducibilities；
Seventh, SSCP technologies；1989, Japanese Scientists Orita had polymorphism using DNA single stranded conformationals, non denatured
When polyacrylamide gel electrophoresis the difference of mobility come analyze DNA it is single-stranded in gene mutation and propose SSCP (Single-
Strand Conformation Polymorphism) concept.SSCP technologies are applied to the composition of microbiologic population by Lee etc.
Analysis.The argentation of the sensitivity such as Orita directly dyes the gel after electrophoresis, establishes PCR-SSCP analytic approach, carries
The high simplicity and sensitivity of detection mutation method.PCR-SSCP method advantages：1) it can be detected with non-isotopic methods, PCR productions
It can direct electrophoresis after object denaturation；2) experiment is not high to DNA original material purity requirements, and requirement is few, cost is relatively low；3) it is suitable for full-page proof
This screening, without knowing DNA sequence dna in advance；4) polymorphism and the mutation on any sites DNA are can detect, sensitivity is high.But it should
Also there are many deficiencies, such as analysis result to be influenced by many factors for technology, and different experiment conditions may cause entirely different
Result；
Eigth, AFLP technologies；AFLP (Amplified Fragment Length Polymorphism) technology 1993 by
Dutch Zabeau and Vos is created, and is combined RFLP and RAPD technical characterstics, is overcome RFLP technical sophistications, has radiological hazard
With the shortcomings that RAPD technical stabilities are poor, recessive inheritance is presented in label, while have concurrently RAPD high efficiency and RFLP it is repeatable
Property feature.The technology is considered as the technology that polymorphism is the abundantest in current DNA fingerprinting technology, main excellent
Point：Required amount of DNA is few, reproducible, polymorphism is strong, high resolution, need not hybridize.But somewhat expensive, the purity to DNA
It is higher etc. with the quality requirement of restriction endonuclease.
But above-mentioned all technologies can not all accomplish micro- life during two hours or so the quick measurement liquor fermentations in scene
The composed structure of object group.
Invention content
The present invention is to provide a kind of quantitative judge significant effect to avoid above-mentioned deficiency of the prior art, prepare
The method of easy, low-cost quantitative determination liquor fermentation process microbiologic population composition.
The present invention is to solve technical problem to adopt the following technical scheme that：
The present invention, which quickly measures the characteristics of method that microbiologic population forms during liquor fermentation, is：By anti-in PCR
It answers and specificity is carried out to the specific microorganism species of some during reaching liquor fermentation using door specific primer in system
Amplification, and quantitative amplification is carried out using qPCR systems, obtain quantitative judge.
The present invention quickly measures the characteristics of method that microbiologic population forms during liquor fermentation and lies also in：By following step
Suddenly door specific primer is prepared：
Step a, the macro genome of extraction brewing microorganism group sample is as PCR amplification template；
Step b, the rDNA expanded in template is expanded close to overall length 16s rDNA sequences, amplimer 27F
(5 '-AGA GTT TGA TCC TGG CTC AG-3 ') and 1492R (5 '-GGT TAC CTT GTT ACG ACTT-3 '), is obtained
Obtain amplified production；
Step c, double by carrying out TA clones, blue hickie screening and hickie bacterium colony successively after purification for the amplified production
To sequencing, no less than 200 effective clones are obtained, effective clone refers to that last bidirectional sequencing sequence passes through sequence ratio
To really belonging to 16s rDNA sequences；
Step d, design of primers is completed by phylogenetic analysis for effective clone's bidirectional sequencing sequence.
The present invention quickly measures the characteristics of method that microbiologic population forms during liquor fermentation and lies also in：
Configuration to the qPCR systems：It is to use qPCR kits and qPCR instruments, and require qPCR instruments that can examine
The fluorescent material that corresponding kit includes is surveyed, the fluorescent material includes EvaGreen and SYBR green；
Setting and operation for the condition of the qPCR systems；Expanded according to different microorganisms group and different doors
The adjusting and optimizing of increasing condition, the primer that whens different doors of the same group of quantitative judge is designed have similar Tm values as possible,
To be tested and applied in identical amplification condition；
Detection for the product of the qPCR systems：During method is established, detected by agarose gel electrophoresis
The purity and quality of amplified production；When amplified production reaches single master tape and the analysis of Tm values has single product peak, no longer need
Glue detection product is run, qPCR only needs to analyze and determine by Tm values when use, and qPCR is exported simultaneously using qPCR instruments
Amplification curve, quantitative result and Tm value analysis results.
The present invention quickly measures the characteristics of method that microbiologic population forms during liquor fermentation and lies also in：
The door specific primer is Bacteroidetes specific primer, and primer pair nucleotides sequence is classified as:
320 (5'-CGC ACG GGT GAG TAA CAC GTAT-3'), 321 (5'-GGG GAT AAA TCC TCT
CAG TTC CCCT-3')；
The door specific primer or the specific primer for Proteobacteria, primer pair nucleotides sequence are classified as:
317 (5'-CCG TAG CTG GTC TGA GAG GAT GAT AA-3'), 319 (5'-CAC GCT TTA CGC
CCA GTA ATT CCYA-3')；
The door specific primer or the specific primer for Firmicutes, primer pair nucleotides sequence are classified as：
J1 (5'-CAG CAG TGG GGA ATC TTCC-3'), J2 (5'-TAG CCG GGG CTT CCT CCT-3')；
The door specific primer is mutual bacteria door specific primer, and primer pair nucleotides sequence is classified as：
J7 (5'-AAA CCG CTT TCA GCA GGG AC-3'), J8 (5'-TAA CTC CCG ACC AAA CAA
GC-3')。
The door specific primer is actinomyces door specific primer, and primer pair nucleotide sequence is as follows:
J9 (5'-AGC GAA CGG GAT TAG ATA CCC C-3'), J10 (5'-AGG TAA GGT TCT TCG
GTT TGC A-3')。
Compared with the prior art, the present invention has the beneficial effect that：
1, the most of bacterium of Pseudomonas involved in liquor fermentation process are unknown microorganism or Anticipated transient without scram, are directly being planted
Classification level on quantitative judge a batch content is higher but strain that its ribosomal dna sequence is very close is also relatively difficult,
And the quantitative amplification based on door specific primer can quickly investigate the macrostructure variation of microbiologic population in the level of door,
Not only simple but also practical, it can be used for measuring random time point or the macroscopic view knot of the microbiologic population in process procedure in fermentation process
Structure change, such as yeast preparation process, fermented grain fermentation process, pit mud fermentation process structure of community composition state.
2, since the method for the present invention can be completed to measure at two hours or so scenes, the micro- life of Rapid Accumulation fermentation can be used for
Object structure of community quantitative data and go out the relation data between wine efficiency, including fermenting speed, quality, yield and flavor etc., is
Persistence improves out wine efficiency and ensures that production efficiency provides technical support.
3, the method for the present invention can be with the absolute content of the main door of microbiologic population in fast quantification fermentation process or opposite
Content has significant effect, advantage of low cost.
4, the consumptive material involved by the implementation of the method for the present invention is all common molecular biology reagents, is conveniently easy to get.Wherein
QPCR system configurations can use any common kit.The composition of qPCR systems is shown in Table 1 in the present invention：
Table 1：QPCR systems form
| 2 × PCR buffer solutions (contain fluorescent dye and PCR synergist, have thermal starting property) | 6μL |
| Primer 1 (2 μM) | 1μL |
| Primer 2 (2 μM) | 1μL |
| Template+H2O | 3.8μL |
| Taq enzyme (5U/ μ L) | 0.2μL |
| Total volume is 12 μ L |
Description of the drawings
Fig. 1 is the biological community structure quantitative result of five doors of the four class pit muds completed using the method for the present invention.
Specific implementation mode
It is by PCR reactants that the method that microbiologic population forms during liquor fermentation is quickly measured in the present embodiment
Specific amplification is carried out to the specific microorganism species of some during reaching liquor fermentation using door specific primer in system,
And quantitative amplification is carried out using qPCR systems, obtain quantitative judge.
A specific primer is prepared as follows：
Step a, the macro genome of extraction brewing microorganism group sample is as PCR amplification template；
Step b, the rDNA expanded in template is expanded close to overall length 16s rDNA sequences, amplimer 27F
(5 '-AGA GTT TGA TCC TGG CTC AG-3 ') and 1492R (5 '-GGT TAC CTT GTT ACG ACTT-3 '), is obtained
Obtain amplified production；
Step c, double by carrying out TA clones, blue hickie screening and hickie bacterium colony successively after purification for the amplified production
To sequencing, no less than 200 effective clones are obtained, effective clone refers to that last bidirectional sequencing sequence passes through sequence ratio
To really belonging to 16s rDNA sequences；
Step d, design of primers is completed by phylogenetic analysis for effective clone's bidirectional sequencing sequence.
Configuration to qPCR systems：It is to use qPCR kits and qPCR instruments, and require qPCR instruments that can detect pair
It includes EvaGreen and SYBR green to answer the fluorescent material that kit includes, the fluorescent material.
Setting and operation for the condition of qPCR systems：Amplification item is carried out according to different microorganisms group and different doors
The adjusting and optimizing of part, the primer that whens different doors of the same group of quantitative judge is designed have similar Tm values as possible, so as to
It is tested and is applied in identical amplification condition.
Detection for the product of qPCR systems：During method is established, is detected and expanded by agarose gel electrophoresis
The purity and quality of product；When amplified production reaches single master tape and the analysis of Tm values has single product peak, it is no longer necessary to run
Glue detects product, and qPCR only needs to analyze and determine by Tm values when use, exports qPCR simultaneously using qPCR instruments and expands
Curve, quantitative result and Tm value analysis results.
The information of major microorganisms in fermentation process, including 5-10 common door such as Bacteroidetes Bacteroidetes,
Firmicutes Firmicutes, Proteobacteria Proteobacteria, mutual bacteria door Synergistetes, actinomyces door
Actinobacteria.By comparison and phylogenetic analysis, identical door is returned together, the sequence difference between different doors
By SAP technical finesses, the door specific primer of single base difference resolution ratio is obtained.
SAP technical finesse processes：Artificial mispairing is introduced in 3 ' end bases second from the bottom of certain primers, which does not influence
The amplification of target sequence can but destroy the amplification of non-target sequences, enhance the specificity of primer.These primer pairs are with each
TA clone's pure plasmids of practical 16S sequences are that template carries out PCR verifications and all hybrid template verifications and qPCR verifications.
Door specific primer in the present embodiment can be Bacteroidetes specific primer, primer pair nucleotide sequence
For:
320 (5'-CGC ACG GGT GAG TAA CAC GTAT-3'), 321 (5'-GGG GAT AAA TCC TCT
CAG TTC CCCT-3')；
Door specific primer or the specific primer for Proteobacteria, primer pair nucleotides sequence are classified as:
317 (5'-CCG TAG CTG GTC TGA GAG GAT GAT AA-3'), 319 (5'-CAC GCT TTA CGC
CCA GTA ATT CCYA-3')；
Door specific primer or the specific primer for Firmicutes, primer pair nucleotides sequence are classified as：
J1 (5'-CAG CAG TGG GGA ATC TTCC-3'), J2 (5'-TAG CCG GGG CTT CCT CCT-3')；
Door specific primer is mutual bacteria door specific primer, and primer pair nucleotides sequence is classified as：
J7 (5'-AAA CCG CTT TCA GCA GGG AC-3'), J8 (5'-TAA CTC CCG ACC AAA CAA
GC-3')。
Door specific primer is actinomyces door specific primer, and primer pair nucleotide sequence is as follows:
J9 (5'-AGC GAA CGG GAT TAG ATA CCC C-3'), J10 (5'-AGG TAA GGT TCT TCG
GTT TGC A-3')。
Experimentation：
1. sample genome sample extraction of fermenting
Each fresh pit mud sample of certain brand white wine weighs 200 milligrams, using Solarbio soil genomic kit or
Shanghai gives birth to work EZUP pillar soil genome extraction agent cassette methods and extracts genome, and last elution volume is 100 μ L.Genome
- 80 DEG C of refrigerations of template sample (10-50ng/uL) are spare.Pit mud soil sample can use plastic bag sealing to be preserved 1 year in -80 DEG C of refrigerators.
2. biological community structure measures
By the amplification of 16s rDNA (amplimer be 27F (5 '-AGA GTT TGA TCC TGG CTC AG-3 ') and
1492R (5 '-GGT TAC CTT GTT ACG ACTT-3 '), amplification template are the cellar for storing things of certain brand White wine pit pool wall and pit bottom
Mud sample product) and TA cloning and sequencings, 793 effectively clones are obtained, the results are shown in Table 2, wherein non-perfect fungus accounts for 60.78%,
In 5 clones cannot get any comparison information, account for 0.63%.Perfect fungus is belonging respectively to Bacteroidetes for remaining
(3.79%), Firmicutes (31.16%), Proteobacteria (3.55%), Synergistetes (0.63%),
The Atopobium (0.13%) of Actinobacteria, it is 318 that perfect fungus, which has effectively cloned number, in other words, wherein
Bacteroidetes Bacteroidetes, Firmicutes Firmicutes, Proteobacteria Proteobacterias, Synergistetes
Mutual bacteria door and Actinobacteria actinomyces doors are respectively 12,99,11,2 and 1 comprising effective colony counts.
Table 2：Certain brand white spirit pit mud Phylogenetic diversity of bacteria statistics (counts) according to BLAST results
The configuration of 3.qPCR systems
QPCR systems can use most business archaeal dna polymerases or qPCR kits, and the composition of qPCR systems is referring to table 1.
Used door specific primer is as follows：
Bacteroidetes specific primer：320 (5'-CGC ACG GGT GAG TAA CAC GTA T-3'), 321 (5'-
GGG GAT AAA TCC TCT CAG TTC CCC T-3')。
The specific primer of Proteobacteria：317 (5'-CCG TAG CTG GTC TGA GAG GAT GAT AA-3'),
319(5'-CAC GCT TTA CGC CCA GTA ATT CCYA-3')。
The specific primer sequence of Firmicutes：J1 (5'-CAG CAG TGG GGA ATC TTC C-3'), J2 (5'-
TAG CCG GGG CTT CCT CCT-3')。
Mutual bacteria door specific primer sequence:J7 (5'-AAA CCG CTT TCA GCA GGG AC-3'), J8 (5'-TAA
CTC CCG ACC AAA CAA GC-3')。
Actinomyces door specific primer:J9(5'-AGC GAA CGG GAT TAG ATA CCC C-3')J10(5'-AGG
TAA GGT TCT TCG GTT TGCA-3')。
4. quantitative criterion：The quantitative criterion substance of each door is the pure plasmid stoste sample that TA is cloned, and measures its standard
True concentration, and it is diluted to 10 by 10 times-10It is secondary.Because the sized molecules amount of above-mentioned plasmid is unique, mass concentration and molar concentration are all
It can accurately measure, for single qPCR products, use quality concentration and molar concentration can；And in this technology
QPCR reaction in, standard sample amplification be single product, and sample amplification be many kinds of some mixing
Product, thus the concentration of standard sample must use molar concentration, sample determine come result be also molar concentration or
Reflected with Molecules.So quantitative result belongs to absolute quantitation.The pure plasmid sample of 5 doors is according to extraction of plasmid DNA
The requirement of kit is extracted, 100 microlitres of elution volume.Plasmid stock carries out doubling dilution (10 times of dilution every time) and is used as each door
Standard quantitative qPCR.The amplification of specificity ensures that the melt curve analysis of qPCR products only connects there are one unimodal or thaw temperature very much
Closely one group of peak of (difference is usually no more than 5 degree)；According to the door specific primer design of the art of this patent, by being produced to the amplification
It is the kind of target door that the cloning and sequencing of object, which can see amplified production all,.The specificity and door specific criteria sample of amplified production
The configuration of product will be possible to ensure that the quantitative validity of qPCR.The quantitative plasmid by each door oneself of each door is as quantitative mark
Standard, and door specific primer ensures that amplified production is the kind sequence of target door, can so ensure quantitative effective of qPCR
Property.5 the above-mentioned of door can quantitatively be cooked respectively, and test condition can also by means such as the numbers of reduction repeated sample after stablizing
It is done together in a qPCR, so as to once complete quantifying for 5 doors or more door, then quickly obtains microorganism
The quantitative result of structure of community.If the number of the door for the microbiologic population being related to is relatively more, such as 7-12, or needs to survey
Fixed sample is more, then it is contemplated that being loaded using 384 orifice plates and the volley of rifle fire.
5. using Bacteroidetes specific primer four kinds of pit mud samples of certain brand white wine are carried out with the quantitative ratio of Bacteroidetes
Compared with.Bacteroidetes primer specific primer amplification length is about 185 base-pairs.
(1) 10 times of dilutions of Bacteroidetes plasmid gradients：2 μ L of plasmid mother liquor are taken to be added in 18 μ L ddH2O
It mixes well, vortex centrifugal mixing is repeated 2 times, and obtains 10-1Grade doubling dilution, so goes down, respectively obtains 10-2、10-3、10-4、
10-5、10-6、10-7、10-8。
(2) Mix1 is prepared：(12 μ L systems) 10-3-10-8(6)
Vortex centrifugal mixing is repeated twice after adding Taq enzyme
(3) the respective Mix2 of whole samples is prepared：10-3、10-4、10-5、10-6、10-7、10-8, sample to be tested 1. 2. 3.
4., NTC (negative control is not added with template)；(totally 11) above-mentioned each sample is a Mix2 (being repeated 3 times enough) of oneself
It is as follows：
Mix1：11 × 3.5 μ L=38.5 μ L
Each self-template of sample：1 × 3.5 μ L=3.5 μ L
The above operation is intended to carry out in ice bath.The equal vortex centrifugal mixing of 11 samples is repeated twice.
(4) eight union loadings of QPCR
| 10-3 | 10-3 | 10-3 | 10-4 | 10-4 | 10-4 | 10-5 | 10-5 |
| 10-5 | 10-6 | 10-6 | 10-6 | 10-7 | 10-7 | 10-7 | 10-8 |
| 10-8 | 10-8 | ① | ① | ① | ② | ② | ② |
| ③ | ③ | ③ | ④ | ④ | ④ | NTC | NTC |
It is tested according to the step-one QPCR programs set and exports quantitation curves.Basic program is as follows：
(5) experimental result and analysis：
Standard plasmid mother liquor DNA molecular a concentration of 75649000 × 108Copy/L, dilution gradient are as follows：
| Gradient dilution | 10-3 | 10-4 | 10-5 | 10-6 | 10-7 | 10-8 |
| Concentration (108Copy/L) | 75649 | 7564 | 756 | 75 | 7 | 0.7 |
The absolute concentration average value that sample calculates：
| Sample | Pit mud sample A | Pit mud sample B | Pit mud sample C | Pit mud sample D |
| Concentration (108Copy/L) | 2 | 744 | 14163 | 851 |
Mutual bacteria door is quantitative determined using above-mentioned identical program, amplification length is about 208 base-pairs.
Mutual bacteria door standard plasmid mother liquor DNA molecular concentration is determined to 67323000 × 108Copy/L, dilution gradient is such as
Under：
| Gradient dilution | 10-3 | 10-4 | 10-5 | 10-6 | 10-7 | 10-8 |
| Concentration (108Copy/L) | 67323 | 6732 | 673 | 67 | 6 | 0.6 |
The absolute concentration that sample calculates：
| Sample | Pit mud sample A | Pit mud sample B | Pit mud sample C | Pit mud sample D |
| Concentration (108Copy/L) | 0 | 140 | 385 | 4 |
Actinomyces door is quantitative determined using above-mentioned identical program, amplification length is about 157 base-pairs.
Actinomyces door standard plasmid mother liquor DNA molecular concentration is determined to 1134140 × 108Copy/L, dilution gradient is such as
Under：
| Gradient dilution | 10-1 | 10-2 | 10-3 | 10-4 | 10-5 | 10-6 |
| Concentration (108Copy/L) | 113414 | 11341 | 1134 | 113 | 11.3 | 1.13 |
The absolute concentration that sample calculates：
| Sample | Pit mud sample A | Pit mud sample B | Pit mud sample C | Pit mud sample D |
| Concentration (108Copy/L) | 0 | 298986 | 13 | 0 |
Firmicutes are quantitative determined using above-mentioned identical program, amplification length is about 135 base-pairs.
Firmicutes standard plasmid mother liquor DNA molecular concentration is determined to 195071744 × 108Copy/L, dilution gradient
It is as follows：
| Gradient dilution | 100 | 10-1 | 10-2 | 10-3 | 10-4 | 10-5 |
| Concentration (108Copy/L) | 195071744 | 19507176 | 1950717 | 195071 | 19507 | 1950 |
The absolute concentration that sample calculates：
| Sample | Pit mud sample A | Pit mud sample B | Pit mud sample C | Pit mud sample D |
| Concentration (108Copy/L) | 122505 | 37972420 | 597973824 | 6704090 |
Proteobacteria is quantitative determined using above-mentioned identical program, amplification length is about 264 base-pairs.
Proteobacteria standard plasmid mother liquor DNA molecular concentration is determined to 3666710 × 1010Copy/L, dilution gradient is such as
Under：
| Gradient dilution | 10-1 | 10-2 | 10-3 | 10-4 | 10-5 | 10-6 |
| Concentration (1010Copy/L) | 366671 | 36667 | 3666 | 366 | 36 | 3 |
The absolute concentration that sample calculates：
| Sample | Pit mud sample A | Pit mud sample B | Pit mud sample C | Pit mud sample D |
| Concentration (1010Copy/L) | 673 | 2818 | 1946 | 9 |
The biological community structure that five doors of four classes pit mud shown in FIG. 1 can be obtained according to above-mentioned flow testing is quantitative
It completed at 2 hours or so as a result, each QPCR quantitatively takes control, is found out by Fig. 1：Pit mud sample A, two of which door are
The species content of Syn and Act is seldom；Pit mud sample B, close with the comparison between community structures of pit mud sample C, species content is richer
It is rich；Pit mud sample D, wherein actinomyces Act contents are less.
Claims (3)
1. a kind of method that microbiologic population forms during quick measurement liquor fermentation, it is characterized in that：By in PCR reactants
Specific amplification is carried out to the specific microorganism species of some during reaching liquor fermentation using door specific primer in system,
And quantitative amplification is carried out using qPCR systems, obtain quantitative judge；
The door specific primer by Bacteroidetes specific primer, the specific primer of Proteobacteria, Firmicutes it is special
Property primer, mutual bacteria door specific primer and actinomyces door specific primer composition；
The Bacteroidetes specific primer, primer pair nucleotides sequence are classified as：
320：5'-CGC ACG GGT GAG TAA CAC GTAT-3',
321：5'-GGG GAT AAA TCC TCT CAG TTC CCCT-3'；
The specific primer of the Proteobacteria, primer pair nucleotides sequence are classified as：
317：5'-CCG TAG CTG GTC TGA GAG GAT GAT AA-3',
319：5'-CAC GCT TTA CGC CCA GTA ATT CCYA-3'；
The specific primer of the Firmicutes, primer pair nucleotides sequence are classified as：
J1：5'-CAG CAG TGG GGA ATC TTCC-3', J2：5'-TAG CCG GGG CTT CCT CCT-3'；
The mutual bacteria door specific primer, primer pair nucleotides sequence are classified as：
J7:5'-AAA CCG CTT TCA GCA GGG AC-3', J8:5'-TAA CTC CCG ACC AAA CAA GC-3'；
The actinomyces door specific primer, primer pair nucleotides sequence are classified as：
J9：5'-AGC GAA CGG GAT TAG ATA CCC C-3',
J10：5'-AGG TAA GGT TCT TCG GTT TGC A-3'.
2. the method that microbiologic population forms during quick measurement liquor fermentation according to claim 1, it is characterized in that：
A specific primer is prepared as follows：
Step a, the macro genome of extraction brewing microorganism group sample is as PCR amplification template；
Step b, the rDNA in the amplification template is expanded close to overall length 16s rDNA sequences, and amplimer is
27F：5 '-AGA GTT TGA TCC TGG CTC AG-3 ' and
1492R：5 '-GGT TAC CTT GTT ACG ACTT-3 ' obtain amplified production；
Step c, for the amplified production by carrying out TA clones, blue hickie screening and the two-way survey of hickie bacterium colony successively after purification
Sequence, obtains no less than 200 effective clones, and effective clone refers to that last bidirectional sequencing sequence is true by sequence alignment
Belong to 16s rDNA sequences in fact；
Step d, design of primers is completed by phylogenetic analysis for effective clone's bidirectional sequencing sequence.
3. the method that microbiologic population forms during quick measurement liquor fermentation according to claim 1, it is characterized in that：
Configuration to the qPCR systems：It is to use qPCR kits and qPCR instruments, and require qPCR instruments that can detect pair
It includes EvaGreen and SYBR green to answer the fluorescent material that kit includes, the fluorescent material；
Setting and operation for the condition of the qPCR systems；Amplification item is carried out according to different microorganisms group and different doors
The adjusting and optimizing of part, the primer that whens different doors of the same group of quantitative judge is designed have similar Tm values as possible, so as to
It is tested and is applied in identical amplification condition；
Detection for the product of the qPCR systems：During method is established, is detected and expanded by agarose gel electrophoresis
The purity and quality of product；When amplified production reaches single master tape and the analysis of Tm values has single product peak, it is no longer necessary to run
Glue detects product, and qPCR only needs to analyze and determine by Tm values when use, exports qPCR simultaneously using qPCR instruments and expands
Curve, quantitative result and Tm value analysis results.
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Non-Patent Citations (2)
| Title |
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| 冠中土壤微生物群落的组成鉴定及定量识别技术的研制;董思斯;《中国优秀硕士学位论文全文数据库——农业科技辑》;20150228(第2期);摘要第2、3段，第14页倒数第1段-第15页第2段，第19页倒数第1段-第21页第5段，第29页倒数第1段，第36页第2段-第37页第2段，第57页第2段，第59页倒数第1段 * |
| 古井贡酒窖泥中微生物群落结构快速定量检测技术的研发及应用;周庆伍 等;《安徽省科技成果登记管理系统》;20141111;项目简介 * |
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