CN105331702A - Method for fast and quantitatively measuring microbial community composition in fermentation process of baijiu - Google Patents
Method for fast and quantitatively measuring microbial community composition in fermentation process of baijiu Download PDFInfo
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- CN105331702A CN105331702A CN201510766757.6A CN201510766757A CN105331702A CN 105331702 A CN105331702 A CN 105331702A CN 201510766757 A CN201510766757 A CN 201510766757A CN 105331702 A CN105331702 A CN 105331702A
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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
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q1/6851—Quantitative amplification
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Abstract
The invention relates to a method for fast and quantitatively measuring the microbial community composition in the fermentation process of baijiu. The method is characterized in that phylum specific primers are used in a PCR system so that special amplification of microbial communities of a certain specific phylum can be performed in the fermentation process of baijiu, quantitative amplification is performed through a qPCR system, and quantitative recognition is achieved. According to the category of known microorganisms, primers of the bacteroidetes, proteobacteria, firmicutes, synergistetes and actinobacteria are respectively designed, and the microstructure change of microbial communities can be fast inspected in the baijiu fermentation process in the level of the phylum on the basis of quantitative amplification of the phylum specific primers.
Description
Technical field
The present invention relates to the microflora's working method in biological technical field, based on door Auele Specific Primer (phylum-specificprimer, PSP), the quick PSP-qPCR quantivative approach of microflora's composition structure in liquor fermentation process is measured.
Background technology
Natural complicated microflora generally by tens, the even more not same species of microorganism of hundreds of form, and this composition can occurrence dynamics change under different envrionment conditionss.Most of mankind of nearly all bacterial classification included by natural microbial group understand very few, so carry out scene to natural microbial group in liquor fermentation process, structure composition is quantitatively also very difficult fast.Technology common at present comprises:
One is classical culture protocols; Such as store most of microorganism in mud to belong to and do not cultivate bacterium, use conventional medium to cultivate cellar for storing things mud microorganism speed slow, qualification workload is very large;
Two is tetra-sodium high-flux sequence methods; This is at present popular both at home and abroad a kind of structure of community method for quantitatively determining, has generally been come by biotech company's Delegation Server, expensive, the order-checking number that can return up to hundreds of thousands of bar, generally can qualitative, quantitative to belonging to;
Three is FISH technology; FISH and fluorescenceinsituhybridization technology is the principle based on base complementrity, by an a bit of (usual 15-30 base, such as 16SrRNA fragment) fluorescently-labeled nucleotide sequence is as probe, hybridize with the target sample on slide glass, combination single-minded with target sequence, shows existence and the quantity of specific nucleotide sequence by detecting hybridization site fluorescence;
Four is DGGE/TGGE technology; DGGE and denaturedgradientgelelectrophoresis technology is proposed at first for detecting DNA mutation by Fischer and Lerman1979.Myers etc. use " GC clamping plate " and heteroduplex technology in 1985 in DGGE.Muzyer etc. confirm that this technology is very effective in microorganisms group genetic diversity and group difference for 1993.The temperature gradient gel elec-trophoresis (TGGE) (temperaturegradientgelelectrophoresis, TGGE) replacing chemical denaturant is afterwards deriving technology.DGGE/TGGE is the difference melting behavior according to double-stranded DNA segment, is separated similar length in PCR primer but the different DNA marker segment (rRNA or rDNA) of sequence.A band on DGGE/TGGE figure can represent a microbe groups, and categorization levels can reach kind, can conveniently judge dominant bacteria or function yeast;
Five is T-RFL technology; T-RFLP and Terminal-Restrictionfragmentlengthpoly-morphism technology is developed by RFLP.T-RFLP know-why is by PCR primer mark fluorescence, by pcr amplification product digestion with restriction enzyme, produce the restriction fragment of different lengths, wherein a part is containing fluorescence, form T-RFLP collection of illustrative plates, disclose the information such as kind, quantity of microorganism in sample;
Six is RAPD technology; RAPD is the polymorphic Monitoring techniques of a kind of DNA, and by pcr amplification, amplified production, after agarose gel electrophoresis separation, ethidium bromide staining, detects DNA fragmentation polymorphism.It is the molecular engineering that a kind of genome to whole unknown nucleotide sequence carries out polymorphism analysis.RAPD technology unique distinction is: 1) to pull the requirement of DNA few for mould, and purity requirement is low, adopts random primer; 2) fast easy and simple to handle, do not need the steps such as molecular hybridization, directly carry out DNA polymorphism analysis; 3) required primer is short, and the binding site in whole genome is many, and fraction of coverage is large, and primer can be used with, and multiple primer pair genome can be used to carry out large-area Polymorphism Analysis; 4) recall rate is high.But RAPD result poor reproducibility;
Seven is SSCP technology; 1989, Japanese Scientists Orita utilizes DNA single chain conformation to have polymorphism, and when native polyacrylamide gel electrophoresis, the difference of mobility is carried out the transgenation in analyzing DNA strand and proposed the concept of SSCP (Single-StrandConformationPolymorphism).SSCP technology is applied to the compositional analysis of microflora by Lee etc.The responsive argentation such as Orita directly dyes to the gel after electrophoresis, establishes PCR-SSCP analytical method, improves the simplicity and susceptibility that detect mutation method.PCR-SSCP method advantage: 1) available non-isotopic methods detects, can direct electrophoresis after PCR primer sex change; 2) experiment is not high to the purity requirement of DNA starting materials, and requirement is few, cost is lower; 3) large sample examination is suitable for, without the need to knowing DNA sequence dna in advance; 4) can detect the polymorphism on any DNA site and sudden change, susceptibility is high.But this technology also has many deficiencies, such as analytical results is by the impact of many factors, and different experiment conditions may cause diverse result;
Eight is AFLP technology; AFLP (AmplifiedFragmentLengthPolymorphism) technology is created by Dutch Zabeau and Vos for 1993, combine RFLP and RAPD technical characterstic, overcome RFLP technical sophistication, have radiological hazard and RAPD technical stability is poor, mark presents the shortcoming of recessive inheritance, have the high efficiency of RAPD and the repeatable feature of RFLP simultaneously concurrently.This technology is considered to the technology that in current DNA fingerprinting technology, polymorphism is the abundantest, its major advantage: required DNA amount less, reproducible, polymorphism is strong, resolving power is high, do not need hybridization etc.But somewhat expensive, to the purity of DNA and the specification of quality of restriction endonuclease more high.
But above-mentioned all technology all cannot accomplish the composition structure of microflora in the Fast Measurement liquor fermentation process of on-the-spot two hours.
Summary of the invention
The present invention is the deficiency for avoiding existing for above-mentioned prior art, provides a kind of quantitative judge Be very effective, prepares the method for quantitative determination liquor fermentation process microflora composition easy, with low cost.
The present invention is that technical solution problem adopts following technical scheme:
In Fast Measurement liquor fermentation process of the present invention, the feature of method of microflora's composition is: reach in liquor fermentation process by using door Auele Specific Primer in PCR reaction system and carry out specific amplification to certain microorganism species concrete, and use qPCR system to carry out quantitative amplification, obtain quantitative judge.
In Fast Measurement liquor fermentation process of the present invention, the feature of the method for microflora's composition is also: prepare door Auele Specific Primer as follows:
The grand genome of step a, extraction brewing microorganism group sample is as polymerase chain reaction (PCR) amplification template;
Step b, the rDNA that increases in described amplification template are close to total length 16srDNA sequence, amplimer is 27F (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1492R (5 '-GGTTACCTTGTTACGACTT-3 '), obtains amplified production;
Step c, described amplified production carried out after purifying to TA clone, the screening of blue hickie and the two-way order-checking of hickie bacterium colony successively, obtain the effective clone being no less than 200, described effective clone refers to that last two-way sequencing sequence belongs to 16srDNA sequence really through sequence alignment;
Steps d, for the two-way sequencing sequence of described effective clone by phylogenetic analysis, complete design of primers.
In Fast Measurement liquor fermentation process of the present invention, the feature of the method for microflora's composition is also:
Configuration to described qPCR system: be use qPCR test kit and qPCR instrument, and the fluorescent substance requiring qPCR instrument can detect corresponding test kit to comprise, described fluorescent substance comprises EvaGreen and SYBRgreen;
For setting and the operation of the condition of described qPCR system; Carry out the adjusting and optimizing of amplification condition according to different microorganisms group and different doors, the primer designed during the different door of the same group of quantitative judge possesses close Tm value, to carry out testing and applying at identical amplification condition as far as possible;
Detection for the product of described qPCR system: in method establishment process, detects purity and the quality of amplified production by agarose gel electrophoresis; When amplified production reaches single master tape and the analysis of Tm value possesses single product peak, no longer need to run glue and detect product, during use, qPCR only needs to be judged by the analysis of Tm value, utilizes qPCR instrument to derive qPCR amplification curve, quantitative result and Tm value analytical results simultaneously.
In Fast Measurement liquor fermentation process of the present invention, the feature of the method for microflora's composition is also:
Described door Auele Specific Primer is Bacteroidetes Auele Specific Primer, and its primer pair nucleotides sequence is classified as:
320(5'-CGCACGGGTGAGTAACACGTAT-3')，321(5'-GGGGATAAATCCTCTCAGTTCCCCT-3')；
Described door Auele Specific Primer or be the Auele Specific Primer of Proteobacteria, its primer pair nucleotides sequence is classified as:
317(5'-CCGTAGCTGGTCTGAGAGGATGATAA-3')，319(5'-CACGCTTTACGCCCAGTAATTCCYA-3')；
Described door Auele Specific Primer or be the Auele Specific Primer of Firmicutes, its primer pair nucleotides sequence is classified as:
J1(5'-CAGCAGTGGGGAATCTTCC-3')，J2(5'-TAGCCGGGGCTTCCTCCT-3')；
Described door Auele Specific Primer or for syntrophism bacterium door Auele Specific Primer, its primer pair nucleotides sequence is classified as:
J7(5'-AAACCGCTTTCAGCAGGGAC-3')，J8(5'-TAACTCCCGACCAAACAAGC-3')。
Described door Auele Specific Primer or be actinomycetes door Auele Specific Primer, its primer pair nucleotide sequence is as follows:
J9(5'-AGCGAACGGGATTAGATACCCC-3')，J10(5'-AGGTAAGGTTCTTCGGTTTGCA-3')。
Compared with the prior art, beneficial effect of the present invention is embodied in:
1, the most of bacterium of the Pseudomonas related in liquor fermentation process is unknown microorganism or Anticipated transient without scram, but directly in the classification aspect of planting, a collection of content of quantitative judge its ribosomal dna sequence higher bacterial classification is closely also more difficult, and in the aspect of door, the macrostructure change of microflora can be investigated fast based on the quantitative amplification of door Auele Specific Primer, not only simple but also practical, can be used for measuring the macrostructure change of the microflora in random time point in fermenting process or process procedure, as Daqu preparation process, wine unstrained spirits fermenting process, the state of the structure of community composition of cellar for storing things mud fermenting process etc.
2, because the inventive method can complete mensuration at two hours scenes, can be used for Rapid Accumulation fermentation flora structure quantitative data with go out wine efficiency, comprise the relation data between fermenting speed, quality, output and local flavor etc., wine efficiency for persistence improves and ensure that production efficiency provides technical support.
3, the inventive method can the absolute content of the main door of microflora or relative content in fast quantification fermenting process, has Be very effective, advantage with low cost.
4, the consumptive material involved by enforcement of the inventive method is all common molecular biology reagents, is conveniently easy to get.Wherein qPCR system configurations can use any common test kit.In the present invention, the composition of qPCR system is in table 1:
Table 1:qPCR system composition
| 2 × PCR damping fluid (containing fluorescence dye and PCR synergistic agent, possessing warm start character) | 6μL |
| Primer 1 (2 μMs) | 1μL |
| Primer 2 (2 μMs) | 1μL |
| Template+H 2O | 3.8μL |
| Taq enzyme (5U/ μ L) | 0.2μL |
| Cumulative volume is 12 μ L |
Accompanying drawing explanation
Fig. 1 is the biological community structure quantitative result of five doors of the four class cellar for storing things mud utilizing the inventive method to complete.
Embodiment
In the present embodiment, in Fast Measurement liquor fermentation process, the method for microflora's composition carries out specific amplification by using door Auele Specific Primer to reach in liquor fermentation process in PCR reaction system to certain microorganism species concrete, and use qPCR system to carry out quantitative amplification, obtain quantitative judge.
Prepare door Auele Specific Primer as follows:
The grand genome of step a, extraction brewing microorganism group sample is as polymerase chain reaction (PCR) amplification template;
Step b, the rDNA that increases in described amplification template are close to total length 16srDNA sequence, amplimer is 27F (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1492R (5 '-GGTTACCTTGTTACGACTT-3 '), obtains amplified production;
Step c, described amplified production carried out after purifying to TA clone, the screening of blue hickie and the two-way order-checking of hickie bacterium colony successively, obtain the effective clone being no less than 200, described effective clone refers to that last two-way sequencing sequence belongs to 16srDNA sequence really through sequence alignment;
Steps d, for the two-way sequencing sequence of described effective clone by phylogenetic analysis, complete design of primers.
Configuration to qPCR system: be use qPCR test kit and qPCR instrument, and the fluorescent substance requiring qPCR instrument can detect corresponding test kit to comprise, described fluorescent substance comprises EvaGreen and SYBRgreen.
Setting and operation for the condition of qPCR system: the adjusting and optimizing carrying out amplification condition according to different microorganisms group and different doors, the primer designed during the different door of the same group of quantitative judge possesses close Tm value, to carry out testing and applying at identical amplification condition as far as possible.
Detection for the product of qPCR system: in method establishment process, detects purity and the quality of amplified production by agarose gel electrophoresis; When amplified production reaches single master tape and the analysis of Tm value possesses single product peak, no longer need to run glue and detect product, during use, qPCR only needs to be judged by the analysis of Tm value, utilizes qPCR instrument to derive qPCR amplification curve, quantitative result and Tm value analytical results simultaneously.
The information of major microorganisms in fermenting process, comprises a common 5-10 door as Bacteroidetes Bacteroidetes, Firmicutes Firmicutes, Proteobacteria Proteobacteria, syntrophism bacterium door Synergistetes, actinomycetes door Actinobacteria.Through comparison and phylogenetic analysis, identical door is returned together, the sequence difference between different doors, by SAP technical finesse, obtains the door Auele Specific Primer of single base difference resolving power.
SAP technical finesse process: hold base second from the bottom to introduce people for mispairing at 3 ' of some primer, this mispairing does not affect the amplification of target sequence, but can destroy the amplification of non-target sequences, enhances the specificity of primer.These primer pairs with the TA of the actual 16S sequence of each clone pure plasmid for template carry out PCR checking and all door hybrid templates checking, and qPCR checking.
Door Auele Specific Primer in the present embodiment can be Bacteroidetes Auele Specific Primer, and its primer pair nucleotides sequence is classified as:
320(5'-CGCACGGGTGAGTAACACGTAT-3')，321(5'-GGGGATAAATCCTCTCAGTTCCCCT-3')；
Door Auele Specific Primer or be the Auele Specific Primer of Proteobacteria, its primer pair nucleotides sequence is classified as:
317(5'-CCGTAGCTGGTCTGAGAGGATGATAA-3')，319(5'-CACGCTTTACGCCCAGTAATTCCYA-3')；
Door Auele Specific Primer or be the Auele Specific Primer of Firmicutes, its primer pair nucleotides sequence is classified as:
J1(5'-CAGCAGTGGGGAATCTTCC-3')，J2(5'-TAGCCGGGGCTTCCTCCT-3')；
Door Auele Specific Primer or for syntrophism bacterium door Auele Specific Primer, its primer pair nucleotides sequence is classified as:
J7(5'-AAACCGCTTTCAGCAGGGAC-3')，J8(5'-TAACTCCCGACCAAACAAGC-3')。
Door Auele Specific Primer or be actinomycetes door Auele Specific Primer, its primer pair nucleotide sequence is as follows:
J9(5'-AGCGAACGGGATTAGATACCCC-3')，J10(5'-AGGTAAGGTTCTTCGGTTTGCA-3')。
Experimentation:
1. fermentation sample genome sample extraction
Certain brand white wine is often planted fresh cellar for storing things mud sample and is taken 200 milligrams, and adopt Solarbio soil genomic kit or Shanghai raw work EZUP pillar soil genome extraction agent cassette method to extract genome, last elution volume is 100 μ L.The refrigeration of-80 DEG C, genomic templates sample (10-50ng/uL) is for subsequent use.Cellar for storing things earth sample can with plastic bag sealing-80 DEG C of Refrigerator stores 1 year.
2. biological community structure measures
By the amplification of 16srDNA, (amplimer is 27F (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1492R (5 '-GGTTACCTTGTTACGACTT-3 '), amplification template is certain brand White wine pit pool wall and cellar for storing things cellar for storing things mud sample at the bottom of pond) and TA cloning and sequencing, obtain 793 effectively clones, result is as shown in table 2, wherein non-perfect fungus accounts for 60.78%, wherein 5 clones can not get any comparison information, account for 0.63%.All the other known mushrooms belong to Bacteroidetes (3.79%) respectively, Firmicutes (31.16%), Proteobacteria (3.55%), Synergistetes (0.63%), the Atopobium (0.13%) of Actinobacteria, perfect fungus has effectively cloned number is in other words 318, wherein Bacteroidetes Bacteroidetes, Firmicutes Firmicutes, Proteobacteria Proteobacteria, Synergistetes syntrophism bacterium door and Actinobacteria actinomycetes door comprise effective colony counts and are respectively 12, 99, 11, 2 and 1.
Table 2: certain brand white spirit pit mud Phylogenetic diversity of bacteria statistics (according to BLAST result statistics)
The configuration of 3.qPCR system
QPCR system can use most business archaeal dna polymerase or qPCR test kit, and the composition of qPCR system is see table 1.
The door Auele Specific Primer used is as follows:
Bacteroidetes Auele Specific Primer: 320 (5'-CGCACGGGTGAGTAACACGTAT-3'), 321 (5'-GGGGATAAATCCTCTCAGTTCCCCT-3').
The Auele Specific Primer of Proteobacteria: 317 (5'-CCGTAGCTGGTCTGAGAGGATGATAA-3'), 319 (5'-CACGCTTTACGCCCAGTAATTCCYA-3').
The specific primer sequence of Firmicutes: J1 (5'-CAGCAGTGGGGAATCTTCC-3'), J2 (5'-TAGCCGGGGCTTCCTCCT-3').
Syntrophism bacterium door specific primer sequence: J7 (5'-AAACCGCTTTCAGCAGGGAC-3'), J8 (5'-TAACTCCCGACCAAACAAGC-3').
Actinomycetes door Auele Specific Primer: J9 (5'-AGCGAACGGGATTAGATACCCC-3') J10 (5'-AGGTAAGGTTCTTCGGTTTGCA-3').
4. quantitative criterion: the quantitative criterion material of each is that TA clones the pure plasmid stoste sample obtained, and measures its actual concentrations, and is diluted to 10 by 10 times
-10secondary.Because the sized molecules amount of above-mentioned plasmid is unique, mass concentration and volumetric molar concentration can Accurate Measurements, and for single qPCR product, functional quality concentration and volumetric molar concentration can; And in qPCR reaction in this technology, what standard model increased is single product, and sample amplification is the mix products of the many kinds of certain, so the concentration of standard model must use volumetric molar concentration, the result that sample measures out is also volumetric molar concentration or reflects with Molecules.So quantitative result belongs to absolute quantitation.The pure plasmid sample of 5 doors extracts according to the requirement of extraction of plasmid DNA test kit, elution volume 100 microlitre.Plasmid stock carries out doubling dilution (each dilution 10 times) as the quantitative standard of each qPCR.Specific amplification ensures that the melt curve analysis of qPCR product only has one group of peak of a unimodal or melting temp closely (difference is generally no more than 5 degree); According to the door specific primer design of the art of this patent, by seeing that amplified production is all the kind of target door to the cloning and sequencing of this amplified production.The specificity of amplified production and the configuration of door specific criteria sample can ensure the validity that qPCR is quantitative.The quantitative plasmid by each door oneself of each is as quantitative criterion, and door Auele Specific Primer ensures that amplified production is the kind sequence of target door, so just can ensure the validity that qPCR is quantitative.The above-mentioned of 5 doors quantitatively can be cooked respectively, test condition also can done with in a qPCR together by reducing the means such as the number of times of repeated sample after stablizing, the quantitative of 5 doors or more door once can be completed, then obtain the quantitative result of biological community structure fast.If the number of the door of the microflora related to is many, such as 7-12, or need the sample of mensuration more, then can consider use 384 orifice plate and volley of rifle fire application of sample.
5. utilize Bacteroidetes Auele Specific Primer to carry out the quantitative comparison of Bacteroidetes to certain brand white wine four kinds cellar for storing things mud sample.Bacteroidetes primer specific primer amplification length is about 185 base pairs.
(1) the plasmid gradient 10 times dilution of Bacteroidetes door: get plasmid mother liquor 2 μ L and join in 18 μ LddH2O and fully mix, vortex centrifugal mixing repetition 2 times, obtains 10
-1level doubling dilution, so goes down, obtains 10 respectively
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, 10
-8.
(2) Mix1:(12 μ L system is prepared) 10
-3-10
-8(6)
To add after Taq enzyme vortex centrifugal mixing repetition twice
(3) whole sample Mix2:10 is separately prepared
-3, 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, testing sample 1. 2. 3. 4., NTC (negative control does not add template); It is as follows that (totally 11) above-mentioned each sample is oneself a Mix2 (enough repeat 3 times):
Mix1：11×3.5μL＝38.5μL
The each self-template of sample: 1 × 3.5 μ L=3.5 μ L
More than operate and all will carry out in ice bath.11 sample equal vortex centrifugal mixing repetitions twice.
(4) QPCR eight union loading
| 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 |
Carry out testing and deriving quantitation curves according to the step-oneQPCR program set.Base program is as follows:
(5) experimental result and analysis:
Standard plasmid mother liquor DNA molecular concentration is 75649000 × 10
8copy/L, dilution gradient is as follows:
| Gradient dilution | 10 -3 | 10 -4 | 10 -5 | 10 -6 | 10 -7 | 10 -8 |
| Concentration (10 8Copy/L) | 75649 | 7564 | 756 | 75 | 7 | 0.7 |
The absolute concentration mean value that sample calculates:
| Sample | Cellar for storing things mud sample A | Cellar for storing things mud sample B | Cellar for storing things mud sample C | Cellar for storing things mud sample D |
| Concentration (10 8Copy/L) | 2 | 744 | 14163 | 851 |
Utilize above-mentioned same program to carry out quantitative assay to syntrophism bacterium door, amplification length is about 208 base pairs.
Syntrophism bacterium door standard plasmid mother liquor DNA molecular concentration is 67323000 × 10 after measured
8copy/L, dilution gradient is as follows:
| Gradient dilution | 10 -3 | 10 -4 | 10 -5 | 10 -6 | 10 -7 | 10 -8 |
| Concentration (10 8Copy/L) | 67323 | 6732 | 673 | 67 | 6 | 0.6 |
The absolute concentration that sample calculates:
| Sample | Cellar for storing things mud sample A | Cellar for storing things mud sample B | Cellar for storing things mud sample C | Cellar for storing things mud sample D |
| Concentration (10 8Copy/L) | 0 | 140 | 385 | 4 |
Utilize above-mentioned same program to carry out quantitative assay to actinomycetes door, amplification length is about 157 base pairs.
Actinomycetes door standard plasmid mother liquor DNA molecular concentration is 1134140 × 10 after measured
8copy/L, dilution gradient is as follows:
| Gradient dilution | 10 -1 | 10 -2 | 10 -3 | 10 -4 | 10 -5 | 10 -6 |
| Concentration (10 8Copy/L) | 113414 | 11341 | 1134 | 113 | 11.3 | 1.13 |
The absolute concentration that sample calculates:
| Sample | Cellar for storing things mud sample A | Cellar for storing things mud sample B | Cellar for storing things mud sample C | Cellar for storing things mud sample D |
| Concentration (10 8Copy/L) | 0 | 298986 | 13 | 0 |
Utilize above-mentioned same program to carry out quantitative assay to Firmicutes, amplification length is about 135 base pairs.
Firmicutes standard plasmid mother liquor DNA molecular concentration is 195071744 × 10 after measured
8copy/L, dilution gradient is as follows:
| Gradient dilution | 10 0 | 10 -1 | 10 -2 | 10 -3 | 10 -4 | 10 -5 |
| Concentration (10 8Copy/L) | 195071744 | 19507176 | 1950717 | 195071 | 19507 | 1950 |
The absolute concentration that sample calculates:
| Sample | Cellar for storing things mud sample A | Cellar for storing things mud sample B | Cellar for storing things mud sample C | Cellar for storing things mud sample D |
| Concentration (10 8Copy/L) | 122505 | 37972420 | 597973824 | 6704090 |
Utilize above-mentioned same program to carry out quantitative assay to Proteobacteria, amplification length is about 264 base pairs.
Proteobacteria standard plasmid mother liquor DNA molecular concentration is 3666710 × 10 after measured
10copy/L, dilution gradient is as follows:
| Gradient dilution | 10 -1 | 10 -2 | 10 -3 | 10 -4 | 10 -5 | 10 -6 |
| Concentration (10 10Copy/L) | 366671 | 36667 | 3666 | 366 | 36 | 3 |
The absolute concentration that sample calculates:
| Sample | Cellar for storing things mud sample A | Cellar for storing things mud sample B | Cellar for storing things mud sample C | Cellar for storing things mud sample D |
| Concentration (10 10Copy/L) | 673 | 2818 | 1946 | 9 |
The biological community structure quantitative result of five doors of four class cellar for storing things mud shown in Fig. 1 can be obtained according to above-mentioned flow testing, quantitatively consuming time control of each QPCR completes at 2 hours, found out by Fig. 1: cellar for storing things mud sample A, wherein the species content of two doors and Syn and Act is little; Cellar for storing things mud sample B, it is close with the comparison between community structures of cellar for storing things mud sample C, and species content is abundanter; Cellar for storing things mud sample D, wherein actinomycetes door Act content is less.
Claims (4)
1. the method that in a Fast Measurement liquor fermentation process, microflora forms, it is characterized in that: reach in liquor fermentation process by using door Auele Specific Primer in PCR reaction system and specific amplification is carried out to certain microorganism species concrete, and use qPCR system to carry out quantitative amplification, obtain quantitative judge.
2. the method that in Fast Measurement liquor fermentation process according to claim 1, microflora forms, is characterized in that: prepare door Auele Specific Primer as follows:
The grand genome of step a, extraction brewing microorganism group sample is as polymerase chain reaction (PCR) amplification template;
Step b, the rDNA that increases in described amplification template are close to total length 16srDNA sequence, amplimer is 27F (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1492R (5 '-GGTTACCTTGTTACGACTT-3 '), obtains amplified production;
Step c, described amplified production carried out after purifying to TA clone, the screening of blue hickie and the two-way order-checking of hickie bacterium colony successively, obtain the effective clone being no less than 200, described effective clone refers to that last two-way sequencing sequence belongs to 16srDNA sequence really through sequence alignment;
Steps d, for the two-way sequencing sequence of described effective clone by phylogenetic analysis, complete design of primers.
3. the method that in Fast Measurement liquor fermentation process according to claim 1, microflora forms, is characterized in that:
Configuration to described qPCR system: be use qPCR test kit and qPCR instrument, and the fluorescent substance requiring qPCR instrument can detect corresponding test kit to comprise, described fluorescent substance comprises EvaGreen and SYBRgreen;
For setting and the operation of the condition of described qPCR system; Carry out the adjusting and optimizing of amplification condition according to different microorganisms group and different doors, the primer designed during the different door of the same group of quantitative judge possesses close Tm value, to carry out testing and applying at identical amplification condition as far as possible;
Detection for the product of described qPCR system: in method establishment process, detects purity and the quality of amplified production by agarose gel electrophoresis; When amplified production reaches single master tape and the analysis of Tm value possesses single product peak, no longer need to run glue and detect product, during use, qPCR only needs to be judged by the analysis of Tm value, utilizes qPCR instrument to derive qPCR amplification curve, quantitative result and Tm value analytical results simultaneously.
4. the method for microflora's composition in Fast Measurement liquor fermentation process according to claim 1, is characterized in that:
Described door Auele Specific Primer is Bacteroidetes Auele Specific Primer, and its primer pair nucleotides sequence is classified as:
320(5'-CGCACGGGTGAGTAACACGTAT-3')，321(5'-GGGGATAAATCCTCTCAGTTCCCCT-3')；
Described door Auele Specific Primer or be the Auele Specific Primer of Proteobacteria, its primer pair nucleotides sequence is classified as:
317(5'-CCGTAGCTGGTCTGAGAGGATGATAA-3')，319(5'-CACGCTTTACGCCCAGTAATTCCYA-3')；
Described door Auele Specific Primer or be the Auele Specific Primer of Firmicutes, its primer pair nucleotides sequence is classified as:
J1(5'-CAGCAGTGGGGAATCTTCC-3')，J2(5'-TAGCCGGGGCTTCCTCCT-3')；
Described door Auele Specific Primer or for syntrophism bacterium door Auele Specific Primer, its primer pair nucleotides sequence is classified as:
J7(5'-AAACCGCTTTCAGCAGGGAC-3')，J8(5'-TAACTCCCGACCAAACAAGC-3')。
Described door Auele Specific Primer or be actinomycetes door Auele Specific Primer, its primer pair nucleotide sequence is as follows:
J9(5'-AGCGAACGGGATTAGATACCCC-3')，J10(5'-AGGTAAGGTTCTTCGGTTTGCA-3')。
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| CN108559771A (en) * | 2018-04-04 | 2018-09-21 | 贵州省产品质量监督检验院 | The multifarious detection method of microbe colony during a kind of brewed spirit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107699610A (en) * | 2017-10-13 | 2018-02-16 | 哈尔滨工业大学（威海） | For detecting two pairs of specific primers of acidproof Bacillus acidi lactici content and its application during liquor fermentation |
| CN108559771A (en) * | 2018-04-04 | 2018-09-21 | 贵州省产品质量监督检验院 | The multifarious detection method of microbe colony during a kind of brewed spirit |
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