CN109251989A - A kind of method of methane bacterial content in quantitative detection pit mud - Google Patents
A kind of method of methane bacterial content in quantitative detection pit mud Download PDFInfo
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- CN109251989A CN109251989A CN201811403485.3A CN201811403485A CN109251989A CN 109251989 A CN109251989 A CN 109251989A CN 201811403485 A CN201811403485 A CN 201811403485A CN 109251989 A CN109251989 A CN 109251989A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C—CHEMISTRY; METALLURGY
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention belongs to biotechnology and field of food fermentation, it is a kind of method for relating to the use of methane backeria in specific primer quantitative detection Luzhou-flavor liquor pit mud, the present invention designs the specific primer of target methane bacterium for the specificity of target methane bacterium 16S rDNA in some classification levels (door detailed outline section belongs to kinds of six classification levels can be with).The method includes the steps of: (1) design of target methane bacterium specific primer；(2) acquisition of different pit mud samples and its macro genome extract；(3) QPCR (quantitative polymerase chain reaction) amplification is carried out come the content of methane backeria in quantitative detection difference Luzhou-flavor liquor pit mud sample to said gene group sample using target methane bacterium specific primer.The present invention is based on the specific SNP of target methane bacterium 16S rDNA (single nucleotide polymorphism, single nucleotide polymorphism) and SAP (simple allele discriminating PCR) Technology design category specific PCR (polymerase chain reaction) primer method, provide and a kind of feasible utilize the molecular biology method of specific primer detection methane backeria from complicated flora.
Description
Technical field
The invention belongs to biotechnology and field of food fermentation, it is white to be that one kind is related to specific primer quantitative detection Luzhou-flavor
The method of methane backeria in the mud of wine cellar.
Background technique
In the brewing process of white wine, pit mud plays an important role as microorganism carrier.It is various in pit mud
Microorganism mutually maintains the balance of group under the anaerobic condition of fermentation, forms the special ecosystem, for the hair of drinks
Ferment degree and flavor, which are moulded, plays conclusive effect.With the development of molecular biology, since 16S rRNA gene is in species
Have well-conserved in evolutionary process, people actively determine the microorganism door classification such as detailed outline kind using 16S rRNA gene
Information is learned, carrying out PCR amplification to the microorganism 16S rRNA gene do not cultivated or cannot cultivated becomes main method, especially
It is the microorganism of extreme environment growth, such as methane backeria.Methane backeria is obligate strict anaerobes, and growth and breeding is especially slow,
Culture of isolated is relatively difficult, and in laboratory simulation, their growth conditions is highly difficult；And in the past to Luzhou-flavor liquor pit
The serial analysis of mud flora the result shows that, methane backeria plays a significant role in producing the fermentation process such as perfume (or spice), thus need to its into
Row further investigation.
Content distribution situation of the methane Pseudomonas in Luzhou-flavor liquor pit mud is similar, and existing detection report is usually
3-6 category.For example once reported 4 advantage methane Pseudomonas in the pit mud of five-Grain Liquor wine, respectively Methanobacterium,
Methanobrevibacter, Methanoculleus and Methanosarcina, wherein Methanosarcina can be sharp simultaneously
With acetic acid and H2.Contain at least five methane Pseudomonas in the pit mud of Mianyang, Sichuan Feng Gu the wine industry, is Methanoculleus respectively
(Methanoculleus, herein referred to as methane capsule Pseudomonas, referring to Baidupedia), Methanosarcina (Sarcina),
Methanobacterium (Methanobacterium), Methanocorpusculum (methane grain Pseudomonas) and
Methanomicrobiaceae (methane germ category).At least 6 methane are found in Luzhou, Sichuan company liquor production pit
Pseudomonas, be respectively Methanobrevibacter, Methanosata, Methanoculleus, Methanobacterium,
Methanocorpusculum and Methanothri, and methane backeria at least different containing 4 classes in the century-old pit mud of Luzhou Old Cellar
Belong to, is methane brevibacterium (Methanobrevibacter), Methanobacterium (Methanobacterium), methane hair on the neck respectively
Pseudomonas (Methanosaeta) and methane capsule Pseudomonas (Methanoculleus).Obviously, different Luzhou-flavor liquos is in daily matter
It needs to design specific (quick) detection technique to the methane Pseudomonas of various combination during control.
The specific detection of methane backeria has had many reports.It is produced in root canal infection for example, Wei Yanxia et al. attempts to study
The diversity of methane archeobacteria utilizes methyl coenzyme M reductase (methyl coenzyme M reductase, MCR) gene α
The specific primer of subunit (mcrA) finds that the diversity of methane phase archeobacteria in selected sample is confined to class oral cavity methane quarter butt
Bacterium sequence type.Hua Jinling etc. has found the methane backeria master in the white Rumen of the Yellow River and Huai He River also with MCR gene specific gene primer
If cud methagen.Lan Afeng etc. determines open country using methane backeria 16S rDNA specific primer Met86F and Met1340R
Methane backeria type in raw Qinling Mountains takin cud, discovery includes at least three section, several categories, and diversity is relatively abundanter.
The methane backeria specific primer designed using genes such as MCR, and using methane backeria ribosomal gene Met86F and
The detection method of the specific primers such as Met1340R is ideally suited for most of methanogens, it can be difficult to different first
Alkane strain category distinguishes.Also there are the report of several methane backeria specific detection technologies, such as Luo Qingchun in white wine research field
Containing for several main methanogens in the mud of different year river wine cellar is determined Deng with known specific primer and quantitative fluorescent PCR
Amount, with the increase in pit mud time, 3 kinds of methane phase Pseudomonas methane mane Pseudomonas (Methanosaeta), methane eight fold ball for discovery
The content of Pseudomonas (Methanosarcina) and Methanobacterium (Methanobacter) is in the increase trend of different amplitudes.
Find that they are the spies in mesh (order) classification level when but reconnoitring to the original of specific primer known to these
Specific primer, and do not explain the basic verification data of primer specificity (for example basic PCR runs glue and amplified production sequencing
Etc. data).For another example Hu shellfish etc. devises the Genus-specific primers of 3 pairs of methane backerias, respectively corresponds Methanobrevibacter,
Methanoculleus and Methanosarcina, they have found Methanoculleus, and this belongs to the Time Change of content
There is apparent difference in Jian Nan Chun and the respective pit mud of Shuijingfang.The discovery after specificity checks of this 3 pairs of primers, which meets, to be set
Meter requires, and finds it is all the target dna sequence designed by sequence verification using these pit muds DNA as the amplified production of template.?
High-flux sequence that can be quick is the results show that the methane Pseudomonas at least Methanosarcina (1- that Ancient Well Imperial Liquor pit mud includes
458),unkown genus-1(1-2),unkown genus-2(2-8),Methanofollis(3-896),
Methanoculleus(16-14155),Methanocorpusculum(6-2180),Methanococcus(2-10),
Methanobrevibacter (3-903) and Methanobacterium (5-1514), two numbers inside bracket are respectively
It is the number and its sequence total number of respective OTU (operating taxonomic unit), shows in Ancient Well Imperial Liquor pit mud
The sequence complexity and Pseudomonas content of this category of Methanoculleus are all the largest relatively in whole methane backerias.In pit mud
In the method system of quality evaluation, the method for detecting specificity of methane Pseudomonas belongs to important construction content.
It is generally believed that methane backeria utilizes H2Restore CO2Generate CH4, balance bacterial metabolism raw to promotion organic acid, alcohol
At direction conversion, promote in fermented grain acid and ester content, also improve liquor output rate.According to " Interspecies H_2 transfer " principle,
The hydrogen that methane Pseudomonas utilizes caproic acid bacteria fermentation to generate is used to generate methane, so that caproic acid bacteria is eliminated the inhibition of hydrogen and promotes to produce acid,
Then improving ethyl hexanoate content, still the above process can only occur in the limited position of pit, because only that certain depth
Under pit sealed bottom fermentation a period of time after be likely to meet the anaerobic growth conditions of methane Pseudomonas.Methane backeria promotes white
The approach of wine fermentation quality be certainly it is various, theoretically different methane Pseudomonas is in pit mud-yellow water-fermented grain interface anaerobism
The effect played in fermentation process will be different with mode.But these methane Pseudomonas pass through which biological pathway is sent out on earth
Wave between effect, methane Pseudomonas that there are the problems such as which important interaction also to need largely to study and could gradually get clear
Chu needs to detect changes of contents of each methane Pseudomonas in fermentation dynamic process.So design a batch is directed to each methane backeria
Specific primer on the category or kind of category or other classification levels is necessary.
SNP (single nucleotide polymorphism) label is used as third generation molecular labeling, in molecular genetic
, pharmacogenetics, medical jurisprudence and diagnosing and treating of disease etc. play an important role.This patent is based on object bacteria
The SNP site belonged in 16S rRNA is established in conjunction with MAFFT Multiple Sequence Alignment technology and scalability SAP design of primers technology
The specific primer design method of any methane Pseudomonas in complicated flora.
Summary of the invention
For the deficiency for making up existing methane Pseudomonas Molecular Identification and quantitative measurement technology, the present invention provides one kind to be related to benefit
With the method for methane backeria in specific primer quantitative detection Luzhou-flavor liquor pit mud, the present invention is for target methane bacterium in some point
The specificity of 16S rDNA, designs the spy of target methane bacterium in class level (door detailed outline section belongs to kinds of six classification levels can be with)
Specific primer.The method includes the steps of: (1) design of target methane bacterium specific primer；(2) different pit mud samples are adopted
Collection and its macro genome extract；(3) QPCR is carried out to said gene group sample using target methane bacterium specific primer
(quantitative polymerase chain reaction) amplification carrys out quantitative detection difference Luzhou-flavor liquor pit mud sample
The content of middle methane backeria.The present invention is based on specific SNP (the single nucleotide of target methane bacterium 16S rDNA
Polymorphism, single nucleotide polymorphism) and SAP (simple allele discriminating PCR) Technology design category
The method of specific PCR (polymerase chain reaction) primer provides a kind of feasible sharp from complicated flora
With the molecular biology method of specific primer detection methane backeria.Specific several correlation steps are described below:
1) pit mud sample
9 point samplings are carried out in Chinese Luzhou-flavor, four wall center of pit respectively takes a bit, and bottom of pond quadrangle respectively takes a bit, pond
Bottom center takes a bit.2 centimetres of depths of every sampling, 2 centimeter squares.9 points of pit mud blocks sampled are mixed mixing as one
A aggregate sample.The macro genome DNA of pit mud is extracted using Ezup pillar genome extraction kit (B518251, the raw work in Shanghai),
The genomic DNA of pit mud from 200mg is dissolved in 100uL TE buffer, be further diluted to 500uL as PCR or
The template of QPCR, what QPCR was measured is the copy Particle density of methane Pseudomonas 16S rDNA in the 500uL solution.Utilize QPCR result
Whole test process can be taken into account the content that reduction calculates methane Pseudomonas in pit mud itself.
2) main agents and software
PCR kit NPK02 (regular-PCR kit) and NPK62 (QPCR kit): Weihai Xiao Dong biotinylated biomolecule is limited
Company；Marker D: the raw work biology Co., Ltd in Shanghai；Specific primer: the raw work biology Co., Ltd synthesis in Shanghai；MAFFT
Online Multiple Sequence Alignment tool.Other biochemical reagents are purchased from the raw work in Shanghai.
3) regular-PCR detection primer specificity is utilized
The specificity of primer is tested using polymerase chain reaction (Polymerase Chain Reaction, PCR)
Card.Using pit mud genomic DNA mixed in equal amounts as hybrid template, PCR reaction system are as follows: 6 μ L of NPK02buffer (2 ×), object
1.5 μ L of species-specific primer, 1 μ L of template, 0.3 3.2 μ L of μ L, ddH2O of Taq enzyme.PCR cycle parameter: 94 DEG C of 4min；94℃
30s, 60 DEG C of 30s, 72 DEG C of 45s, 37 circulations；72℃-2min.Take 8 μ L reaction solutions carry out 1% Ago-Gel (100V,
30min) electrophoresis.
4) 16S rRNA sequence alignment analysis and the confirmation of methane capsule Pseudomonas SNP site
The 16S of highest 30 Pseudomonas of content and target methane Pseudomonas (such as methane capsule Pseudomonas) in cellar mud microorganisms flora
RRNA full length sequence is tested by high-flux sequence and is obtained；MAFFT is a kind of higher comparison method of Multiple Sequence Alignment accuracy,
Using MAFFT to above-mentioned sequence and the 210 target Pseudomonas obtained from Genbank database search (such as methane capsule Pseudomonas
Methanoculleus 16S rRNA (all taking full length sequence) gene order) is compared, and selects respective representative sequence
Column, to carry out the SNP discriminance analysis between each category.Here full length sequence actually refers to that ribosomal RNA gene is all variable
The sequence that area all includes.SNP marker is as third generation molecular labeling, in molecular genetics, pharmacogenetics, medical jurisprudence and disease
The diagnosing and treating etc. of disease plays an important role.Searching is compared to sequence by MAFFT Multiple Sequence Alignment tool
SNP site returns in target Pseudomonas to all SNP sites listed and carries out SNP verification with itself remaining sequence, it is desirable that the position
Point base is identical as remaining sequence position base overwhelming majority；It is investigated again with remaining sequence alignment of each Pseudomonas of flora, it is desirable that with
The position base overwhelming majority is different, to select the best SNP site of theoretically effect.
5) design of methane backeria Genus-specific primers and QPCR are quantitative
The length of PCR primer need to be between 20~25bp, and annealing temperature need to be at 60 DEG C or so, and G/C content should be in 40-60%
Between, the stability of primer is kept with this.According to the method that is typically designed of SAP theory, 3 ' end ends of primer should be SNP
Point, in the artificial introducing mispairing in the previous site (penultimate of primer) of SNP site, so primer and target sequence
(target methane Pseudomonas) only has a mispairing in penultimate, and and non-target sequences in most latter two base position be all mispairing.
So non-target sequences can be prevented from effectively being expanded by suitable amplification condition.This research is based on the theory and exists
The position of mispairing, which has been done, slightly to be adjusted, and the primer of design is shown in following result.Suitable content in design is that SNP site only needs
It is placed on the last bit (being not necessarily last position, can be penultimate) at the end of primer 3 ', mismatch site is also placed in last bit, no
It must be penultimate (being also possible near antepenulatimate), objective is to make primer and target sequence several in 3 ' ends of primer
The pairing intensity of a base is greater than non-target sequences.Inventor laboratory, which has been done, in this respect several gropes to test.Draw
The specificity of object passes through PCR amplification and product sequence verification, determines that the errorless primer of specificity can be further used for QPCR again
Verifying.It is required that the welding curve of QPCR is unimodal and expands without apparent dimer.It is special with designed two pairs of methane Pseudomonas
The PCR product (measurement DNA concentration) of the specific primer standard substance quantitative as QPCR, is serially diluted for 10 times and does 7 gradients altogether
Dilution, respectively from 10-1It is diluted to 10-7Make standard curve.QPCR reaction condition is arranged in thermodynamic parameter according to primer.
62 × NPK62Buffer of μ L of primer reaction system, 1.75 μ L primers (respectively final concentration of 2 μM of upstream and downstream), the 4 macro bases of μ L pit mud
Because of a group DNA profiling, 0.25 μ L Taq enzyme.Grope to after best Q PCR condition, each pair of primer is repeated 3 times at optimum conditions.
DNA copy amount calculation formula
C in formula: for original liquid concentration
L: for pcr amplification product length
Primer designed by the present invention can be used in target methane Pseudomonas in Luzhou-flavor liquo brewing by QPCR technology
Quick and precisely quantitative, quantitative accuracy meets the required precision of the daily pit mud Quality Control of enterprise.
Primer design method provided by the invention is suitble under complicated flora background be that any methane Pseudomonas (can be a guiding principle
Mesh section belongs to kind of any one Pseudomonas level of six classification levels) the relatively high primer of design a batch specificity, this batch of primer warp
Crossing practical application may further determine that best primer.
Attached drawing table explanation
Fig. 1: the pcr amplification product electrophoresis verifying of specific primer pair；
The melting curve of Fig. 2: two couples of primer QPCR experiment；
The QPCR result of Fig. 3: two pairs of primers (Met1 and Met2).
Table 1: belong in embodiment and represent sequence selection result
Table 2: two pairs of primer information in embodiment
Table 3: the unidirectional sequencing result of PCR product in embodiment
Specific embodiment
Below by taking specific primer design and QPCR the quantitative test application of methane capsule Pseudomonas in Ancient Well Imperial Liquor pit mud as an example
Illustrate the implementation process of above-mentioned patented technology.
Embodiment 1: the design of methane capsule bacterium specific primer pair
Belong to the 16S rRNA overall length of (table 1) using MAFFT 30 highest to content in ancient well cellar mud microorganisms flora
The 16S rRNA of gene order and the 210 methane capsule Pseudomonas (Methanoculleus) obtained from Genbank database search
(some are overall lengths) gene order is compared, and selects respective representative series.By using MAFFT Multiple Sequence Alignment work
Tool represents sequence to methane capsule Pseudomonas and respectively belongs to the total 1+30x2=61 sequence of representative series with Gu well flora and be compared, just
Step has found 114 SNP sites of methane capsule bacterium.Feasibility in methane capsule Pseudomonas is carried out to SNP site later to verify, and is remained
41 SNP sites；Then 41 SNP sites are carried out belonging to outer feasibility verifying, it is (total with 30 category all sequences of ancient well flora
5499) after comparison, have found three best SNP sites of effect, site TCCAGGThe representativeness that belongs to of CCC is
94.3% and he to belong to representativeness be 0.4%, site AAAACTTT's belongs to that representativeness is 94.8% and he belongs to representativeness and is
0.8%, site TTCGACAGTo be 94.8% (be equivalent to based on its amplifying specific of the primer of the site design representativeness that belongs to of T
Property be not less than 90%, the daily Quality Control assessment that can satisfy food fermentation enterprise pit mud quality requires) and he belong to representativeness and be
0.4%.Select TTCGACAGThis SNP site of T devises two pairs of primers, and particular sequence and amplification sub-information are shown in Table lattice 2.
Belong in 1 embodiment of table and represents sequence selection result
Note: * g_unidentified:k_Bacteria, p_Firmicutes, c_Clostridia, o_
Clostridiales、f_Lachospiraceae.
Two pairs of primer information in 2 embodiment of table
Embodiment 2: the verifying of methane capsule bacterium specific primer pair
Designed above-mentioned two pairs of primers are subjected to PCR amplification experiment, PCR reaction system (12 μ L) are as follows: NPK02buffer
(the bright mixing of the macro genome DNA lamp of pit mud sample extraction is used as template by (2X) 6 μ L, 1.5 μ L of Species-specific primer, 1 μ L of template
DNA), 0.3 μ L, dd H of Taq enzyme2O 3.2μL.PCR reaction cycle parameter: 94 DEG C of 4min, [94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C
45s, 37cycles], 72 DEG C of 2min.Agarose gel electrophoresis: 1% Ago-Gel, Marker D20004 μ L, amplification produce
Appropriate EB is added in buffer in 8 μ L+ sample-loading buffer of object, 3 μ L.Voltage is seen in 40mA or more every 20min in 110V, electric current
Examine primary, about 40min stopping electrophoresis.The specificity of primer is observed by gel imager, electrophoresis result is as shown in Figure 1, electricity
Band of swimming is substantially in the same size with prediction, shows designed specific primer to the specificity having to methane capsule bacterium.It will be upper
It states pcr amplification product purifying submitting sequencing and obtains two sections of sequences (being unidirectionally sequenced using Met1-f and Met2-f respectively), sequencing knot
Fruit is as shown in table 3.1 sequence of table is subjected to BLAST verifying in NCBI, searches its similar sequences, result is target sequence.
The unidirectional sequencing result of PCR product in 3 embodiment of table
Embodiment 3: the QPCR of methane capsule bacterium in Luzhou-flavor liquor pit mud
18 ancient well pit mud samples, including 6, A plant area (40 years pits, sample 1-6), 3, B plant area (a century have been used altogether
Pit, sample 7-9), 9, C plant area (5 years pits, sample 10-18 are shown in Fig. 3).It extracts macro in Luzhou-flavor liquor pit mud sample
Genome, genome concentration are 6ng/ μ L.Use Met1-f/Met1-r, genome of the Met2-f/Met2-r primer pair to extraction
Carry out QPCR.QPCR overall reaction system (12 μ L) are as follows: 2 × NPK62Buffer6 μ L, primer (respectively final concentration of 2 μM of upstream and downstream)
1.75 μ L, 4 μ L of pit mud macro genome DNA template, 0.25 μ L of Taq enzyme.QPCR reaction condition are as follows: the 1st pair of primer Q-PCR reaction interval
Sequence is 95 DEG C of 4min, [95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 42cycles], 72 DEG C of 2min；2nd pair of primer Q-PCR reaction interval
Sequence is 95 DEG C of 4min, [95 DEG C of 30s, 65 DEG C of 30s, 72 DEG C of 30s, 42cycles], 72 DEG C of 2min.QPCR welding curve is shown in figure
The melting curve of 2.QPCR is an important indicator for measuring primer specificity.Melting curve expected from the present embodiment is a master
The secondary peak for having many fusing points very close under peak.This is because what is amplified is that one group of size and fusing point are all very close
DNA fragmentation.Fig. 2 shows the reasonable welding curve of two pairs of primers.QPCR quantifies calculated result and sees Fig. 3, two pairs of primers
The basic trend of quantitative result be also consistent, the visible trend for being exactly methane capsule bacterial content content in the sample is old pit
The new pit of >.
From the point of view of the high-flux sequence result of the macro genome of pit mud, abundance of the methane capsule bacterium in Ancient Well Imperial Liquor pit mud sample
Ranking is about the 7th, belongs to high abundance Pseudomonas.But other kinds of methane Pseudomonas abundance ranking is all after 45, than
If Methanocorpusculum arranges the 45th, Methanobacterium arranges the 62nd, and Methanobrevibacter arranges
86, Methanofollis arranges the 88th.It should be giving off a strong fragrance that this abundance precedence data, which prompts this category of Methanoculleus,
" Interspecies H_2 transfer " is primarily involved in Pseudomonas during type brewed spirit.Preliminary inspection of the present embodiment to 18 ancient well pit mud samples
Survey the result shows that, the copy number of methane capsule bacterium DNA is probably 2000 × 10 in the corresponding Genomic DNA solution of old pit pit mud8
A/L (Fig. 3) is finally converting the result is that the number of methane capsule bacterium DNA target fragment is about 1.5 × 10 in every milligram of pit mud6
It is a.
The specific primer of known methane capsule bacterium is considerably less.Two pairs of primers of the present embodiment provide more for researcher
Selection.It is very unique in view of effect of the methane Pseudomonas in Luzhou-flavor liquo fermentation process.Subsequent needs are to different methane backerias
Category will design more better PCR primers.There is no relatively more known methane capsule bacterium primer and 2 pairs of primers herein for the present embodiment
Measuring specific difference and QPCR quantitative result difference in 18 samples.Main reason is that the different hairs of different white wine
The composition of flora differs greatly in ferment sample, when several pairs of primers are widely different towards flora genome background in different samples,
The degrees of specificity respectively showed is different certainly.In other words, the specificity of which pair of primers is desirably dependent on earth
The specific sample used, research staff need to find the best primer for being suitble to sample used in actual test process.
Sequence table
<110>Harbin Institute (Weihai) of Technology
<120>in a kind of quantitative detection pit mud methane bacterial content method
<130> Demo reference number
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 545
<212> DNA
<213>Methanoculleus-specific 16S rDNA sequence-1 (Met1-f sequencing gained sequence)
<400> 1
gattagatac ccgggtagtc ccagccgtaa actatgcgcg ttaggtgtat cggtgaccac 60
gagttaccga ggtgccgaag ccaaaccgtg aaacttcctc ctgttaagta cggtcgcaag 120
gatgaaactt aaaggaattg gcgggcgagc accacaacag tggagcctgc ggtttaattg 180
ggctcaactt cgcaaagctc atcggataag acagcggaat gattgctgca ctgaagactc 240
tgcatgactg gctgagagtc ggtgcatgga tctctccagt gcgtactgtg aagcatcttg 300
ttaagttagg ccacgagcaa gacccacgcc aaccggtgcc agcatgccct ccggactgat 360
gttaacactg ttgctaccgc ctctgcttcc gaggaggaag gaatgagcaa cgctagttca 420
gcatgccccg aattatccgc gctacacgcg ggctacaatg ggcaggacaa tgggcatcga 480
caccgaatct gaaggcaatc tcttaaacct gtccttagtt cggattgtgg gtgtaacttg 540
gcaaa 545
<210> 2
<211> 357
<212> DNA
<213>Methanoculleus-specific 16S rDNA sequence-2 (Met2-f sequencing gained sequence)
<400> 2
gaaaacttta caatgcgggc aaccgtgata agggaacctc gagtgcctgt aaatgcaggc 60
tgttcaggtg cctaaaacac acctgaagaa agggccgggc aagaccggtg ccagccgccg 120
cgataatacc ggcggctcga gtggtggccg cttttattgg gcttaaagcg ttcgtagctg 180
ggttgttaag tctctgggga aatctggcgg cttaaccgtc agccgtctaa gggatactgg 240
caatcttgga accgggagag gtgaggggta cttcgggggt aggagtgaaa tcctgtaatc 300
ctcgagggac cacctgtggc gaaggcgcct caccagaacg gcttcgacag cgaagcg 357
Claims (5)
1. a kind of method of methane backeria in quantitative detection Luzhou-flavor liquor pit mud, it is characterised in that the method includes the steps of:
(1) design of target methane bacterium specific primer；
(2) acquisition of different pit mud samples and its macro genome extract；
(3) QPCR amplification is carried out come quantitative detection difference sample to said gene group sample using target methane bacterium specific primer
The content of middle methane backeria.It is any one that target methane bacterium or target methane Pseudomonas herein refer to six classification levels (door detailed outline section belongs to kind)
One group of methane backeria in a level can be with.Pseudomonas wording is popular appellation, it is not limited to belong to this classification level.
2. method according to claim 1, it is characterised in that target methane bacterium specific primer, design method is such as
Under: using the specific methane Pseudomonas of some in Luzhou-flavor liquor pit mud as research object, by content in pit mud at most before
The 16SrRNA overall length of 30 Pseudomonas represents sequence, the 16SrRNA overall length of target methane Pseudomonas represents sequence and database search
The target methane Pseudomonas 16SrRNA overall length other sequences selected use MAFFT (MultipleAlignmentusingFastFou
RierTransform, https: //www.ebi.ac.uk/Tools/msa/mafft/) Multiple Sequence Alignment analysis, find out target
Site whole singlenucleicpolymorphism (SNP) of methane Pseudomonas.Target methane bacterium is carried out to whole SNP sites
It is verified in belonging to the outer feasibility of Pseudomonas, 3-5 best SNP sites is found out, with reselection on the basis of this optimal one SNP
Point carries out specific primer design.Multipair target methane Pseudomonas specific primer can be designed in principle.
3. the method according to claim 1, wherein using SNP site and scalability SAP
(simpleallelediscriminatingPCR) method design primer.It is typically designed method according to SAP theory, primer
3 ' ends end should be SNP site, in the artificial introducing mispairing in the previous site (penultimate of primer) of SNP site, this
Sample one, which comes primer and target sequence (target methane Pseudomonas), only has a mispairing in penultimate, and and non-target sequences most latter two
Base position is all mispairing.So non-target sequences can be prevented from effectively being expanded by suitable amplification condition.
This patent has done slightly scalability adjustment in the position of mispairing based on the theory, i.e. SNP site has only to be placed at the end of primer 3 '
2, end (being not necessarily last position, can be penultimate), mismatch site is also placed in last bit, is not necessarily reciprocal the
Two (being also possible near antepenulatimate), objective is to allow primer and target sequence matching in the base of 3 ' end 2-4 of primer
Non-target sequences is greater than to intensity.The specificity of primer passes through PCR amplification and product sequence verification, determines that specificity is errorless
Primer can be further used for QPCR (quantitativePCR) and verify again, it is desirable that the welding curve of QPCR is unimodal and does not have
Apparent dimer amplification.
4. the method according to claim 1, wherein being one group of specific methane backeria (methane according to the above method
Capsule Pseudomonas) devise two pairs of primers, nucleotide sequence are as follows:
Met1-f5 '-TCACCAGAACGGCTTCGATAGT-3 ',
Met1-r5'-GCGAGTTACAGCCCACAATCC-3'；
Met2-f5 '-CGGAGTTGGATTCTGAGACACG-3 ',
Met2-r5’-CCAGCTTTCGTCCTTCGCT-3’。
5. application according to claim 1, which is characterized in that the field of the application is field of food fermentation.
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