CN106434914A - Key microbial functional genome detection method in pepper peeling process - Google Patents
Key microbial functional genome detection method in pepper peeling process Download PDFInfo
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Abstract
The invention discloses a key microbial functional genome detection method in a pepper peeling process. According to the key microbial functional genome detection method in the pepper peeling process, from the perspective of micro-ecosystem, pepper epidermis microorganisms in the peeling process are taken as an object; metagenome DNA of pepper epidermis bacteria is extracted and purified and a variable region sequence is amplified, and then the variable region sequence of the epidermis bacteria is read with the application of a high-throughput sequencing technology; by virtue of a bioinformatics platform, the sequence is subjected to quality control and species annotation and function prediction are completed, and on the basis, a microorganism species, which plays a vital role in the pepper peeling process, in fresh fruit epidermis and a core metabolic pathway thereof are comprehensively and objectively revealed depending on a statistical method. The detection method provided by the invention simplifies experimental operations and improves a sequencing efficiency, so that the development of metagenomics is greatly promoted.
Description
Technical field
The invention belongs to gene engineering technology field, key microorganisms function base in more particularly, to a kind of Fructus Piperiss exfoliating process
Detection method because of group.
Background technology
Fructus Piperiss are recorded in Tang earliest and show what Su Jing et al. between celebrating year write《Tang materia medica》, it is that Piperaceae Piper is perennial
Evergreen liana.Fructus Piperiss are suitable for growing in moistening, high temperature, calm and fertile soil area, and therefore north and south latitude is attached under the line
Area plantation within part height above sea level 500m is the most extensive.As the topmost torrid zone in world spice crop, Fructus Piperiss produce earliest
In India, with the popularizing planting of Fructus Piperiss, this is made object depth and is liked by the people of the world, and has widely in medical science and field of food
Purposes.The Ming Dynasty, Li Shizhen (1518-1593 A.D.) once existed《Compendium of Materia Medica》In refer to：" Fructus Piperiss, because its pungent like green pepper, therefore green pepper name, real non-green pepper.Recklessly
The hot pure sun of green pepper, flat help fire, confused mesh sends out skin ulcer." and modern medicine shows, Fructus Piperiss have treatment gastrofrigid vomiting stomachache and have loose bowels, food
It is intended to depressed, the wonderful effect of epilepsy abundant expectoration.By, being shown, its main chemical compositions includes to the chemical constituents determination of pepper fruits：Raw
Alkaloids (mainly pyrrolidines amide alkaloid), volatile oil, organic acid, lignanoid, phenolic compound and trace element etc., and
The most active component of content is piperine.Because Fructus Piperiss enjoy the good reputation of the king of spice always, international market is to recklessly in recent years
The demand of green pepper keeps sustainable growth.In international trade, black pepper total trade volume accounts for 80%, and Radix orixae japonicae accounts for 20%.And in BAIHU
In the green pepper course of processing, peeling technology is particularly important, and the process of decortication determines the quality of Radix orixae japonicae, directly affects Radix orixae japonicae
The whether pure white fragrance of color, smell and taste and strong, and then be also the quality assurance of follow-up Fructus Piperiss deep processed product.Take off currently for Fructus Piperiss
The research of skin is also in the exploratory stage, and conventional Fructus Piperiss peeling method mainly has immersion decortication peeling, machinery after acid-alkali treatment
Decortication, the biological decortication that enzyme process is peeled and is left to be desired.Fructus Piperiss peeling method the most traditional is to soak to remove seed-skin method, and also referred to as macerates skin
Method.Even to this day, the method is still favored by Fructus Piperiss Planting household deeply.Than remaining several method, the maximum advantage of the method
It is simple, directly Fructus Piperiss fresh fruit is placed in circulating water and soaks, first soaking rear peel is become the Fructus Piperiss fruit rotted in water
Directly fully trampled with foot in pond, then repeatedly rinse and remove the debris such as carpopodium with water until fully clean, then dry in the sun.
Although simple, the method exist in itself process-cycle length, floor space is big, water consumption is big, high labor intensive, macerate smelly
The defects such as highly seasoned and processing cost height, and Fructus Piperiss decortication is thorough, end product quality is unstable, and Radix orixae japonicae yield is relatively low.With
The prevailing of machining, some academys and business tie-up have developed Black pepper husking machine, its principle is first by Fructus Piperiss fresh fruit
It is positioned over decortication in tumbler after high temperature steams or dilute hydrochloric acid soaks.Although the method decortication ratio is more thoroughly, deposit
In the problem that heat and consumption of raw materials are too high, and Fructus Piperiss are easily impaired, and waste liquid of peeling is hardly consistent with national discharge of wastewater mark
Standard, severe contamination environment, finally make the method to be difficult to promote.And enzyme process decortication mainly uses pectin enzymatic degradation cortex institute
The pectin class gelling substance containing.Add the ferment treatment such as pectase, cellulase, xylanase and mannase at room temperature recklessly
Green pepper, makes fresh fruit epidermis degradable by zymolysis in the state of standing or stirring.The method does not cause environment dirty
Dye, finished product quality is higher.But the use of various enzymes makes this method cost too high, therefore also cannot popularize.Fructus Piperiss
The Pectin, proto of fresh fruit epidermis plays crosslinked action, and Pectin, proto is mainly with the poly D- galacturonic acid of (Isosorbide-5-Nitrae) key bonding as base
The polysaccharose substance of this structure.It is to be directed to the new approaches that Fructus Piperiss are peeled in the last few years that biology removes seed-skin method, and this method utilizes high yield
The microbiologic population of pectase ferments to Fructus Piperiss epidermis, and decomposing Pectin, proto makes peel separate and then reach the effect of decortication
Really.Compare with traditional peeling technology, its decortication production cycle is relatively short, and it is relatively low also to have an energy consumption, less investment of peeling, end product quality
Height, the advantage such as pollutant emission is few.However, the method still suffers from some bottlenecks, the Fructus Piperiss epidermis glue of complexity in extension process
Matter composition needs complicated enzyme system accordingly, and such enzyme system is not yet studied clear at present, and microorganism culturing technological requirement is relatively
High.Research in terms of microbial degumming for the China achieves great successes, but lacks the application test in Fructus Piperiss decortication.Recklessly
Green pepper is the tropical agricultural product of torrid areas, is the important crops of vast farmerses extra earning, is also the one of torrid areas economic development
Big specialty industries.At present the main pepper products of China are Radix orixae japonicaes, therefore the preliminary working peeling method of Fructus Piperiss and Radix orixae japonicae
Quality just becomes impact Hainan Province or even the national vital factor of Fructus Piperiss industry.But the Fructus Piperiss decortication preliminary working of current China
Also rest on the level of traditional infusion process, the Fructus Piperiss quality that this method obtains is low, not only color and luster burnt hair, and with serious different
Taste.Therefore continue to optimize microorganism and remove seed-skin method, invent a whole set of fast and accurately in Fructus Piperiss exfoliating process key microorganisms and
The discriminating method of its functional gene, is subsequently to compound out to improve Fructus Piperiss quality, reduce production cost, be easily achieved industrial metaplasia
The Research foundation of the microbial ecological agent producing, therefore meaning is especially great.
In sum, the Fructus Piperiss decortication preliminary working of current China also rests on the level of traditional infusion process, the Fructus Piperiss obtaining
Quality is low, not only color and luster burnt hair, and with serious abnormal flavour.
Content of the invention
It is an object of the invention to provide in a kind of Fructus Piperiss exfoliating process key microorganisms functional genome detection method,
Aim to solve the problem that the Fructus Piperiss decortication preliminary working of current China also rests on the level of traditional infusion process, the Fructus Piperiss quality obtaining is low, no
Only color and luster burnt hair, and the problem with serious abnormal flavour.
The present invention is achieved in that a kind of detection method of key microorganisms functional genome in Fructus Piperiss exfoliating process,
In described Fructus Piperiss exfoliating process, the detection method of key microorganisms functional genome is from the angle of microecosystem, with exfoliating process
Middle Fructus Piperiss epidermis microorganism is object；By extracting macro genome DNA the purification of Fructus Piperiss epidermis antibacterial, expand variable region sequences
Afterwards, application high throughput sequencing technologies complete the reading of epidermis antibacterial variable region sequences, using wide bioinformatics platform, sequence are entered
Row Quality Control simultaneously completes species annotation and function prediction, by fresh fruit table in statistical method comprehensively objective announcement Fructus Piperiss exfoliating process
Microbial species and its core metabolic pathway that skin plays a crucial role.
Further, in described Fructus Piperiss exfoliating process, the detection method of key microorganisms functional genome specifically includes following step
Suddenly：
Step one, from Fructus Piperiss epidermis enriched microorganism, the method acquisition antibacterial macro genome DNA of application frozen-thawed is simultaneously pure
Change；
Step 2, PCR expands, and with bacterial 16 S rDNA V3-V4 variable region for purpose fragment, application universal primer completes
The amplification of this variable region fragment and purification；
Step 3, application is read based on the sequence that the high-flux sequence instrument of secondary sequencing principle completes amplified fragments；
Step 4, is understood to measuring sequence by wide bioinformatics platform, completes annotation and the work(of microbial species
Can gene prediction, simultaneously Statistics Application method screen the microbial species that play a crucial role in Fructus Piperiss exfoliating process and its
Core Feature gene expression.
Further, the extraction of Fructus Piperiss epidermis microorganism macro genome DNA：Increase frozen-thawed adds ceramic bead and hits before extraction
Beat crumbling method release cellular content, then application CTAB method is extracted, and uses agarose gel electrophoresiies and micro purple after obtaining DNA
Outer spectrophotometer detects DNA mass.
Further, Fructus Piperiss epidermis antibacterial variable region amplification includes：
1) design of sample specific barcode primer：With primer-design software, design sample V3-V4 variable region is special
Property bar code high-flux sequence primer；
2) bacterial 16 S rRNA V3-V4 variable region amplification：With microorganism macro genome DNA as template, with above-mentioned design
Sample specific barcode high-flux sequence primer enters performing PCR amplification.
Further, PCR primer, after electrophoresis detection and quantitation, applies secondary high-flux sequence platform to carry out bridge-type to sample
PCR high-flux sequence, each sample guarantees to obtain 20,000 high-quality sequence.
Further, based on wide bioinformatics platform, application is increased income, and raw letter analysis software is long to primitive sequencer reading first to be carried out
Quality Control, removes low quality sequence and retains high-quality sequence；Run Analysis of Microbial Diversity program on the server, through choosing
Representative series and with reference to bacterium storehouse compare after obtain species diversity information, based on Unifrac evolutionary distance analysis sample it
Between beta diversity and explain between difference put sample Community composition dynamic change；Application random forest machine learning model meter
Calculate weight and the Changing Pattern of each strain, using function prediction open source software, function base is carried out to regular change microorganism
Cause and the annotation of metabolic pathway.
Further, amplimer：Sequence is：SEQ ID NO：1 and SEQ ID NO：2.
Another object of the present invention is to providing one kind using key microorganisms functional gene in described Fructus Piperiss exfoliating process
The Fructus Piperiss peeling method of the detection method of group.
The detection method of key microorganisms functional genome in the Fructus Piperiss exfoliating process that the present invention provides, to collection from difference
The Fructus Piperiss of environment carry out exfoliating process dynamic monitoring, can complete to close in exfoliating process within 24 hours (1 working day)
Key microorganism and its examination of functional genome, the biology for Fructus Piperiss removes seed-skin method offer microorganism preferred strain reference, and for recklessly
Green pepper preliminary working industrialized development provides technological guidance；Can on the basis of not carrying out microorganism fungus kind culture, easy, quick,
Qualitative, quantitative screens the core microorganism in Fructus Piperiss exfoliating process exactly, and detection resolution is high, obtains the micro- life of core quick
Disclose its major metabolic pathways and Core Feature gene, for follow-up Fructus Piperiss biological decortication Tiny ecosystem system while the strain of thing comprehensively
The exploitation of agent provides reference data.Inventor once did excessive quantifier elimination in terms of Fructus Piperiss fermentable decortication, and attempted using
Aspergillus niger solid fermentation Fructus Piperiss are peeled, and decortication time efficiency is greatly improved；And in this heuristic process, gradually
It is realized that the complicated enzyme system that Fructus Piperiss exfoliating process needs is far from what single microorganism effect disclosure satisfy that, and Fructus Piperiss are natural
In exfoliating process be also complicated microbiologic population mutually regulate and control, coefficient result.Therefore, in Fructus Piperiss microorganism decortication
It has breakthrough in method, first have to make complicated biological community structure relation in Fructus Piperiss nature exfoliating process clear, and these
The functional metabolism regulatory pathway of microorganism, screen out on this basis in Fructus Piperiss exfoliating process play mastery reaction microorganism and
Its ratio, is then proportionally configured to microorganism formulation by separation and amplification culture mode, finally acts on Fructus Piperiss again
Epidermis, significantly improves Radix orixae japonicae quality while accelerating Fructus Piperiss decortication.
The method of traditional microbiologic population's identification based on pure culture, due to the restriction of culture medium and condition of culture, is only capable of
Enough parts disclose microbial diversity in microecological environment sample it is impossible to the explaination Community composition of objective system.Secondary sequencing
The appearance of platform and the research reaching its maturity as microecosystem microbiologic population field are filled with one fresh blood, adjoint
Developing rapidly of second filial generation sequencing technologies and advancing by leaps and bounds of bioinformatics technique, based on Tiny ecosystem sample biome knot
The research of structure and its functional gene and metabolic pathway is possibly realized.In known microecosystem, with Fructus Piperiss dynamic fermentation
During the biological community structure of its epidermis compare, what human intestine's flora will be complicated is many.Even if but so complicated
Microecosystem in, the metagenomics technology based on secondary microarray dataset still can easily be tackled and accurately dissect.Grand
Genome refers to the summation of whole microorganism hereditary materials in specific environment.Metagenomics research and utilization sequencing technologies are to environment
In sample, the genome of whole microorganisms is measured, to analyze genomic constitution and function and the metabolic pathway of micropopulation,
Understand multiformity and the abundance of micropopulation, explore the relation between microorganism and environment and host, excavate and research is new
There is gene of specific function etc..Compared with traditional method, clone need not be built based on the grand genome research of high-flux sequence
Library, it is to avoid the system deviation using Host Strains, sample cloned and caused in library construction process, simplifies experiment
Operation, improves sequencing efficiency, thus being greatly promoted the development of metagenomics；Therefore apply the present invention to Fructus Piperiss to take off
During skin, the dynamic studies of its epidermis biological community structure and functional metabolism are feasible.
Brief description
Fig. 1 is the detection method stream of key microorganisms functional genome in Fructus Piperiss exfoliating process provided in an embodiment of the present invention
Cheng Tu.
Fig. 2 is the grand gene DNA electrophoretogram of Fructus Piperiss sample epidermis microorganism provided in an embodiment of the present invention.
Fig. 3 is that Fructus Piperiss sample 16S rRNA V3-V4 variable region sequences PCR electrophoretogram provided in an embodiment of the present invention is illustrated
Figure.
Specific embodiment
In order that the objects, technical solutions and advantages of the present invention become more apparent, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not used to
Limit the present invention.
The present invention from the angle of microecosystem, with exfoliating process the microorganism of Fructus Piperiss epidermis as object of study, by carrying
Take macro genome DNA the purification of Fructus Piperiss epidermis antibacterial.After expanding its variable region sequences, application high throughput sequencing technologies complete table
The reading of skin antibacterial variable region sequences, using wide bioinformatics platform sequence is carried out Quality Control and complete species annotation and function pre-
Survey, again on the basis of disclose micro- life of playing a crucial role of fresh fruit epidermis in Fructus Piperiss exfoliating process by statistical method is comprehensively objective
Thing species and its core metabolic pathway.
Below in conjunction with the accompanying drawings the application principle of the present invention is explained in detail.
As shown in figure 1, in Fructus Piperiss exfoliating process provided in an embodiment of the present invention key microorganisms functional genome detection
Method comprises the following steps：
S101：From Fructus Piperiss epidermis enriched microorganism, the method acquisition antibacterial macro genome DNA of application frozen-thawed is simultaneously pure
Change；
S102：PCR expands, and with bacterial 16 S rDNA V3-V4 variable region for purpose fragment, application universal primer completes this
The amplification of variable region fragment and purification；
S103：Application is read based on the sequence that the high-flux sequence instrument of secondary sequencing principle completes amplified fragments；
S104：Understood to measuring sequence by wide bioinformatics platform, complete annotation and the function of microbial species
The prediction of gene, microbial species and its core that Statistics Application method examination simultaneously plays a crucial role in Fructus Piperiss exfoliating process
Cardiac function gene expression.
In Fructus Piperiss exfoliating process provided in an embodiment of the present invention, the detection method of key microorganisms functional genome is specifically wrapped
Include following steps：
1st, the extraction of Fructus Piperiss epidermis microorganism macro genome DNA：
The key of application macro genomics technologies microorganisms group is that in sample, microorganism macro genome DNA extracts matter
The quality of amount.Grope through early stage and test it has been found that if it is desired to comprehensively obtaining the something lost of all microorganisms of sample as far as possible
Pass material, need to increase before extraction more strong frozen-thawed and add ceramic bead impact crumbling method release cellular content,
Then application CTAB method is extracted, and uses agarose gel electrophoresiies and micro UV spectrophotometer measuring DNA mass after obtaining DNA.
2nd, Fructus Piperiss epidermis antibacterial variable region amplification：
Fructus Piperiss exfoliating process mesocuticle microorganism is mainly made up of antibacterial, directed toward bacteria we intend expanding its 16S rRNA
V1-V3 variable region sequences carry out diversity analysis.1) design of sample specific barcode primer：With primer-design software,
Design sample V3-V4 variable region specific barcode high-flux sequence primer.2) bacterial 16 S rRNA V3-V4 variable region amplification：
With mentioned microorganism macro genome DNA as template, carried out with the sample specific barcode high-flux sequence primer of above-mentioned design
PCR expands.
3rd, pcr amplification product purification, quantitation and pyrosequencing：
PCR primer, after electrophoresis detection and quantitation, applies secondary high-flux sequence platform to carry out bridge-type PCR high pass to sample
Measure sequence, each sample guarantees to obtain 20,000 high-quality sequence, can cover the alkali of more than 99% microbial information in sample
Basic sequence is constituted.
4th, wide bioinformatics platform analysis microbial diversity and functional genome：
Based on wide bioinformatics platform, application is increased income, and raw letter analysis software is long to primitive sequencer reading first to carry out Quality Control, goes
Except low quality sequence retains high-quality sequence.Then on self-built raw letter platform, run Analysis of Microbial Diversity program, pass through
Choose representative series and obtain species diversity information after comparing with reference bacterium storehouse, sample is analyzed based on Unifrac evolutionary distance
Beta diversity between product simultaneously explains the Community composition dynamic change putting sample between difference.Based on the above results, apply gloomy at random
Woods machine learning model calculates weight and the Changing Pattern of each strain, finally using function prediction open source software to regularity
Change microorganism carries out the annotation of functional gene and metabolic pathway.
With reference to specific embodiment, the application effect of the present invention is explained in detail.
Embodiment 1：The detection of key microorganisms and its functional gene in the Fructus Piperiss exfoliating process of Hainan Province Qionghai area
1st, the extraction of Fructus Piperiss epidermis microorganism macro genome DNA：
With 8 parts of Fructus Piperiss samples in the decortication stage from Hainan Province's Qionghai area for the collection as object of study, extract front increase relatively
Add ceramic bead impact crumbling method release cellular content for strong frozen-thawed, then application CTAB method is extracted, specifically side
Method is as follows：Take 10.0g sample Fructus Piperiss sample carrying out washing treatment, by the thalline of eluting in 1.5mL centrifuge tube, be immediately placed in liquid nitrogen
Fully charge, puts into thawing (about 5min) in 60 DEG C of water-baths, multigelation 4 times, is incubated 4h in 37 DEG C of constant-temperature tables after taking-up,
12000g centrifugation 10min under room temperature, collects supernatant and is transferred in another centrifuge tube.Supernatant and isopyknic chloroform in
12000g centrifugation 10min (in order that precipitation, aqueous phase and organic faciess layering), Aspirate supernatant is transferred in another centrifuge tube and carries out
Phenol chloroform 2 times, the Sodium Acetate Trihydrate through 0.1 times of volume, the ice isopropanol precipitating STb gene of 1 times of volume, then 70% washing with alcohol
Precipitation 2 times, back dissolving is standby.Agarose gel electrophoresiies and micro UV spectrophotometer measuring DNA mass is used after obtaining DNA.Obtain
The DNA gel electrophoretogram obtaining is as shown in Figure 2.
2nd, Fructus Piperiss epidermis antibacterial variable region amplification：
Fructus Piperiss exfoliating process mesocuticle microorganism is mainly made up of antibacterial, directed toward bacteria we intend expanding its 16S rRNA
V3-V4 variable region sequences carry out diversity analysis.Amplimer：With antibacterial V3-V4 area universal primer as amplimer, primer sequence
It is classified as：SEQ ID NO：1 338F(5′-ACTCCTACGGGAGGCAGCA-3′)；SEQ ID NO：2 806R(5′-
GGACTACHVGGGTWTCTAAT-3′).Amplification system：Reaction system 100 μ L：10 × PCR Buffer 10 μ L, 25mmo1/L
Mg2+2.0 μ L, 2.5mmol/L dNTPs 8 μ L, each 4 μ L of 5mmol/L upstream and downstream primer, DNA profiling 100ng about, 5U/ μ L
Taq enzyme (Promega) 1.5uL, dd H2O complements to 100 μ L.Amplification condition：94 DEG C of degeneration 5min；Circulation 30 times：94 DEG C of degeneration
1min, 55 DEG C of annealing 45s, 72 DEG C of extension 1min, then 72 DEG C of extension 7min, 4 DEG C of preservations.Amplified production is entered with performing PCR electrophoresis,
Electrophoretogram such as Fig. 3.
3rd, pcr amplification product purification, quantitation and pyrosequencing：
PCR primer, after electrophoresis detection and quantitation, applies secondary high-flux sequence platform to carry out bridge-type PCR high pass to sample
Measure sequence, each sample guarantees to obtain 20,000 high-quality sequence, can cover the alkali of more than 99% microbial information in sample
Basic sequence is constituted.
4th, wide bioinformatics platform analysis microbial diversity and functional genome：
Based on wide bioinformatics platform, application is increased income, and raw letter analysis software is long to primitive sequencer reading first to carry out Quality Control, goes
Except low quality sequence retains high-quality sequence.Then on self-built raw letter platform, run Analysis of Microbial Diversity program, pass through
Choose representative series and obtain species diversity information after comparing with reference bacterium storehouse, sample is analyzed based on Unifrac evolutionary distance
Beta diversity between product simultaneously explains the Community composition dynamic change putting sample between difference.Based on the above results, apply gloomy at random
Woods machine learning model calculates weight and the Changing Pattern of each strain, finally using function prediction open source software to regularity
Change microorganism carries out the annotation of functional gene and metabolic pathway.By annotation, Fructus Piperiss epidermis microbial diversity and its metabolism
Path contribution rate is as shown in table 1：
Superior microorganism Pseudomonas and its Core Feature gene content distribution (%) in table 1 Fructus Piperiss exfoliating process
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all essences in the present invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.
Claims (8)
1. in a kind of Fructus Piperiss exfoliating process key microorganisms functional genome detection method it is characterised in that described Fructus Piperiss take off
During skin, the detection method of key microorganisms functional genome is from the angle of microecosystem, with Fructus Piperiss epidermis in exfoliating process
Microorganism is object；By extracting macro genome DNA the purification of Fructus Piperiss epidermis antibacterial, after amplification variable region sequences, application is high
Flux sequencing technologies complete the reading of epidermis antibacterial variable region sequences, using wide bioinformatics platform, sequence are carried out with Quality Control complete
Become species annotation and function prediction, play crucial work by fresh fruit epidermis in statistical method comprehensively objective announcement Fructus Piperiss exfoliating process
Microbial species and its core metabolic pathway.
2. in Fructus Piperiss exfoliating process as claimed in claim 1 key microorganisms functional genome detection method, its feature exists
In in described Fructus Piperiss exfoliating process, the detection method of key microorganisms functional genome specifically includes following steps：
Step one, from Fructus Piperiss epidermis enriched microorganism, applies the method for frozen-thawed to obtain antibacterial macro genome DNA purification；
Step 2, PCR expands, and with bacterial 16 S rDNA V3-V4 variable region for purpose fragment, application universal primer completes this can
Become amplification and the purification of area's fragment；
Step 3, application is read based on the sequence that the high-flux sequence instrument of secondary sequencing principle completes amplified fragments；
Step 4, is understood to measuring sequence by wide bioinformatics platform, completes annotation and the function base of microbial species
The prediction of cause, microbial species and its core that Statistics Application method examination simultaneously plays a crucial role in Fructus Piperiss exfoliating process
Functional gene is expressed.
3. in Fructus Piperiss exfoliating process as claimed in claim 2 key microorganisms functional genome detection method, its feature exists
In the extraction of Fructus Piperiss epidermis microorganism macro genome DNA：Increase frozen-thawed adds ceramic bead impact crumbling method and releases before extraction
Put cellular content, then application CTAB method is extracted, after obtaining DNA, use agarose gel electrophoresiies and micro ultraviolet spectrophotometer
Detection DNA mass.
4. in Fructus Piperiss exfoliating process as claimed in claim 2 key microorganisms functional genome detection method, its feature exists
In the amplification of Fructus Piperiss epidermis antibacterial variable region includes：
1) design of sample specific barcode primer：With primer-design software, design sample V3-V4 variable region specificity bar
Shape code high-flux sequence primer；
2) bacterial 16 S rRNA V3-V4 variable region amplification：With microorganism macro genome DNA as template, with the sample of above-mentioned design
Specific barcode high-flux sequence primer enters performing PCR amplification.
5. in Fructus Piperiss exfoliating process as claimed in claim 2 key microorganisms functional genome detection method, its feature exists
In PCR primer, after electrophoresis detection and quantitation, is applied secondary high-flux sequence platform that sample is carried out with bridge-type PCR high pass and measured
Sequence, each sample guarantees to obtain 20,000 high-quality sequence.
6. in Fructus Piperiss exfoliating process as claimed in claim 2 key microorganisms functional genome detection method, its feature exists
In based on wide bioinformatics platform, application is increased income, and raw letter analysis software is long to primitive sequencer reading first to carry out Quality Control, removes low-quality
Amount sequence retains high-quality sequence；Run Analysis of Microbial Diversity program on self-built raw letter platform, representative through choosing
Sequence and with reference to bacterium storehouse compare after obtain species diversity information, based on Unifrac evolutionary distance analyze sample between β many
Sample simultaneously explains the Community composition dynamic change putting sample between difference；Application random forest machine learning model calculates each
The weight of strain and Changing Pattern, carry out functional gene and metabolism using function prediction open source software to regular change microorganism
The annotation of path.
7. in Fructus Piperiss exfoliating process as claimed in claim 2 key microorganisms functional genome detection method, its feature exists
In amplimer：Sequence is：SEQ ID NO：1 and SEQ ID NO：2.
8. in Fructus Piperiss exfoliating process described in a kind of utilization claim 1～7 any one key microorganisms functional genome inspection
The Fructus Piperiss of survey method decortication.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109112225A (en) * | 2018-08-30 | 2019-01-01 | 海南大学 | Key microorganisms are screened and the method for screening in a kind of beautiful natural fermentation process of promise |
| CN110846424A (en) * | 2019-11-05 | 2020-02-28 | 烟台大学 | Rapid inspection and quarantine method for entry and exit port microorganisms |
| CN111276185A (en) * | 2020-02-18 | 2020-06-12 | 上海桑格信息技术有限公司 | Microorganism identification and analysis system and device based on second-generation high-throughput sequencing |
| CN114334003A (en) * | 2021-12-22 | 2022-04-12 | 中国水产科学研究院南海水产研究所 | Fermented golden pomfret deep learning quality discrimination method and system based on single molecule sequencing |
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2016
- 2016-09-26 CN CN201610850856.7A patent/CN106434914A/en active Pending
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| 佟婷婷等: "基于宏基因组分析四川泡菜母水作引子的泡菜发酵过程中细菌多样性变化", 《食品工业科技》 * |
| 刘莉扬等: "高通量测序技术在宏基因组学中的应用", 《中国医药生物技术》 * |
| 史敏等: "胡椒脱皮及其机理的研究进展", 《安徽农业科学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109112225A (en) * | 2018-08-30 | 2019-01-01 | 海南大学 | Key microorganisms are screened and the method for screening in a kind of beautiful natural fermentation process of promise |
| CN110846424A (en) * | 2019-11-05 | 2020-02-28 | 烟台大学 | Rapid inspection and quarantine method for entry and exit port microorganisms |
| CN111276185A (en) * | 2020-02-18 | 2020-06-12 | 上海桑格信息技术有限公司 | Microorganism identification and analysis system and device based on second-generation high-throughput sequencing |
| CN111276185B (en) * | 2020-02-18 | 2023-11-03 | 上海桑格信息技术有限公司 | Microorganism identification analysis system and device based on second-generation high-throughput sequencing |
| CN114334003A (en) * | 2021-12-22 | 2022-04-12 | 中国水产科学研究院南海水产研究所 | Fermented golden pomfret deep learning quality discrimination method and system based on single molecule sequencing |
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