CN109402223A - A kind of method and its application based on animal wastes sample building sequencing library - Google Patents
A kind of method and its application based on animal wastes sample building sequencing library Download PDFInfo
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- CN109402223A CN109402223A CN201811337191.5A CN201811337191A CN109402223A CN 109402223 A CN109402223 A CN 109402223A CN 201811337191 A CN201811337191 A CN 201811337191A CN 109402223 A CN109402223 A CN 109402223A
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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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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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
- C12Q1/6869—Methods for sequencing
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- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
Abstract
The present invention relates to molecular biology fields, more particularly to a kind of method and its application based on animal wastes sample building sequencing library, the method based on animal wastes sample building sequencing library includes the following steps: that (1) pre-processes fecal specimens, prepares sample suspension；(2) RNA, the Nong Du≤25ng/ μ L for the RNA that Detection and Extraction obtain are extracted from sample suspension；(3) reverse transcription；(4) sequencing library is constructed.The method based on animal wastes sample building sequencing library can be applied to viral diagnosis, viruses indentification, the discovery of unknown virus, public hygienic environment detection, environmental protection and feed addictive selection etc..
Description
Technical field
The invention belongs to technical field of molecular biology, and in particular to one kind is based on animal wastes sample building sequencing text
The method and its application in library.
Background technique
With the change of the factors such as temperature, season and environment, animal is easy the pathogenic microorganisms such as virus infection, and
Most of viruses have infectiousness, not only result in the damage of animal, it is also possible to can damage to ecological environment.In addition,
Virus has latency, and can hide for a long time can't detect in animal body.Currently, commonly detecting the side of virus in industry
Method is usually that can cause suffering to animal in sampling, the difficulty of sampling is relatively large using serum or animal tissue as sample.
Excrement is simultaneously a kind of special material as a kind of Non invasive sampling method, undoubtedly a kind of ideal sample material, but excrement
Material, containing more complicated substance, is influenced, microorganism in excrement by exposure duration, store method, temperature and sampling method etc.
RNA is easy to degrade, and has an adverse effect to subsequent experimental, even results in failure.Furthermore collected fecal sample is not
It can be directly used in the extraction of RNA or DNA, just can be used for extracting whole nucleic acid therein after needing to be homogenized.
Currently used method for detecting virus is to be detected using the method for elisa, PCR or kit, above-mentioned detection side
Method is general only to can detect that one or more of viruses every time, and detection efficiency is low.By taking the detection of swine disease poison as an example, common method master
There are 3 kinds: first is that detection card, such as swine fever, porcine pseudorabies, pig blue-ear disease, it is divided into antigen detection card and antibody test card,
It is short the time required to detection, but its sensibility is lower, and testing result can only provide the sxemiquantitative knot of the positive, weakly positive, feminine gender
Fruit can not provide numerical testing result；Second is that antibody (antigen) detects ELISA kit, currently, common swine fever, pig
Pseudoabies, pig blue-ear disease, Porcine circovirus desease, Schweineseuche etc., it is existing domestic, also there is import, easy to operate, inspection
It is fast to survey result, but needs microplate reader, and specific virus can only be detected.Third is that PCR detection method, special by PCR amplification
Fixed virus primer amplification is viral accordingly, and the viral species of detection are few, and testing conditions require high.
High throughput sequencing technologies can detecte over one hundred a sample, realize once to ten tens of thousands of to millions of DNA moleculars into
Row sequencing, wide coverage, flux is high, and whole hereditary information in energy test sample, accuracy is high, easy to operate, and
Feasibility is high.And the premise for carrying out high-flux sequence is to obtain the sequencing library based on sequencing technologies platform.Due to animal wastes
Sample composition is complicated, nucleic acid content is low, and influence factor is more, needs to grope successfully to construct sequencing library by cumbersome, and
Success rate is low.
Summary of the invention
The present invention provides a kind of method based on animal wastes sample building sequencing library, to solve the above problems.The base
Nong Du≤25ng/ μ the L for meeting RNA is only needed in the method for animal wastes sample building sequencing library, constructs sequencing library
Success rate it is high.
Method based on animal wastes sample building sequencing library of the invention adopts the following technical scheme that one kind is based on
The method of animal wastes sample building sequencing library, which comprises the steps of: (1) fecal specimens are located in advance
Reason prepares sample suspension；(2) RNA, the Nong Du≤25ng/ μ L for the RNA that Detection and Extraction obtain are extracted from sample suspension；(3)
Reverse transcription；(4) sequencing library is constructed.
Preferably, the sample suspension the preparation method is as follows: PBS buffer solution is added into fecal specimens, machinery concussion
Device shakes to solid sample and sufficiently dissolves；10min is centrifuged under the conditions of 12000rpm with refrigerated centrifuge；Supernatant is drawn, is used in combination
0.45 μm of aqueous filter membrane is filtered supernatant, is repeated once, and collects filtrate to get the sample suspension is arrived.
Preferably, the ratio between dosage of the fecal specimens and PBS buffer solution be (5-7): (3-5), the fecal specimens with
G meter, the PBS buffer solution is in terms of mL.
Preferably, the animal wastes sample is pig manure sample, cow dung sample or sheep dung sample.
Preferably, the animal wastes sample is by choosing out fresh excreta surface, picking inside insertion excrement with sample spoon
It obtains or is derived from -80 DEG C of environment to freeze fecal specimens.
Preferably, further include following steps: the concentration of RNA in sample suspension being detected after preparing sample suspension, if
RNA concentration in detected sample suspension is more than or equal to 35ng/ μ L, is mentioned using Tiangeng viral RNA/DNA extraction kit
Take the RNA in sample suspension；If the RNA concentration in sample suspension is less than 35ng/ μ L, sample suspension is extracted using Trizol method
In RNA.
Preferably, the method for the reverse transcription is as follows: carrying out one chain of reversion first, reversion one is taken out from -20 DEG C of refrigerators
The reagent and enzyme of chain, mix well, and reagent and enzyme are placed on ice chest by centrifugation, prepare reaction system:
Then it is uniformly mixed, after micro- centrifugation, 70 DEG C of incubation 10min, then rapid ice bath 2min, adds 5*RT
Buffer 4 μ l, DTT 2 μ l, 42 DEG C of incubation 2min be eventually adding 1 μ l of HiFiscript, after mixing, 42 DEG C incubation
50min, 85 DEG C of incubation 5min.Then the reagent for taking out two chains of reversion from -20 DEG C again is placed on ice, prepares reaction system,
After the completion of preparation, it is uniformly mixed, 5 μ L0.5M EDTA are added in of short duration centrifugation, 15 DEG C of incubation 2h after completion of the reaction
(PH8.0), and with magnetic bead purify, obtain sample DNA；One chain of the reversion is closed using the first chain of HiFiScript cDNA
At kit, two chains of the reversion use the second chain of cDNA synthetic agent box.
Preferably, the method for the building sequencing library is as follows: carrying out end to the sample DNA that reverse transcription obtains first
It repairs, end is taken out from -20 DEG C of refrigerators and repairs buffer solution, is mixed well, buffer is repaired into end and end is repaired
Enzyme is placed on ice chest, prepares reaction system,
After the completion of preparation, it is uniformly mixed, 12 DEG C of incubations 15min, 37 DEG C of incubations 15min, 72 DEG C of incubations 20min, HOLD
ON 4℃.After reaction, following preparation coupled reaction system is added in of short duration centrifugation:
After the completion of preparation, it is uniformly mixed, of short duration centrifugation, 20 DEG C of incubation 15min are carried out pure after reaction with magnetic bead
Change.The reaction solution for connecting connector after purification is expanded again, prepares amplification reaction system:
After the completion of PCR reaction system is prepared, sample is placed in PCR instrument, runs following PCR by uniformly mixed and micro- centrifugation
Response procedures:
It after PCR response procedures, is purified using magnetic bead, recycles DNA, and be dissolved in the TE buffer of 10 μ L, examined
The concentration of DNA library is surveyed, and gel electrophoresis is carried out to DNA library.
Preferably, the inspection of the RNA Concentration Testing, the concentration of the RNA extracted from sample suspension of the sample suspension
It surveys and the Concentration Testing of sequencing library is all made of Qubit method.
The method application based on animal wastes sample building sequencing library of the invention: viral diagnosis, viruses indentification,
Application in terms of discovery, public hygienic environment detection, environmental protection and the feed addictive selection of unknown virus.
The beneficial effects of the present invention are: the present invention is using animal wastes as sample, the content of animal wastes amplifying nucleic acid relative to
The sample sizes such as blood are few, extract difficulty it is big, the present invention by the way that suitable extracting method is pre-processed and selected to sample,
It can be enriched with from animal wastes and obtain required RNA；In addition, improving the convenience of sampling using animal wastes as sample, keep away
Exempt from that animal is caused to damage.The sequencing library that the present invention constructs was used for for two generations by sequencing library construction method through the invention
Sequencing technologies can detect a variety of viruses simultaneously, and the frequency that can be occurred according to virus determines the relative amount of virus.The present invention
Sequencing library construction method the concentration of RNA is required it is low, it is only necessary to RNA concentration, which is greater than 25ng/ μ L, can build Kucheng's function, success
Rate is high；It is more that method based on animal wastes sample building sequencing library of the invention is suitable for pig, ox, sheep and pet cat and dog etc.
Kind animal, and the success rate for constructing sequencing library is high.
By carrying out initial survey to the concentration of the RNA in sample suspension before RNA is extracted, determined according to initial survey result from sample
The method that RNA is extracted in product suspension helps to reduce experimental cost.
Of the invention pre-processes fecal specimens, first mixes fecal specimens according to specific ratio and PBS buffer
It closes, be centrifuged, filtering is made sample suspension and extracts RNA from sample suspension again, the processing suitable for many animals fecal specimens.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, below will to embodiment or
Attached drawing needed to be used in the description of the prior art is briefly described, it should be apparent that, the accompanying drawings in the following description is only
Some embodiments of the present invention, for those of ordinary skill in the art, without any creative labor,
It is also possible to obtain other drawings based on these drawings.
Fig. 1 is the gel electrophoresis figure that the embodiment of the present invention one constructs obtained sequencing library；
Fig. 2 is in the embodiment of the present invention two based on the RNA that Trizol method the is extracted sequencing library constructed
Gel electrophoresis figure；
Fig. 3 is gel electrophoresis of the embodiment of the present invention two using the RNA of the various concentration sequencing library constructed
Figure；
Fig. 4 is the gel electrophoresis figure that the embodiment of the present invention three constructs obtained sequencing library；
In Fig. 1: M Marker, 1- sample number into spectrum 1,2- sample number into spectrum 2,3- sample number into spectrum 3；
In Fig. 2: M Marker, 1- sample number into spectrum；
In Fig. 3: M Marker, 1- sample number into spectrum, 2- sample number into spectrum, 3- sample number into spectrum；
In Fig. 4: M Marker, 1- sample number into spectrum, 2- sample number into spectrum.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts it is all its
His embodiment, shall fall within the protection scope of the present invention.
Embodiment one: it is taken from the different collected fecal specimens in pig farm, at step as described below respectively
Reason.
1. the preparation of sample suspension: firstly, taking 30g ± 5g fecal specimens, 20ml ± 5mL PBS buffer solution is added, is vortexed
Oscillation, until fecal specimens state viscosity is appropriate, 12000rpm is centrifuged 20 minutes.Aspirate supernatant, with 0.45 micron membrane filter
It is filtered 2 times, in Qubit3.0 (brand: Thermo Fisher model: 3.0Fluorometer) test sample suspension
The concentration of RNA.The suspension of preparation is temporarily stored into -80 DEG C of refrigerators.
2, RNA is extracted from sample suspension:
RNA isolation kit: the Proteinase K of 50 μ l and the GB of 500 μ l is added in the sample suspension for taking 500 μ L to extract first
Buffer, 56 DEG C of incubation 15min；Secondly 250 μ L dehydrated alcohols are added to be vortexed, mix, stands 5min and liquid is transferred to absorption
In column, 8000rpm is centrifuged 1min；Then 500 μ LGD and 8000rpm centrifugation 1min is added；600 μ L PW are added and stand 2min,
8000rpm is centrifuged 1min and repetition；It adds 500 μ L dehydrated alcohol 8000rpm centrifugation 1min and 12000rpm is centrifuged
2min；Adsorption column is moved into new centrifuge tube, 3min is placed at room temperature for, it is rear that 20 μ LRNase-Free ddH2O are added, it is placed at room temperature for
5min, 12000rpm are centrifuged 1min；And using the RNA concentration after Q μ bit Detection and Extraction, with the micro light splitting of nanodrop2000
The purity for the RNA that photometer Detection and Extraction obtain.Wherein, Proteinase K, GB buffer, solution GD, solution PW and RNase-
Free ddH2O is selected from the virus genom DNA/RNA extracts kit (centrifugal column of TIANGEN Biotech (Beijing) Co., Ltd.
Type), catalog number (Cat.No.): DP315, version number: DP140916.
3, reverse transcription:
(1) it carries out one chain of reversion: taking out the reagent and enzyme of one chain of reversion from -20 DEG C of refrigerators, mix well, be centrifuged；
Reagent and enzyme are placed on ice chest, prepare reaction system according to table 1:
Table 1: one chain reaction system of reversion
| Component | Reaction system (μ L) |
| dNTP-mix | 4 |
| Primer Mix | 2 |
| RNA template | 7 |
| Total system | 13 |
Then it is uniformly mixed, after micro- centrifugation, 70 DEG C of incubation 10min, then rapid ice bath 2min, adds 5*RT
buffer 4μL,DTT 2μL；42 DEG C of incubation 2min, are eventually adding 1 μ L of HiFiscript, after mixing, 42 DEG C of incubations
50min, 85 DEG C of incubation 5min.Wherein dNTP-mix, Primer Mix, HiFiscript (200U/ μ L), 5*RT buffer and
DTT be selected from the first chain of HiFiScript cDNA synthetic agent box (hundred AudioCodes Biotechnology Co., Ltd of Tianjin, catalog number (Cat.No.):
CW2569, version number:
05/2014；) synthesis, RNA template is the RNA solution extracted from sample suspension.
(2) two chains of reversion are carried out: taking out the reagent of two chains of reversion from -20 DEG C of refrigerators, is placed on ice, is prepared according to table 2
Reaction system；
Table 2: two chain reaction systems of reversion
| Component | Reaction system (μ L) |
| The deionized water of nuclease free | 68 |
| Reaction Buffer | 8 |
| DNA PolymeraseI | 3 |
| RNase H | 1 |
| Total system | 80 |
After the completion of preparation, it is uniformly mixed, 5 μ L EDTA are added after completion of the reaction, are used in combination by of short duration centrifugation, 15 DEG C of incubation 2h
Magnetic bead is purified to get sample DNA.Wherein, Reaction Buffer (10X), RNase H (1U/ μ L) and DNA
PolymeraseI (10U/ μ L) be selected from the second chain of cDNA synthetic agent box (green skies Bioisystech Co., Ltd, specification:
D7172。)
4, sequencing library constructs: progress end reparation first takes out end from -20 DEG C of refrigerators and repairs buffer solution,
It mixes well, buffer and end repair enzyme is repaired into end, is placed on ice chest, prepare reaction system according to table 3；
Table 3: reaction system is repaired in end
| Component | Reaction system (μ L) |
| Sample DNA | 11.3 |
| Repair buffer in end | 1.3 |
| End repair enzyme | 0.4 |
| Total system | 13 |
After the completion of preparation, it is uniformly mixed, 12 DEG C of incubations 15min, 37 DEG C of incubations 15min, 72 DEG C of incubations 20min, HOLD
ON 4℃.After reaction, of short duration centrifugation is added according to table 4 and prepares coupled reaction system
Table 4: coupled reaction system
| Component | Reaction system (μ L) |
| Repair reactant in end | 13 |
| Ligase | 0.4 |
| Connect buffer | 2.8 |
| Adapter for illumine | 1 |
| Total system | 17.2 |
After the completion of preparation, it is uniformly mixed, of short duration centrifugation, 20 DEG C of incubation 15min are carried out pure after reaction with magnetic bead
Change.The reaction solution for connecting connector after purification is expanded again, prepares amplification reaction system according to table 5
Table 5: amplification reaction system
Note: used in table 4 Adapter for illumine (Adapter1:
GATCGGAAGAGCACACGTCTGAACTCCAGT*C, as shown in SEQ ID No.52；Adapter2:ACACTCTTTCCCTAC
ACGACGCTCTTCCGATC*T, as shown in SEQ ID No.53) and table 5 in Univesial Primer (AATGAT
ACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T, as shown in SEQ ID No.51) and
The sequence of Index Primer (SEQ ID No.1-50) is provided by illumine microarray dataset, the raw work bioengineering in Shanghai
Technology Co., Ltd.'s synthesis；Repair buffer and end repair enzyme, the ligase used in table 4 and company in the end used in table 3
It meets the HiFidelity 2X PCR Master Mix used in buffer and table 5 and builds library examination selected from two generations sequencing rapid DNA
Agent box (health is century Biotechnology Co., Ltd, specification: CW2585)
After the completion of PCR reaction system is prepared, sample is placed in PCR instrument by uniformly mixed and micro- centrifugation, table 6
PCR response procedures:
Table 6:PCR response procedures
It after PCR response procedures, is purified using magnetic bead, gel electrophoresis, obtains the DNA piece of 200-350bp or so
Section, and be dissolved in the TE buffer of 10 μ L, with the concentration of the DNA library of Qubit detection after purification.
Sample suspension is successively prepared according to above-mentioned steps (1-4), RNA, reverse transcription and Jian Ku are extracted from sample suspension,
And in prepared sample suspension RNA concentration, extract the obtained concentration of RNA and library DNA concentration after purification uses
The detection of Qubit method.Electrophoresis (1.5% agarose gel electrophoresis, voltage: 100V, 45 minutes) result of DNA library after purification
See Fig. 1, testing result see the table below table 7
The sequencing library of 7 swine excrement sample of table constructs relevant experimental data
Gel electrophoresis result shows that the DNA sequencing library constructed has the segment of 200-350bp or so, builds Kucheng
Function.
It is dense according to each sample DNA by sequencing library that the method according to the invention constructs after library result is built in acquisition
The relationship of degree relationship and sequencing data amount carries out sample mixing, sequencing to sample.Bioinformatics can be carried out to sequencing result later
Analysis.Such as: sequencing data is passed through into preliminary treatment first, low quality, joint sequence are removed, according to the number of FASTQ-Clean
According to removal repetitive sequence；Then it is sequenced, comparing is carried out according to virus database known to sequencing result and platform,
The experimental data after comparing is arranged, obtains report, identifies whether to carry certain pathogen in sample with this.
Specific example is as follows: the fecal sample on Xinxiang City, Henan Province Huixian City pig farm of diarrhea occurs for acquisition, according to this
The method of the building sequencing library of invention constructs sequencing library, and is sequenced, and carries out bioinformatic analysis, knot to sequencing result
Fruit shows the full base of Pseudorabies virus, porcine encephalomyelitis virus, pig circular ring virus, Porcine epidemic diarrhea virus and swine fever virus
Because group frequency occurred is higher.Meanwhile in terms of sequencing result, also detected that in the fecal sample on the pig farm various other
Virus relevant to pig, but the frequency occurred is lower, such as swine fever virus, the separation strains of Bungowannah, pig parvoviral
Disease 2, pig transmissible encephalomyelitis virus, pig blue-ear disease are malicious, foot and mouth disease virus-is O-shaped, classical swine fever virus, chitling virus B, pig
Poxvirus, pig parainfluenza virus, pig mumps virus, porcine endogenous retrovirus E, porcine torovirus, pig Sa disease Peyronie
Poison, inclusion-body rhinitis virus of pigs, pig coronavirus, pig circular ring virus, pig ridge virus, pig astrovirus 2, pig astrovirus 3, pig star
Shape virus-4, pig astrovirus 5, pig parvoviral 1, pig parvoviral 4, pig parvoviral 5, pig parvoviral 6, porcine adenovirus
3, over one hundred kind of porcine adenovirus C, pig bocavirus 4-1, pig bocavirus 1, pig bocavirus 3 and pig bocavirus 5 etc. and pig phase
The virus of pass.
Since virus has certain infectiousness, it is easy that the other biological in environment is caused to infect or environment is caused
Pollution, it is therefore contemplated that, the method for the invention based on animal wastes sample building sequencing library is in viral diagnosis, disease
Malicious identification, the discovery of unknown virus, public hygienic environment detection, environmental protection and feed addictive selection etc. all have
Wide application prospect.
Embodiment two:
1. the fecal specimens on pair certain pig farm carry out sample suspension preparation, extract RNA, reverse transcription and construct sequencing library,
Middle sample suspension preparation constructs the same embodiment step of sequencing library with one step 2 of embodiment with one step 1 of embodiment, reverse transcription
4, extract RNA method be Trizol method, the specific steps are as follows: take 600 μ L extract sample suspension, after being divided to two pipes, respectively plus
Enter 900 μ L Trizol to mix, be placed at room temperature for 5min；Add 180 μ L chloroforms, sufficiently oscillation mix, 12000rpm, 4 DEG C from
Heart 15min；It takes 600 μ L upper strata aqueous phases and 600 μ L isopropanols is added, mix, be placed at room temperature for 10min, 12000rpm, 4 DEG C of centrifugations
10min；Supernatant is abandoned, 75% ethyl alcohol 10000rpm 4min of 1ml is added；Supernatant is abandoned, 75% ethyl alcohol 10000rpm is added
4min, brief centrifugation, the opening 10min that dries in the air dry；The dissolution of 20 μ L DEPC water is added.Trizol used in the above method is selected from
Trizol total RNA extraction reagent box (health is century Biotechnology Co., Ltd, specification: CW0508A).
The concentration and purifying for the RNA that the RNA concentration in prepared sample suspension, extraction are obtained during the experiment
Library DNA concentration afterwards is detected using Qubit method.The electrophoresis result of DNA library after purification is shown in Fig. 2, and testing result see the table below
Table 8
Table 8
From upper table 8 it is found that the RNA purity extracted using Trizol method is low, reverse transcription and sequencing according to the invention
The concentration for the DNA library after purification that library constructing method obtains is relatively low.In conjunction with Fig. 2 it is found that the present embodiment constructed
Sequencing library has the segment of 200-350bp size, can be used for DNA sequencing, sequencing library constructs successfully.
2. being pre-processed according to the method for embodiment one to collected pig manure sample, RNA is extracted, reverse transcription and survey
Sequence library construction.
The concentration and purifying for the RNA that the RNA concentration in prepared sample suspension, extraction are obtained during the experiment
Library DNA concentration afterwards is detected using Qubit method.The electrophoresis result of DNA library after purification is shown in Fig. 3, and testing result see the table below
Table 9
Table 9
Referring to upper table 9 and Fig. 3 it is found that the concentration for extracting obtained RNA is real to subsequent reverse transcription and building sequencing library
It tests and has a major impact.
Embodiment three: sequencing library is constructed based on sheep dung sample and cow dung sample
The preparation for carrying out sample suspension to collected sheep dung and cow dung sample (is hanged with one step 1) of embodiment, from sample
The (((same to implement with one step 3) of embodiment and sequencing library building with one step 2) of embodiment, reverse transcription of RNA is extracted in liquid
One step 4) of example.
The concentration and purifying for the RNA that the RNA concentration in prepared sample suspension, extraction are obtained during the experiment
Library DNA concentration afterwards is detected using Qubit method.The electrophoresis result of DNA library after purification is shown in Fig. 3, and testing result see the table below
Table 10
Table 10
From upper table 10 it is found that the DNA sequencing library that the method according to the invention is constructed based on cow dung and sheep dung sample
Concentration it is high, in conjunction with Fig. 4 it is found that the sequencing library that the present embodiment constructs has the segment of 200-350bp size, can use
In DNA sequencing, sequencing library is constructed successfully.
The experiment proved that method of the invention is also applied for the building of the fecal specimens based on other animals such as pet cat and dog
Sequencing library.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
The SEQ ID No.1-SEQ ID No.53 mentioned in description of the invention is as follows:
SEQ ID No.1 CAAGCAGAAGACGGCATACGAGATTGTGTTAAGTGACTGGAGTTCAGACGTGTGCT
CTTCCGATC*T
SEQ ID No.2 CAAGCAGAAGACGGCATACGAGATTTGCTACGGTGACTGGAGTTCAGACGTGTGCT
CTTCCGATC*T
SEQ ID No.3 CAAGCAGAAGACGGCATACGAGATAAGACGTCGTGACTGGAGTTCAGACGTGTGCT
CTTCCGATC*T
SEQ ID No.4 CAAGCAGAAGACGGCATACGAGATTCCGACGGGTGACTGGAGTTCAGACGTGTGCT
CTTCCGATC*T
SEQ ID No.5 CAAGCAGAAGACGGCATACGAGATGCAGGCATGTGACTGGAGTTCAGACGTGTGCT
CTTCCGATC*T
SEQ ID No.6 CAAGCAGAAGACGGCATACGAGATCACACTGGGTGACTGGAGTTCAGACGTGTGCT
CTTCCGATC*T
SEQ ID No.7 CAAGCAGAAGACGGCATACGAGATGGCCGGTTGTGACTGGAGTTCAGACGTGTGCT
CTTCCGATC*T
SEQ ID No.8 CAAGCAGAAGACGGCATACGAGATGGCCTCGCGTGACTGGAGTTCAGACGTGTGCT
CTTCCGATC*T
SEQ ID No.9 CAAGCAGAAGACGGCATACGAGATCATGCGGCGTGACTGGAGTTCAGACGTGTGCT
CTTCCGATC*T
SEQ ID No.10 CAAGCAGAAGACGGCATACGAGATGGCAACAGGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.11 CAAGCAGAAGACGGCATACGAGATCGGCCAATGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.12 CAAGCAGAAGACGGCATACGAGATAGCCGTCCGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.13 CAAGCAGAAGACGGCATACGAGATACAGAGTGGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.14 CAAGCAGAAGACGGCATACGAGATTGAATCATGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.15 CAAGCAGAAGACGGCATACGAGATTGACAGACGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.16 CAAGCAGAAGACGGCATACGAGATGTGGTCGTGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.17 CAAGCAGAAGACGGCATACGAGATCCGGCTAAGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.18 CAAGCAGAAGACGGCATACGAGATCGATCCTGGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.19 CAAGCAGAAGACGGCATACGAGATGGCTCTTCGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.20 CAAGCAGAAGACGGCATACGAGATGCTCTCTAGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.21 CAAGCAGAAGACGGCATACGAGATTGTCGCGTGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.22 CAAGCAGAAGACGGCATACGAGATAGGTGCCGGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.23 CAAGCAGAAGACGGCATACGAGATGATAGCCGGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.24 CAAGCAGAAGACGGCATACGAGATTAACACCGGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.25 CAAGCAGAAGACGGCATACGAGATGGAGTAGAGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.26 CAAGCAGAAGACGGCATACGAGATCGGATTAGGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.27 CAAGCAGAAGACGGCATACGAGATCGGACGGAGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.28 CAAGCAGAAGACGGCATACGAGATGTGTGTTAGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.29 CAAGCAGAAGACGGCATACGAGATCTCGTCCGGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.30 CAAGCAGAAGACGGCATACGAGATTGGAGAGGGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.31 CAAGCAGAAGACGGCATACGAGATATTGCGTTGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.32 CAAGCAGAAGACGGCATACGAGATTCGTAAGCGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.33 CAAGCAGAAGACGGCATACGAGATCCGTCACGGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.34 CAAGCAGAAGACGGCATACGAGATGCGAAGTAGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.35 CAAGCAGAAGACGGCATACGAGATGGACTGCGGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.36 CAAGCAGAAGACGGCATACGAGATGAGCATTGGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.37 CAAGCAGAAGACGGCATACGAGATTCGCCGTGGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.38 CAAGCAGAAGACGGCATACGAGATCAGCGGCGGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.39 CAAGCAGAAGACGGCATACGAGATAAGGATGCGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.40 CAAGCAGAAGACGGCATACGAGATTTAGACAAGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.41 CAAGCAGAAGACGGCATACGAGATGTCCAGAAGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.42 CAAGCAGAAGACGGCATACGAGATATCTATCGGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.43 CAAGCAGAAGACGGCATACGAGATTTACTGTTGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.44 CAAGCAGAAGACGGCATACGAGATTGGAATTCGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.45 CAAGCAGAAGACGGCATACGAGATTTGGCGCCGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.46 CAAGCAGAAGACGGCATACGAGATGCCTTAATGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.47 CAAGCAGAAGACGGCATACGAGATAAGCGATTGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.48 CAAGCAGAAGACGGCATACGAGATAACCGCAAGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.49 CAAGCAGAAGACGGCATACGAGATGCAATGGCGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.50 CAAGCAGAAGACGGCATACGAGATGTATTCTCGTGACTGGAGTTCAGACGTGTGC
TCTTCCGATC*T
SEQ ID No.51(Universal) AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGA
CGCTCTTCCGATC*T
SEQ ID No.52 (Adapter1) GATCGGAAGAGCACACGTCTGAACTCCAGT*C
SEQ ID No.53 (Adapter2) ACACTCTTTCCCTACACGACGCTCTTCCGATC*T
Remarks: (1) " * " in any of the above-described sequence has phosphorothioate bond modification before representing end T；
(2) above-mentioned sequence is only one of embodiment of the invention, and those skilled in the art should learn label sequence
Column, joint sequence, the selection of universal primer sequence are in close relations with selected microarray dataset and method, when the different surveys of selection
When sequence platform is sequenced, adaptation is carried out according to the requirement of selected microarray dataset.
Sequence table
<110>Henan is great rises agriculture Co., Ltd
<120>a kind of method and its application based on animal wastes sample building sequencing library
<160> 53
<170> SIPOSequenceListing 1.0
<210> 1
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
caagcagaag acggcatacg agattgtgtt aagtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 2
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
caagcagaag acggcatacg agatttgcta cggtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 3
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
caagcagaag acggcatacg agataagacg tcgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 4
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
caagcagaag acggcatacg agattccgac gggtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 5
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
caagcagaag acggcatacg agatgcaggc atgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 6
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
caagcagaag acggcatacg agatcacact gggtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 7
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
caagcagaag acggcatacg agatggccgg ttgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 8
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
caagcagaag acggcatacg agatggcctc gcgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 9
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
caagcagaag acggcatacg agatcatgcg gcgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 10
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
caagcagaag acggcatacg agatggcaac aggtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 11
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
caagcagaag acggcatacg agatcggcca atgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 12
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
caagcagaag acggcatacg agatagccgt ccgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 13
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
caagcagaag acggcatacg agatacagag tggtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 14
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
caagcagaag acggcatacg agattgaatc atgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 15
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
caagcagaag acggcatacg agattgacag acgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 16
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
caagcagaag acggcatacg agatgtggtc gtgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 17
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
caagcagaag acggcatacg agatccggct aagtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 18
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
caagcagaag acggcatacg agatcgatcc tggtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 19
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
caagcagaag acggcatacg agatggctct tcgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 20
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
caagcagaag acggcatacg agatgctctc tagtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 21
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
caagcagaag acggcatacg agattgtcgc gtgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 22
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
caagcagaag acggcatacg agataggtgc cggtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 23
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
caagcagaag acggcatacg agatgatagc cggtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 24
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
caagcagaag acggcatacg agattaacac cggtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 25
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
caagcagaag acggcatacg agatggagta gagtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 26
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
caagcagaag acggcatacg agatcggatt aggtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 27
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
caagcagaag acggcatacg agatcggacg gagtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 28
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
caagcagaag acggcatacg agatgtgtgt tagtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 29
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
caagcagaag acggcatacg agatctcgtc cggtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 30
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
caagcagaag acggcatacg agattggaga gggtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 31
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
caagcagaag acggcatacg agatattgcg ttgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 32
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
caagcagaag acggcatacg agattcgtaa gcgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 33
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
caagcagaag acggcatacg agatccgtca cggtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 34
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
caagcagaag acggcatacg agatgcgaag tagtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 35
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
caagcagaag acggcatacg agatggactg cggtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 36
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
caagcagaag acggcatacg agatgagcat tggtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 37
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
caagcagaag acggcatacg agattcgccg tggtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 38
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
caagcagaag acggcatacg agatcagcgg cggtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 39
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
caagcagaag acggcatacg agataaggat gcgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 40
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
caagcagaag acggcatacg agatttagac aagtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 41
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
caagcagaag acggcatacg agatgtccag aagtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 42
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
caagcagaag acggcatacg agatatctat cggtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 43
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 43
caagcagaag acggcatacg agatttactg ttgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 44
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 44
caagcagaag acggcatacg agattggaat tcgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 45
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 45
caagcagaag acggcatacg agatttggcg ccgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 46
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 46
caagcagaag acggcatacg agatgcctta atgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 47
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 47
caagcagaag acggcatacg agataagcga ttgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 48
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 48
caagcagaag acggcatacg agataaccgc aagtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 49
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 49
caagcagaag acggcatacg agatgcaatg gcgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 50
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 50
caagcagaag acggcatacg agatgtattc tcgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 51
<211> 58
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 51
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatct 58
<210> 52
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 52
gatcggaaga gcacacgtct gaactccagt c 31
<210> 53
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 53
acactctttc cctacacgac gctcttccga tct 33
Claims (9)
1. a kind of method based on animal wastes sample building sequencing library, which comprises the steps of: (1) to excrement
Just sample is pre-processed, and prepares sample suspension；(2) RNA, the concentration for the RNA that Detection and Extraction obtain are extracted from sample suspension
≤ 25ng/ μ L (3) reverse transcription；(4) sequencing library is constructed.
2. the method according to claim 1 based on animal wastes sample building sequencing library, which is characterized in that the sample
Product suspension the preparation method is as follows: PBS buffer solution is added into fecal specimens, mechanical oscillator is shaken sufficiently molten to solid sample
Solution；10min is centrifuged under the conditions of 12000rpm with refrigerated centrifuge；Draw supernatant, and with 0.45 μm of aqueous filter membrane to supernatant into
Row filtering, is repeated once, and collects filtrate to get the sample suspension is arrived.
3. the method according to claim 2 based on animal wastes sample building sequencing library, which is characterized in that the excrement
Just the ratio between dosage of sample and PBS buffer solution is (5-7): (3-5), the fecal specimens are in terms of g, and the PBS buffer solution is with mL
Meter.
4. the method based on animal wastes sample building sequencing library according to claim 1 to 3, feature
It is, the animal wastes sample is pig manure sample, cow dung sample or sheep dung sample.
5. the method according to claim 4 based on animal wastes sample building sequencing library, which is characterized in that described dynamic
Object fecal specimens are by choosing out fresh excreta surface with sample spoon, and picking is obtained or is derived from -80 DEG C of environment inside insertion excrement
Freeze fecal specimens.
6. the method based on animal wastes sample building sequencing library described according to claim 1 or 2 or 3 or 5 any one,
It is characterized in that, further including following steps: being detected after preparing sample suspension to the concentration of RNA in sample suspension, if being examined
RNA concentration in the sample suspension measured is more than or equal to 35ng/ μ L, extracts sample using Tiangeng viral RNA/DNA extraction kit
RNA in product suspension；If the RNA concentration in sample suspension is less than 35ng/ μ L, extracted in sample suspension using Trizol method
RNA。
7. the method according to claim 1 based on animal wastes sample building sequencing library, which is characterized in that described anti-
The method of transcription is as follows: one chain of reversion carried out first, and the reagent and enzyme of one chain of reversion are taken out from -20 DEG C of refrigerators, it is sufficiently mixed
Even, reagent and enzyme are placed on ice chest by centrifugation, prepare reaction system:
Then it is uniformly mixed, after micro- centrifugation, 70 DEG C of incubation 10min, then rapid ice bath 2min, adds 4 μ of 5*RT buffer
L, DTT 2 μ l, 42 DEG C of incubation 2min are eventually adding 1 μ l of HiFiscript, and after mixing, 42 DEG C of incubation 50min, 85 DEG C incubate
Educate 5min.Then the reagent for taking out two chains of reversion from -20 DEG C again is placed on ice, prepares reaction system,
After the completion of preparation, it is uniformly mixed, 5 μ L0.5M EDTA are added in of short duration centrifugation, 15 DEG C of incubation 2h after completion of the reaction
(PH8.0), and with magnetic bead purify, obtain sample DNA；One chain of the reversion is closed using the first chain of HiFiScript cDNA
At kit, two chains of the reversion use the second chain of cDNA synthetic agent box.
8. the method according to claim 1 based on animal wastes sample building sequencing library, which is characterized in that the structure
The method for building sequencing library is as follows: carrying out end reparation to the sample DNA that reverse transcription obtains first, takes out from -20 DEG C of refrigerators
Buffer solution is repaired in end, mixes well, and buffer and end repair enzyme are repaired in end, is placed on ice chest, reaction is prepared
System,
After the completion of preparation, it is uniformly mixed, 12 DEG C of incubations 15min, 37 DEG C of incubations 15min, 72 DEG C of incubations 20min, HOLD ON 4
℃.After reaction, following preparation coupled reaction system is added in of short duration centrifugation:
After the completion of preparation, it is uniformly mixed, of short duration centrifugation, 20 DEG C of incubation 15min are purified with magnetic bead after reaction.It will be pure
The reaction solution that connector is connected after change is expanded again, prepares amplification reaction system:
After the completion of PCR reaction system is prepared, sample is placed in PCR instrument by uniformly mixed and micro- centrifugation, runs following PCR reaction
Program: initial denaturation: 98 DEG C, 30s；Denaturation: 98 DEG C, 10s；Annealing: 65 DEG C, 30s；Extend: 72 DEG C, 30s, wherein denaturation, annealing
15 circulations are repeated with extending；Extend eventually: 72 DEG C, 5min；
It after PCR response procedures, is purified using magnetic bead, recycles DNA, and be dissolved in the TE buffer of 10 μ L, detect DNA
The concentration in library, and gel electrophoresis is carried out to DNA library.
9. described in claim 1-8 any one based on animal wastes sample building sequencing library method viral diagnosis,
Application in terms of viruses indentification, the discovery of unknown virus, public hygienic environment detection, environmental protection and feed addictive selection.
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