CN109266768A - It is a kind of for identifying the screening technique of the nucleotide fragments of nearly edge microorganism - Google Patents
It is a kind of for identifying the screening technique of the nucleotide fragments of nearly edge microorganism Download PDFInfo
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- 239000012634 fragment Substances 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 25
- 238000012216 screening Methods 0.000 title claims abstract description 18
- 239000002773 nucleotide Substances 0.000 title claims abstract description 16
- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 16
- 244000005700 microbiome Species 0.000 title claims abstract description 14
- 108020004414 DNA Proteins 0.000 claims abstract description 71
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 71
- 230000001580 bacterial effect Effects 0.000 claims abstract description 57
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 33
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 33
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 20
- 238000013461 design Methods 0.000 claims abstract description 13
- 238000005516 engineering process Methods 0.000 claims abstract description 7
- 238000012408 PCR amplification Methods 0.000 claims abstract description 5
- 230000003252 repetitive effect Effects 0.000 claims abstract description 5
- 230000004069 differentiation Effects 0.000 claims abstract description 4
- 241001482686 Azospirillum sp. TSA2s Species 0.000 claims description 22
- 241000053914 Arthrobacter sp. 2 Species 0.000 claims description 21
- 241000589941 Azospirillum Species 0.000 claims description 9
- 108091034117 Oligonucleotide Proteins 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 4
- 238000001962 electrophoresis Methods 0.000 claims description 4
- 229920000936 Agarose Polymers 0.000 claims description 2
- 241000186063 Arthrobacter Species 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 238000012360 testing method Methods 0.000 claims description 2
- 239000000284 extract Substances 0.000 abstract description 7
- 230000000813 microbial effect Effects 0.000 abstract description 7
- 230000008569 process Effects 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 2
- 239000008223 sterile water Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000002689 soil Substances 0.000 description 5
- 238000011529 RT qPCR Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
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- 239000000539 dimer Substances 0.000 description 1
- 238000012850 discrimination method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- PWPJGUXAGUPAHP-UHFFFAOYSA-N lufenuron Chemical compound C1=C(Cl)C(OC(F)(F)C(C(F)(F)F)F)=CC(Cl)=C1NC(=O)NC(=O)C1=C(F)C=CC=C1F PWPJGUXAGUPAHP-UHFFFAOYSA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
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- 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
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The invention discloses a kind of for identifying the screening technique of the nucleotide fragments of nearly edge microorganism, and this method is that the complete genome sequence of objective microbe is cut into the nucleic acid small fragment containing overlay region using electronic shearography technology；The fasta file of nucleic acid small fragment compares in NCBI NT database；Hit table I is obtained, repetitive sequence is deleted, and then obtains hit nucleic acid sequence；Hit nucleic acid sequence I is compared with the nucleic acid small fragment containing overlay region, obtains unnatural death more control sequences；It repeats the above steps primary, designs PCR primer, extract 10 kinds of bacterial strain or more of genome DNA of category or relative genus where objective microbe bacterial strain, construct nucleic acid pool, PCR amplification verifying.The specific nucleic acid sequence of purpose microbial strains expeditiously can be largely obtained provided by the present invention for the specific nucleic acid screening technique of nearly edge Strain differentiation, the reproducibility of experimental result is high, simplifies whole experiment process, has good applicability.
Description
Technical field
The invention belongs to microbial information technical fields, and in particular to a kind of for identifying the nucleic acid piece of nearly edge microorganism
The screening technique of section.
Technical background
The identification of microorganism closer for affiliation is always Microbial Breeding scholar, especially progress plant growth-promoting bacteria
The important process of the expert of research.Have by traditional discrimination method based on form, physio-biochemical characteristics very much
Defect, such as: form, the physio-biochemical characteristics of the microorganism in the same category are substantially similar, it is difficult to identify.
With the development of molecular biology and bioinformatics technique, and more and more microbial genome sequencings
It completes, continues to develop out in the various methods of DNA fragmentation as the molecular labeling for identifying species.These technologies have specificity
By force, the features such as accuracy rate is high, repeatability is strong, the time is short, has been widely used for all kinds of microorganisms of curing the disease, environmental microorganism etc.
Analyze and identify.And these technologies are difficult to distinguish same kind to the nucleic acid sequence of the high degree of specificity for specific kind
Interior different strains, for a certain specific objective microorganism specific nucleic acid sequence screening there are certain difficulty.
Summary of the invention
The object of the present invention is to provide a kind of for identifying the screening technique of the nucleic acid fragment of nearly edge microorganism, existing to solve
There is the problem that same kind different strains identification is difficult in technology.
In order to solve the above technical problems, the present invention adopts the following technical scheme:
It is a kind of for identifying the screening technique of the nucleotide fragments of nearly edge microorganism, which is characterized in that this method include such as
Lower step:
(1) complete genome sequence of objective microbe is obtained；
(2) the electronic shearography technology for utilizing splitter online software, the genome sequence of purpose bacterial strain is cut into and is contained
200~1500bp nucleic acid small fragment of the overlay region 100~500bp；The network address of the splitter online software are as follows: http: //
www.bioinformatics.nl/emboss-explorer/。
(3) the fasta file that step (2) is obtained to the nucleic acid small fragment containing overlay region compares in NCBI NT database；
(4) step (3) obtains hit table I after comparing, and deletes and repeats repetitive sequence, and then obtains hit nucleic acid sequence I；
(5) by hit nucleic acid sequence I that step (4) obtains and the nucleic acid small fragment containing overlay region that step (2) obtain into
Row compares, and obtains unnatural death more control sequences I；
(6) the unnatural death more control sequences I obtained in step (5) are compared again in NCBI NT database, are obtained
Table II is hit, deletes and repeats repetitive sequence, and then obtains hit nucleic acid sequence II；
(7) step (5) are repeated, by hit nucleic acid sequence II that step (6) obtain and step (2) obtain containing overlay region
Nucleic acid small fragment is compared, and obtains unnatural death more control sequences II, and the unnatural death amplifying nucleic acid sequence II column are the possible micro- life of target
The strain specificity nucleic acid fragment of object；
(8) the strain specificity nucleic acid fragment of the possible objective microbe obtained using step (7) designs PCR as template
Primer；
(9) 10 kinds of bacterial strain or more of genome DNA of category or relative genus where extracting objective microbe bacterial strain constructs core
Sour pond；
(10) nucleic acid pool obtained respectively using the genomic DNA of objective microbe bacterial strain and step (9) is template, with step
(8) primer designed is that primer carries out PCR amplification, and amplified production carries out agarose electrophoresis detection；
If using the genomic DNA of objective microbe bacterial strain as the PCR test positive of template, and obtained with step (9)
Nucleic acid pool is that the PCR of template is detected as the negative corresponding nucleic acid fragment of primer, as objective microbe Strain differentiation
Nucleic acid specific fragment.
Nucleotide sequence is submitted to NCBI NT database (https: //blast.ncbi.nlm.nih.gov/
Blast.cgi? PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK LOC=blasthome) it is compared
When, the corresponding category of selection target microbial strains, other parameters are set as default value.
Selected gene is genomic DNA in the present invention.
It is miscellaneous that the objective microbe bacterial strain specific nucleic acid squences of acquisition can be used for qPCR, Southern marking, Southern
The quantity of the methods of friendship measurement aimed strain.
It is a kind of for identifying the oligonucleotide fragment of Azospirillum sp.TSA2S bacterial strain, including SEQ ID NO.2,
SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.7, any one nucleosides shown in SEQ ID NO.8
Acid sequence.
It is a kind of for identifying the primer of Azospirillum sp.TSA2S bacterial strain, with SEQ ID NO.2, SEQ ID
Nucleotides sequence shown in NO.3, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 are any is classified as mould
Plate, the primer designed；
Including any pair in following primer pair:
2sFw12:GAAGAACAGGACACGTGCAA, 2sRv12:TCAAATGAGTGCCCTAAGCA；
2sFw13:AATGGATTGCAGCAAAAAGC, 2sRv13:GATGCAGGAGTGCCAAAAGT；
2sFw14:TTCGAGCCCTAACAGGAGAA, 2sRv14:TAAAGGGCAGACGCAATAGG；
2sFw16:TGTTCGACTACGCTCATGCT, 2sRv16:GAGAGCTGCACCGACAGTTT；
2sFw17:TTTCCCCTGACTGCGTATCT, 2sRv17:CTGAATCCGTTCACAGCAAA；
2sFw18:TCAGATGTTTTGGCACATCG, 2sRv18:CTATCGTGCTCATTGCGGTA.
The above-mentioned oligonucleotide fragment for identifying Azospirillum sp.TSA2S bacterial strain is in identification Azospirillum
Application in sp.TSA2S bacterial strain.
The above-mentioned primer for identifying Azospirillum sp.TSA2S bacterial strain is in identification Azospirillum
Application in sp.TSA2S bacterial strain.
It is a kind of for identifying the oligonucleotide fragment of Arthrobacter sp.2 bacterial strain, including NO:17~18 SEQ ID
Shown in any one nucleotide sequence.
It is a kind of for identifying the primer of Arthrobacter sp.2 bacterial strain, with SEQ ID NO 17~18 it is any shown in
Nucleotides sequence is classified as the primer that stencil design obtains；
Including any pair in following primer pair:
A2Fw3:ACAGGAAACGGAAATCGTTG, A2Rv3:GTTCTTCCTGCGAAGCTCAT；
A2Fw4:TTTTTCTCGGCGTCCATATC, A2Rv4:TGTTCCAGTGGTGCTTTGTC；
A2Fw5:TTCCCGCAGACCAGTTTATC, A2Rv5:GCACTATTATCCGCGTTCGT；
A2Fw6:TGGAGCAGGGACTTAGATGG, A2Rv6:TACATGGGCTCGGTGTATCC.
The above-mentioned oligonucleotide fragment for identifying Arthrobacter sp.2 bacterial strain is in identification Arthrobacter
Application in sp.2 bacterial strain.
The above-mentioned primer for identifying Arthrobacter sp.2 bacterial strain is in identification Arthrobacter sp.2 bacterial strain
Application.
The utility model has the advantages that
A kind of nucleic acid fragment screening technique identified for microbial strains provided by the invention and application, it is micro- using target
The genomic information of biological bacterial strain establishes being compared for aimed strain nucleic acid fragment library and the genomic information of non-targeted bacterial strain,
Comparison hit sequence is obtained, and then obtains sequence in unnatural death；PCR primer is designed using conventional primer design method, constructs target
The nearly edge nucleic acid pool of bacterial strain further passes through the method bacterium specific nucleic acid sequence of PCR.Provided by the present invention for nearly edge
The specific nucleic acid screening technique of Strain differentiation expeditiously can largely obtain the specific nucleic acid sequence of purpose microbial strains
Column, the reproducibility of experimental result is high, meanwhile, preliminary analysis is carried out by bioinformatics means, simplifies entire experiment
Process.In addition, the specific nucleic acid sequence screened using the method for the present invention, can also further pass through PCR, qPCR and nucleic acid probe
Deng design, quantitatively or semi-quantitatively detect objective microbe bacterial strain in the rich of the ecosystem (such as in soil or plant rhizosphere)
Degree.Experimentation uses general software, and method not high to the cost requirement of experimental facilities therefore of the invention has good
Applicability.
Detailed description of the invention
Figure 1A zospirillum sp.TSA2S strain specificity primer PCR electrophoretogram (duct M:DL2000 molecular weight,
Duct 1-8: nucleic acid pool 1-8, duct 9:Azospirillum sp.TSA2S, duct 10: sterile water).
Strain specificity primer PCR electrophoretogram (duct M:DL2000 molecular weight, the duct of Fig. 2A rthrobacter sp.2
1-8: nucleic acid pool 1-8, duct 9:Azospirillum sp.TSA2S, duct 10: sterile water).
Fig. 3 strain specificity primer 2 sFw 13/2sRv 13 is quantitative determined in soil and is inoculated with Azospirillum
The survival condition of bacterial strain after sp.TSA2S bacterial strain 22 days, 34 days and 41 days.
Fig. 4 strain specificity primer 2 sFw 14/2sRv 14 is quantitative determined in soil and is inoculated with Azospirillum
The survival condition of bacterial strain after sp.TSA2S bacterial strain 22 days, 34 days and 41 days.
Fig. 5 strain specificity primer 2 sFw 20/2sRv 20 is quantitative determined in soil and is inoculated with Azospirillum
The survival condition of bacterial strain after sp.TSA2S bacterial strain 22 days, 34 days and 41 days.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
It applies content described in example and is merely to illustrate the present invention, without sheet described in detail in claims should will not be limited
Invention.
The strain specific segments of the design of embodiment 1 Azospirillum sp.TSA2S.
Carry out Azospirillum sp.TSA2S full-length genome be finely sequenced, with electronics cutting implement splitter into
Row electronic shearography generates the 300bp segment being overlapped containing 100bp.The 300bp segment of acquisition establishes nucleic acid fragment database.?
In NCBI NT database, using nucleic acid fragment as querying condition, selection species are Azospirillum (taxid:191), displaying
Maximum target is 10, other use default parameters, carries out BLASTN search.Downloading hit table, extracts hit sequence, and further
Table in unnatural death is obtained, sequence in unnatural death is extracted in nucleic acid fragment database.It is inquiry item with sequence in the unnatural death extracted
Part, the maximum target of displaying are 10, other use default parameters, carry out BLASTN search.Downloading hit table, extracts hit sequence
Column, and table in unnatural death is further obtained, sequence in unnatural death, as possible bacterial strain are extracted second in nucleic acid fragment database
Specific fragment.The part strain specific segments of acquisition are as shown in NO.1~14 SEQ ID.
The strain specificity primer of the design of embodiment 2 Azospirillum sp.TSA2S.
Using strain specific segments shown in NO.1~15 SEQ ID as template, strain specificity is designed according to following standard
QPCR primer: PCR product is no more than 280bp, and primer length is not about 56-60 DEG C in 18-27bp, primer Tm, two primers
Less than 2 DEG C, hairpin structure, repetition and the primer dimer that primer is not previously predicted are formed Tm difference.The Azospirillum of design
The strain specificity primer of sp.TSA2S such as table 1.
The strain specificity primer of table 1Azospirillum sp.TSA2S
The strain specificity primer of the screening of embodiment 3 Azospirillum sp.TSA2S.
Azospirillum sp.TSA2S total genomic dna is extracted, with the possible Azospirillum in table 2
The strain specificity primer of sp.TSA2S is constructed the genomic nucleic acids pond of non-targeted bacterial strain by table 3, right using sterile water as feminine gender
According to.
Strain specificity primer information of the table 2 for the possible Azospirillum sp.TSA2S of verifying
The genomic nucleic acids pond information of the non-targeted bacterial strain of table 3
With 2720Thermal cycler (Applied Biosystems, Foster City, CA, USA) PCR instrument, use
PCR amplification is carried out according to the following conditions.
Reaction system:
Response parameter:
PCR product detection is carried out with 2% agarose gel electrophoresis.If Successful amplification Azospirillum
Sp.TSA2S, fail to expand the primer of sterile water and non-targeted nucleic acid pool, as strain specificity primer.In attached drawing 1
2sFw12Rv12,2sFw13Rv13,2sFw14Rv14,2sFw16Rv16,2sFw17Rv17 and 2sFw18Rv18.
Embodiment 4
The strain specificity primer for screening obtained Azospirillum sp.TSA2S is converted into qPCR primer, and carries out
Azospirillum sp.TSA2S bacterial strain quantitative measurement in pedotheque.
Embodiment 5
It is miscellaneous that the strain specificity nucleic acid sequence of the Azospirillum sp.TSA2S obtained according to screening designs Southern
It hands over and explores, and carry out the semiquantitative determination of Azospirillum sp.TSA2S bacterial strain quantity in pedotheque.
The strain specific segments of the design of embodiment 6 Arthrobacter sp.2.
The full-length genome for carrying out Arthrobacter sp.2 is finely sequenced, and carries out electricity with electronics cutting implement splitter
Son shearing, generates the 300bp segment being overlapped containing 100bp.The 300bp segment of acquisition establishes nucleic acid fragment database.In NCBI NT
In database, using nucleic acid fragment as querying condition, the maximum target of displaying is 10, other use default parameters, carries out BLASTN
Search.Downloading hit table, extracts hit sequence, and further obtain table in unnatural death, extracts unnatural death in nucleic acid fragment database
Middle sequence.It is querying condition with sequence in the unnatural death extracted, the maximum target of displaying is 10, other use default parameters,
Carry out BLASTN search.Downloading hit table, extracts hit sequence, and further obtain table in unnatural death, in nucleic acid fragment database
In second extract sequence in unnatural death, as possible strain specific segments.The part strain specific segments such as SEQ of acquisition
Shown in NO.15~20 ID.
Embodiment 7:
It is arranged with nucleotides sequence shown in NO.1~6 SEQ ID as template, the bacterial strain of the Arthrobacter sp.2 of design
Specific primer is as shown in table 4.
The strain specificity primer of table 4Arthrobacter sp.2
Embodiment 8
Arthrobacter sp.2 total genomic dna is extracted, with the bacterium of the possible Arthrobacter sp.2 in table 5
Strain specific primer, the genomic nucleic acids pond of non-targeted bacterial strain is constructed by table 6, using sterile water as negative control.
Strain specificity primer information of the table 5 for the possible Arthrobacter sp.2 of verifying
The genomic nucleic acids pond information of the non-targeted bacterial strain of table 6
With 2720Thermal cycler (Applied Biosystems, Foster City, CA, USA) PCR instrument, use
PCR amplification is carried out according to the following conditions.
Reaction system:
Response parameter:
PCR product detection is carried out with 2% agarose gel electrophoresis.If Successful amplification Arthrobacter sp.2, not
The primer of sterile water and non-targeted nucleic acid pool, as strain specificity primer can be expanded.As in attached drawing 2 A2Fw3Rv3,
A2Fw4Rv4, A2Fw5Rv5 and A2Fw6Rv6.
Embodiment 9
The strain specificity primer for screening obtained Arthrobacter sp.2 is converted into qPCR primer, and carries out soil
Arthrobacter sp.2 bacterial strain quantitative measurement in sample.
Embodiment 10
The strain specificity nucleic acid sequence design Southern hybridization of the Arthrobacter sp.2 obtained according to screening is visited
Rope, and carry out the semiquantitative determination of Arthrobacter sp.2 bacterial strain quantity in pedotheque.
Sequence table
<110>Nanjing University of Technology
Nanjing Information engineering Univ
<120>a kind of for identifying the screening technique of the nucleotide fragments of nearly edge microorganism
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gcactacatg agaatataca ttggtggcaa catatcggat caacggctgg ctttatcttt 180
agcttagcag ggccaatgca atttcatgca aattatggac atcttaaaaa ttttcttaag 240
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ttctccggag aagtttgaag aaaaagccgg taatatttat tttgagtgct tagggcactc 240
atttgatata acttatggaa atgcaataac caccttgatt gcaacgcttg acatgtacaa 300
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ggattgcagc aaaaagcgtt tttaaatacg atcttgaatg gctttcaaaa aagcacgagt 180
accctgtaat gaaaggttat atagatgatc tttttatcca aacttttggc actcctgcat 240
cagctttcga atatgtaagg tattcctgat aatgtcagac ttacttagga gccccaggta 300
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acctattgcg tctgcccttt acgattcact aaaaactcac cgttcggagc gagttcgtca 180
atatccccaa tatagtgagt caagaattag tcgatacact atttcatacg gtgaattgtg 240
ccgtatggct gaagttccaa ttcagccaat tggaattggc aagtatcttg gtgaagtggc 300
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ttctgaaaag tacgtggctg gcaaattcgg tgacagcaga ccatactcct ttgaaggaga 180
ttatgattcg aatgaagata acaacaatgt ggttgtgtat tccggtttga acccgtttgt 240
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<211> 300
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
tctaaattca tcatctcatc attctttatc ccaatagtcg tttcgctcgg cccaaataca 60
attgtttccc ctgactgcgt atctgtcaga gaaactatgt cgacgacata accaaccaca 120
gcctccaggc aagctgcgtc tgtaacttgc tgtataaagt cacctaaaac attttttgct 180
gtgaacggat tcagaatctt agtttctgac acaagaatgc atgtaatttg atttttatag 240
attgttagac gaatatcacc atgaataccc gcctcgggat ggcggaattt aacaccaaat 300
<210> 7
<211> 300
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
cggaaacttt ggatgggaac ctcttataag ccgaaccgac tttaaggagc gtttcttaga 60
cttcaggtct aaagttttcg ccctcacatt tatgcgtcta cgacgaacgc acaccctttt 120
ggctctaaaa ggctgccatg gcgaaataat cagatgtttt ggcacatcgc gaatagtgca 180
tacatttact gtaacatttt ccccttcaac cccatcccca cgtgttgcag caagtacaag 240
cctgccccat tcataccgca atgagcacga tagaccatca atctttggct cagcaacgaa 300
<210> 8
<211> 300
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
tctccaatta ttctgcctcc gttcggatta aattacattt ttgaaggaca agcaagattt 60
tctcaaatac aatatctttc actttcctca aataagaaat ttgattttga tcatgcaaat 120
tctgcaggaa tgatgtcaga gctttataca aaagctttca atttttacat tgagaaaacg 180
ggcataccat atccttcttc aatactagat aattctgtaa acttgtttct tctggcatgc 240
gatatatctc tgaatactac tgcaggattt ccattatcat acacgacaga agacgaaacg 300
<210> 9
<211> 300
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
caagaattag tcgatacact atttcatacg gtgaattgtg ccgtatggct gaagttccaa 60
ttcagccaat tggaattggc aagtatcttg gtgaagtggc aacccactgt aatgaacacg 120
gatttcctcc catcaatgca cttgcggtca acggagacag tggattgcct ggtgacagtt 180
acgatcaagc gggagacggg ctttgctccc ttgctaattg gccaaaggag ctagaaagct 240
gcattaatga acagaggcaa tacccataaa ataatattga attatatttt catgtctctg 300
<210> 10
<211> 300
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
ttcaggtttg gcaaccgaaa gcagcaagac actgatccga gggttgcaaa atgacattct 60
cgcagatctt ccttccacat ccgcctgcag atgccgtcac ccctggcaaa tcatactcaa 120
tccaagcccc caattttccg gaaaagcaaa agctaattct cggctatttt cagcaaggac 180
gccaagagga cgcacctcaa aaccaaagcg ttttcagaag gatttcatat atcagtccct 240
gctcaggggg aacaattttg ctgcaaatcc tgcaatagct tttttcagta tttctaggtc 300
<210> 11
<211> 300
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
aaaaagccgg taatatttat tttgagtgct tagggcactc atttgatata acttatggaa 60
atgcaataac caccttgatt gcaacgcttg acatgtacaa agaaggggaa gatattgaat 120
ctcttttccc taacgcaaac ataatggcag aaataaattc tgaaagaaaa agcaaaaagg 180
aaaataacta ctaccatggc tctccaatta ttctgcctcc gttcggatta aattacattt 240
ttgaaggaca agcaagattt tctcaaatac aatatctttc actttcctca aataagaaat 300
<210> 12
<211> 300
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
gcgcccgagt tgcgtttgga gattacttaa cactcacaag taatcgccga aaatatagtc 60
tagaatgaag ctttcttctt ctttttttct tgctctcttc tgtcactaag agtttcactc 120
cattctggcc cttcatatgt gatagaaatc caaccttccc ctctccttcc gagggacttt 180
atctccatca tgataaccat actctgtaat tcccaaagcg taaaccaata cccgcagccg 240
tcgtaattaa ggcattgata aaactcatca ctctctttgt agagctttaa cccgtaattc 300
<210> 13
<211> 300
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
aatacaaaga gaaatccgaa ttggagaaag aagttaaaga aaatcctcta tacgactatg 60
gcataaaatc taccatgcgg gaagaggaaa gctctggcgt aaaaagctat tttcagatga 120
tcgacagaat aggaattgtt aagatagttg agaaggaaat actctcaact atcgtgaatt 180
ttcttgaaga gcgtggcgtg gacacgtcag agcttaagga acggcagaca tctatcctga 240
ataacggaat tatcatctct ggtggaaatg taaatgcgaa cggaattgtg gctggaaaca 300
<210> 14
<211> 300
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
ggtgctgccg tcgacatccc attcagatca ccagtggcga aggatgcaga tcgaaaaatg 60
tttggccgcg ctttttccat atggtgccga ggagcctggg tccgtgcaga ctctccctct 120
agactcatac tcttgctgtg atgtgttcga ctacgctcat gctgccgaaa tagttatgga 180
acaagttgaa agtcagggag gttgcgccaa catatctttg atacacatgg gcgcaaagct 240
ccaaactgtc ggtgcagctc tcgcattagc tgcgcgccca gaagttgccc tcgtgggtgc 300
<210> 15
<211> 300
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
cacggctctc ttaccctgcc aatagttgac ggttatttag cttcacccgg tcgggtgttg 60
ctcctggtat ccaccgcacc cctcatccgg acccgggggg ccggaccgta agaggcgcag 120
tcctccatac ccggaagccg ggcttcggac agggagggct ggatacgcgt agtcagccac 180
tgctcccgca ctcctgaatg agacgtgctg cttgtgtccg cgtcagcgga ctaggcatga 240
gggcacagca aactgctcca ccaagtgtac tacagcggac ctgcacggaa ggacggtttc 300
<210> 16
<211> 300
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
atcagggacg tgagtgattt cattgcttcc ccctgcaacg cgtcacacac gtgcccggca 60
tcagactgcc cacaaccgcg catcacacgg tcacggcacg tgaatgtgaa aaatagccct 120
gtctcgtcag tgcttcatca cgcgacagcc cgtgcaacgg tgctgaggtt cgcgcagtca 180
gccactgatt tcgggcccgc ccattcctga accgaagact tggcgggtcg tttgggccca 240
ctcgaaaatc tacctacggc atggagcacg aacgcgagta gttgttacct gatttagtga 300
<210> 17
<211> 300
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
agccagctca ccgggggtca gcccgtcctg agcatatcga gctatgtcct ctcgtatgga 60
tgattcgatc tctgacagga aacggaaatc gttggccaac gaggcggtgg tccacactcg 120
cgggaggtct gcgtagccag ttcgatagcc aaaccaacgg cccatttgga gcgccgaatc 180
gtacatcgat gagcttcgca ggaagaacga cgagatcagt ccttccaagg tcagcccgcg 240
agacaaggta ttcccgccca cagcgatgac cgtagcaggc tcgtccgtat agatgagacg 300
<210> 18
<211> 300
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
tgcttcggtc agccgggagg ccaggggtat ctggtctatt ccctgcgacg cctcccgcat 60
tgccctcctg ggacgctccc cggtggaccg agtcctcgct aacttttttc tcggcgtcca 120
tatccctccc tgccccaccc ggttcttcac gtcccgcagc gatgcgacgg actggctcaa 180
tgacgaccgg taggtgccgt gttctgccct gcggcggacg atccgaggcg gacaaagcac 240
cactggaaca acactcggat cggcgcgcgt gagcccagca gaaggcgctg aggatccccg 300
<210> 19
<211> 300
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
tggtgagggt aaactactgt ctcgccgacc tcaaaaacca tgtggacatt cccctttccc 60
gcagaccagt ttatcacgat taaggcatac aactgccatg gatcgtgctc ggaaaccgca 120
tgaatccgcg gaaaacggcc cgaatcggcc cttaaaaccc cttgacgaac gcggataata 180
gtgcattggg caccctctgc caagggccgc cgggtacgac ggcggacggt caccgaatcc 240
gcgtttgagc gccattgcgg ataggctatg cctgaataat gtttcaagac tctcaaggag 300
<210> 20
<211> 300
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
agggcagcaa cgggaaggtg tggactcgat tttctcacgt caggcatcac gaccacacga 60
cgactgatgc ctacattcgc cgagaagcga cgatcgaccc tcgcataccg tttcacatgg 120
agcagggact tagatgggta tccgcggacg tttacgccga gtcccaggcc gcagacttgt 180
acggcgggtt tctgaaagct gcgttgtggc cgagcgggga attcggatac accgagccca 240
tgtatcttcg gacggtatgg caccaaataa ggaacagcga aagttgtgcg attccattag 300
<210> 21
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
ttctgaaagt ggcattcacg 20
<210> 22
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
aagataaagc cagccgttga 20
<210> 23
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
gaagaacagg acacgtgcaa 20
<210> 24
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
tcaaatgagt gccctaagca 20
<210> 25
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
aatggattgc agcaaaaagc 20
<210> 26
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
gatgcaggag tgccaaaagt 20
<210> 27
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
ttcgagccct aacaggagaa 20
<210> 28
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
taaagggcag acgcaatagg 20
<210> 29
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
agtacgtggc tggcaaattc 20
<210> 30
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
ttattcccgg atccaacaaa 20
<210> 31
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
tgttcgacta cgctcatgct 20
<210> 32
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
gagagctgca ccgacagttt 20
<210> 33
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
tttcccctga ctgcgtatct 20
<210> 34
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
ctgaatccgt tcacagcaaa 20
<210> 35
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
tcagatgttt tggcacatcg 20
<210> 36
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
ctatcgtgct cattgcggta 20
<210> 37
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
tgcaggaatg atgtcagagc 20
<210> 38
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
tatcgcatgc cagaagaaac 20
<210> 39
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
tggcaaccca ctgtaatgaa 20
<210> 40
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
ccaattagca agggagcaaa 20
<210> 41
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
ctcgcagatc ttccttccac 20
<210> 42
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
gcgtccttgc tgaaaatagc 20
<210> 43
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 43
taaccacctt gattgcaacg 20
<210> 44
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 44
cggaggcaga ataattggag 20
<210> 45
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 45
ctccattctg gcccttcata 20
<210> 46
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 46
ttggtttacg ctttgggaat 20
<210> 47
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 47
gaggaaagct ctggcgtaaa 20
<210> 48
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 48
ccgttcctta agctctgacg 20
<210> 49
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 49
ggctctctta ccctgccaat 20
<210> 50
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 50
gtatggagga ctgcgcctct 20
<210> 51
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 51
cagggacgtg agtgatttca t 21
<210> 52
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 52
tgaagcactg acgagacagg 20
<210> 53
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 53
acaggaaacg gaaatcgttg 20
<210> 54
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 54
gttcttcctg cgaagctcat 20
<210> 55
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 55
tttttctcgg cgtccatatc 20
<210> 56
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 56
tgttccagtg gtgctttgtc 20
<210> 56
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 56
ttcccgcaga ccagtttatc 20
<210> 58
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 58
gcactattat ccgcgttcgt 20
<210> 59
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 59
tggagcaggg acttagatgg 20
<210> 60
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 60
tacatgggct cggtgtatcc 20
Claims (10)
1. a kind of for identifying the screening technique of the nucleotide fragments of nearly edge microorganism, which is characterized in that this method includes as follows
Step:
(1) complete genome sequence of objective microbe is obtained；
(2) the electronic shearography technology for utilizing splitter online software, the genome sequence of purpose bacterial strain is cut into containing overlapping
The nucleic acid small fragment in area；
(3) the fasta file that step (2) is obtained to the nucleic acid small fragment containing overlay region compares in NCBI NT database；
(4) step (3) obtains hit table I after comparing, and deletes the repetitive sequence in hit table I, and then obtains hit nucleic acid sequence
Ⅰ；
(5) the nucleic acid small fragment containing overlay region that the hit nucleic acid sequence I that step (4) obtains is obtained with step (2) is compared
It is right, obtain unnatural death more control sequences I；
(6) the unnatural death more control sequences I obtained in step (5) are compared again in NCBI NT database, are hit
Table II deletes the repetition repetitive sequence in hit table II, and then obtains hit nucleic acid sequence II；
(7) step (5) are repeated, by the hit nucleic acid sequence II that step (6) obtain and the nucleic acid containing overlay region that step (2) obtain
Small fragment is compared, and obtains unnatural death more control sequences II, and the unnatural death amplifying nucleic acid sequence II column are possible objective microbe
Strain specificity nucleic acid fragment；
(8) the strain specificity nucleic acid fragment of the possible objective microbe obtained using step (7) designs PCR primer as template；
(9) 10 kinds of bacterial strain or more of genome DNA of category or relative genus where extracting objective microbe bacterial strain constructs nucleic acid
Pond；
(10) nucleic acid pool obtained respectively using the genomic DNA of objective microbe bacterial strain and step (9) utilizes step as template
(8) primer designed carries out PCR amplification, and amplified production carries out agarose electrophoresis detection；
If using the genomic DNA of objective microbe bacterial strain as the PCR test positive of template, and the nucleic acid obtained with step (9)
Pond is that the PCR of template is detected as the negative corresponding nucleic acid fragment of primer, as the special of objective microbe Strain differentiation
Property nucleic acid fragment.
2. according to claim 1 for identifying the screening technique of the nucleotide fragments of nearly edge microorganism, which is characterized in that institute
State the nucleic acid small fragment containing overlay region, the length of overlay region is 100~500bp, the length of the nucleic acid small fragment is 200~
1500bp。
3. a kind of for identifying the oligonucleotide fragment of Azospirillum sp.TSA2S bacterial strain, which is characterized in that including SEQ
It is ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.7, any shown in SEQ ID NO.8
A kind of nucleotide sequence.
4. a kind of for identifying the primer of Azospirillum sp.TSA2S bacterial strain, which is characterized in that with SEQ ID NO.2,
Nucleotides sequence shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 are any
It is classified as template, the primer designed；
Including any pair in following primer pair:
2sFw12:GAAGAACAGGACACGTGCAA, 2sRv12:TCAAATGAGTGCCCTAAGCA；
2sFw13:AATGGATTGCAGCAAAAAGC, 2sRv13:GATGCAGGAGTGCCAAAAGT；
2sFw14:TTCGAGCCCTAACAGGAGAA, 2sRv14:TAAAGGGCAGACGCAATAGG；
2sFw16:TGTTCGACTACGCTCATGCT, 2sRv16:GAGAGCTGCACCGACAGTTT；
2sFw17:TTTCCCCTGACTGCGTATCT, 2sRv17:CTGAATCCGTTCACAGCAAA；
2sFw18:TCAGATGTTTTGGCACATCG, 2sRv18:CTATCGTGCTCATTGCGGTA.
5. for identifying that the oligonucleotide fragment of Azospirillum sp.TSA2S bacterial strain is being identified described in claim 3
Application in Azospirillum sp.TSA2S bacterial strain.
6. for identifying the primer of Azospirillum sp.TSA2S bacterial strain in identification Azospirillum described in claim 4
Application in sp.TSA2S bacterial strain.
7. a kind of for identifying the oligonucleotide fragment of Arthrobacter sp.2 bacterial strain, which is characterized in that including SEQ ID
Any one nucleotide sequence shown in NO:17~18.
8. a kind of for identifying the primer of Arthrobacter sp.2 bacterial strain, which is characterized in that with NO17~18 times SEQ ID
Nucleotides sequence shown in one is classified as the primer that stencil design obtains；
Including any pair in following primer pair:
A2Fw3:ACAGGAAACGGAAATCGTTG, A2Rv3:GTTCTTCCTGCGAAGCTCAT；
A2Fw4:TTTTTCTCGGCGTCCATATC, A2Rv4:TGTTCCAGTGGTGCTTTGTC；
A2Fw5:TTCCCGCAGACCAGTTTATC, A2Rv5:GCACTATTATCCGCGTTCGT；
A2Fw6:TGGAGCAGGGACTTAGATGG, A2Rv6:TACATGGGCTCGGTGTATCC.
9. for identifying that the oligonucleotide fragment of Arthrobacter sp.2 bacterial strain is being identified described in claim 7
Application in Arthrobacter sp.2 bacterial strain.
10. for identifying the primer of Arthrobacter sp.2 bacterial strain in identification Arthrobacter described in claim 8
Application in sp.2 bacterial strain.
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| CN201811373222.2A CN109266768A (en) | 2018-11-19 | 2018-11-19 | It is a kind of for identifying the screening technique of the nucleotide fragments of nearly edge microorganism |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112331268A (en) * | 2020-10-19 | 2021-02-05 | 成都基因坊科技有限公司 | Method for obtaining specific sequence of target species and method for detecting target species |
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| CN101684481A (en) * | 2009-03-30 | 2010-03-31 | 电子科技大学 | Method for preparing salvia miltiorrhiza EST-SSR molecular mark, specific primer and application thereof |
| CN103243164A (en) * | 2013-05-06 | 2013-08-14 | 西北农林科技大学 | Method for identifying psathyrostachys huashanica allogenic material in wheat genome |
| CN103646193A (en) * | 2013-12-24 | 2014-03-19 | 辽宁大学 | PCR primer design method for identification of closely related species |
| CN103714267A (en) * | 2013-12-27 | 2014-04-09 | 中国人民解放军军事医学科学院生物工程研究所 | Detection or assisted detection method for bacterial strains to be detected based on species specific sequence |
| CN105274092A (en) * | 2015-11-30 | 2016-01-27 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Batch acquiring method for specific isothermal oligonucleotide probes |
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2018
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| CN101684481A (en) * | 2009-03-30 | 2010-03-31 | 电子科技大学 | Method for preparing salvia miltiorrhiza EST-SSR molecular mark, specific primer and application thereof |
| CN103243164A (en) * | 2013-05-06 | 2013-08-14 | 西北农林科技大学 | Method for identifying psathyrostachys huashanica allogenic material in wheat genome |
| CN103646193A (en) * | 2013-12-24 | 2014-03-19 | 辽宁大学 | PCR primer design method for identification of closely related species |
| CN103714267A (en) * | 2013-12-27 | 2014-04-09 | 中国人民解放军军事医学科学院生物工程研究所 | Detection or assisted detection method for bacterial strains to be detected based on species specific sequence |
| CN105274092A (en) * | 2015-11-30 | 2016-01-27 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Batch acquiring method for specific isothermal oligonucleotide probes |
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| CN112331268A (en) * | 2020-10-19 | 2021-02-05 | 成都基因坊科技有限公司 | Method for obtaining specific sequence of target species and method for detecting target species |
| CN112331268B (en) * | 2020-10-19 | 2023-04-14 | 成都基因坊科技有限公司 | Method for obtaining specific sequence of target species and method for detecting target species |
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