CN101012263A - General purpose primer for cloning bacteria gyrase-gyrB gene - Google Patents
General purpose primer for cloning bacteria gyrase-gyrB gene Download PDFInfo
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- CN101012263A CN101012263A CN 200610136701 CN200610136701A CN101012263A CN 101012263 A CN101012263 A CN 101012263A CN 200610136701 CN200610136701 CN 200610136701 CN 200610136701 A CN200610136701 A CN 200610136701A CN 101012263 A CN101012263 A CN 101012263A
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Abstract
The invention discloses a general primer of cloned bacterium spiral-accelerating enzyme-gyrB gene, whose nucleic sequence of upstream primer is 5'-CC(ATGC)GG(ATGC)ATG TA(CT) AT(ATC)GG-3' and the nucleic sequence of downstream primer is 5'-CAT (CT) TC (ATGC) CC (ATGC) A (AG) (ATGC) CC (CT) TT (AG) (AT) A (ATGC) C (GT) (CT) TG-3'), wherein the whole sequence of PCR product approximates to the whole sequence of gyrB gene, which possesses stronger specificity and sensitivity.
Description
Technical field
The present invention relates to microflora and grow the molecular marked compound field, refer more particularly to the universal primer of bacteria gyrase-gyrB gene.
Background technology
On modern microbial taxonomy, 16S rDNA has been widely used in the phylogeny status of research bacterium, but a large amount of studies show that, and for the nearer bacterial strain of sibship, it is not enough relying on 16S rDNA homology between bacterial strain to plant calmly merely.The gyrB gene is the gene of coding DNA gyrase (a kind of protokaryon II type topoisomerase) B subunit, and as a kind of new molecule marker, this gene has been widely used in the detection of microorganism under the evaluation of bacterium, fungi kind level level and the natural environmental condition at present.The research that the phylogenetic systematics of Aeromonas, Rhodopseudomonas and Shiva Bordetella etc. is identified has proved that for the evaluation between each bacterial classification in the more approaching genus of sibship, this molecular classification index of gyrB gene has stronger feasibility.Tracing it to its cause, be because this gene has higher base to replace frequency than 16S rDNA, and the average base replacement rate of 16S rDNA is per 5,000 ten thousand 1% on the one hand, and gyrB is per 1,000,000 years 0.7%-0.8%.On the other hand, gyrB gene ubiquity and almost all occurring in bacterium with single copy form, and 16S rDNA has different copy numbers in different bacterium, and there is nature difference between the different copies, therefore, the research method based on gyrB more is applicable to quantitative analysis than the research method based on 16S rDNA.
At present, most widely used a pair of gyrB gene universal primer is designed in nineteen ninety-five by Yamamoto and Harayama at the detection of microorganism and microbial species and aspect the evaluation between planting, this primer can amplify the gyrB gene from most of bacterial genomes DNA, amplified production and length are about 1.2Kb.
The primer of Yamamoto and Harayama research, its upstream primer sequence is:
5-GAAGTCATCATGACCGTTCTGCA (TC) GC (TCAG) GG (TCAG) GG (TCAG) AA (AG) TT (TC) GA-3, the downstream primer sequence is:
5-AGCAGGGTACGGATGTGCGAGCC(AG)TC(TCAG)AC(AG)TC(TCAG)GC(AG)TC(TCAG)GTCAT-3。
This primer is to being according to intestinal bacteria (GYRB_E.COLI; ACCESSIONNUMBER:P06982), pseudomonas (GYRB_PSEPU; ACCESSION NUMBER:P13364) and subtilis (GYRB_BASCUS; Two amino acid conserved sequences of dna gyrase B subunit ACCESSION NUMBER:P05652) design, with the length of this primer institute amplification PCR products generally about 1.2Kb.And in fact, about the about 2.0Kb of length of the gyrB gene of many bacteriums.Therefore, when the bacterium between to not homophyletic of the same race being carried out somatotype, the genetic information that sequence comprised of 1.2Kb left and right sides length was limited.
Summary of the invention
The object of the invention is to provide a kind of universal primer of new gyrB gene, solves the limited problem of genetic information amount that existing gyrB gene primer is comprised.This new molecular marked compound that the present invention obtains, with more help carrying out the environmental microorganism ecological diversity and to different strain, with bacterial classification not the bacterium between the homophyletic system carry out the research of somatotype.
Detailed technology scheme of the present invention is:
Analyze two comparatively conservative aminoacid sequence PGMYIG according to 26 kinds of known DNA of bacteria gyrase B subunits, QRYKGLGEM designs and synthesizes out the pair of degenerate primers of gyrB gene according to these two aminoacid sequences.
The general degenerated primer of the cloning bacteria gyrase that the present invention obtains-gyrB gene, the nucleotides sequence of its upstream degenerated primer JGYP1 is classified as: 5 '-CC (ATGC) GG (ATGC) ATGTA (CT) AT (ATC) GG-3 ', the nucleotides sequence of its downstream degenerated primer JGYP2 is classified as: 5 '-CAT (CT) TC (ATGC) CC (ATGC) A (AG) is (AT) (CT) TG-3 ' of A (ATGC) C (GT) of CC (CT) TT (AG) (ATGC)).
By this to primer, have a liking for the ferrous thiobacillus of acid oxidase (ATCC 23270 and DQ062115 (the 16S rDNA sequence accession number of this bacterial strain is DQ672266)) from what the thiobacillus ferrooxidans belonged to (Acidithiobacillus) respectively, (the 16S rDNA sequence accession number of these bacterial strains is respectively five strain leptospirillum ferriphilums in the hook end spiral Pseudomonas (Leptospirillum): DQ343299, EF025341, EF025340, EF025337, DQ451017 and EF025342) and iron protoxide hook end spirobacteria (ATCC 53992), the bacterial strain (ATCC 15859) that Sphingomonas XJ-1 of sphingosine monospore Pseudomonas (Sphingomonas) (the 16S rDNA sequence accession number of this bacterial strain is DQ672266) and Acidiphilum belong to has amplified the gyrB gene respectively in the genomic dna of totally 4 genus 11 bacterial strains.Show that thus this primer can amplify the gyrB gene from most of bacterial genomes DNA.The length of the gyrB gene that this primer amplification goes out is about 2.2Kb.Carry out somatotype and classification according to having amplified the gyrB gene respectively among the 12 strain bacterial genomes DNA to 5 genus, meet existing research the somatotype between the bacterial strain of the same race in the evaluation of these bacterial strains and the varying environment.
Compare with the degenerated primer of Harayama design with above-mentioned Yamamoto, universal primer of the present invention is because based on two in 26 kinds of DNA of bacteria gyrase B subunit sequences comparatively conservative aminoacid sequences, therefore, comprise more genetic information amount, have more ubiquity.Length with primer of the present invention institute amplification PCR products is about 2.2Kb, the full length sequence of the gyrB gene of this and many bacteriums is approaching, substantially can reflect the entrained genetic information of this gene really, with the Yamamoto primer extension product that research obtains with Harayama is that 1.2Kb compares, and it has solved well and has comprised the insufficient problem of genetic information.Therefore, when the bacterium between application universal primer of the present invention to not homophyletic of the same race is carried out somatotype, its specificity and susceptibility were stronger.
Description of drawings
Two comparatively conservative aminoacid sequences of the DNA of bacteria gyrase B subunit that Fig. 1 searches out from 26 kinds of known bacteriums;
Fig. 2 uses the electrophorogram of primer amplification iron protoxide hook end spirobacteria of the present invention (ATCC 53992) gyrB gene.
Embodiment
From the Internet database, derive the aminoacid sequence of 26 kinds of known bacteriums (seeing Table 1) dna gyrase B subunit, utilize submodule AlignX among the biosoftware Vector NTI Suite 7 to search out two comparatively conservative aminoacid sequence (PGMYIG of DNA of bacteria gyrase B subunit, QRYKGLGEM) (as accompanying drawing 1), obtain the nucleotide sequence of the pair of degenerate primers of gyrB gene according to these two aminoacid sequences, its upstream degenerated primer (JGYP1) sequence is: 5 '-CC (ATGC) GG (ATGC) ATGTA (CT) AT (ATC) GG-3 ', downstream degenerated primer (JGYP2) sequence is: 5 '-CAT (CT) TC (ATGC) CC (ATGC) A (AG) is (AT) (CT) TG-3 ' of A (ATGC) C (GT) of CC (CT) TT (AG) (ATGC)).With existing DNA synthetic technology,, synthesize this degenerated primer as automatic dna synthesizer.
26 kinds of known bacteria names of table 1:gyrB gene degenerated primer designing institute foundation and the acceptance of gyrB subunit sequence in the Internet database number
| Strain name | The acceptance of gyrB subunit sequence in the INTERNET database number |
| Myxococcus xanthus | AJ000543.1 |
| Haemophilus influenzae 86-028NP | CP000057.1 |
| Rhizobium etli CFN 42 | CP000133.1 |
| Yersinia pestis KIM | AE009952.1 |
| Buchnera aphidicola | P29435 |
| Vibrio parahaemolyticus | O51859 |
| E.coli | P0AES7 |
| Shigella flexneri | P0AES8 |
| Campylobacter jejuni | AL139074.2 |
| Pseudomonas putida | P13364 |
| Salmonella enterica serovar Typhi | AL627280.1 |
| Neisseria meningitides Z2491 | AL162752.2 |
| Bordetella pertussis Tohama I | BX640412.1 |
| Mycobacterium leprae | AL583917.1 |
| Mannheimia succiniciproducens | AE016827.1 |
| Brucella abortus | AE017223.1 |
| Nitrosomonas europaea ATCC 19718 | BX321856.1 |
| Synechocystis | P77966 |
| Mycobacterium tuberculosis H37Rv | BX842572.1 |
| Neisseria gonorrhoeae FA 1090 | AE004969.1 |
| Pasteurella multocida | AE006184.1 |
| Wigglesworthia glossinidia endosymbiont of Glossina brevipalpis | BA000021.3 |
| Yersinia pestis CO92 | CAC93544.1 |
| Streptomyces coelicolor A3(2) | CAB92994.1 |
| Ehrlichia ruminantium str. Welgevonden | CAI26937.1 |
| Mycobacterium bovis AF2122/97 | CAD92867.1 |
Utilize this to primer, by round pcr amplification iron protoxide hook end spirobacteria (ATCC 53992) gyrB gene.
Employed PCR reaction conditions is (a 50uL system):
5uL 10 * PCR reaction buffer
4uL 25mM Mg2+(MgC12)
2uL 10mM (dATP, dTTP, the concentration of dGTP and dCTP)
2.5 U archaeal dna polymerase
5uL 20mM primer JGYP1
5uL 20mM primer JGYP2
2uL (dna profiling is an amount of)
Add deionized water to 50uL
The PCR response procedures is:
1) 93 ℃ of pre-sex change of DNA are 5 minutes,
2) 93 ℃ of pre-sex change of DNA are 1 minute,
3) annealing temperature: 63 ℃ of starting temperatures, each circulation reduces by 0.6 ℃, and temperature is held constant at 57 ℃ after 10 circulations
Annealing time: 1 minute,
4) 72 ℃ were extended 2 minutes 30 seconds,
5) 72 ℃ were extended 10 minutes,
Circulation step 2), 3), 4) 35 times, obtains amplified production.Owing in the PCR reaction factor of amplification influence is mainly contained the influence of primer concentration and annealing temperature, for can greater efficiency amplify the purpose fragment, adopted the primer and the touchdown PCR program of high density.
Detect the PCR reaction product with 1.0% agarose gel electrophoresis, electrophoresis result as shown in Figure 2, wherein band 1 is contrast, band 2 is gyrB target gene fragment (2193bp), band 3 is a 200bp dna molecular amount standard.
Embodiment 2:
By these two primers, have a liking for the ferrous thiobacillus of acid oxidase (ATCC 23270 and DQ062115 (the 16S rDNA sequence accession number of this bacterial strain is DQ672266)) from what the thiobacillus ferrooxidans belonged to (Acidithiobacillus) respectively, (the 16S rDNA sequence accession number of these bacterial strains is respectively five strain leptospirillum ferriphilums in the hook end spiral Pseudomonas (Leptospirillum): DQ343299, EF025341, EF025340, EF025337, DQ451017 and EF025342) and iron protoxide hook end spirobacteria (ATCC 53992), the bacterial strain (ATCC 15859) that Sphingomonas XJ-1 of sphingosine monospore Pseudomonas (SPhingomonas) (the 16S rDNA sequence accession number of this bacterial strain is DQ672266) and Acidiphilum belong to has amplified gyrB gene (as shown in table 2) respectively in the genomic dna of totally 4 genus 11 bacterial strains.
Table 2 utilizes the gyrB gene of the bacterial strain of primer amplification of the present invention
| Belong to and strain name | The gyrB mrna length (bp) of amplification | |
| The thiobacillus ferrooxidans belongs to | ATCC 23270 | 2146 |
| DQ062115 | 2186 | |
| Hook end spiral Pseudomonas | DQ343299 | 2193 |
| EF025341 | 2194 | |
| EF025340 | 2193 | |
| EF025337 | 2192 | |
| DQ451017 | 2194 | |
| EF025342 | 2189 | |
| ATCC 53992 | 2193 | |
| Sphingosine monospore Pseudomonas | SPhingomonas XJ-1 | 2403 |
| Acidiphilum belongs to | ATCC 15859 | 1944 |
Claims (1)
1. the universal primer of cloning bacteria gyrase-gyrB gene, comprise upstream primer and downstream primer, the nucleotides sequence that it is characterized in that described upstream primer is classified as: 5 '-CC (ATGC) GG (ATGC) ATG TA (CT) AT (ATC) GG-3 ', the nucleotides sequence of described downstream primer is classified as: 5 '-CAT (CT) TC (ATGC) CC (ATGC) A (AG) is (AT) (CT) TG-3 ' of A (ATGC) C (GT) of CC (CT) TT (AG) (ATGC)).
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101492731B (en) * | 2008-12-04 | 2011-05-11 | 浙江理工大学 | Quick PCR detection method for colloid bacillus cereus |
| CN109385420A (en) * | 2018-10-26 | 2019-02-26 | 湖北工业大学 | The method for cloning the A Erduo ketoreductase gene of unknown bacterium |
| CN114703290A (en) * | 2022-01-25 | 2022-07-05 | 中国科学院南海海洋研究所 | General amplification primer, amplification method and application of sea cucumber |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4021285B2 (en) * | 2002-08-30 | 2007-12-12 | 株式会社ニチレイフーズ | Primer and probe for detection of Vibrio vulnificus and detection method using them |
| KR20050075454A (en) * | 2002-12-13 | 2005-07-20 | 니찌레이 푸드 인코포레이티드 | Primer and probe for detecting vibrio cholerae or vibrio mimicus and detection method using the same |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101492731B (en) * | 2008-12-04 | 2011-05-11 | 浙江理工大学 | Quick PCR detection method for colloid bacillus cereus |
| CN109385420A (en) * | 2018-10-26 | 2019-02-26 | 湖北工业大学 | The method for cloning the A Erduo ketoreductase gene of unknown bacterium |
| CN114703290A (en) * | 2022-01-25 | 2022-07-05 | 中国科学院南海海洋研究所 | General amplification primer, amplification method and application of sea cucumber |
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