CN104120168B - Fusarium sambucinum detection kit and detection method - Google Patents
Fusarium sambucinum detection kit and detection method Download PDFInfo
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- CN104120168B CN104120168B CN201310145104.7A CN201310145104A CN104120168B CN 104120168 B CN104120168 B CN 104120168B CN 201310145104 A CN201310145104 A CN 201310145104A CN 104120168 B CN104120168 B CN 104120168B
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- fusarium sambucinum
- fusarium
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- 241000221779 Fusarium sambucinum Species 0.000 title claims abstract description 34
- 238000001514 detection method Methods 0.000 title claims abstract description 23
- 238000003752 polymerase chain reaction Methods 0.000 claims abstract description 10
- 238000012360 testing method Methods 0.000 claims abstract description 5
- 230000003321 amplification Effects 0.000 claims abstract description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 4
- 108020004414 DNA Proteins 0.000 claims description 24
- 239000000203 mixture Substances 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 5
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 4
- 238000004925 denaturation Methods 0.000 claims description 4
- 230000036425 denaturation Effects 0.000 claims description 4
- 244000052616 bacterial pathogen Species 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 229920004890 Triton X-100 Polymers 0.000 claims description 2
- 239000013504 Triton X-100 Substances 0.000 claims description 2
- 238000000137 annealing Methods 0.000 claims description 2
- 230000004087 circulation Effects 0.000 claims description 2
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 claims description 2
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 claims description 2
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 claims description 2
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 claims description 2
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 claims description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 239000003381 stabilizer Substances 0.000 claims description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 1
- 230000001580 bacterial effect Effects 0.000 claims 1
- 238000012408 PCR amplification Methods 0.000 abstract description 4
- 239000012634 fragment Substances 0.000 abstract description 4
- 238000012797 qualification Methods 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 3
- 244000000004 fungal plant pathogen Species 0.000 abstract description 2
- 241000894007 species Species 0.000 abstract description 2
- 241000223218 Fusarium Species 0.000 description 19
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 18
- 238000000034 method Methods 0.000 description 6
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 238000013461 design Methods 0.000 description 4
- 241000526118 Fusarium solani f. radicicola Species 0.000 description 3
- 101000597041 Gallus gallus Transcriptional enhancer factor TEF-3 Proteins 0.000 description 3
- 101000653735 Homo sapiens Transcriptional enhancer factor TEF-1 Proteins 0.000 description 3
- 102100029898 Transcriptional enhancer factor TEF-1 Human genes 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 101150087698 alpha gene Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000003113 dilution method Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
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- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 239000002023 wood Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 102000002508 Peptide Elongation Factors Human genes 0.000 description 1
- 108010068204 Peptide Elongation Factors Proteins 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
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- 238000005286 illumination Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention belongs to the species identification technology field of plant pathogenic fungi.Purpose be to provide a kind of simple and rapid detection plant pathogenic fungi fusarium sambucinum based on polymerase chain reaction (PCR) (Fusarium sambucinum) test kit and detection method.Detection kit includes specific primer sequences and the Mix of a pair qualification fusarium sambucinum, and pair of primers sequence is respectively as follows: Fs F:5 ' GAC CCA AAT CTA AGC TCG CC 3 '；Fs R:5 ' GAA GGG CAT GTC GCT CAA G 3 '.Detection method is: utilize Fs F and Fs R primer that the genomic DNA of fusarium sambucinum is carried out PCR amplification, it is possible to obtain the specific amplification fragment of a length of 309 bp, and non-fusarium sambucinum can not expand this fragment of acquisition.PCR reacts the detectable limit to fusarium sambucinum genomic DNA concentration up to 70 100 pg/ μ L.Utilize the present invention, fusarium sambucinum can be detected accurately, easily and quickly.
Description
Technical field
The present invention relates to the species identification technology field of plant pathogenic fungi.
Background technology
Dry rot of potato is one of world's storage of potato phase important disease, by multiple Fusarium spp. (Fusarium
Spp.) cause.Wherein fusarium sambucinum strong adaptability, widely distributed, pathogenicity are strong, always North America and Some European area
The Dominantpathogen of dry rot of potato, and the advantage of the ground dry rot of potato such as Ye Shi China Hebei, Gansu and Inner Mongol
Pathogen.Tradition Identification of The Genus Fusarium uses morphological method to carry out.But, the various complexity of Fusarium spp. categorizing system, and temperature,
The change of the condition of culture such as humidity, pH value, illumination, all can cause the morphological characteristic generation significant change of Fusarium spp., in addition form
Qualification ratio itself is relatively time-consuming, the precise Identification faces enormous challenge of Fusarium spp..In recent years, along with the development of molecular biology,
The enriching constantly of fungal sequence in particularly GenBank GenBank, the qualification accurately and rapidly for Fusarium spp. is opened up
New direction.
Translation elongation factor (Translation elongation factor-l alpha, TEF-l α) gene comprises extensively
General phylogenetics information, on Identification of The Genus Fusarium, TEF-l α sequence has the biggest advantage.First, TEF-l α sequence exists
The horizontal information content of Fusarium Species is high；Secondly, in Fusarium, the gene copy of non-homology it is not detected by.The present invention is led to
Cross the data set to Fusarium TEF-1 α full sequence composition and compare analysis, protect in have found fusarium sambucinum kind
Keep, and the DNA region of variability that there are differences with other kind Fusarium spp., design the special primer of fusarium sambucinum, and set up
The test kit of detection fusarium sambucinum accurately, easily and fast and detection method.
Summary of the invention
It is an object of the invention to provide a kind of detection kit identifying fusarium sambucinum accurately, easily and fast and inspection
Survey method.Specifically include Specific PCR primers and the Mix of a pair autonomous Design, and a whole set of PCR evaluation program and step.
1. technical scheme is as follows: appear download Fusarium from Genebank GenBank for the first time
The all sequences of TEF-1 α gene.After the data set forming download sequence is compared and analyzed, find out fusarium sambucinum
Guard in kind, and the region of DNA territory that there are differences with other kind Fusarium spp., design the special of bone wood Fusarium spp. according to sequence difference
Primers F s-F and Fs-R.Extract pathogenic bacteria genomic DNA and measure concentration, utilizing the specific primer pair of designed, designed, given
Under the conditions of carry out PCR reaction, detect whether that through agarose gel electrophoresis the DNA fragmentation of 309 bp occurs, just can be accurate and differentiate
Agarose gel electrophoresis result.Meanwhile, by the fusarium sambucinum genomic DNA of concentration known by 10 times of gradient dilution method dilutions
Laggard performing PCR expands, and determines the limting concentration that fusarium sambucinum genomic DNA is detected by the method.
2. authentication method is to use following steps:
(1) extraction of mycelia DNA；
(2) amplification of DNA fragments, i.e. carries out polymerase chain reaction, for the DNA of the pair of primers of polymerase chain reaction
Sequence is:
Fs-F:5’-GAC CCA AAT CTA AGC TCG CC-3’；
Fs-R:5’-GAA GGG CAT GTC GCT CAA G-3’；
(3) agarose gel electrophoresis analysis；
(4) judgement of qualification result: if pcr amplification product occurs the DNA band of expection 309 bp length on agarose,
Then supplying test sample is fusarium sambucinum；
(5) by the fusarium sambucinum genomic DNA of concentration known by the dilution of 10 times of gradient dilution methods, with each of dilution
The DNA of concentration is that template carries out PCR amplification, determines that the limting concentration that fusarium sambucinum genomic DNA is detected by the method is
70-100 pg/μL；
This invention is compared with Morphological Identification, affected by environment little, can more accurately, conveniently and quickly identify Ramulus Sambuci Williamsii
Fusarium spp., and be that the carry disease germs detection of potato seed provides strong technical support.
3. detailed description of the invention:
(1) acquisition of fusarium sambucinum special primer Fs-F/ Fs-R sequence: from Genebank GenBank
The all sequences of middle download Fusarium TEF-1 α gene.With Clustal X, download sequence is compared, and use BioEdit
Manually adjust, the data set of all sequences composition is analyzed, conservative in have found fusarium sambucinum kind, and and its
The DNA region of variability that his kind Fusarium spp. there are differences, designs the special primer of fusarium sambucinum, sequence be Fs-F:5 '-
GAC CCA AAT CTA AGC TCG CC-3’；Fs-R:5 '-GAA GGG CAT GTC GCT CAA G-3 ', this primer is theoretical
Amplified fragments is 309 bp；
(2) Kit components includes: primers F s-F and Fs-R (each 10 μMs), Mix [Taq enzyme (0.1-0.3 U/ μ L),
dATP (0.03-0.10 mM)、dTTP (0.03-0.10 mM)、dGTP(0.03-0.10 mM)、dCTP(0.03-0.10
mM)、MgCl2(0.3-1.0 mM)、KCl(130-150 mM)、Tris-HCl(20-30 mM, pH 9.0-9.5)、1%
Triton X-100 and stabilizer], ddH2O；
(3) extraction and the concentration of genomic DNA measures: uses CTAB method to extract fusarium sambucinum genomic DNA, utilizes
Ultraviolet spectrophotometer measures DNA concentration；
(4) polymerase chain reaction:
A. reference reaction system is 25 μ L, and each reactant liquor consists of:
Mix 12.5 μL；
Forward primer Fs-F 1 μ L；
Downstream primer Fs-R 1 μ L；
Template DNA 1 μ L；
Aseptic ddH2O 9.5 μL；
B. polymerase chain reaction is carried out in PCR instrument, and set program is:
The initial denaturation stage: 94 DEG C, 4 min；
Denaturation stage: 94 DEG C, 30-40 s；
Annealing stage: 61 DEG C, 30-40 s；
The extension stage: 72 DEG C, 20-25 s；
Cycle-index: 28 circulations；
Finally extend the stage: 72 DEG C, 7-10 min, reaction terminate after 4 DEG C of preservations；
(5) electrophoresis detection and result judge: take 5 μ L PCR primer and carry out electrophoresis detection in 1.2% agarose gel.Synthetism
All there is the specific band of 309 bp in wood Fusarium spp., and non-fusarium sambucinum is all without this band；
(6) determination of DNA concentration detectable limit: by the aseptic ddH of fusarium sambucinum genomic DNA of concentration known2O
Carry out 10 times of gradient dilutions, carry out PCR amplification and electrophoresis detection with the DNA of each concentration of dilution for template, amplification condition with
Electrophoretic detection ibid 2 step, determines that the limting concentration of fusarium sambucinum DNA detection is 70-100 pg/ μ L.
Claims (5)
- The detection kit of the most a set of fusarium sambucinum, it is characterised in that: test kit include a pair specific primer sequence and Mix；Primers DNA sequences is: Fs-F:5 '-GAC CCA AAT CTA AGC TCG CC-3 ', Fs-R:5 '-GAA GGG CAT GTC GCT CAA G-3’；In test kit the composition of Mix be Taq enzyme 0.1-0.3U/ μ L, dATP 0.03-0.10mM, dTTP 0.03-0.10mM, dGTP 0.03-0.10mM、dCTP 0.03-0.10mM、MgCl2 0.3-1.0mM、KCl 130-150mM、pH 9.0- 9.5Tris-HCl 20-30mM, 1%Triton X-100 and stabilizer.
- 2. the detection kit of fusarium sambucinum as claimed in claim 1, it is characterised in that: polymerase chain reaction is detected The concentration limit value of fusarium sambucinum genomic DNA is 70-100pg/ μ L.
- 3. utilize the detection method of the fusarium sambucinum of detection kit described in claim 1, it is characterised in that: extract pathogenic bacteria Genomic DNA also measures concentration, utilizes special primer, carries out PCR reaction under prescribed conditions, examines through agarose gel electrophoresis Survey, occur that the bacterial strain of 309bp specific amplification band is fusarium sambucinum.
- 4. detection method as claimed in claim 3, it is characterised in that: identify the polymerase chain reaction system of fusarium sambucinum It is 25 μ L:
- 5. detection method as claimed in claim 3, it is characterised in that: identify the polymerase chain reaction bar of fusarium sambucinum Part is:The initial denaturation stage: 94 DEG C, 4min；Denaturation stage: 94 DEG C, 30-40s；Annealing stage: 61 DEG C, 30-40s；The extension stage: 72 DEG C, 20-25s；Cycle-index: 28 circulations；Finally extend the stage: 72 DEG C, 7-10min, reaction terminate after 4 DEG C of preservations.
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| CN201310145104.7A CN104120168B (en) | 2013-04-24 | 2013-04-24 | Fusarium sambucinum detection kit and detection method |
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| CN104120168B true CN104120168B (en) | 2016-08-10 |
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| CN106520570B (en) * | 2016-11-10 | 2019-10-29 | 红河学院 | Raw hook-shaped trichoderma strain and its application in a kind of loquat |
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Non-Patent Citations (6)
| Title |
|---|
| A DNA sequence database for identifying Fusarium;David M. Geiser等;《European Journal of Plant Pathology》;20041231;第110卷(第5期);473-479 * |
| David M. Geiser等.A DNA sequence database for identifying Fusarium.《European Journal of Plant Pathology》.2004,第110卷(第5期),473-479. * |
| 杜密茹.马铃薯干腐病病原菌鉴定及病害防治的研究.《中国优秀硕士学位论文全文数据库农业科技辑》.2011,(第S1期),D046-77. * |
| 柳凤等.现代生物技术在镰刀菌分类学中的应用.《中国农学通报》.2012,第28卷(第30期),166-170. * |
| 现代生物技术在镰刀菌分类学中的应用;柳凤等;《中国农学通报》;20121231;第28卷(第30期);166-170 * |
| 马铃薯干腐病病原菌鉴定及病害防治的研究;杜密茹;《中国优秀硕士学位论文全文数据库农业科技辑》;20111215(第S1期);D046-77 * |
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