CN104120168A - Fusarium sambucinum detection kit and detection method thereof - Google Patents
Fusarium sambucinum detection kit and detection method thereof Download PDFInfo
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- CN104120168A CN104120168A CN201310145104.7A CN201310145104A CN104120168A CN 104120168 A CN104120168 A CN 104120168A CN 201310145104 A CN201310145104 A CN 201310145104A CN 104120168 A CN104120168 A CN 104120168A
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- 241000221779 Fusarium sambucinum Species 0.000 title claims abstract description 38
- 238000001514 detection method Methods 0.000 title claims abstract description 26
- 238000003752 polymerase chain reaction Methods 0.000 claims abstract description 10
- 238000006243 chemical reaction Methods 0.000 claims abstract description 8
- 108020004414 DNA Proteins 0.000 claims description 24
- 239000000203 mixture Substances 0.000 claims description 11
- 238000012360 testing method Methods 0.000 claims description 5
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 4
- 230000003321 amplification Effects 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 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 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 108091028043 Nucleic acid sequence Proteins 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
- 238000004321 preservation Methods 0.000 claims description 2
- 238000011144 upstream manufacturing Methods 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 claims 1
- 238000010998 test method Methods 0.000 claims 1
- 241000894006 Bacteria Species 0.000 abstract description 10
- 238000000034 method Methods 0.000 abstract description 7
- 238000012408 PCR amplification Methods 0.000 abstract description 5
- 239000012634 fragment Substances 0.000 abstract description 4
- 244000000004 fungal plant pathogen Species 0.000 abstract description 2
- 241000223218 Fusarium Species 0.000 description 7
- 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 6
- 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
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 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
- 238000000605 extraction Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 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
- 241000233866 Fungi Species 0.000 description 1
- 102000002508 Peptide Elongation Factors Human genes 0.000 description 1
- 108010068204 Peptide Elongation Factors Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
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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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention belongs to the technical field of kind and molecule identification of plant pathogenic fungi, and aims to provide a polymerase chain reaction (PCR)-based simple and rapid plant pathogenic fungus Fusarium sambucinum detection kit and a detection method thereof. The kit includes a pair of specific primer sequences for identifying Fusarium sambucinum, and Mix, the pair of primer sequences comprise Fs-F:5'-GAC CCA AAT CTA AGC TCG CC-3' and Fs-R:5'-GAA GGG CAT GTC GCT CAA G-3'. The method is characterized in that PCR amplification of the genome DNA of Fusarium sambucinum is carried out by using the Fs-F and Fs-R primers to obtain a specific applied fragment with the length of 309bps, and bacteria not the Fusarium sambucinum cannot be amplified to obtain the fragment. The detection limit of the PCR reaction on the genome DNA concentration of the Fusarium sambucinum can reach 70-100pg/[mu]L. The Fusarium sambucinum can be accurately, conveniently and rapidly detected through the kit and the method.
Description
Technical field
The present invention relates to the kind Molecular Identification technical field of plant pathogenic fungi.
Background technology
Dry rot of potato is one of world's storage of potato important disease of phase, by multiple sickle-like bacteria (
fusariumspp.) cause.Wherein fusarium sambucinum strong adaptability, widely distributed, virulence is strong, is the Dominantpathogen of North America and Some European area dry rot of potato always, and the Dominantpathogen of the ground dry rot of potato such as Ye Shi China Hebei, Gansu and the Inner Mongol.Tradition Identification of The Genus Fusarium adopts morphological method to carry out.But, the various complexity of sickle-like bacteria categorizing system, and the change of the culture condition such as temperature, humidity, pH value, illumination, all can cause the morphological specificity generation considerable change of sickle-like bacteria, in addition identification of morphology itself is more consuming time, the precise Identification faces enormous challenge of sickle-like bacteria.In recent years, along with molecular biological development, particularly the enriching constantly of fungi sequence in GenBank GenBank, for new direction has been opened up in the qualification accurately and rapidly of sickle-like bacteria.
Translation elongation factor (Translation elongation factor-l alpha, TEF-l α) gene comprises phylogenetics information widely, and on Identification of The Genus Fusarium, TEF-l α sequence has very large advantage.First, TEF-l α sequence is high at the horizontal information content of Fusarium Species; Secondly the gene copy of non-homology, in Fusarium, do not detected.The present invention is by comparing analysis to the data set of Fusarium TEF-1 α full sequence composition, find in fusarium sambucinum kind conservative, and the DNA region of variability there are differences with other kind sickle-like bacteria, design the special primer of fusarium sambucinum, and set up test kit and the detection method of detection fusarium sambucinum accurately, easily and fast.
Summary of the invention
The object of this invention is to provide a kind of detection kit and detection method of identifying accurately, easily and fast fusarium sambucinum.Specifically comprise specific PCR primer and the Mix of a pair of autonomous design, and a whole set of PCR qualification program and step.
1. technical scheme of the present invention is as follows: appear all sequences of downloading Fusarium TEF-1 α gene from Genebank GenBank for the first time.After the data set of download sequence composition is compared and analyzed, find out in fusarium sambucinum kind conservatively, and the DNA region there are differences with other kind sickle-like bacteria, according to special primer Fs-F and the Fs-R of the wooden sickle-like bacteria of sequence difference design bone.Extract germ genomic dna and measure concentration, utilizing the Auele Specific Primer pair of designed, designed, under specified criteria, carrying out PCR reaction, detecting and whether have the DNA fragmentation of 309 bp to occur through agarose gel electrophoresis, just can accurate and discriminating agarose gel electrophoresis result.Meanwhile, the fusarium sambucinum genomic dna of concentration known is diluted to laggard performing PCR amplification by 10 times of gradient dilution methods, determine the threshold concentration that the method detects fusarium sambucinum genomic dna.
2. authentication method is to adopt following steps:
(1) extraction of mycelia DNA;
(2) amplification of DNA fragments, carries out polymerase chain reaction, for the DNA sequence dna of the pair of primers of polymerase chain reaction 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 the DNA band of 309 bp length appears expecting in pcr amplification product on agarose, be fusarium sambucinum for test sample;
(5) the fusarium sambucinum genomic dna of concentration known is diluted by 10 times of gradient dilution methods, the DNA of each concentration taking dilution carries out pcr amplification as template, determines that the threshold concentration that the method detects fusarium sambucinum genomic dna is 70-100 pg/ μ L;
This invention is compared with Morphological Identification, affected by environment little, can be more accurately, convenient and identify rapidly fusarium sambucinum, and provide strong technical support for the detection of the potato seed of carrying disease germs.
3. embodiment:
(1) acquisition of fusarium sambucinum special primer Fs-F/ Fs-R sequence: all sequences of downloading Fusarium TEF-1 α gene from Genebank GenBank.Download sequence is compared with Clustal X, and manually adjust with BioEdit, data set to all sequences composition is analyzed, find in fusarium sambucinum kind conservative, and the DNA region of variability there are differences with other kind sickle-like bacteria, design the special primer of fusarium sambucinum, sequence is Fs-F:5 '-GAC CCA AAT CTA AGC TCG CC-3 '; Fs-R:5 '-GAA GGG CAT GTC GCT CAA G-3 ', the theoretical amplified fragments of this primer is 309 bp;
(2) test kit composition comprises: primers F s-F and Fs-R (each 10 μ M), 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), MgCl
2(0.3-1.0 mM), KCl (130-150 mM), Tris-HCl (20-30 mM, pH 9.0-9.5), 1% Triton X-100 and stablizer], ddH
2o;
(3) extraction of genomic dna and concentration determination: adopt CTAB method to extract fusarium sambucinum genomic dna, utilize ultraviolet spectrophotometer to measure DNA concentration;
(4) polymerase chain reaction:
A. reference reaction system is 25 μ L, and each reaction solution consists of:
Mix 12.5 μL；
Upstream primer Fs-F 1 μ L;
Downstream primer Fs-R 1 μ L;
Template DNA 1 μ L;
Aseptic ddH
2o 9.5 μ L;
B. carry out on PCR instrument polymerase chain reaction, and set program is:
The initial sex change stage: 94 DEG C, 4 min;
The sex change 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 finish after 4 DEG C of preservations;
(5) electrophoresis detection and result judgement: get 5 μ L PCR products and carry out electrophoresis detection in 1.2% sepharose.All there is the specific band of 309 bp in fusarium sambucinum, non-fusarium sambucinum is all without this band;
(6) determining of DNA concentration limit of detection: by the aseptic ddH of fusarium sambucinum genomic dna of concentration known
2o carries out 10 times of gradient dilutions, carries out pcr amplification and electrophoresis detection taking the DNA of each concentration of diluting as template, the same 2 steps of amplification condition and electrophoretic detection, and the threshold concentration of determining fusarium sambucinum DNA detection is 70-100 pg/ μ L.
Claims (7)
1. the detection kit of a set of fusarium sambucinum, is characterized in that: test kit comprises a pair of specific primer sequence and Mix.
2. the detection kit of fusarium sambucinum as claimed in claim 1, it is characterized in that: a pair of specificity PCR primed DNA 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. the detection kit of fusarium sambucinum as claimed in claim 1, is characterized in that: the composition of test kit Mix is 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), MgCl
2(0.3-1.0 mM), KCl (130-150 mM), Tris-HCl (20-30 mM, pH 9.0-9.5), 1% Triton X-100 and stablizer.
4. the detection kit detection method of fusarium sambucinum as claimed in claim 1, it is characterized in that: extract germ genomic dna and measure concentration, utilize special primer, under specified criteria, carry out PCR reaction, detect through agarose gel electrophoresis, occur that the bacterial strain of 309 bp specific amplification bands is fusarium sambucinum.
5. the detection kit detection method of fusarium sambucinum as claimed in claim 4, is characterized in that: the polymerase chain reaction system of identifying fusarium sambucinum is (reference reaction system is 25 μ L):
Mix 12.5 μL；
Upstream primer Fs-F 1 μ L;
Downstream primer Fs-R 1 μ L;
Template DNA 1 μ L;
Aseptic ddH
2o 9.5 μ L.
6. the kit test method as described in right 4, is characterized in that: the polymerase chain reaction condition of identifying fusarium sambucinum is:
The initial sex change stage: 94 DEG C, 4 min;
The sex change 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 finish after 4 DEG C of preservations.
7. the detection kit of fusarium sambucinum as claimed in claim 1, is characterized in that: the concentration limit value that fusarium sambucinum genomic dna is detected in polymerase chain reaction is 70-100 pg/ μ L.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310145104.7A CN104120168B (en) | 2013-04-24 | 2013-04-24 | Fusarium sambucinum detection kit and detection method |
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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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| Publication Number | Publication Date |
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| CN104120168A true CN104120168A (en) | 2014-10-29 |
| CN104120168B CN104120168B (en) | 2016-08-10 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106520570A (en) * | 2016-11-10 | 2017-03-22 | 红河学院 | Loquat endophytic unciform trichoderma strain and applications thereof |
-
2013
- 2013-04-24 CN CN201310145104.7A patent/CN104120168B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| DAVID M. GEISER等: "A DNA sequence database for identifying Fusarium", 《EUROPEAN JOURNAL OF PLANT PATHOLOGY》 * |
| 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》 * |
| 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》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106520570A (en) * | 2016-11-10 | 2017-03-22 | 红河学院 | Loquat endophytic unciform trichoderma strain and applications thereof |
| 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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| Publication number | Publication date |
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| CN104120168B (en) | 2016-08-10 |
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