CN102634509B - Method for quickly and efficiently extracting deoxyribonucleic acid (DNA) of wheat stripe rust directly from infected wheat leaf blades - Google Patents
Method for quickly and efficiently extracting deoxyribonucleic acid (DNA) of wheat stripe rust directly from infected wheat leaf blades Download PDFInfo
- Publication number
- CN102634509B CN102634509B CN201210126926.6A CN201210126926A CN102634509B CN 102634509 B CN102634509 B CN 102634509B CN 201210126926 A CN201210126926 A CN 201210126926A CN 102634509 B CN102634509 B CN 102634509B
- Authority
- CN
- China
- Prior art keywords
- chelex
- wheat
- pcr
- dna
- leaf blades
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for quickly and efficiently extracting deoxyribonucleic acid (DNA) of wheat stripe rust directly from infected wheat leaf blades. The method is characterized in that 20mu l of prepared 20% chelex-100 suspension is drawn and injected into a 100mu l polymerase chain reaction (PCR) tube; 5mu l of chelex-100 supernate is drawn and dripped on the wheat leaf blades with wheat stripe rust uredispore and the spots of the leaf blades on which the chelex-100 supernate is dripped are smeared repeatedly to wash off the aecidiospore; the chelex-100 supernate containing the aecidiospore on the leaf blades is drawn back and added into the PCR tube containing 20mu l of 20% chelex-100 suspension; the mixture is drawn repeatedly and mixed evenly by a liquid-moving device; the mixture is put in boiling water bath for water bath for 2 minutes and then put in a vortex mixer to be oscillated and mixed evenly for 10 seconds; the mixture is subjected to water bath for 5 minutes and preserved at the temperature of minus 20 DEG C for later use; and the supernate can be used as templates of PCR. The invention provides an efficient and rapid method for extracting DNA of wheat stripe rust which is used as PCR amplification templates directly from infected wheat leaf blades.
Description
Technical field
The present invention relates to a kind of directly method of efficient Rapid Isolation of Wheat strip rust bacteria DNA from susceptible wheat leaf blade, belong to plant protection field.
Background technology
The stripe rust of wheat being caused by wheat stripe rust (Puccinia striiformis f.sp.tritici) is one of Major Diseases of each wheat belt, the world.Along with molecular biological development, the exploration of increasing researchist in wheat stripe rust related protein and DNA molecular level increases rapidly, and the content of PCR-based research is also enriched gradually.And the prerequisite of carrying out pcr amplification is to extract more complete genomic dna.The method of extracting fungal DNA both at home and abroad mainly contains: CTAB method, and SDS extraction method, the chemical processes such as urea extraction method, Benzyl Chloride extraction method are abolished cell walls, then use organic solvent extracting DNA.All there is complex operation step in these methods, wastes time and energy, and thalline requirement is large, and DNA pick-up rate is low etc.And for obligatory parasitism fungi---the puccinia striiformis that can not carry out artificial culture, when extracting monospore heap DNA, need to pile and separate inoculation by monospore, enough puccinia striiformis uredospores are obtained in the breeding cultivation of strip rust bacteria etc., and because uredospore cell walls is thicker, when extracting its genomic dna, more general fungi is loaded down with trivial details and difficult.Up to the present, although many to the report of wheat stripe rust DNA extraction Research on Methods, still a lot of to the uredinial demand of wheat stripe rust in these reports.Therefore, exploring a kind of DNA extraction method that efficient, quick, required sample size is few is very necessary for wheat stripe rust bacteria molecalar detection.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of directly method of efficient Rapid Isolation of Wheat strip rust bacteria DNA from susceptible wheat leaf blade for the deficiencies in the prior art.
Technical scheme of the present invention is as follows:
A kind of directly method of efficient Rapid Isolation of Wheat strip rust bacteria DNA from susceptible wheat leaf blade, it is characterized in that, 20% the chelex-100 suspension of drawing that 20 μ l prepare is in 100 μ l PCR pipes, the Chelex-100 supernatant liquor of drawing 5 μ l has on wheat stripe rust uredospore wheat leaf blade in long, and drip have the scab place of Chelex-100 supernatant liquor repeatedly to smear blade repeatedly, by spring spore wash lower after, suck back the Chelex-100 supernatant liquor that contains spring spore on blade and join in the PCR pipe that 20 μ l 20%chelex-100 suspensions are housed, repeatedly draw and mix with pipettor, put into boiling water bath water-bath 2min, be put into again vibration on vortex mixer and mix 10s, after water-bath 5min, be stored in-20 ℃ standby, supernatant liquor all can be used as the template of PCR, fungi rrna rDNA-ITS primer: F:5 '-TCCGTAGGTGAACCTGCGG-3 ', R:5 '-TCCTCCGCTTATTGATATGC-3 ', puccinia striiformis Auele Specific Primer CYR33:F:5 '-TGTCGTCTCGCCAATCTTT-3 ', R:5 '-GCGGGTGTCAGTTTCTCC-3 ', carries out pcr amplification, pcr amplification system is 10 μ l, wherein with 1 μ l supernatant liquor, does DNA profiling, the supperMix of 5 μ l, the primer of 0.8 μ l, ddH
2o 3.2 μ l, amplification program is 94 ℃ of 3min, 94 ℃ of 50s, 53 ℃ or 51 ℃ of 1min, 72 ℃ of 1min, 30 circulations, 72 ℃ of 10min, sepharose with 1.5% detects PCR product
The present invention utilizes chelex-100 directly the blade that contains wheat stripe rust to be carried out after pathogenic bacteria gene group DNA extraction, carries out PCR detection with fungi rrna rDNA-ITS primer and puccinia striiformis Auele Specific Primer.Set up a set of efficient, fast, can directly from the plant of feeling puccinia striiformis, extract the method for puccinia striiformis DNA as pcr amplification template.
Accompanying drawing explanation
Fig. 1 is that ITS amplified production detects; M is DL2000Marker, and 1-6 represents respectively 1-6 puccinia striiformis sample, the Bipolaris fungi contrast that C is artificial culture, the chelex-100 of the negative contrast 20% of CK.
Fig. 2 is that CYR33 amplified production detects; M is DL2000Marker, and 1-6 represents respectively 1-6 puccinia striiformis sample, the Bipolaris fungi contrast that C is artificial culture, the chelex-100 of the negative contrast 20% of CK.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
The directly method of efficient Rapid Isolation of Wheat strip rust bacteria DNA from susceptible wheat leaf blade:
The chelex-100 of 20g is dissolved in the distilled water of 100mL sterilizing, is made into 20% chelex-100 suspension standby.After fully mixing, draw chelex-100 suspension that 20 μ l prepare in 100 μ l PCR pipes, the Chelex-100 supernatant liquor of drawing 5 μ l has on wheat stripe rust uredospore wheat leaf blade in long, and drip have the scab place of Chelex-100 supernatant liquor repeatedly to smear blade repeatedly, by spring spore wash lower after, suck back the Chelex-100 supernatant liquor that contains spring spore on blade and join in the PCR pipe that 20 μ l 20%chelex-100 suspensions are housed, repeatedly draw and mix with pipettor, put into boiling water bath water-bath 2min, be put into again vibration on vortex mixer and mix 10s, after water-bath 5min, be stored in-20 ℃ standby, supernatant liquor all can be used as the template of PCR.
Fungi rrna rDNA-ITS primer (F:5 '-TCCGTAGGTGAACCTGCGG-3 ', R:5 '-TCCTCCGCTTATTGATATGC-3 ') and puccinia striiformis Auele Specific Primer CYR33 (F:5 '-TGTCGTCTCGCCAATCTTT-3 ', R:5 '-GCGGGTGTCAGTTTCTCC-3 ') carry out pcr amplification, pcr amplification system is 10 μ l, wherein with 1 μ l supernatant liquor, do DNA profiling, the supperMix of 5 μ l, the primer of 0.8 μ l, ddH
2o3.2 μ l.Amplification program is 94 ℃ of 3min, 94 ℃ of 50s, 53 ℃ or 51 ℃ of 1min, 72 ℃ of 1min (30 circulation), 72 ℃ of 10min.Sepharose with 1.5% detects PCR product.
Result shows, the DNA sample of the Bipolaris fungi of all puccinia striiformis standard specimens that extract through Chelex-100 method and artificial culture all can amplify rDNA-ITS fragment, clip size, in 600bp (Fig. 1) left and right, conforms to expectation clip size, and band is also more clear.With puccinia striiformis physiological strain special primer, CYR33 detects it, and its result shows, all can amplify the target stripe (Fig. 2) of CYR33 through the puccinia striiformis standard specimen of Chelex-100 method extraction, and band is very clear; And Bipolaris fungi and negative control water all fail to amplify target stripe.As can be seen here, use this method directly from susceptible wheat leaf blade, to extract the DNA of wheat stripe rust and be feasible for PCR.
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (1)
1. a direct method of efficient Rapid Isolation of Wheat strip rust bacteria DNA from susceptible wheat leaf blade, it is characterized in that, 20% the chelex-100 suspension of drawing that 20 μ l prepare is in 100 μ l PCR pipes, the Chelex-100 supernatant liquor of drawing 5 μ l has on wheat stripe rust uredospore wheat leaf blade in long, and drip have the scab place of Chelex-100 supernatant liquor repeatedly to smear blade repeatedly, by spring spore wash lower after, suck back the Chelex-100 supernatant liquor that contains spring spore on blade and join in the PCR pipe that 20 μ l 20%chelex-100 suspensions are housed, repeatedly draw and mix with pipettor, put into boiling water bath water-bath 2min, be put into again vibration on vortex mixer and mix 10s, after water-bath 5min, be stored in-20 ℃ standby, supernatant liquor all can be used as the template of PCR, fungi rrna rDNA-ITS primer: F:5 '-TCCGTAGGTGAACCTGCGG-3 ', R:5 '-TCCTCCGCTTATTGATATGC-3 ', puccinia striiformis Auele Specific Primer CYR33:F:5 '-TGTCGTCTCGCCAATCTTT-3 ', R:5 '-GCGGGTGTCAGTTTCTCC-3 ', carries out pcr amplification, pcr amplification system is 10 μ l, wherein with 1 μ l supernatant liquor, does DNA profiling, the supperMix of 5 μ l, the primer of 0.8 μ l, ddH
2o 3.2 μ l, amplification program is 94 ℃ of 3min, 94 ℃ of 50s, 53 ℃ or 51 ℃ of 1min, 72 ℃ of 1min, 30 circulations, 72 ℃ of 10min, sepharose with 1.5% detects PCR product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210126926.6A CN102634509B (en) | 2012-04-27 | 2012-04-27 | Method for quickly and efficiently extracting deoxyribonucleic acid (DNA) of wheat stripe rust directly from infected wheat leaf blades |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210126926.6A CN102634509B (en) | 2012-04-27 | 2012-04-27 | Method for quickly and efficiently extracting deoxyribonucleic acid (DNA) of wheat stripe rust directly from infected wheat leaf blades |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102634509A CN102634509A (en) | 2012-08-15 |
| CN102634509B true CN102634509B (en) | 2014-04-16 |
Family
ID=46619086
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210126926.6A Expired - Fee Related CN102634509B (en) | 2012-04-27 | 2012-04-27 | Method for quickly and efficiently extracting deoxyribonucleic acid (DNA) of wheat stripe rust directly from infected wheat leaf blades |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102634509B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498473A (en) * | 2014-11-23 | 2015-04-08 | 云南省农业科学院农业环境资源研究所 | Method for rapid separation of pathogen DNA from rice blast single scab |
| CN104498474A (en) * | 2014-11-23 | 2015-04-08 | 云南省农业科学院农业环境资源研究所 | Method for direct extraction of pathogen DNA from pathogenic tissue of Graminaceous crop |
| CN107460191A (en) * | 2017-09-15 | 2017-12-12 | 长江大学 | It is a kind of directly from the method for high efficiency extraction wheat powdery mildew DNA on susceptible wheat leaf blade |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1733936A (en) * | 2005-07-13 | 2006-02-15 | 南京师范大学 | Spiroplasma pathogenic microorganism PCR fast checking technique |
| CN102321618A (en) * | 2011-09-30 | 2012-01-18 | 生工生物工程(上海)有限公司 | Method and kit for directly amplifying DNA (deoxyribonucleic acid) segment from biological material |
-
2012
- 2012-04-27 CN CN201210126926.6A patent/CN102634509B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1733936A (en) * | 2005-07-13 | 2006-02-15 | 南京师范大学 | Spiroplasma pathogenic microorganism PCR fast checking technique |
| CN102321618A (en) * | 2011-09-30 | 2012-01-18 | 生工生物工程(上海)有限公司 | Method and kit for directly amplifying DNA (deoxyribonucleic acid) segment from biological material |
Non-Patent Citations (5)
| Title |
|---|
| 余仲东等.锈菌夏孢子DNA的微量快速提取方法.《生物技术通讯》.2005,第16卷(第1期),第48-50页. * |
| 小麦条锈菌水源11类群的RAPD分析及SCAR标记的建立;郝保军等;《植物病理学报》;20101231;第40卷(第1期);第2页1.2部分，第4-5页2.3-2.4部分 * |
| 张庆等.微量提取小麦叶片中条锈菌DNA的方法初探.《西南农 业学报》.2010,第23卷(第6期),第1887-1890页. * |
| 郝保军等.小麦条锈菌水源11类群的RAPD分析及SCAR标记的建立.《植物病理学报》.2010,第40卷(第1期),第2页1.2部分，第4-5页2.3-2.4部分. |
| 陈吉良等.一种快速高效提取病原真菌DNA作为PCR模板的方法.《菌物学报》.2011,第30卷(第1期),第148页1"材料与方法"部分. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102634509A (en) | 2012-08-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101935710B (en) | Pigeon sex discriminating method | |
| CN104046700A (en) | Detection kit for quickly identifying donkey skin, horse skin and mule skin | |
| CN102634509B (en) | Method for quickly and efficiently extracting deoxyribonucleic acid (DNA) of wheat stripe rust directly from infected wheat leaf blades | |
| CN104480202B (en) | A kind of Fructus Luffae reference gene and application thereof | |
| CN103233062B (en) | Duplex PCR authentication method of cordyceps sinensis original powder | |
| CN104561332A (en) | SSR molecular marker for identifying aspen gender and application of SSR molecular marker | |
| CN102559848A (en) | Simple preparation method of fungi molecular biological identification DNA template, and PCR amplification method | |
| CN103103258A (en) | Method for analyzing generating trend of aflatoxin B1 in peanut meal by multiple PCR (Polymerase Chain Reaction) technology | |
| CN103243164A (en) | Method for identifying psathyrostachys huashanica allogenic material in wheat genome | |
| CN104372073A (en) | Reagent and detection method capable of specifically detecting Mur antigen gene | |
| CN109022457A (en) | A kind of * DNA bar code sequence and its application | |
| CN104099423B (en) | For the molecular labeling of cutter long-tailed anchovy different ecological type population identification | |
| CN105200133A (en) | ISSR marker for authenticating high-quality agaricus bisporus bacterial strain and application | |
| CN104073562B (en) | A kind of molecular marker for cutter long-tailed anchovy different ecological type population identification | |
| CN105648049A (en) | Applications of agarose in PCR amplification, PCR amplification kit, and PCR amplification method | |
| CN102925580A (en) | Quick detection method for Notch1 gene mutation | |
| CN101948828A (en) | Trace animal cell genomic DNA template preparation solution and corresponding DNA template preparation method | |
| CN110423823A (en) | A kind of C. guichenoti DNA bar code sequence and its application | |
| CN101942507A (en) | Method for rapidly detecting chain alexandrum by adopting LAMP (Loop-Mediated Isothermal Amplification) technology | |
| CN109055407A (en) | A kind of erythroculter ilishaeformis DNA bar code sequence and its application | |
| CN102719551B (en) | Loop-mediated isothermal amplification detection primer of vibrio shilonii, detection kit and detection method | |
| CN107460191A (en) | It is a kind of directly from the method for high efficiency extraction wheat powdery mildew DNA on susceptible wheat leaf blade | |
| CN103352085A (en) | Primers and probe of PCR detection for a transformant of insect resistant genetically modified cotton MON531 | |
| CN102418151B (en) | Method for constructing cDNA (complementary DNA) library of soil microbe | |
| CN102559862A (en) | Dominant fungi in microbial community of puer tea and spectrum thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140416 Termination date: 20150427 |
|
| EXPY | Termination of patent right or utility model |