CN104630369B - The PCR detection method of fusarium moniliforme - Google Patents
The PCR detection method of fusarium moniliforme Download PDFInfo
- Publication number
- CN104630369B CN104630369B CN201510076984.6A CN201510076984A CN104630369B CN 104630369 B CN104630369 B CN 104630369B CN 201510076984 A CN201510076984 A CN 201510076984A CN 104630369 B CN104630369 B CN 104630369B
- Authority
- CN
- China
- Prior art keywords
- fusarium moniliforme
- primer
- detection method
- pcr
- auele specific
- 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
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
- C12Q1/6876—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
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- 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]
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A PCR detection method for fusarium moniliforme carries out pcr amplification with fusarium moniliforme Auele Specific Primer to testing sample, last electroresis appraisal; This Auele Specific Primer is the nucleotide sequence based on fusarium moniliforme gibberellin biological synthesis related gene, the gibberellin biological synthesis related gene being fusarium moniliforme is sequence-specific, this detection method use this other detection method is different to primer, may be used for rapid detection and go out paddy rice, thus can detect whether rice paddy seed carries Fusarium moniliforme Sheld.
Description
Technical field
The present invention relates to a kind of detection method of rice disease, espespecially a kind of PCR (polymerase chain reaction) method detecting rice bakanae disease (ricebakanae).
Background technology
Rice bakanae disease is one of rice disease, infect by rattan logical sequence gibberella (Invisible element is rattan logical sequence Fusariumsp) fungal disease caused, this sick common sympton is rice strain excessive growth, causes diseased plant and around plant mildew is withered after Rise's boot period, has had a strong impact on the output of paddy rice.
Research shows that fusarium moniliforme can pass band through seed, and therefore Seed-associated fungi just seems especially important.Whether pathogenic fungi being carried for rice paddy seed, in the past mainly through carrying out separation and Culture to the microorganism entrained by seed, then identifying according to the spore-bearing morphological specificity of culture.This method not only the separation and Culture time long, simultaneously because living contaminants affects separating effect, detector efficiency is not high yet.Therefore, the rapid molecular detection method setting up a kind of energy precise Identification fusarium moniliforme is necessary.
Summary of the invention
Technical problem to be solved by this invention is: for above-mentioned the deficiencies in the prior art, provides a kind of PCR detection method of energy precise Identification fusarium moniliforme.The pair of primers that the method uses is special to fusarium moniliforme, this detection method use this other detection method is different to primer, rapid detection can go out Fusarium moniliforme Sheld in rice paddy seed.
In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is: a kind of Auele Specific Primer detecting fusarium moniliforme for PCR, and described Auele Specific Primer is:
Forward primer: 5-TGCCCGGGAATGACTTTGAA-3, SEQIDNo.1;
Reverse primer: 5-GCATCACAGCAATGGAGGAC-3, SEQIDNo.2.
The design of above-mentioned Auele Specific Primer is the nucleotide sequence based on fusarium moniliforme gibberellin biological synthesis related gene.
The present invention provides a kind of PCR detection method of fusarium moniliforme simultaneously, and the method carries out pcr amplification to testing sample, last electroresis appraisal with the fusarium moniliforme Auele Specific Primer of above-mentioned design.
The inventive method is described in detail as follows:
One, design of primers:
The present inventor utilizes the conservative property of eukaryote evolutionary process more control sequences, searches for and download nucleotide sequence (the sequence accession number emb|HF679025.1 of the gibberellin biological synthesis related gene (gibberellinbiosynthesis-relatedgene) of fusarium moniliforme (Fusariumfujikuroi) fungus strain IMI58289 from NCBI
|), use primerBLAST to design primer, and test through BLASTn, find out the primer candidate regions that other fungal sequence is different all with it, design pair of primers (being synthesized by precious biotechnology (Dalian) company limited):
Forward primer: 5-TGCCCGGGAATGACTTTGAA-3 (SEQIDNo.1);
Reverse primer: 5-GCATCACAGCAATGGAGGAC-3 (SEQIDNo.2);
By forward primer and reverse primer respectively through BLAST retrieval, in conjunction with see Fig. 1 and Fig. 2, result shows, to cover with the sequence 100% of reverse primer with forward primer and 100% identical whole are nucleotide sequences of fusarium moniliforme.
Two, the extraction of DNA:
Following method is taked to extract the rice paddy seed DNA of band fusarium moniliforme:
(1) SDS dissociating buffer is placed in 60 DEG C of water-bath preheatings.
(2) take 0.5g mycelia, be placed in the mortar of precooling, pour liquid nitrogen into, as early as possible mycelia is ground.(or adding the SDS dissociating buffer of preheating, with quartzite sand grind)
(3) powder is loaded in the centrifuge tube of aseptic 1.5mL, add in the SDS dissociating buffer of 600uL preheating, rotate gently and make it mixing.
(4) sample is in 65 DEG C of insulation 10min.
(5) add the NaCL of isopyknic 7.5mol/L, put upside down mixing gently.
(6) the centrifugal 5min of ice bath 8min, 10000rpm.
(7) get 600uL supernatant liquor in EP pipe, add the chloroform-isoamyl alcohol (24:1) of equal-volume (600uL), put upside down mixing gently, centrifugal 10 minutes of 10000rpm.
(8) sucked mutually by upper water in another clean centrifuge tube, add the NaAc of 0.1 times of volume and the precooling dehydrated alcohol (or pre-cold isopropanol of 1 times of volume) of 2 volumes, mix gently, 1h placed by-20 DEG C of refrigerators, and nucleic acid is precipitated.
(9) collect the centrifugal 10min of DNA:10000rpm, remove supernatant.
(10) 70% ethanol wash, wash 2 times.
(11) after alcohol volatilization, with 0.1 times of TE or ddH
2o dissolves, and-20 DEG C of packing save backup.
Three, PCR process:
The DNA of above-mentioned primer pair to the rice paddy seed of band fusarium moniliforme is adopted to carry out PCR detection.Expection PCR primer is 295bp.
PCR reaction system is 25 μ l, specifically sees the following form 1:
The table each main component of 1PCR reaction system and consumption
| Component | Starting point concentration | Consumption |
| PCR Buffer is (containing Mg 2+) | 10× | 2.5μl |
| dNTPs | 10mmol/L | 0.5μl |
| Forward primer | 12.5pmol/μl | 2.0μl |
| Reverse primer | 12.5pmol/μl | 2.0μl |
| TaqDNA enzyme | 5U/μl | 0.2μl |
| DNA profiling | 50ng/μl | 2.0μl |
| ddH 2O | 15.8μl |
Reaction conditions: 95 DEG C of denaturation 3min; Then 94 DEG C of sex change 1min, 58 DEG C of annealing 1min, 72 DEG C extend 20sec, carry out 33 circulations with this understanding; Last 72 DEG C extend 5min again.
In the 1.2% agarose glue being cooled to 50-60 DEG C, add ethidium bromide (EB) solution makes its final concentration be 0.5 μ g/ml, after glue solidifies completely, transfer to comb, in groove, then add 0.5 × TBE dilution buffer just do not have offset plate upper surface to liquid level.10 μ lPCR amplified productions and 2 μ l6 × sample-loading buffer are mixed, carefully add in groove with micro-adjustable pipette, using 100bpDNAMarker as standard molecular weight, voltage remains on 60-80V, and electric current carries out electrophoresis at more than 40mA.When tetrabromophenol sulfonphthalein band move to apart from gel front be about 2cm time, stop electrophoresis, observe with gel imaging instrument.The band that result amplifies conforms to the size of expection.
By the fragment that increases reclaim, and be connected on T-carrier, be transformed in intestinal bacteria (Escherichiacoli) DH5 α competence again, select white colony to check order, sequence is carried out BLAST (http://blast.ncbi.nlm.nih.gov/) comparison, result shows increased sequence and Sarocladiumattenuatum) fungus strain CBS414.81 has good consistence, and only Individual base there occurs change.Show that this may be used for rapid detection fusarium moniliforme completely to primer.
Compared with prior art, advantage of the present invention is: the primer pair that present method uses is that the gibberellin biological synthesis related gene of fusarium moniliforme is special, this primer pair is different from other detection method, may be used for rapid detection fusarium moniliforme, thus rapid detection can go out rice paddy seed and whether carry Fusarium moniliforme Sheld, for increasing production of rice is laid a good foundation.
Accompanying drawing explanation
Fig. 1 is the BLAST result of fusarium moniliforme forward primer.
Fig. 2 is the BLAST result of fusarium moniliforme reverse primer.
Fig. 3 adopts Auele Specific Primer of the present invention to the electrophoresis result figure making PCR.
Wherein, M is molecular weight marker; 1 is rice bakanae disease diseased plant; 2 is rice seedling blight strain; 3 is the rice leaf not with Fusarium moniliforme Sheld.
Fig. 4 adopts Auele Specific Primer of the present invention to the electrophoresis result figure carrying out PCR detection with Fusarium moniliforme Sheld rice paddy seed.
Wherein, M is molecular weight marker; 1 is the rice leaf without Fusarium moniliforme Sheld; 2-10 is the rice paddy seed of band Fusarium moniliforme Sheld; 11-12 is the rice paddy seed without Fusarium moniliforme Sheld.
Embodiment
Embodiment 1
The Auele Specific Primer in the present invention is adopted to carry out PCR detection to the rice bakanae disease diseased plant from liuyang hunan, using the rice seedling damping-off strain from Liuyang and the rice leaf not with Fusarium moniliforme Sheld as negative control, the results are shown in Figure 3, rice bakanae disease diseased plant is positive, amplifies the band of 295bp.
Embodiment 2
Adopt the Auele Specific Primer in the present invention to from Liuyang with in the rice varieties of Fusarium moniliforme Sheld early the seed of 39 carry out PCR detection, using weed from Hunan agricultural university the rice leaf of garden teaching base not with fusarium moniliforme and not with in Fusarium moniliforme Sheld early the seed of 39 kinds as negative control, see Fig. 4.Result shows, and the seed of band fusarium moniliforme is positive, amplify the band of 295 bases, and contrast is all without band.
Illustrate of the present invention this be special to Auele Specific Primer to fusarium moniliforme, rapid detection can go out rice bakanae disease.
Claims (4)
1. detect an Auele Specific Primer for fusarium moniliforme for PCR, it is characterized in that, described Auele Specific Primer is:
Forward primer: 5-TGCCCGGGAATGACTTTGAA-3;
Reverse primer: 5-GCATCACAGCAATGGAGGAC-3.
2. detect the Auele Specific Primer of fusarium moniliforme as claimed in claim 1 for PCR, it is characterized in that, the design of described Auele Specific Primer is the nucleotide sequence based on fusarium moniliforme gibberellin biological synthesis related gene.
3. a PCR detection method for fusarium moniliforme, is characterized in that: the method carries out pcr amplification to testing sample, last electroresis appraisal with the fusarium moniliforme Auele Specific Primer according to any one of claim 1 or 2.
4. the PCR detection method of fusarium moniliforme as claimed in claim 3, is characterized in that: described PCR primer size is 295bp.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510076984.6A CN104630369B (en) | 2015-02-13 | 2015-02-13 | The PCR detection method of fusarium moniliforme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510076984.6A CN104630369B (en) | 2015-02-13 | 2015-02-13 | The PCR detection method of fusarium moniliforme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104630369A CN104630369A (en) | 2015-05-20 |
| CN104630369B true CN104630369B (en) | 2016-02-24 |
Family
ID=53209613
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510076984.6A Expired - Fee Related CN104630369B (en) | 2015-02-13 | 2015-02-13 | The PCR detection method of fusarium moniliforme |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104630369B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107022624B (en) * | 2017-05-08 | 2021-06-11 | 浙江农林大学 | LAMP method and kit for rapidly detecting bakanae disease bacteria of rice from rice seeds |
| CN109609543A (en) * | 2018-12-29 | 2019-04-12 | 青岛袁策集团有限公司 | It is a kind of for identifying rice to the transient expression vector of bakanae disease resistance |
| CN110527630B (en) * | 2019-05-24 | 2021-04-20 | 浙江工业大学 | Aleurites lutescens mutant strain bred by ARTP mutagenesis technology and application thereof |
| CN110117592A (en) * | 2019-06-04 | 2019-08-13 | 中国农业大学 | A method of quickly detecting fusarium moniliforme rattan storehouse fusarium based on RPA |
| CN111378776A (en) * | 2019-10-25 | 2020-07-07 | 安徽省农业科学院植物保护与农产品质量安全研究所 | RPA primer and detection method for detecting rice bakanae disease Gibberella fujikuei |
| CN111876514B (en) * | 2020-07-24 | 2023-06-06 | 中国水稻研究所 | Rapid detection method for gibberellin miniascape generated in bakanae disease germ of rice |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5585238A (en) * | 1994-04-25 | 1996-12-17 | Ciba-Geigy Corporation | Detection of fungal pathogens using the polymerase chain reaction |
| US5827695A (en) * | 1997-08-04 | 1998-10-27 | Novartis Finance Corporation | Detection of wheat fungal pathogens using the polymerase chain reaction |
-
2015
- 2015-02-13 CN CN201510076984.6A patent/CN104630369B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN104630369A (en) | 2015-05-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104630369B (en) | The PCR detection method of fusarium moniliforme | |
| Umpiérrez-Failache et al. | Regional differences in species composition and toxigenic potential among Fusarium head blight isolates from Uruguay indicate a risk of nivalenol contamination in new wheat production areas | |
| Delavenne et al. | Fungal diversity in cow, goat and ewe milk | |
| CN101649350B (en) | Molecular detection method for rapidly distinguishing mating types of Chinese caterpillar fungus | |
| Waite et al. | Design and evaluation of nematode 18S rDNA primers for PCR and denaturing gradient gel electrophoresis (DGGE) of soil community DNA | |
| CN102796812B (en) | PCR (Polymerase chain reaction) primer capable of simultaneously detecting five soil-borne fungal diseases of wheat paddock and detecting method thereof | |
| CN102965438B (en) | Duplex polymerase chain reaction (PCR) detection primer group, kit and method for pathogenic channa source aeromonas schubertii | |
| CN104263813A (en) | Sequences of primer for identifying fusarium solani and/or fusarium oxysporum, kit and method thereof | |
| WO2020176834A1 (en) | Methods, apparatuses, and systems for improving microbial preservation yield through rescue and serial passage of preserved cells | |
| CN103045747B (en) | Molecular detection primer for sweet potato black rot germs and application of molecular detection primer | |
| CN102220247B (en) | Glycyrrhiza endophytic fungi for producing glycyrrhetic acid | |
| CN104131115A (en) | Gene chip for detecting pathogenic fungi of poplar canker and application thereof | |
| CN102643756A (en) | Endophytic fungus for improving content of glycyrrhetinic acid by fermenting liquorice | |
| CN104120124B (en) | SCAR marker for specifically detecting wheat stripe rust and detection method | |
| CN102220320A (en) | Specific molecular marker of volvariella volvacea V23 strain as well as obtaining method and application thereof | |
| CN104498593A (en) | Primer pair and kit for identification or assisted identification of stored bean weevils | |
| Zhu et al. | Molecular specificity and detection for Pseudanabaena (cyanobacteria) species based on rbcLX sequences | |
| CN101760554A (en) | Primer sequence for detecting resistance frequency of botrytis cinerea population to strobilurin fungicide and method thereof | |
| CN101402995B (en) | Method for technical evaluation of hedgehog fungus bacterial Houwang with molecular marker technology | |
| Hesham et al. | Use of PCR-denaturing gradient gel electrophoresis for the discrimination of Candida species isolated from natural habitats | |
| CN105018575A (en) | Application of multiplex PCR (polymerase chain reaction) in fungal detection of septicemia | |
| CN104046681A (en) | Detection method for rapid identification of aspergillus fumigatus | |
| CN103122383B (en) | Horizontal fluorescent quantitation polymerase chain reaction (PCR) detection method of streptococcus suis strain | |
| CN103290000B (en) | SCAR marker of biocontrol Hypocrea virens, its application and quantitative detection method | |
| Gu et al. | Molecular characterization and toxin quantification of Microcystis panniformis: A microcystin producer in Lake Taihu, China |
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: 20160224 Termination date: 20180213 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |