CN106701970A - Molecular biological method for identifying three Botryosphaeria on apples - Google Patents
Molecular biological method for identifying three Botryosphaeria on apples Download PDFInfo
- Publication number
- CN106701970A CN106701970A CN201710046559.1A CN201710046559A CN106701970A CN 106701970 A CN106701970 A CN 106701970A CN 201710046559 A CN201710046559 A CN 201710046559A CN 106701970 A CN106701970 A CN 106701970A
- Authority
- CN
- China
- Prior art keywords
- botryosphaeria
- checked
- bacterial strain
- dna
- seat chamber
- 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.)
- Pending
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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Mycology (AREA)
- Botany (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicines Containing Plant Substances (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a molecular biological method for identifying three Botryosphaeria on apples and belongs to the technical field of biology. The molecular biological method for identifying the three Botryosphaeria on the apples comprises the step of carrying out PCR (Polymerase Chain Reaction) amplification by taking DNA (Deoxyribonucleic Acid) of a to-be-detected strain belonging to Bbotryosphaeria.Sp as a template and adopting primers EF1-S and EF1-A; if an amplified product only contains 230 to 250bp of DNA fragments, the to-be-detected strain is Botryosphaeria dothidea; if the amplified product only contains 140 to 160bp of DNA fragments, the to-be-detected strain is Botryosphaeria stevensii; if the amplified product only contains 170 to 180bp of DNA fragments, the to-be-detected strain is Botryosphaeria obtusa; the sequences of the primers EF1-S and EF1-A are respectively shown as SEQ ID NO:1 and SEQ ID NO:2. The identification method provided by the invention has the advantages of simplicity, convenience, quickness, high specificity and good generality.
Description
Technical field
Three kinds of molecular biology methods of grape seat chamber bacterium on apple are identified the present invention relates to a kind of, belongs to biotechnology neck
Domain.
Background technology
Botryosphaeria（Botryosphaeria.Sp）Middle apple shell color list is every spore ulcer bacteria
（Botryosphaeria stevensii）, grape seat chamber bacterium（Botryosphaeriadothidea）With blunt grape seat chamber bacterium
（Botryosphaeriaobtusa）It is three kinds of important pathogen fungies in apple disease.These three disease funguses can all endanger
The limb of the fruit trees such as apple, pears and fruit, form the symptoms such as ulcer, wheel line on limb, fruit, are to have a strong impact on apple in the world
The important pathogen of fruit production（Slippers &Wingfield 2004；Wang Fan etc. 2013）.Botryosphaeria
（Botryosphaeria.Sp）Middle apple shell color list is every spore ulcer bacteria（B. stevensii）It is the inward plant quarantine of China
Property harmful organism, host range is very wide, in addition to apple, pears, can also endanger 11 sections such as grape, Oak Tree, elm, loquat,
18 plants of category（Van Niekerket al. 2004）.And cause of disease is relied primarily at present for the detection of the category pathogen
The hazard symptoms of bacterium, colony characteristicses, the phorozoon of germ and epigamous morphological feature, but be difficult to make a distinction them and
Identify kind.
The content of the invention
To overcome the above-mentioned problems in the prior art, the invention provides three kinds of grape seat chambers on one kind identification apple
The molecular biology method of bacterium, simple and efficient, specific high, versatility is good, can rapidly and accurately identify Botryosphaeria
（Botryosphaeria.Sp）In three kinds of important pathogen apple shell color lists every spore ulcer bacteria（Botryosphaeria stevensii）, grape seat chamber bacterium（Botryosphaeriadothidea）With blunt grape seat chamber bacterium
（Botryosphaeriaobtusa）, inspection and quarantining for import/export is particularly well-suited to importing and exporting such disease fungus that apple is carried
Rapid identification.
The purpose of the present invention adopts the following technical scheme that realization：
Three kinds of molecular biology methods of grape seat chamber bacterium on a kind of identification apple, including to belong to Botryosphaeria
（Botryosphaeria.Sp）Bacterial strain DNA to be checked be template, using primer EF1-S and EF1-A enter performing PCR amplification step
Suddenly；If the amplified production only DNA fragmentation containing 230-250bp, bacterial strain to be checked is grape seat chamber bacterium
（Botryosphaeriadothidea）；If the amplified production only DNA fragmentation containing 140-160bp, bacterial strain to be checked is apple
Shell color list is every spore ulcer bacteria（Botryosphaeria stevensii）；If DNA piece of the amplified production only containing 170-180bp
Section, then bacterial strain to be checked is blunt grape seat chamber bacterium（Botryosphaeriaobtusa）；The sequence of the primer EF1-S and EF1-A
Respectively such as SEQ ID NO:1 and SEQ ID NO:Shown in 2.
In the present invention, the PCR amplification system contains the μ L of 2 × Taq DNA polymerase buffer solution 12.5, primer EF1-S
0.5 μ L, the μ L of primer EF1-A 0.5, the μ L of bacterial strain DNA to be checked 1 and the μ L of aseptic double-distilled water 10.5.
In the present invention, the PCR amplification programs are as follows：94 DEG C of predegeneration 5min；94 DEG C of denaturation 30s, 48 DEG C of renaturation
30s, 72 DEG C of extension 30s, 35 circulations；72 DEG C of extension 10min.
In the present invention, the DNA with bacterial strain to be checked is as template, using the universal primer ITS-F and ITS- of ITS genetic fragments
R differentiates whether bacterial strain to be checked belongs to Botryosphaeria（Botryosphaeria.Sp）, the primer I TS-F's and ITS-R
Sequence is respectively such as: SEQ ID NO:3 and SEQ ID NO:Shown in 4.
The present invention with the Botryosphaeria fungi of China's intercept and capture as test material, by ITS and EF-1αTwo genes
Collectively as Botryosphaeria（Botryosphaeria.Sp）In three kinds of important pathogen apple shell color lists every spore ulcer
Germ（Botryosphaeria stevensii）, grape seat chamber bacterium（Botryosphaeriadothidea）With blunt grape seat chamber
Bacterium（Botryosphaeriaobtusa）Standard DNA bar code.First, differentiate that pathogen belongs to Portugal by ITS universal primers
Grape seat chamber Pseudomonas（Botryosphaeria.Sp）, then by the specific primer for EF-1 α genes, differentiate that pathogen is
No is one of above-mentioned three kinds.Authentication method of the present invention is simple and efficient, specific high, versatility is good, can rapidly and accurately identify
Whether it is above-mentioned three kinds of pathogens, is particularly well-suited to inspection and quarantining for import/export to importing and exporting such disease fungus that apple is carried
Rapid identification.
Brief description of the drawings
Fig. 1 is the EF-1 α gene amplification fragments of bacterial strain to be checked, and wherein swimming lane 1-3 is apple shell color list every spore ulcer bacteria
（Botryosphaeria stevensii）DNA cloning product, swimming lane 4-6 is grape seat chamber bacterium
（Botryosphaeriadothidea）DNA cloning product, swimming lane 7-9 is blunt grape seat chamber bacterium
（Botryosphaeriaobtusa）DNA cloning product.
Specific embodiment
Protection content of the invention is not limited to following examples.Under the spirit and scope without departing substantially from inventive concept, this
Art personnel it is conceivable that change and advantage be all included in the present invention, and with appending claims be protect
Shield scope.Implement process of the invention, condition, reagent, experimental technique etc., in addition to the following special content for referring to, be this
The universal knowledege and common knowledge in field, the present invention are not particularly limited content.
1st, specific primer design
The present invention belongs to fungal sequence ITS, SSU, LSU, EF-1 with by comparing existing grape seat chamber in NCBI gene poolsα,Point
Safety pin can simultaneously differentiate Botryosphaeria to above-mentioned sequences Design（Botryosphaeria.Sp）In three kinds of important diseases
Opportunistic pathogen apple shell color list is every spore ulcer bacteria（Botryosphaeria stevensii）, grape seat chamber bacterium
（Botryosphaeriadothidea）With blunt grape seat chamber bacterium（Botryosphaeriaobtusa）Multiple primers.Through excessive
The screening of amount, it is found that differentiating by ITS universal primers be Botryosphaeria（Botryosphaeria.Sp）Afterwards, using pin
To the primer pair EF1-S and EF1-A of EF-1 α genes, it is possible to authenticate whether sample is Botryosphaeria
（Botryosphaeria.Sp）In three kinds of bacterium.
The sequence of special primer EF1-S and EF1-A is as follows：
EF1-S（SEQ ID NO:1）:5'-GCCACGACGGATCTCCTTGA-3 ',
EF1-A（SEQ ID NO:2）:5'-AGATTGGCGGTATTGGCACG-3’.
2. whether discriminating bacterial strain to be checked is apple shell color list every spore ulcer bacteria（Botryosphaeria stevensii）、
Grape seat chamber bacterium（Botryosphaeriadothidea）With blunt grape seat chamber bacterium（Botryosphaeriaobtusa）It is specific
Method
Fungi is belonged to the Botryosphaeria for being isolated from apple Apple of Chinese intercept and capture：Apple shell color list is every spore ulcer
Germ（Botryosphaeria stevensii）, grape seat chamber bacterium（Botryosphaeriadothidea）With blunt grape seat chamber
Bacterium（Botryosphaeriaobtusa）It is bacterial strain to be checked.
（1）DNA profiling is extracted
Bacterial strain to be checked is placed in potato dextrose agar（Potato Dextrose Agar; PDA）On culture medium, 25 DEG C of perseverances
Temperature culture 3-5 days, after reaching logarithmic phase to fungi growth, picking colony carries out the extracting of DNA.
Fungal DNA is extracted according to corrected cetyl trimethyl ammonium（Cetyltrimethyl Ammonium
Bromide；CTAB）Method is carried out.First, the mycelium grown on a small amount of PDA plate is scraped, 2mL sterile centrifugations are transferred to
Guan Zhong.Then, (sodium dodecyl sulfate, the sodium salt of 40 μ L 10% are added in centrifuge tube；SDS) ten
The small steel ball of sodium dialkyl sulfate solution and a diameter of 0.5cm 2, uses ball milling instrument（German Retsch, Lay is speeded MM400）With 30
Secondary/second frequency oscillation treatment 5-10min；Add the CTAB lysates of 600 μ L 1%（The Tris-Hcl of 0.05mol/L, pH8.0 delays
Contain 0.7mol/L NaCl and 0.01mol/L EDTA in fliud flushing）Concussion is mixed；It is put into isothermal vibration water-bath and is incubated 0.5-2h
（350r/min, 65 DEG C）.Add phenol solution, chloroform, the iso pentane alcohol mixture of 600 μ L saturations（The phenol solution of saturation,
Chloroform and isoamyl alcohol volume ratio are 25：24：1）, 12000r/min high speed centrifugations 5min after fully mixing.400 μ L of supernatant liquid are taken,
Add isometric chloroform, 12000r/min high speed centrifugations 5min after fully mixing.Supernatant is taken, -20 DEG C of isopropyls of precooling are added
The μ L of alcohol 300, fully mix, 12000r/min centrifugations 10min.Supernatant is discarded, -20 DEG C of μ of 75% ethanol solution 600 of precooling are added
L, 12000r/min are centrifuged 5min.Supernatant is abandoned, 100uL ddH are added after natural drying2O, concussion is mixed, and obtains final product bacterial strain to be checked
DNA, puts -20 DEG C of preservations.
（2）PCR is expanded
Differentiate whether bacterial strain to be checked belongs to Botryosphaeria（Botryosphaeria.Sp）
DNA with bacterial strain to be checked enters performing PCR and expands as template, using the universal primer ITS-F and ITS-R of ITS genetic fragments.If
Bacterial strain to be checked is Botryosphaeria（Botryosphaeria.Sp）, then can expand to the size about fragment of 300bp.
The sequence of primer I TS-F（SEQ ID NO:3）：5'-CTTGGTCATTTAGAGGAAGTAA-3', the sequence of primer I TS-R（SEQ
ID NO:4）：5'-GCTGCGTTCTTCATCGATGC-3'.
This experiment uses the PCR reaction systems of 50 μ l, including 10 μm of each 1 μ l of ol/L upstream and downstream primers, 0.25 μ l
Taq archaeal dna polymerases（Qiagen）、5μl 10×Buffer（Qiagen）, the dNTP of 1 μ l 10mmol/L, 3.5 μ l MgCl2With
And 100-200ng DNA profilings, it is eventually adding ddH2O supplies volume to 50 μ l.PCR reaction conditions are 95 DEG C of predegenerations 5
min；25 circular responses：95 DEG C of denaturation 30 s, 50 DEG C of annealing 30s, 72 DEG C of extension 40s；72 DEG C of 7 min of extension.PCR primer is used
0.8% agarose gel electrophoresis is detected.With apple shell color list every spore ulcer bacteria（Botryosphaeria stevensii）, Portugal
Grape seat chamber bacterium（Botryosphaeriadothidea）With blunt grape seat chamber bacterium（Botryosphaeriaobtusa）DNA is mould
Plate, enters performing PCR and expands using primer I TS-F and ITS-R, has amplified the size about band of 300bp, it was demonstrated that bacterial strain to be checked
Belong to Botryosphaeria（Botryosphaeria.Sp）.
Differentiate whether bacterial strain to be checked is apple shell color list every spore ulcer bacteria（Botryosphaeria stevensii）、
Grape seat chamber bacterium（Botryosphaeriadothidea）Or blunt grape seat chamber bacterium（Botryosphaeriaobtusa）
Because bacterial strain to be checked belongs to Botryosphaeria（Botryosphaeria.Sp）, therefore adopt and differentiate with the following method to be checked
Whether bacterial strain is one of above-mentioned three kinds of fungies：DNA with bacterial strain to be checked is expanded as template using primer EF1-S and EF1-A PCR
EF-1 alpha gene fragments.If the amplified production only DNA fragmentation containing 230-250bp, bacterial strain to be checked is grape seat chamber bacterium
（Botryosphaeriadothidea）；If the amplified production only DNA fragmentation containing 140-160bp, it is determined that bacterial strain to be checked
It is apple shell color list every spore ulcer bacteria（Botryosphaeria stevensii）；If amplified production only contains 170-
The DNA fragmentation of 180bp, it is determined that bacterial strain to be checked is blunt grape seat chamber bacterium（Botryosphaeriaobtusa）.
PCR reactions are carried out in 200 μ L PCR reaction tubes, react the μ L of cumulative volume 25, including following reagent：
2 × Taq DNA polymerase buffer solution（Qiagen）12.5 μ L,
Primer EF1-S（10umol/L）0.5 μ L,
Primer EF1-A（10umol/L）0.5 μ L,
Template DNA（150ng/ul）1 μ L,
The μ L of aseptic double-distilled water 10.5.
There are false positive results to prevent experiment, Setup Experiments negative control, with aseptic double-distilled water alternate template DNA, is pressed
Same method carries out DNA and extracts and PCR amplifications.
After PCR reaction solutions have been prepared, 4000rpm centrifugation 10s, by PCR pipe insertion PCR instrument, are reacted as follows：94℃
Predegeneration 5min；94 DEG C of denaturation 30s, 48 DEG C of renaturation 30s, 72 DEG C of extension 30s, 35 circulations；72 DEG C of extension 10min.
Take 5 μ LPCR products using 1% agarose gel electrophoresis separate purpose band, the electrophoresis 30min under the conditions of 100V,
Using the analysis detection imaging of ultraviolet gel imaging instrument.Result such as Fig. 1.With grape seat chamber bacterium（Botryosphaeriadothidea）
DNA is template, the only DNA fragmentation containing 230-250bp in the amplified production of acquisition, it was demonstrated that the bacterial strain is grape seat chamber bacterium
（Botryosphaeriadothidea）.With apple shell color list every spore ulcer bacteria（Botryosphaeria stevensii）'s
DNA is template, the only DNA fragmentation containing 140-160bp in the amplified production of acquisition, it was demonstrated that the bacterial strain is apple shell color list every spore
Ulcer bacteria（Botryosphaeria stevensii）.With blunt grape seat chamber bacterium（Botryosphaeriaobtusa）DNA is
Template, the only DNA fragmentation containing 170-180bp in the amplified production of acquisition, it was demonstrated that the bacterial strain is blunt grape seat chamber bacterium
（Botryosphaeriaobtusa）.
SEQUENCE LISTING
<110>Propagation and Food Test Center of Jiangsu Entry-Exit Inspection and Quarantine Bureau
Agricultural University Of Nanjing
<120>Three kinds of molecular biology methods of grape seat chamber bacterium on a kind of identification apple
<130> 20170119
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> artificial
<220>
<223> EF1-S
<400> 1
gccacgacgg atctccttga 20
<210> 2
<211> 20
<212> DNA
<213> artificial
<220>
<223> EF1-A
<400> 2
agattggcgg tattggcacg 20
<210> 3
<211> 22
<212> DNA
<213> artificial
<220>
<223> ITS-F
<400> 3
cttggtcatt tagaggaagt aa 22
<210> 4
<211> 20
<212> DNA
<213> artificial
<220>
<223> ITS-R
<400> 4
gctgcgttct tcatcgatgc 20
Claims (4)
1. it is a kind of to identify three kinds of molecular biology methods of grape seat chamber bacterium on apple, including to belong to Botryosphaeria
（Botryosphaeria.Sp）Bacterial strain DNA to be checked be template, using primer EF1-S and EF1-A enter performing PCR amplification step
Suddenly；If the amplified production only DNA fragmentation containing 230-250bp, bacterial strain to be checked is grape seat chamber bacterium（Botryosphaeria dothidea）；If the amplified production only DNA fragmentation containing 140-160bp, bacterial strain to be checked is apple shell color list every spore canker
Bacterium（Botryosphaeria stevensii）；If the amplified production only DNA fragmentation containing 170-180bp, bacterial strain to be checked is
Blunt grape seat chamber bacterium（Botryosphaeria obtusa）；The sequence of the primer EF1-S and EF1-A is respectively such as SEQ ID
NO:1 and SEQ ID NO:Shown in 2.
2. three kinds of molecular biology methods of grape seat chamber bacterium on apple are identified according to claim 1, it is characterised in that institute
State PCR amplification system and contain the μ L of 2 × Taq DNA polymerase buffer solution 12.5, the μ L of primer EF1-S 0.5, the μ of primer EF1-A 0.5
L, the μ L of bacterial strain DNA to be checked 1 and the μ L of aseptic double-distilled water 10.5.
3. three kinds of molecular biology methods of grape seat chamber bacterium on apple are identified according to claim 2, it is characterised in that institute
State PCR amplification programs as follows：94 DEG C of predegeneration 5min；94 DEG C of denaturation 30s, 48 DEG C of renaturation 30s, 72 DEG C of extension 30s, 35 are followed
Ring；72 DEG C of extension 10min.
4. according to three kinds of molecular biology methods of grape seat chamber bacterium, its feature on one of the claim 1-3 identification apples
Be the DNA with bacterial strain to be checked as template, differentiate that bacterial strain to be checked is using the universal primer ITS-F and ITS-R of ITS genetic fragments
It is no to belong to Botryosphaeria（Botryosphaeria.Sp）, the sequence of the primer I TS-F and ITS-R is respectively such as SEQ ID
NO:3 and SEQ ID NO:Shown in 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710046559.1A CN106701970A (en) | 2017-01-22 | 2017-01-22 | Molecular biological method for identifying three Botryosphaeria on apples |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710046559.1A CN106701970A (en) | 2017-01-22 | 2017-01-22 | Molecular biological method for identifying three Botryosphaeria on apples |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106701970A true CN106701970A (en) | 2017-05-24 |
Family
ID=58910141
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710046559.1A Pending CN106701970A (en) | 2017-01-22 | 2017-01-22 | Molecular biological method for identifying three Botryosphaeria on apples |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106701970A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877463A (en) * | 2021-03-11 | 2021-06-01 | 中国海关科学技术研究中心 | Primer probe set, kit and detection method for real-time fluorescent PCR (polymerase chain reaction) detection of apple shell color monascus dissimilatons |
| CN115820925A (en) * | 2023-01-07 | 2023-03-21 | 沈阳农业大学 | Primer group, kit, method and application for detecting pathogenic bacteria of brown spot of oak leaves |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102747078A (en) * | 2012-07-03 | 2012-10-24 | 北京市农林科学院 | Primers used for rapid detection of Neofusicoccum parvum and application thereof |
| CN103966362A (en) * | 2014-05-12 | 2014-08-06 | 河北农业大学 | Method for synchronously detecting four apple viruses |
| CN104774955A (en) * | 2015-04-25 | 2015-07-15 | 向华 | Botryosphaeria dothidea detection method |
| CN105177148A (en) * | 2015-09-23 | 2015-12-23 | 北京市农林科学院 | Dual PCR primer for detecting two types of pathogens of grape canker at same time and application of dual PCR primer |
| EP2478090B1 (en) * | 2009-09-17 | 2016-09-07 | University Of Saskatchewan | Method for increasing plant growth using the fungus trichoderma harzianum |
| CN106350453A (en) * | 2016-11-22 | 2017-01-25 | 中国农业科学院北京畜牧兽医研究所 | Separation/purification and pathogenicity identification method for pathogenic bacteria of fusarium root rot of Medicago sativa |
-
2017
- 2017-01-22 CN CN201710046559.1A patent/CN106701970A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2478090B1 (en) * | 2009-09-17 | 2016-09-07 | University Of Saskatchewan | Method for increasing plant growth using the fungus trichoderma harzianum |
| CN102747078A (en) * | 2012-07-03 | 2012-10-24 | 北京市农林科学院 | Primers used for rapid detection of Neofusicoccum parvum and application thereof |
| CN103966362A (en) * | 2014-05-12 | 2014-08-06 | 河北农业大学 | Method for synchronously detecting four apple viruses |
| CN104774955A (en) * | 2015-04-25 | 2015-07-15 | 向华 | Botryosphaeria dothidea detection method |
| CN105177148A (en) * | 2015-09-23 | 2015-12-23 | 北京市农林科学院 | Dual PCR primer for detecting two types of pathogens of grape canker at same time and application of dual PCR primer |
| CN106350453A (en) * | 2016-11-22 | 2017-01-25 | 中国农业科学院北京畜牧兽医研究所 | Separation/purification and pathogenicity identification method for pathogenic bacteria of fusarium root rot of Medicago sativa |
Non-Patent Citations (2)
| Title |
|---|
| SLIPPERS B ET.AL: "Combined multiple gene genealogies and phenotypic characters differentiate several species previously identified as Botryosphaeria dothidea．", 《MYCOLOGIA》 * |
| 冯小慧等: "杨树溃疡病病原的多重PCR检测技术", 《林业科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877463A (en) * | 2021-03-11 | 2021-06-01 | 中国海关科学技术研究中心 | Primer probe set, kit and detection method for real-time fluorescent PCR (polymerase chain reaction) detection of apple shell color monascus dissimilatons |
| CN115820925A (en) * | 2023-01-07 | 2023-03-21 | 沈阳农业大学 | Primer group, kit, method and application for detecting pathogenic bacteria of brown spot of oak leaves |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106399490B (en) | LAMP primer group for detecting phytoplasma and kit and application thereof | |
| Chakraborty et al. | Molecular characterization of Trichoderma viride and Trichoderma harzianum isolated from soils of North Bengal based on rDNA markers and analysis of their PCR-RAPD profiles | |
| Schena et al. | Rapid and sensitive detection of Rosellinia necatrlxin roots and soils by real time scorpion-PCR | |
| CN108707687B (en) | PCR detection and identification method capable of distinguishing blast bacteria and rice blast bacteria | |
| CN103602738B (en) | Multiple PCR (Polymerase Chain Reaction) primer and method for quickly identifying variety of scallops | |
| CN105063219B (en) | Guava anthrax bacteria specific PCR detection primer and its detection method | |
| Javanmardi et al. | Identification of Safflower as a fraud in commercial Saffron using RAPD/SCAR | |
| CN108676910A (en) | A kind of LAMP detection primer of fusarium prolifertum and its application | |
| CN102605092B (en) | LAMP (Loop-mediated isothermal amplification) rapid detection method of Citrus huanglongbing | |
| CN108220474A (en) | A kind of LAMP detection primer of Fusarium graminearum and its application | |
| CN102094080B (en) | Quick molecular detection method for simultaneously detecting three kinds of fusarium toxins and application thereof | |
| Shen et al. | Development of a loop‐mediated isothermal amplification assay for rapid and sensitive detection of Sporisorium scitamineum in sugarcane | |
| CN106701970A (en) | Molecular biological method for identifying three Botryosphaeria on apples | |
| CN104830984B (en) | The fluorescence PCR detecting method and the primer and probe of melon anthrax bacteria | |
| CN106868138A (en) | Primer and kit for identifying the pathogen of tomato neckrot root rot and droop | |
| CN104611424B (en) | The PCR RFLP methods of quick discriminating Radix Apioris Fortunei (Radix Lespedezae Buergeri) and its various pseudo- mixed product | |
| Lahuf et al. | A simple, rapid, safe and low-cost method to extract DNA from phytopathogenic fungi. | |
| Grover et al. | Rapid method for isolation of PCR amplifiable genomic DNA of Ralstonia solanacearum infested in potato tubers | |
| CN108315469A (en) | Primer composition and kit of the pathogenic sickle-like bacteria of ring mediated isothermal amplification method detection and application thereof | |
| CN108531639B (en) | It is a kind of for detecting the specific primer and rapid detection method of Botryodiplodia theo-bromae | |
| Pallez et al. | Validation of a quick PCR method suitable for direct sequencing: identification of Fusarium fungal species and chemotypes for preventive approaches in food safety | |
| Qiao et al. | Development of nested PCR, multiplex PCR, and loop-mediated isothermal amplification assays for rapid detection of Cylindrocladium scoparium on Eucalyptus | |
| CN103898225B (en) | A kind of primer and discrimination method differentiating the hidden kind of Bemisia tabaci and trialeurodes vaporariorum | |
| CN106434879B (en) | Rapidly and efficiently detect the method for Cordyceps militaris different strain mating type | |
| CN109517911B (en) | Dual PCR detection primer and detection method for melon bacterial fruit blotch and leaf blotch |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170524 |
|
| WD01 | Invention patent application deemed withdrawn after publication |