CN103088138B - Detection primer and molecular detection method of wheat black point dominant pathogen alternaria - Google Patents
Detection primer and molecular detection method of wheat black point dominant pathogen alternaria Download PDFInfo
- Publication number
- CN103088138B CN103088138B CN201310028328.XA CN201310028328A CN103088138B CN 103088138 B CN103088138 B CN 103088138B CN 201310028328 A CN201310028328 A CN 201310028328A CN 103088138 B CN103088138 B CN 103088138B
- Authority
- CN
- China
- Prior art keywords
- wheat
- primer
- disease
- pcr amplification
- detection
- 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 belongs to the technical field of crop disease detection and specifically discloses a detection primer and a molecular detection method of wheat black point dominant pathogen alternaria. A pair of detection primers of wheat black point dominant pathogen alternaria is design in the invention, wherein the nucleotide sequences of the detection primers are as follows: the upper stream primer Aa1F is 5'-TATGAAGGCGGGCTGGAA-3', and the down stream primer Aa1R is 5'-TTTGCTGATAGAGAGTGCGC-3'; and the specific primer has very high specificity to the wheat pathogen alternaria and can make a distinction between the wheat pathogen alternaria and other main pathogens; the invention further provides a molecular detection method of alternaria carried by various tissues of the wheat by using THE said specific primer pair; the molecular detection method is easy and convenient to operate and is fast, the sensitivity can reach 0.05ng/mu L, the detection cost is relatively low, and the detection method can be used for detecting the alternaria carried by various tissues of seeds, roots, stems, leaves and spikes of wheat and has significant practical significance and economic value.
Description
Technical field
The invention belongs to corps diseases detection technique field, be specifically related to a kind of detection primer and molecular detecting method of wheat black advantage cause of disease alternaric bacteria.
Background technology
Wheat black is the disease of the pathologies such as the caryopsis surface of the class wheat black that has different shapes and degree, brown, pink, redness, canescence, and the cause of disease is complicated.Research shows to cause that the main pathogenic fungi of wheat black has alternaric bacteria, wheat class root-rot mould etc. from the spore bacterium that wriggles, cereal reaping hook, and wherein alternaric bacteria is the Dominantpathogen of the black embryo disease of Wheat in Henan Province.
Alternaric bacteria not only infects wheat grain, at root, stem, blade, can cause different syndromes, serious to wheat harm.The wheat seed that content of molds is large can cause that seedling is weak, root-rot, stem rot, affects growth and the output of wheat, and severe patient even can cause the withered death of wheat seeding.Alternaric bacteria infects blade can cause wheat leaf blight, and the initial stage forms less yellow chlorisis spot on blade, after extend to central authorities and become beige, edge tawny Long Circle scab, when moist, scab surface can form the mould layer of grey black.
The wheat black being caused by alternaric bacteria affects yield and quality of wheat, more seriously alternaric bacteria produces chain lattice spore toxin, as chain lattice spore phenol, chain lattice spore phenol MEE, tenuazonic acid, chain lattice p0-357, thin lattice verticillium toxin I, II, III and AAL toxin.The long-term edible food containing these toxin can cause chronic poisoning, bring out canceration.
Setting up cause of disease detection technique is fast and accurately the needs of disease-resistant wheat genetic research, breeding for disease resistance, disease control.To the detection method of wheat black Dominantpathogen, be mainly dull and stereotyped bacterium colony separation, the microscopic examination identification method of cultivating at present, be based on colonial morphology and mycelia and spore structure, identify complicated operation, step is many, cycle is long, is difficult to quantitatively, without molecular detection technology, report.To the Molecular Detection of alternaria nees fungus, in brassicaceous vegetable, pomegranate, Radix Dauci Sativae, there are reports.Yet, though above-mentioned species cause of disease alternaric bacteria and wheat cause of disease alternaric bacteria belong to normalizing, but be different microspecies, because alternaric bacteria infects host's specialization, cause of disease on other species does not all infect wheat, its gene order also has significant difference, so these methods can not be for the Molecular Detection of wheat cause of disease alternaric bacteria.
Summary of the invention
For problems such as the black embryo disease pathogen of conventional wheat dientification of bacteria technological operation are complicated, step is many, the cycle is long, efficiency is low, the invention provides a kind of detection primer of wheat black advantage cause of disease alternaric bacteria, this detection primer pair wheat cause of disease alternaric bacteria has very strong specificity, the present invention also provides the quick nest-type PRC detection technique of a grow wheat cause of disease alternaric bacteria, and this molecular detecting method has high specificity, highly sensitive feature.
The present invention includes following technical scheme:
The invention provides a kind of detection primer of wheat black advantage cause of disease alternaric bacteria, the nucleotides sequence of described primer is classified as: upstream primer Aa1F:5 '-TATGAAGGCGGGCTGGAA-3 ', downstream primer Aa1R:5 '-TTTGCTGATAGAGAGTGCGAC-3 '.
The invention provides above-mentioned primer in the application to the discriminating of wheat cause of disease alternaric bacteria or in detecting.
The present invention also provides a kind of method of utilizing above-mentioned primer to detect wheat cause of disease alternaric bacteria, comprises the following steps:
(1) extract the genomic dna of tissue to be detected on wheat seed or plant;
(2) take described DNA carries out first round pcr amplification as pathogenic fungi universal primer ITS1 5 '-TCCGTAGGTGAACCTGCGG-3 ' for template and ITS4 5 '-TCCTCCGCTTATTGATATGC-3 ': pcr amplification adopts 25 μ L reaction systems, comprising: the 5 U/ μ L Taq archaeal dna polymerases of 0.25 μ L, 2.5 μ L are containing 15mmol/L MgCl
210 * PCR Buffer, the 2.5 mmol/L dNTP Mixtrue of 2 μ L, 10 μ mol/L ITS1 and the ITS4 primer of each 1 μ L, the 50 ng/ μ L DNA of 1 μ L, the ddH of 17.25 μ L
2o; Pcr amplification program: 95 ℃ of denaturation 5 min, 95 ℃ of sex change 30 s of 30 circulations, 58 ℃ of annealing 30 s and 72 ℃ of extension 50 S, finally extend 10 min at 72 ℃;
(3) with wheat cause of disease alternaric bacteria special primer, carry out second and take turns specific amplification: by 200 times of first round PCR product dilutions, get 1 μ L and carry out second and take turns pcr amplification, pcr amplification adopts 25 μ L reaction systems; Comprising: the 5 U/ μ L Taq archaeal dna polymerases of 0.25 μ L, 2.5 μ L are containing 15mmol/L MgCl
210 * PCR Buffer, the 2.5 mmol/L dNTP Mixtrue of 2 μ L, 10 μ mol/L Aa1F and the Aa1R primer of each 1 μ L, the first round pcr amplification product dilution template of 1 μ L, the ddH of 17.25 μ L
2o; Pcr amplification program: 94 ℃ of denaturation 5 min, 94 ℃ of sex change 30 s of 20 circulations, 56 ℃ of annealing 30 s and 72 ℃ of extension 40 s, finally extend 10 min at 72 ℃;
(4) get the above-mentioned PCR product of 10 μ L, the agarose gel electrophoresis that is 1.5% ~ 2.0% by mass concentration is separated, and electrophoretic buffer is 1 * TAE, and voltage is 4 ~ 5 V/cm, and electrophoresis finishes rear with ethidium bromide staining, gel imaging system analysis; At 443 bp places, there is specific band, illustrate that wheat grain is with cause of disease alternaric bacteria, otherwise do not carry alternaric bacteria.
According to the method for above-mentioned detection wheat cause of disease alternaric bacteria, in described step (4), the mass concentration of agarose gel is 2%, and electrophoretic voltage is 5 V/cm.
the present invention is useful effect actively:
We are separated 7 chain lattice spore microspecies on the sick seed of the black embryo of Wheat in Henan Province, and these microspecies and 1 standard alternaric bacteria microspecies have been carried out to sequencing analysis, and the sequence of reference also has in addition:
alternaria tenuissimastrain EGS 34-015(GenBank query ID: AF347032.1),
alternaria alternatastrain EGS 34-016(GenBank query ID: AF347031),
alternaria tenuiseU732734.1(GenBank query ID: FJ040178.1), designed the primer of energy specificity identification Alternaria triticina bacterium, set up nest-type PRC molecular detection technology of the present invention.
1. Auele Specific Primer of the present invention has very strong specificity to wheat cause of disease alternaric bacteria, can distinguish other the main pathogenic fungi wheat class root-rot of wheat cause of disease alternaric bacteria and wheat from spore bacterium, wheat powdery mildew, Rhizoctonia cerealis, Fusarium graminearum, the fusarium moniliforme of wriggling.
2. easy and simple to handle, quick, the high specificity of molecular detecting method of the present invention, sensitivity reach 0.05 ng/ μ L, can carry out relative quantitative assay, and testing cost is lower, can be used for detecting wheat grain, root, stem, leaf, fringe and respectively organize entrained alternaric bacteria, for Alternaria triticina bacterium, cause the aobvious disease of disease early monitoring before, for definite disease control best period, there is important effect, for the formulation of control strategy provides scientific basis.
3. alternaric bacteria and wheat class root-rot are 2 large the main pathogenic fungi of wheat black from the spore bacterium that wriggles, extensively be present in wheat belt, not fast, specificity identification and relative quantitative assay pathogenic bacteria technology in the situation that, cannot study the specialization disease resistance of these 2 kinds of cause of diseases with regard to wheat.Foundation of the present invention makes the Genetic and gene locating research of the black embryo disease of the anti-chain lattice of wheat spore become possibility, is conducive to advance the sick genetic research of the black embryo of the anti-chain lattice of wheat spore and breeding for disease resistance work.The present invention is the technical barrier of the capturing in order to solve these problems that run in the Genetic and gene locating research that we carry out the black embryo disease of the anti-chain lattice of wheat spore just.
accompanying drawing explanation:
The detection test electrophorogram that Fig. 1 the present invention carries out different samples.In figure, M is molecular weight standard DL2000,1st ~ 8 swimming lanes are respectively alternaric bacteria reference culture EGS34-016, the isolated strains Ta-Aa-1 ~ Ta-Aa-7 of Henan Province, the 9th swimming lane is that wheat class root-rot is from the spore bacterium that wriggles, the 10th swimming lane is wheat powdery mildew, and the 11st swimming lane is Rhizoctonia cerealis, and the 12nd swimming lane is Fusarium graminearum, the 13rd swimming lane is fusarium moniliforme, the 14th swimming lane is that curved spore is mould, and the 15th swimming lane is wheat leaf blade DNA, and the 16th swimming lane is blank water;
Fig. 2 the present invention is to cause of disease alternaric bacteria detection sensitivity test electrophorogram.In figure 1 ~ 8 swimming lane respectively corresponding cause of disease alternaric bacteria DNA concentration be 1 pg/ μ L, 10 pg/ μ L, 50 pg/ μ L, 100 pg/ μ L, 500 pg/ μ L, 1 ng/ μ L, 10 ng/ μ L and 50 ng/ μ L;
The electrophorogram that 2 pairs of alternaric bacteria leaf blight leaf samples of Fig. 3 embodiment of the present invention detect.In figure 1 ~ 5 swimming lane respectively correspondence connect the leaf sample of 0 ~ 4 grade of bacterium sequela, the 6th swimming lane is not for connecing the contrast leaf sample of bacterium;
The detection electrophorogram that the wheat black seed of 1 pair of inoculation alternaric bacteria of Fig. 4 embodiment of the present invention and natural occurrence carries out.1 ~ 5 swimming lane corresponding 0 ~ 4 grade of seed sample of the sick level of black embryo that connects bacterium results respectively in figure, 6th, 7 swimming lanes are 0 grade of natural occurrence, 1 grade of sick seed samples of black embryo, the 8th swimming lane is alternaric bacteria, the 9th swimming lane is that wheat class root-rot is from the spore bacterium that wriggles, the 10th swimming lane is wheat healthy leaves sample, and the 11st swimming lane is blank water.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in more detail, but the invention is not restricted to these embodiment.
The detection primer nucleotides sequence of wheat black advantage cause of disease alternaric bacteria of the present invention is classified as: upstream primer Aa1F:5 '-TATGAAGGCGGGCTGGAA-3 ' and downstream primer Aa1R:5 '-TTTGCTGATAGAGAGTGCGAC-3 '.Utilize this primer can be from wheat cause of disease alternaric bacteria specific amplification go out the product of 443 bp.
Above-mentioned primer carries alternaric bacteria situation molecular detecting method for wheat grain, comprises the following steps:
(1) sample collecting
The wheat grain of Field inoculation alternaric bacteria and the wheat grain of the embryo disease of naturally turning black of choosing results ,-20 ℃ save backup.
(2) sample grinds
Respectively above-mentioned wheat grain is ground in the mortar of sterilizing, in process of lapping, should note avoiding the crossed contamination of sample;
Also can adopt tissue grinder to grind: get one grained wheat seed, first crumb roughly, put into 2 mL sterilizing centrifuge tubes, every pipe is put into the steel ball of diameter 5 mm, and centrifuge tube is put in to precooling in liquid nitrogen, after precooling, with tissue grinder 30 Hz, grinds 1 min.
(3) DNA extraction
1. to the SLS cell pyrolysis liquid (Chen Guangjin that adds 800 μ L in the sample grinding, Li Hongye, Zhang Zhi's virtue. a kind of SLS lysate of rapid extraction fungal DNA and application [P] .CN:200810059177 thereof, 2008-07-30), due to more containing polysaccharide in seed, in SLS lysate, add the polyvinylpyrrolidone (PVP) that accounts for its volume 2%-6%, put into again 65 ℃ of water-bath heating in water bath 10-15 min, slight wobble centrifuge tube during this time, make sample dissociation liquid not fall to the pipe end, so that fully cracking;
2. the phenol, chloroform, the primary isoamyl alcohol mixed solvent that to the volume ratio that adds 800 μ L in centrifuge tube, are 25:24:1, slightly shake 1 min left and right, makes it into milkiness shape, mixes;
3. centrifugal 10 min under 12000 r/min, draw supernatant liquor 600 μ L lightly with the rifle head of cutting, and proceed in the 1.5 mL centrifuge tubes that contain 360 μ L Virahols, by centrifuge tube 30 s that slowly fluctuate, Virahol are fully mixed with supernatant liquor;
4. 10 min precipitation DNA at standing 30 min or-20 ℃ under room temperature;
5. centrifugal 10 min under 12000 r/min, outwell liquid, note DNA precipitation not being poured out;
6. the ethanol that is 75% to the volumetric concentration that adds 1 mL in centrifuge tube, teetertotters, and with finger, flicks tip, and DNA precipitation is suspended in ethanol;
7. centrifugal 5 min under 12000 r/min, outwell liquid, repeat the operation of previous step;
8. centrifugal 3 min under 12000 r/min, the liquid in pipe is thoroughly clean after, seasoning DNA under room temperature;
9. after DNA is dry, add the TE damping fluid of 40 μ L ,-20 ℃ save backup.
(4) pcr amplification detects
1. first round universal primer PCR amplification:
Pcr amplification adopts 25 μ L reaction systems, comprising: the 5 U/ μ L Taq archaeal dna polymerases of 0.25 μ L, 2.5 μ L are containing 15mmol/L MgCl
210 * PCR Buffer, the 2.5 mmol/L dNTP Mixtrue of 2 μ L, 10 μ mol/L ITS1 and the ITS4 primer of each 1 μ L, the 50 ng/ μ L DNA of 1 μ L, the ddH of 17.25 μ L
2o; Pcr amplification program: 95 ℃ of denaturation 5 min; 95 ℃ of sex change 30 s of 30 circulations, 58 ℃ of annealing 30 s and 72 ℃ of extension 50 S; Finally at 72 ℃, extend 10 min; ITS1 5 '-TCCGTAGGTGAACCTGCGG-3 ', (Liu Chun comes ITS4 5 '-TCCTCCGCTTATTG-ATATGC-3 ', Wen Jingzhi, Yang Mingxiu, Li Yonggang. the application of rDNA-ITS in plant pathogenic fungi Molecular Detection. Northeast Agricultural University's journal, 2007,38 (1): 101-106);
2. second take turns specific primer PCR amplification:
By 200 times of first round PCR product dilutions, get 1 μ L and carry out second and take turns pcr amplification; Pcr amplification adopts 25 μ L reaction systems, comprising: the 5 U/ μ L Taq archaeal dna polymerases of 0.25 μ L, 2.5 μ L are containing 10 * PCR Buffer of 15mmol/L MgCl2, the 2.5 mmol/L dNTP Mixtrue of 2 μ L, 10 μ mol/L Aa1F and the Aa1R primer of each 1 μ L, the first round pcr amplification product dilution template of 1 μ L, the ddH of 17.25 μ L
2o; Pcr amplification program: 94 ℃ of denaturation 5 min; 94 ℃ of sex change 30 s of 20 circulations, 56 ℃ of annealing 30 s and 72 ℃ of extension 40 s; Finally at 72 ℃, extend 10 min;
3. electrophoresis detection
Get 10 μ L PCR products, the agarose gel electrophoresis that is 2% by mass concentration is separated, 1 * TAE damping fluid for electrophoretic buffer, and voltage is 5 V/cm, electrophoresis finishes rear with ethidium bromide staining, with gel imaging system, analyzes.
(5) detected result
Electrophoresis result is shown in Fig. 4, and field connects the wheat grain of bacterium and the wheat grain of natural occurrence all with alternaric bacteria, although black embryo symptom does not also appear in 0 grade of wheat grain surface, also has after testing alternaric bacteria.
Above-mentioned primer carries alternaric bacteria situation molecular detecting method for wheat leaf blade, comprises the following steps:
(1) sample collecting
Get and add the alternaric bacteria spore suspension of its volume ratio 0.02% tween to be sprayed onto on the wheat leaf blade of one heart stage of leaf, sterilized water is sprayed in contrast, after dry on blade face, adds the cover that transite plate is made; Every 12 h spray sterilized water with moisturizing to cover inner side; Connect and within the 10th day, get other wheat leaf blade of different onset level after bacterium and normal blade detects.
(2) extract sample DNA
1. 0.05 g morbidity wheat leaf blade is placed in 2 sterilized mL centrifuge tubes, and every pipe is put into the steel ball of diameter 5 mm, and centrifuge tube is put in to precooling in liquid nitrogen, after precooling, with tissue grinder 30 Hz, grinds 1 min; Also can in sterilized mortar, directly grind;
2. to the SLS cell pyrolysis liquid that adds 800 μ L in the sample crushing, put into 65 ℃ of water-bath heating in water bath 10-15 min, during slight wobble centrifuge tube, make sample dissociation liquid not fall to the pipe end, so that fully cracking;
3. to centrifuge tube, add phenol, chloroform, the primary isoamyl alcohol mixed solvent that the volume ratio of 800 μ L is 25:24:1, slightly shake 1 min left and right, make it into milkiness shape, mix;
4. centrifugal 10 min under 12000 r/min; With the rifle head of cutting, draw lightly supernatant liquor 600 μ L, proceed in the 1.5 mL centrifuge tubes that contain 360 μ L Virahols, by centrifuge tube 30 s that slowly fluctuate, Virahol is fully mixed with supernatant liquor;
5. 10 min precipitation DNA at standing 30 min or-20 ℃ under room temperature;
6. centrifugal 10 min under 12000 r/min, outwell liquid, note DNA precipitation not being poured out;
7. the ethanol that is 75% to the volumetric concentration that adds 1 mL in centrifuge tube, teetertotters, and with finger, flicks tip, and DNA precipitation is suspended in ethanol;
8. centrifugal 5 min under 12000 r/min, outwell liquid, repeat the operation of previous step;
9. centrifugal 3 min under 12000 r/min, the liquid in pipe is thoroughly clean after, seasoning DNA under room temperature;
10. after DNA is dry, add the TE damping fluid of 40 μ L ,-20 ℃ save backup.
(3) pcr amplification detects
1. first round universal primer PCR amplification:
Pcr amplification adopts 25 μ L reaction systems, comprising: the 5 U/ μ L Taq archaeal dna polymerases of 0.25 μ L, 2.5 μ L are containing 15mmol/L MgCl
210 * PCR Buffer, the 2.5 mmol/L dNTP Mixtrue of 2 μ L, 10 μ mol/L ITS1 and the ITS4 primer of each 1 μ L, the 50 ng/ μ L DNA of 1 μ L, the ddH of 17.25 μ L
2o; Pcr amplification program: 95 ℃ of denaturation 5 min; 95 ℃ of sex change 30 s of 30 circulations, 58 ℃ of annealing 30 s and 72 ℃ of extension 50 S; Finally at 72 ℃, extend 10 min;
2. second take turns specific primer PCR amplification:
By 200 times of first round PCR product dilutions, get 1 μ L and carry out second and take turns pcr amplification; Pcr amplification adopts 25 μ L reaction systems, comprising: the 5 U/ μ L Taq archaeal dna polymerases of 0.25 μ L, 2.5 μ L are containing 15mmol/L MgCl
210 * PCR Buffer, the 2.5 mmol/L dNTP Mixtrue of 2 μ L, 10 μ mol/L Aa1F and the Aa1R primer of each 1 μ L, the first round pcr amplification product dilution template of 1 μ L, the ddH of 17.25 μ L
2o; Pcr amplification program: 94 ℃ of denaturation 5 min; 94 ℃ of sex change 30 s of 20 circulations, 56 ℃ of annealing 30 s and 72 ℃ of extension 40 s; Finally at 72 ℃, extend 10 min;
3. electrophoresis detection
Get 10 μ L PCR products, the agarose gel electrophoresis that is 2% by mass concentration is separated, 1 * TAE damping fluid for electrophoretic buffer, and voltage is 5 V/cm, electrophoresis finishes rear with ethidium bromide staining, with gel imaging system, analyzes.
(4) detected result
Electrophoresis result is shown in Fig. 3, connect the wheat leaf blade of bacterium all with chain lattice spore, although disease symptom does not appear in the wheat leaf blade surface of 0 grade of morbidity, also have after testing alternaric bacteria, and connect bacterium blade surface, do not fall ill, and PCR detects the existence that alternaric bacteria also do not detected.
SEQUENCE LISTING
<110> Agricultural University Of He'nan
Detection primer and the molecular detecting method of <120> wheat black advantage cause of disease alternaric bacteria
<130> \
<160> 2
<170> PatentIn version 3.2
<210> 1
<211> 18
<212> DNA
<213> manually designs
<400> 1
tatgaaggcg ggctggaa 18
<210> 2
<211> 21
<212> DNA
<213> manually designs
<400> 2
tttgctgata gagagtgcga c 21
Claims (2)
1. a method of utilizing wheat cause of disease alternaric bacteria special primer to detect wheat cause of disease alternaric bacteria, it is characterized in that: the nucleotides sequence of described primer is classified as: upstream primer Aa1F:5 '-TATGAAGGCGGGCTGGAA-3 ', downstream primer Aa1R:5 '-TTTGCTGATAGAGAGTGCGAC-3 ';
Detection method comprises the following steps:
(1) extract the genomic dna of tissue to be detected on wheat seed or plant;
(2) take described DNA carries out first round pcr amplification as pathogenic fungi universal primer ITS1 5 '-TCCGTAGGTGAACCTGCGG-3 ' for template and ITS4 5 '-TCCTCCGCTTATTGATATGC-3 ': pcr amplification adopts 25 μ L reaction systems, comprising: the 5 U/ μ L Taq archaeal dna polymerases of 0.25 μ L, 2.5 μ L are containing 15mmol/L MgCl
210 * PCR Buffer, the 2.5 mmol/L dNTP Mixtrue of 2 μ L, 10 μ mol/L ITS1 and the ITS4 primer of each 1 μ L, the 50 ng/ μ L DNA of 1 μ L, the ddH of 17.25 μ L
2o; Pcr amplification program: 95 ℃ of denaturation 5 min, 95 ℃ of sex change 30 s of 30 circulations, 58 ℃ of annealing 30 s and 72 ℃ of extension 50 S, finally extend 10 min at 72 ℃;
(3) with wheat cause of disease alternaric bacteria special primer, carry out second and take turns specific amplification: by 200 times of first round PCR product dilutions, get 1 μ L and carry out second and take turns pcr amplification, pcr amplification adopts 25 μ L reaction systems; Comprising: the 5 U/ μ L Taq archaeal dna polymerases of 0.25 μ L, 2.5 μ L are containing 15mmol/L MgCl
210 * PCR Buffer, the 2.5 mmol/L dNTP Mixtrue of 2 μ L, 10 μ mol/L Aa1F and the Aa1R primer of each 1 μ L, the first round pcr amplification product dilution template of 1 μ L, the ddH of 17.25 μ L
2o; Pcr amplification program: 94 ℃ of denaturation 5 min, 94 ℃ of sex change 30 s of 20 circulations, 56 ℃ of annealing 30 s and 72 ℃ of extension 40 s, finally extend 10 min at 72 ℃;
(4) get the above-mentioned PCR product of 10 μ L, the agarose gel electrophoresis that is 1.5% ~ 2.0% by mass concentration is separated, and electrophoretic buffer is 1 * TAE, and voltage is 4 ~ 5 V/cm, and electrophoresis finishes rear with ethidium bromide staining, gel imaging system analysis; At 443 bp places, there is specific band, illustrate that wheat grain is with cause of disease alternaric bacteria, otherwise do not carry alternaric bacteria.
2. the method for detection wheat cause of disease alternaric bacteria according to claim 1, is characterized in that: in described step (4), the mass concentration of agarose gel is 2%, and electrophoretic voltage is 5 V/cm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310028328.XA CN103088138B (en) | 2013-01-25 | 2013-01-25 | Detection primer and molecular detection method of wheat black point dominant pathogen alternaria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310028328.XA CN103088138B (en) | 2013-01-25 | 2013-01-25 | Detection primer and molecular detection method of wheat black point dominant pathogen alternaria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103088138A CN103088138A (en) | 2013-05-08 |
| CN103088138B true CN103088138B (en) | 2014-02-12 |
Family
ID=48201265
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310028328.XA Expired - Fee Related CN103088138B (en) | 2013-01-25 | 2013-01-25 | Detection primer and molecular detection method of wheat black point dominant pathogen alternaria |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103088138B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105660222B (en) * | 2016-03-23 | 2019-04-16 | 河南农业大学 | A kind of inoculation identification method that can observe black embryo of wheat disease symptoms in time |
| CN108148924A (en) * | 2018-02-22 | 2018-06-12 | 黑龙江省农业科学院植物脱毒苗木研究所 | The molecular detection primer and its detection method of potato plant tikka class disease alternariosis opportunistic pathogen |
| CN108315472A (en) * | 2018-04-27 | 2018-07-24 | 安徽省农业科学院植物保护与农产品质量安全研究所 | It is a kind of to be used for the Primer composition and its application that alternaric bacteria LAMP is quickly detected |
| CN113999927B (en) * | 2021-02-24 | 2023-10-20 | 四川省农业科学院植物保护研究所 | Primer group and kit for early and rapid detection of apple heart germ, detection method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1293241A (en) * | 2000-09-20 | 2001-05-02 | 南京农业大学 | Method for using crude metabolite of alternaric bacteria in biologic herbiciding |
-
2013
- 2013-01-25 CN CN201310028328.XA patent/CN103088138B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1293241A (en) * | 2000-09-20 | 2001-05-02 | 南京农业大学 | Method for using crude metabolite of alternaric bacteria in biologic herbiciding |
Non-Patent Citations (2)
| Title |
|---|
| Fatma BENSASSI et al..First report of Alternaria species associated with black point of wheat in Tunisia.《 Annals of Microbiology》.2009,第59卷(第3期),摘要、第466页第2、4-5段，第467页第2段. |
| First report of Alternaria species associated with black point of wheat in Tunisia;Fatma BENSASSI et al.;《 Annals of Microbiology》;20090930;第59卷(第3期);摘要、第466页第2、4-5段，第467页第2段 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103088138A (en) | 2013-05-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101974650B (en) | Polymerase chain reaction (PCR) method for detecting fusarium oxysporum and kit | |
| CN103333969B (en) | Method and kit for quickly detecting verticillium dahliae through PCR (polymerase chain reaction) | |
| CN103088138B (en) | Detection primer and molecular detection method of wheat black point dominant pathogen alternaria | |
| CN107760795B (en) | LAMP primer for rapidly detecting anthracnose bacteria of tea trees and detection method | |
| CN103725782B (en) | Method, kit and primer for detecting pathogenic bacteria of lotus fungal rot disease carried by lotus seeds | |
| CN101974651B (en) | Fluorescence quantitative polymerase chain reaction (PCR) detection method and detection kit for phytophthora capsici leonian | |
| CN102321738A (en) | Fluorescence quantitative PCR (polymerase chain reaction) universal premier for detecting pathogenic aspergillus, detection probe and kit | |
| CN107119048B (en) | Pseudocercospora mori rDNA and application thereof in molecular detection of pseudocercospora mori | |
| CN101792795A (en) | Identification method for determining anastomosis groups of rhizoctonia solani | |
| CN101851684B (en) | Fungus colony polymerase chain reaction (PCR) method and pathogenic fungus identification method | |
| CN113025744A (en) | Nested PCR (polymerase chain reaction) specific primer for loquat colletotrichum gloeosporioides as well as detection method and application thereof | |
| CN111850155A (en) | Application of specific target primer in simultaneous and rapid identification of two pathogenic bacteria of strawberry infection | |
| CN104911262B (en) | A kind of dalbergia odorifera tar spot bacterium molecule detection primer and quick determination method | |
| CN104120170B (en) | Potato early blight bacterium detection kit and detection method | |
| CN106868147B (en) | Molecular detection primer for sigatoka bacteria and rapid detection method thereof | |
| CN102382899B (en) | Molecular detection method for fusarium graminearum and application thereof | |
| CN105462971A (en) | Specific molecular marker for rice blast resistance gene Pi2 and dedicated primers thereof | |
| CN102864221A (en) | Polymerase chain reaction (PCR) detection method for colletotrichum falcatum went | |
| CN102925568B (en) | Primer and probe for detecting acidovorax citrulli and detection method for acidovorax citrulli | |
| CN107674923B (en) | Specific molecular detection primer for soybean botrytis cinerea and application thereof | |
| CN107058559B (en) | Molecular detection method and kit for plant pathogenic fungi | |
| CN106244720B (en) | A kind of anthracnose of peach bacterium molecule detection primer and detection method | |
| CN110724757A (en) | Primer for mulberry fertilize large sclerotinia sclerotiorum detection and application thereof | |
| AU2021104196A4 (en) | PCR detection method for detecting pathogen causing sugarcane pokkah boeng | |
| CN113897456B (en) | Real-time fluorescence PCR detection primer probe combination and detection kit for four fir anthracnose pathogens and application of kit |
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: 20140212 Termination date: 20150125 |
|
| EXPY | Termination of patent right or utility model |