CN101691605A - Nested PCR detection method of oak wilt - Google Patents
Nested PCR detection method of oak wilt Download PDFInfo
- Publication number
- CN101691605A CN101691605A CN200910034195A CN200910034195A CN101691605A CN 101691605 A CN101691605 A CN 101691605A CN 200910034195 A CN200910034195 A CN 200910034195A CN 200910034195 A CN200910034195 A CN 200910034195A CN 101691605 A CN101691605 A CN 101691605A
- Authority
- CN
- China
- Prior art keywords
- seconds
- dna
- bretz
- hunt
- primer
- 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.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a nested PCR detection method of oak wilt which can be carried and transferred by log, wooden packing and the like. The method of the invention designs a pair of specific primers CF01/CF02 for distinguishing oak wilt, sample DNA is used as template, universal primers ITS1/ITS4 are adopted to perform the first round PCR amplification, then the product of the first round PCR amplification is used as template to perform the second round PCR amplification of the specific primer, the product of the amplification is detected by electrophoresis test and if 280bp of DNA specific fragments exist, the tested pathogen is detected to be oak wilt. The detection method can be used for detection on the condition of the existence of trace pathogenic bacteria, the sensitivity is 1pg DNA, when the method is used for detecting pathogen in soil, only the existence of one spore of pathogen can realize the detection so that the method has strong reliability and is suitable for the fast oak wilt detection for port nursery stock and wooden packing.
Description
Technical field
The present invention relates to a kind of nested PCR detection method (or claiming Nested PCR method) of detection method, particularly Ceratocystis fagacearum (Bretz) Hunt of mainly carrying the Ceratocystis fagacearum (Bretz) Hunt of propagation by log, Wooden package etc., belong to biological technical field.
Background technology
The oak blight is by Ceratocystis fagacearum (Bretz) Hunt (Ceratocystis fagacearum, be abbreviated as C.fagacearum) a kind of fungal disease of caused main harm oak kind, be distributed in the U.S. of North America at present, states such as Canadian and European Bulgaria, Poland, Romania, domestic discovery as yet.
Oak blight (Ceratocystis fagacearum), nineteen forty-two is at first found in the continent, Wisconsin State, causes the rapid death of local red oak.After this this disease constantly spreads, and geographic more than 20 states, the U.S. central and east all have discovery at present, have become the main destructive disease of the U.S. domestic robur class seeds.There is thousands of robur withered every year in the serious state of Wisconsin and the Minnesota State of harm.At present, it is by China, and European Union (EU) and each member states of European plant protection tissue (EPPO) classify quarantine harmful organisms as.
Main grafting by sick strong root is propagated in this pathogenic bacteria short range, can also propagate on ground by insect vector, wherein most importantly nitidulid and bark beetle.The long-distance communications of germ are then mainly undertaken by germ-carrying nursery stock and moist robur log or the long-distance transport of its product.In recent years, China is from the U.S., the quantity of epidemic-stricken area import nursery stocks such as Canada and Wooden package sharply rises, therefore, in these vegetable matters, very likely carrying pathogenic bacteria, and the host of China distributes and to decide to grow diffusion with the ecoclimate condition all is beneficial to this germ very much, in the face of so severe situation, need set up as early as possible a cover fast, sensitive, reliable detection method, thereby the massive losses of avoiding the intrusion of this germ to be caused for China's Forest Resources and production of forestry.
Up to now, less for the molecular detecting method of Ceratocystis fagacearum (Bretz) Hunt report in the world, 1999, people such as R.C.WITTHUHN set up the PCR-RFLP technology and have distinguished the Phylogenetic Relationships that long beak shell belongs to some important kinds, had wherein also comprised C.fagacearum, but this method cost height, the process complexity, detection sensitivity is low, is difficult to quantitatively, and for the purity requirement of sample DNA than higher, so be not suitable in each quarantine port promotion and application.
In recent years, more and more researchers adopts molecular biological method to detect the forest pathogenic bacteria.Utilize in the nucleus or plastosome in rDNA all have a wide range of applications on the different categorization levels that fungi is identified for the round pcr of detection site.The part on the ribosomal gene wherein, transcribed spacer (ITS1 and ITS2) is because more conservative in planting, but between planting, there is polymorphism, in addition, because nucleus is interior or the interior rDNA of plastosome is a multiple copied, even the sample DNA amount seldom or under the not high situation of quality also can amplify due product, so the foundation of at present a lot of molecular detecting methods all is based on this site.And about the Nested PCR detection method of Ceratocystis fagacearum (Bretz) Hunt, even other molecular detecting method, report is not all arranged both at home and abroad.
Summary of the invention
Nido (Nested) the PCR detection method that the purpose of this invention is to provide the Ceratocystis fagacearum (Bretz) Hunt that uses at a kind of port that is suitable for quarantining so that fast, sensitive, exactly port nursery stock and Wooden package are carried out the detection of Ceratocystis fagacearum (Bretz) Hunt.
The present invention extracts the DNA of germ from mycelia, soil or the wooden unit of germ, as template, carrying out the pcr amplification of first round universal primer with sample DNA, is template with first round PCR product again, carry out second pcr amplification of taking turns Auele Specific Primer, amplified production carries out electrophoresis detection.
Concrete detection method is as follows:
1. extract the DNA of Ceratocystis fagacearum (Bretz) Hunt sample, cryopreservation is standby;
2. design the Auele Specific Primer of a pair of discriminating wilt
Announce based composition difference (accession number: EU918711, AY528959, the DQ318196 of the ITS regional sequence that Ceratocystis belongs to according to GeneBank, AY214001, U75626, U75627, EF042612, EF190968, AY233924, FJ347031, AY233921, DQ318195, EU245018, AY528999, DQ074743, DQ074742, DQ318203, AY821864, AF043607, U75630, DQ318198, EF042613, DQ318202, AY528973, AF275495, DQ318205, U75621, EF408554, AY529004, AY907044, AY907036 DQ061281), utilizes primer-design software (Primer Premier5), design a pair of Auele Specific Primer CF01/CF02 that can differentiate Ceratocystis fagacearum (Bretz) Hunt, its sequence is:
Upstream primer CF01:5 '-GGCAGGGACTTCTTTCTT-3 ';
Downstream primer CF02:5 '-AAGGCTTGAGTGGTGAAA-3 ';
3. utilize universal primer ITS1/ITS4 (reference, White DJ, et al.1990.Amplification anddirect sequencing of fungal ribosomal RNA genes for phylogenetics.) and Auele Specific Primer CF01/CF02, carry out the Nested pcr amplification of Ceratocystis fagacearum (Bretz) Hunt
The sequence of universal primer ITS1/ITS4 is:
ITS1：5’-TCCGTAGGTGAACCTGCGG-3’；
ITS4：5’-TCCTCCGCTTATTGATATGC-3’
Amplification step is as follows:
First round amplification: universal primer PCR amplification
The reaction system cumulative volume is 25 μ l, and reactive component is: 10 * reaction buffer 2.5 μ l, the MgCl of 25mM
22.5 μ l, the dNTP 2.5 μ l of 2.5mM, each 1 μ l of the upstream and downstream primer I TS1/ITS4 of 10 μ M, 5U/ μ l Taq archaeal dna polymerase 0.25 μ l, template Ceratocystis fagacearum (Bretz) Hunt sample DNA extracting solution 1 μ l, aqua sterilisa is supplied 25 μ l; The amplified reaction program is: increase in advance 95 ℃ 180 seconds, then 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 60 seconds, 20 circulations were extended 300 seconds at 72 ℃ at last.
Second takes turns amplification: the special primer pcr amplification
The reaction system cumulative volume is 25 μ l, with 10 times of first round PCR product dilutions, gets 1 μ l as template, other reactive component: 10 * reaction buffer 2.5 μ l, 25mM MgCl
22.5 μ l, 2.5mM dNTP 2.5 μ l, each 1 μ l of the upstream and downstream primer CF01/CF02 of 10 μ M, 5U/ μ l Taq archaeal dna polymerase 0.25 μ l, aqua sterilisa is supplied 25 μ l; The amplified reaction program is: increase in advance 95 ℃ 180 seconds, then 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 60 seconds, 35 circulations were extended 300 seconds at 72 ℃ at last.
4. amplified production carries out electrophoresis detection
Get 8 μ l amplified productions, adopt 1.5% (mass/volume) sepharose, electrophoresis is 30 minutes under voltage 150V, place afterwards in the EB and dyeed 20 minutes, take out, the detection of taking pictures under gel imaging system if there is the DNA specific band of 280bp, determines that then the germ that is detected is a Ceratocystis fagacearum (Bretz) Hunt.
The present invention is directed to mainly log of Ceratocystis fagacearum (Bretz) Hunt by getting dirty, Wooden package, vegetable matter and thereupon the transportation of the soil that carries and the characteristic of carrying out long-distance communications, the media that may carry germ is carried out DNA extraction and Molecular Detection, saved the method for traditional separation and Culture, shortened the quarantine cycle greatly in time, whole testing process can be finished in 1-2 days, program is simple, is easy to promote at each quarantine port.The Auele Specific Primer that present method is used is based on pathogenic bacteria and designs with ITS zone (internal gene transcribed spacers) the based composition difference that belongs to other kind together, and specificity is fine, can be used for distinguishing the little allied species of difference on form.Even this detection method can detect under the situation that micro-pathogenic bacteria exists accurately, sensitivity is 1pg DNA, when being applied in the soil tested for pathogens, as long as there is 1 cause of disease spore to exist, can detect good reliability.
Description of drawings
Fig. 1 is the gel electrophoresis figure that utilizes special primer CF01/CF02 amplification products therefrom, and wherein M is 2000-bpDNA marker; 1 is C.fagacearum bacterial strain 200423; 2 is C.fagacearum bacterial strain 200425; 3～13 is the part bacterial strain in the table 1; 14 negative contrasts.
Fig. 2 utilizes the electrophorogram of special primer CF01/CF02 to different concns DNA cloning products therefrom.
Fig. 3 utilizes different concns DNA to carry out the electrophorogram of Nested PCR products therefrom.
Fig. 4 utilizes Nested PCR to detect the electrophorogram of germ spore count in the soil.
Embodiment
Below by example the present invention is described in further detail.
Nido (Nested) PCR of embodiment 1 Ceratocystis fagacearum (Bretz) Hunt detects
According to the following steps:
1. sample DNA extracts
1.1 extract the mycelia sample DNA
1.1-1 strains tested is cultivated and mycelium is collected
Strains tested is connected on the PDA substratum, and 20-25 ℃ of dark culturing 10 days scrapes mycelia gently with inoculating needle, places centrifuge tube, lyophilize, and-20 ℃ of preservations are standby.
1.1-2 extract mycelia DNA with the CTAB method, concrete operations are as follows:
1) get the mycelia of the freezing mistake of 0.5g, be put in the flat centrifuge tube of 2.0ml, adding 2 diameters is the 3mm steel ball, closes upper tube cap, places on the Oscillating Mill MM400 beveller, and 30f/min ground 3 minutes;
2) take out steel ball, add 500 μ l CTAB, in liquid nitrogen freezing 2 minutes, then 75 ℃ of water-baths were 2 minutes, repeat twice, melt 30 minutes in 75 ℃ of water-baths for the last time;
3) add phenol, chloroform, primary isoamyl alcohol solution (three's volume ratio is 25: 24: 1, and adding total amount is 500 μ l), the mixing that turns upside down, centrifugal 10 minutes of 12000rpm;
4) get supernatant, the ice dehydrated alcohol that adds 2 times of volumes precipitates 1 hour;
5) place on the whizzer centrifugal 10 minutes of 12000rpm;
6) abandon supernatant, precipitation is used 70% washing with alcohol, natural air drying, 50 μ lddH
2The O dissolving DNA ,-20 ℃ of preservations are standby.
1.2 extract the DNA of pedotheque to be detected
1) get 0.3g soil, oven dry grinds with beveller;
2) fine powder after will grinding adds in the centrifuge tube of 2.0ml, adds 0.4% (mass/volume) skim-milk solution of 500 μ l, vortex mixing, 12000rpm, centrifugal 15 minutes;
3) get supernatant, add the Proteinase K damping fluid (50mM Tris-HCl pH=8.0,2.5mM EDTApH=8.0,1%SDS, 10 μ g/ml Proteinase Ks) of 1 times of volume, 55 ℃ water-bath 1-3 hour;
4) press the NH that gained mixture 1/2 volume adds 7.5M
4AC solution, the mixing that turns upside down, centrifugal 15 minutes of 12000rpm;
5) draw supernatant, add the dehydrated alcohol of two volumes, react extremely to precipitate in about 1 hour and all separate out centrifugal 15 minutes of 12000rpm;
6) abandon supernatant, remove natural air drying, 50 μ lddH with the hypsokinesis of 70% washing with alcohol precipitation
2The O dissolving DNA ,-20 ℃ of preservations are standby.
1.3 extract the DNA in the wooden unit sample to be detected
1) with the chopping of wooden unit sample, grinds to form wood chip;
2) get the 0.5g wood chip, add in the centrifuge tube of 2.0ml, add the Proteinase K damping fluid (50mMTris-HCl pH=8.0,2.5mM EDTA pH=8.0,1%SDS, 10 μ g/ml Proteinase Ks) of 500 μ l, 55 ℃ water-bath 1-3 hour;
3) press the NH that gained mixture 1/2 volume adds 7.5M
4AC solution, the mixing that turns upside down, centrifugal 15 minutes of 12000rpm;
4) draw supernatant, the dehydrated alcohol that adds two volumes reacts extremely to precipitate in about 1 hour separates out centrifugal 15 minutes of 12000rpm fully;
5) abandon supernatant, remove natural air drying, 50 μ lddH with the hypsokinesis of 70% washing with alcohol precipitation
2The O dissolving DNA ,-20 ℃ of preservations are standby.
2. carry out first round pcr amplification amplification with universal primer ITS1/ worker TS4
The reaction system cumulative volume is 25 μ l, and reactive component is: 10 * reaction buffer, 2.5 μ l, 25mM Mgcl
22.5 each 1 μ l of the upstream and downstream primer I TS1/ITS4 (authorized company's synthetic) of μ l, 2.5mM dNTP 2.5 μ l, 10 μ M, 5U/ μ l TaqDNA polysaccharase 0.25 μ l, template DNA 10ng, aqua sterilisa is supplied 25 μ l; Replace template DNA with aqua sterilisa, as negative control.Mixed solution increases on the PTC-200PCR instrument, and response procedures is: increase in advance 95 ℃ 180 seconds, then 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 60 seconds, 20 circulations were extended 300 seconds at 72 ℃ at last.
3. carry out second with Auele Specific Primer CF01/CF02 and take turns pcr amplification (germ DNA carries out specific amplification)
With 10 times of first round PCR product dilutions, to get 1 μ l and carry out second as template and take turns pcr amplification, reaction system is 25 μ l.
The reaction system cumulative volume is 25 μ l, and reactive component is: 10 * reaction buffer, 2.5 μ l damping fluids (available from TaKaRa company), 25mMMgCl
22.5 each 1 μ l of the upstream and downstream primer CF01/CF02 of μ l, 2.5mM dNTP 2.5 μ l, 10 μ M, 5U/ μ l Taq archaeal dna polymerase 0.25 μ l, template DNA 10ng, aqua sterilisa complements to 25 μ l; Replace template DNA as negative control with aqua sterilisa.Mixed solution increases on the PTC-200PCR instrument, and response procedures is: increase in advance 95 ℃ 180 seconds, then 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 60 seconds, 35 circulations were extended 300 seconds at 72 ℃ at last.
4. amplified production electrophoresis detection: get second and take turns pcr amplification product 8 μ l, on 1.5% (weight/volume) sepharose, carried out electrophoresis 30 minutes, voltage is 100-150V, electrophoresis is placed in the EB and dyeed 30 minutes, detected result under UV-light, if there is the band of 280bp, then proof has the existence of Ceratocystis fagacearum (Bretz) Hunt.
These routine his-and-hers watches 1 30 listed bacterial strains detect, Nested pcr amplification result shows, have only 2 strains A TCC 200423 of Ceratocystis fagacearum (Bretz) Hunt and the specific band that ATCC 200425 can amplify the 280bp size, and negative control and other bacterial strain do not amplify specific band.
Fig. 1 is seen in the gel electrophoresis of part bacterial strain amplification products therefrom in the table 1.
Among Fig. 1, M is 2000-bp DNA marker; 1 is C.fagacearum strains A TCC 200423; 2 is C.fagacearum strains A TCC 200425; 3-13 is the part bacterial strain in the table 1; 14 negative contrasts.
Table 1 is used for the strains tested of primer specificity checking
The DNA of bacterial strain Ceratocystisfagacearum ATCC 200423 is diluted to the different concns gradient, is respectively 1 * 10
2Pg/ μ l, 10pg/ μ l, 1pg/ μ l, 1 * 10
-1Pg/ μ l, 1 * 10
-2Pg/ μ l, the DNA 1 μ l that gets each concentration respectively is a template, only carries out pcr amplification one time with Auele Specific Primer CF01/CF02 respectively, the amplification rear electrophoresis the results are shown in Figure 2.All obtain equifinality through the triplicate amplification.
Among Fig. 2, M is 2000-bp DNA marker; DNA concentration in 1～5 expression, the 25 μ l reaction systems is respectively the amplified production of 100pg, 10pg, 1pg, 0.1pg, 0.01pg; The 6th, negative control.Fig. 2 shows that single Auele Specific Primer CF01/CF02 that uses carries out pcr amplification one time, and minimum can detected germ DNA concentration be 10pg/ μ l.
The sensitivity test of embodiment 3Nested PCR
With embodiment 2, the DNA of bacterial strain Ceratocystisfagacearum ATCC 200423 is diluted to the different concns gradient, be respectively 1 * 10
2Pg/ μ l, 10pg/ μ l, 1pg/ μ l, 1 * 10
-1Pg/ μ l, 1 * 10
-2Pg/ μ l, getting 1 μ l is template, carries out the first round, second successively by the step 2 of embodiment 1 and step 3 and takes turns the two-wheeled amplification.
Electrophoresis result through two-wheeled amplification products therefrom is seen Fig. 3, and among the figure, M is 2000-bp DNA marker; 1-5 is that DNA concentration is respectively the amplified production of 100pg, 10pg, 1pg, 0.1pg, 0.01pg in the 25 μ l reaction systems; The 6th, negative control.Show that from Fig. 3 through the two-wheeled pcr amplification, minimum can detected germ DNA concentration be 1pg/ μ l.
Compare with Fig. 2 of embodiment 2, visible Nested PCR of the present invention adopts the twice PCR amplification, and its detection sensitivity is higher than embodiment 2 single detection sensitivities of using Auele Specific Primer CF01/CF02 to carry out a pcr amplification.
Embodiment 4Nested PCR method carries out carrying in the soil detection of Ceratocystis fagacearum (Bretz) Hunt
In going out the soil of bacterium, 0.3g added 1 * 10 respectively
5Individual, 1 * 10
4Individual, 1 * 10
3Individual, 1 * 10
2Individual, 10,1 spore suspension of the bacterial strain Ceratocystis fagacearum ATCC 200423 of totally 6 gradients extract DNA, carry out the first round, second successively by the step 2 of embodiment 1 and step 3 and take turns the two-wheeled amplification.
Product electrophoresis result after the two-wheeled amplification is seen Fig. 4.Among Fig. 4, M is 2000-bp DNA marker; The 1st, positive control; Add the DNA cloning result who is extracted behind the withered spore of 100000,10000,1000,100,10,1 oaks respectively in 2～7 expression 0.3g soil, the 8th, negative control.From Fig. 4 as seen, when utilizing Nested PCR to carry out carrying the detection of Ceratocystis fagacearum (Bretz) Hunt in the soil, the lowest detection limit is 1 spore in the 0.3g soil.
SEQUENCE?LISTING
＜110〉Propagation and Food Test Center of Jiangsu Entry-Exit Inspection and Quarantine Bureau
＜120〉nested PCR detection method of Ceratocystis fagacearum (Bretz) Hunt
<130>jscrj0902
<160>2
<170>PatentIn?version?3.3
<210>1
<211>18
<212>DNA
<213>Artificial
<220>
<223>CF01
<400>1
ggcatggact?tctttctt 18
<210>2
<211>18
<212>DNA
<213>Artificial
<220>
<223>CF02
<400>2
aaggcttgag?tggtgaaa 18
Claims (1)
1. the nested PCR detection method of a Ceratocystis fagacearum (Bretz) Hunt extracts the DNA of Ceratocystis fagacearum (Bretz) Hunt sample, and-20 ℃ of preservation are standby, it is characterized in that, and the Auele Specific Primer CF01/CF02 of design discriminating Ceratocystis fagacearum (Bretz) Hunt, its sequence is:
Upstream primer CF01:5 '-GGCAGGGACTTCTTTCTT-3 ';
Downstream primer CF02:5 '-AAGGCTTGAGTGGTGAAA-3 ';
Utilize universal primer ITS1/ITS4 and Auele Specific Primer CF01/CF02, carry out the Nested pcr amplification of Ceratocystis fagacearum (Bretz) Hunt, amplification step is as follows:
First round amplification: universal primer PCR amplification
The reaction system cumulative volume is 25 μ l, and reactive component is: 10 * reaction buffer 2.5 μ l, the MgCl of 25mM
22.5 μ l, the dNTP 2.5 μ l of 2.5mM, each 1 μ l of the upstream and downstream primer I TS1/ITS4 of 10 μ M, 5U/ μ l Taq archaeal dna polymerase 0.25 μ l, template Ceratocystis fagacearum (Bretz) Hunt sample DNA extracting solution 1 μ l, aqua sterilisa is supplied 25 μ l; The amplified reaction program is: increase in advance 95 ℃ 180 seconds, then 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 60 seconds, 20 circulations were extended 300 seconds at 72 ℃ at last;
Second takes turns amplification: the special primer pcr amplification
The reaction system cumulative volume is 25 μ l, with 10 times of first round PCR product dilutions, gets 1 μ l as template, other reactive component: 10 * reaction buffer 2.5 μ l, 25mM MgCl
22.5 μ l, 2.5mM dNTP 2.5 μ l, each 1 μ l of the upstream and downstream primer CF01/CF02 of 10 μ M, 5U/ μ l Taq archaeal dna polymerase 0.25 μ l, aqua sterilisa is supplied 25 μ l; The amplified reaction program is: increase in advance 95 ℃ 180 seconds, then 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 60 seconds, 35 circulations were extended 300 seconds at 72 ℃ at last;
Amplified production carries out electrophoresis detection:
Get 8 μ l second and take turns pcr amplification product, adopt 1.5% (mass/volume) sepharose, electrophoresis is 30 minutes under voltage 150V, place afterwards in the EB and dyeed 20 minutes, take out, the detection of taking pictures under gel imaging system if there is the DNA specific band of 280bp, determines that then the germ that is detected is a Ceratocystis fagacearum (Bretz) Hunt.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100341950A CN101691605B (en) | 2009-09-02 | 2009-09-02 | Nested PCR detection method of oak wilt |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100341950A CN101691605B (en) | 2009-09-02 | 2009-09-02 | Nested PCR detection method of oak wilt |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101691605A true CN101691605A (en) | 2010-04-07 |
| CN101691605B CN101691605B (en) | 2011-09-21 |
Family
ID=42080319
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009100341950A Expired - Fee Related CN101691605B (en) | 2009-09-02 | 2009-09-02 | Nested PCR detection method of oak wilt |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101691605B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966325A (en) * | 2014-04-30 | 2014-08-06 | 四川农业大学 | Chinese chestnut blight nest-type PCR (polymerase chain reaction) detection kit and use method thereof |
| CN104017886A (en) * | 2014-06-18 | 2014-09-03 | 四川农业大学 | Nested polymerase chain reaction (PCR) detection kit for cylindrocladium scoparium and using method of detection kit |
-
2009
- 2009-09-02 CN CN2009100341950A patent/CN101691605B/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966325A (en) * | 2014-04-30 | 2014-08-06 | 四川农业大学 | Chinese chestnut blight nest-type PCR (polymerase chain reaction) detection kit and use method thereof |
| CN103966325B (en) * | 2014-04-30 | 2015-10-28 | 四川农业大学 | A kind of chestnut epidemic disease bacterium nest type PCR detection reagent and using method thereof |
| CN104017886A (en) * | 2014-06-18 | 2014-09-03 | 四川农业大学 | Nested polymerase chain reaction (PCR) detection kit for cylindrocladium scoparium and using method of detection kit |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101691605B (en) | 2011-09-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Khan et al. | Molecular identification and genetic characterization of Macrophomina phaseolina strains causing pathogenicity on sunflower and chickpea | |
| Zheng et al. | Identification of a novel Alternaria alternata strain able to hyperparasitize Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust | |
| Bent et al. | Root-associated ectomycorrhizal fungi shared by various boreal forest seedlings naturally regenerating after a fire in interior Alaska and correlation of different fungi with host growth responses | |
| Cannon et al. | Lectera, a new genus of the Plectosphaerellaceae for the legume pathogen Volutella colletotrichoides | |
| Fasusi et al. | Propagation and characterization of viable arbuscular mycorrhizal fungal spores within maize plant (Zea mays L.) | |
| CN104774955A (en) | Botryosphaeria dothidea detection method | |
| CN103627798B (en) | Primer group, kit and method for detecting pathogenic bacteria of rice by multiplex PCR (Polymerase Chain Reaction) method | |
| CN102094080B (en) | Quick molecular detection method for simultaneously detecting three kinds of fusarium toxins and application thereof | |
| CN108285927A (en) | Molecular labeling Caps7 and its application in a kind of gene of rice Cd accumulation related gene OsHMA3 | |
| CN101691605B (en) | Nested PCR detection method of oak wilt | |
| CN103146812B (en) | Polymerase chain reaction (PCR) detection method of specificity of botrytis cinerea | |
| CN101928779B (en) | Real-time fluorescent RCR molecular detection kit for leptosphaeria maculans and detection method thereof | |
| CN107119048A (en) | Mulberry Femoral pseudoaneurysm rDNA and its application in mulberry Femoral pseudoaneurysm Molecular Detection | |
| CN101086015B (en) | Mushroom 45 bacteria molecular specific mark and its obtaining method and uses | |
| CN102220320B (en) | Specific molecular marker of volvariella volvacea V23 strain as well as obtaining method and application thereof | |
| CN100392103C (en) | Molecular detection method for Fusarium circinatum | |
| CN101475991B (en) | Method for identifying black fungus bacterial strain 185 and gene sequence for identifying black fungus bacterial strain 185 | |
| Zhu et al. | F usarium asiaticum: an Emerging Pathogen Jeopardizing Postharvest Asparagus Spears | |
| CN101265493B (en) | Molecular mark, detecting method and application of mushroom cultivation strain | |
| CN103290000B (en) | SCAR marker of biocontrol Hypocrea virens, its application and quantitative detection method | |
| CN101086017B (en) | Mushroom 507 bacteria molecular specific mark and its obtaining method and uses | |
| CN105420372A (en) | PCR primer for detecting alternaria kikuchiana and detection method using same | |
| CN104046698B (en) | Quadruple PCR (Polymerase Chain Reaction) primers for synchronously detecting three quarantine phytophthoras on malus and amplification method thereof | |
| CN101086019A (en) | Mushroom 'Shenxiang'93 bacteria molecular specific mark, and method and uses | |
| CN101701247A (en) | Real-time fluorescence PCR detection method of Ceratocystis fagacearum (Bretz) Hunt |
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 | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110921 Termination date: 20120902 |