CN104630371A - Molecular detection method for elm blight pathogen - Google Patents
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Abstract
The invention discloses a molecular detection method for elm blight pathogen. The molecular detection method for elm blight pathogen comprises the following steps: by using the DNA of the sample to be detected as a template, performing the first-round reaction of nest PCR via a universal primer ITS1-F/ITS4-B, by taking the product of the first-round reaction as a template and taking the primer OpF and primer OpR as the specific primers, performing the second-round reaction of nest PCR, performing the electrophoresis detection on the reaction product, if the electrophoretogram has 304bp strips, determining that the elm blight pathogen is detected, and otherwise, determining that the elm blight pathogen is not detected, wherein the sequence of the primer OpF is 5'-GCCCGCCACTGGTTTTGA-3'; and the sequence of the primer OpR is 5'-CCGCCCGAACCTTTTCCA-3'. An exact and rapid way is offered for detecting and evaluating the elm blight pathogen by the method, strong specificity is achieved by the primer OpF and the primer OpR, and the detection sensitivity of the method is 10 times of that of the general PCR.
Description
Technical field
The invention belongs to the field of molecular detection of Forest Protection, be specifically related to a kind of Dutch elm disease bacterium (
ophiostoma ulmi(Buisman) Nannf.) molecular detecting method.
Background technology
Dutch elm disease is one of destructive Plant diseases of most, is reported in Belgium and Switzerland the earliest, this disease occurs in succession in the states such as the Belgium greatly about 1881 ~ nineteen twenty-one northwest Europe, France, Holland, has spread all over Italy and Bulgarian to nineteen thirty.The thirties in last century and the seventies this disease there occurs twice large Outbreak, spread all over rapidly Europe, North America and countries and regions, more than 30, the Central Asia, destructive destruction caused to the elm in these areas, brings tremendous economic, Ecological Loss.China territory is vast, elm aboundresources, is distributed widely in China's north and south band, has according to incompletely statistics: 19 kinds, 2 mutation, 6 cultivars and some new cultivated varieties such as American elm, black elm, Huang Yu, Chinese toon elm, Chinese elm.Elm is because its growth is rapid, reproductivity is strong, wide adaptability, be not only the fine tree species that China is township afforestation, or one of chief species of China protection forest, the ecological forest, Timber stands and scenic forest, to China's township afforestation, renewable resources utilizes and improvement of the ecological environment plays a part very important.Dutch elm disease bacterium is mainly caused harm Elm seeds, once import China into, cause very large loss will to the elm kind of China.Now list China's quarantine harmful organisms register in.
At present, in the world for the molecular detecting method of Dutch elm disease bacterium report less, Witthuhn etc. establish PCR-RFLP technology to distinguish the Phylogenetic Relationships that long beak shell belongs to some important kinds, but the method cost is high, process is complicated, and detection sensitivity is low, is difficult to quantitatively, and higher for the purity requirement of sample DNA, therefore be not suitable in each quarantine port promotion and application.
So, be badly in need of setting up a set of detection method fast and accurately to it, the financial loss being difficult to make up caused after entering a country to avoid this pathogenic bacteria.At present, former bacterium can be detected by morphological observation after separate tissue and molecular Biological Detection authentication method disease.But it is large that the former also exists separating difficulty, and the shortcomings such as the cycle is long, inaccurate, in recent years, increasing investigator adopts molecular biological method to detect tree pathogen.As utilized in nucleus or the round pcr that in plastosome, rDNA is detection site all has a wide range of applications in the different categorization levels of Fungal identification.Present stage for Dutch elm disease bacterium need set up a set of fast, accurately, molecular detecting method reliably, pathogenic bacteria that can be entrained in rapid detection immigration timber, in case cause domestic unnecessary loss.
Summary of the invention
Goal of the invention: for the deficiencies in the prior art, the object of this invention is to provide the molecular detecting method of a kind of Dutch elm disease bacterium, to providing one approach accurately and rapidly for the detection of Dutch elm disease bacterium.
Technical scheme: in order to realize foregoing invention object, the technical solution used in the present invention is:
The molecular detecting method of a kind of Dutch elm disease bacterium: with testing sample DNA for template, the first round reaction of nest-type PRC is carried out with universal primer ITS1-F/ITS4-B, with first round reaction product for template, with primer OpF and primer OpR for Auele Specific Primer, carry out nest-type PRC second takes turns reaction, reaction product carries out electrophoresis detection, occurs that 304bp band is and detects Dutch elm disease bacterium, otherwise do not detect in electrophorogram; Wherein, primer OpF sequence is: 5'-GCCCGCCACTGGTTTTGA-3'; Primer OpR sequence is: 5'-CCGCCCGAACCTTTTCCA-3'.
The molecular detecting method of described Dutch elm disease bacterium, concrete steps are as follows:
(1) mycelia of the freezing mistake of 0.5g is got, be put in the flat centrifuge tube of 2mL, adding 2 diameters is 3mm steel ball, closing upper tube cap is placed on beveller, 30f/min, grinding 3min, take out steel ball, add 500 μ L CTAB, freezing 2min in liquid nitrogen, be put in again in 75 DEG C of water-baths and melt 2 minutes, repeat twice, 30min is melted for the last time in 75 DEG C of water-baths, then the phenol of the 25:24:1 of 500 μ L is added: chloroform: primary isoamyl alcohol, to turn upside down mixing, the centrifugal 10min of 12000rpm, get supernatant, add the precooling dehydrated alcohol precipitation 1h of 2 times of volumes, the centrifugal 10min of 12000rpm, abandon supernatant, precipitation uses 70% washing with alcohol, natural air drying, 50 μ L ddH
2o dissolving DNA ,-20 DEG C save backup,
(2) nest-type PRC reaction, the primer that nest-type PRC first round PCR reacts is universal primer ITS1-F/ITS4-B, PCR reaction cumulative volume is 25 μ L, comprise 1 μ L genomic dna, the each 1 μ L of upstream and downstream primer of 10 μMs, 2.5 μ L 10 × PCR buffer, the MgCl of 2 μ L 25mM
2, 2.5 μ L dNTP, 1.25U Taq archaeal dna polymerase, 14.75 μ L ddH
2o; PCR response procedures is: pre-amplification 95 DEG C of 180s, then 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 60s, and 30 circulations, finally extend 10min at 72 DEG C; Nest-type PRC second takes turns reaction with Auele Specific Primer OpF/OpR for primer, nest-type PRC first round PCR primer dilutes 50 times, get 2uL and take turns reaction template as second, PCR reaction system is identical with the first round, and PCR reaction conditions is: pre-amplification 95 DEG C of 180s, then 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 60s, 30 circulations, finally extend 10min at 72 DEG C;
(3) get nested PCR product and carry out agargel electrophoresis detection, occur in electrophorogram that 304bp band is and detect Dutch elm disease bacterium.
In step (1), when carrying out mycelia DNA extraction, after DNA being dissolved in 100uL TE solution, again adding 200uL CTAB and repeating to extract once.
The present invention utilize fungi universal primer ITS1-F/ITS4-B increased Dutch elm disease bacterium nrDNA ITS and carried out sequencing.Pass through gene comparision, devise a pair Dutch elm disease bacterium primer OpF/OpR, designed Auele Specific Primer is put back to the Primer-Blast comparison in GeneBank, and use this to carry out nested PCR amplification to verify the specificity of designed primer to all strains testeds of primer pair.Nest-type PRC is based on internal primer, carries out two-wheeled pcr amplification, can increase the concentration of DNA cloning fragment, plays diluting effect to PCR inhibitory substance, and low DNA concentration and PCR Inhibitor therefore can be avoided the impact detected.
Beneficial effect: the present invention for template, with primer OpF and primer OpR for Auele Specific Primer, carries out nest-type PRC reaction with testing sample DNA, product namely can rapid detection Dutch elm disease bacterium after carrying out electrophoresis detection.The detection that the method is Dutch elm disease bacterium and assessment provide one approach accurately and rapidly, and primer OpF and primer OpR has very strong specificity, and the detection sensitivity of present method can reach 10 times of Standard PCR.
Accompanying drawing explanation
Fig. 1 is 1.2% agargel electrophoresis figure of the DNA sensitivity technique of Auele Specific Primer OpF and primer OpR standard PCR amplification Dutch elm disease bacterium; Wherein, deposition condition, 100v, 57mA, electrophoresis 30min; In figure, swimming lane M is DL2000 DNA Marker; Swimming lane 1 ~ 4 is Dutch elm disease bacteria strain DNA; Swimming lane 5 ~ 8 be Ceratocystis fimbriata Strains (
ceratocystis sensu lato) DNA; Swimming lane 9 ~ 10 be Ceratocystis fimbriata bacterium (
ceratocystis sensu lato) DNA; Swimming lane 11 is distilled water;
Fig. 2 is 1.2% agargel electrophoresis figure of the DNA sensitivity technique of Auele Specific Primer OpF ∕ OpR nested PCR amplification Dutch elm disease bacterium; Wherein, deposition condition, 100v, 57mA, electrophoresis 30min; In figure, swimming lane M is DL2000 DNA Marker; Swimming lane 1 ~ 10 is that swimming lane 11 is distilled water respectively containing DNA profiling 1pg in 20uL PCR reaction system;
Fig. 3 is the 1.2% agargel electrophoresis figure utilizing Auele Specific Primer OpF and primer OpR Standard PCR to detect isolate similar to the colony morphology characteristic and Dutch elm disease bacterium cultural colony that are separated acquisition; Wherein, deposition condition, 100v, 57mA, electrophoresis 30min; In figure, swimming lane M is DL2000 DNA Marker; Swimming lane 1 is Dutch elm disease bacterium positive control; Swimming lane 2 is separation and purification Dutch elm disease bacterium DNA; Swimming lane 3 is bacterial isolate bacterium hybrid dna; Swimming lane 4 is distilled water;
Fig. 4 is 1.2% agargel electrophoresis figure of Dutch elm disease bacterium Auele Specific Primer OpF and primer OpR nest-type PRC response analysis; Wherein, deposition condition, 100v, 57mA, electrophoresis 30min; In figure, swimming lane M is DL2000 DNA Marker; Swimming lane 1 ~ 4 is Dutch elm disease bacteria strain DNA; Swimming lane 5 ~ 8 be Ceratocystis fimbriata Strains (
ceratocystis sensu lato) DNA; Swimming lane 9 ~ 10 be Ceratocystis fimbriata bacterium (
ceratocystis sensu lato) DNA; Swimming lane 11 is distilled water;
Fig. 5 is 1.2% agargel electrophoresis figure of Auele Specific Primer Op1/Op2 and Auele Specific Primer Op3/Op4 nest-type PRC response analysis; Wherein, deposition condition, 100v, 57mA, electrophoresis 30min; In figure, swimming lane M is DL2000 DNA Marker; Swimming lane 1 ~ 4 is Dutch elm disease bacteria strain DNA; Swimming lane 5 ~ 6 be Ceratocystis fimbriata Strains (
ceratocystis sensu lato) DNA; Swimming lane 7 ~ 8 be Ceratocystis fimbriata bacterium (
ceratocystis sensu lato) DNA.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further explained.
Details as Follows for the materials and methods that following examples use:
Confession examination Dutch elm disease bacterium (
ophiostoma ulmi(Buisman) Nannf.) 1 ~ 4 bacterial strain, provide by Entry-Exit Inspection and Quarantine Bureau of Jiangsu Province, 5 ~ No. 10 6 bacterial strains are preserved by Nanjing Forestry University's forest Pathology Lab; Ceratocystis fimbriata Strains (
ceratocystis sensu lato) and Ceratocystis fimbriata bacterium (
ceratocystis sensu lato) as with reference to bacterial strain, be the strain excellent provided by Entry-Exit Inspection and Quarantine Bureau of Jiangsu Province for examination Dutch elm disease bacterium, therefore using Dutch elm disease bacterium as the positive strain detected.
TaKaRa DNA Fragment Purification Kit, E.Z.N.A.
tMcycle-Pure Kit, archaeal dna polymerase, dNTP, pMD19-T carrier, DNA Marker etc. are purchased from the precious biotech firm in Dalian, and primer is synthesized by Nanjing Jin Sirui company.
Embodiment 1
Cetyl trimethylammonium bromide (CTAB) method is adopted to carry out strains tested and mycelia DNA extraction, be specially: the mycelia of getting the freezing mistake of 0.5g, be put in the flat centrifuge tube of 2mL, adding 2 diameters is 3mm steel ball, closing upper tube cap is placed on beveller, 30f/min, grinding 3min, take out steel ball, add 500 μ L CTAB, freezing 2min in liquid nitrogen, be put in again in 75 DEG C of water-baths and melt 2 minutes, repeat twice, 30min is melted for the last time in 75 DEG C of water-baths, then the phenol of the 25:24:1 of 500 μ L is added: chloroform: primary isoamyl alcohol, to turn upside down mixing, the centrifugal 10min of 12000rpm, get supernatant, add the precooling dehydrated alcohol precipitation 1h of 2 times of volumes, the centrifugal 10min of 12000rpm, abandon supernatant, precipitation uses 70% washing with alcohol, natural air drying, 50 μ L ddH
2o dissolving DNA ,-20 DEG C save backup.Wherein, when carrying out mycelia DNA extraction, after DNA being dissolved in 100uL TE solution, again adding 200uL CTAB and repeating to extract once.Extract DNA and detect the requirement all meeting PCR through ultraviolet spectrophotometer and 1.2 % agarose gel electrophoresis.
With the DNA of Dutch elm disease bacterium for template, with fungi universal primer ITS1-F ∕ ITS4-B, pcr amplification is carried out to Dutch elm disease bacterium, reaction system is: it is 25 μ L that each PCR reacts cumulative volume, comprise 25 μ L genomic dnas, the each 1 μ L of upstream and downstream primer of 10 μMs, 2.5 μ L 10 × PCR buffer, the MgCl of 2 μ L 25mM
2, 2.5 μ L dNTP, 1.25 U Taq archaeal dna polymerases (Takara), 14.75 μ L ddH
2o.PCR response procedures is: pre-amplification 95 DEG C of 180s, then 94 DEG C of 30s, and 58 DEG C of 30s, 72 DEG C of 60s, 35 circulations, finally extend 10min at 72 DEG C.Wherein, the sequence of ITS1-F is: the sequence of 5'-CTTGGTCATTTAGAGGAAGTAA-3', ITS4-B is: 5'-CAGGAGACTTGTACACGGTCCAG-3'.
PCR primer is reclaimed through E.Z.N.A.TMCycle-Pure Kit, connect pMD19-T carrier, transform JM109, select positive colony and serve the order-checking of Hai Meiji company after Insert Fragment checking, the sequence of acquisition is carried out Primer-Blast comparison on GeneBank, result is learnt consistent with Dutch elm disease bacterium sequence existing on GeneBank, therefore judges that sequencing result is as Dutch elm disease bacterium.
Embodiment 2
GeneBank obtains Dutch elm disease bacterium and belongs to all the other ITS sequence 9 of planting together, the sequence of Dutch elm disease bacterium is obtained together with embodiment 1, carry out Clustal W Multiple sequence alignments, Primer premier 5.0 is utilized to devise the Auele Specific Primer of a pair Dutch elm disease bacterium: OpF/OpR, OpF sequence is: 5'-GCCCGCCACTGGTTTTGA-3', OpR sequence is: 5'-CCGCCCGAACCTTTTCCA-3', and amplified production size is 304bp.
The primer that nest-type PRC first round PCR reacts is universal primer ITS1-F ∕ ITS4-B, carries out pcr amplification according to the system in embodiment 1 and step.Standard PCR and nest-type PRC second take turns reaction with Auele Specific Primer OpF/OpR for primer, nest-type PRC first round PCR primer dilutes 50 times, get 2uL and take turns reaction template as second, PCR reaction system is identical with the first round, and response procedures is: pre-amplification 95 DEG C of 180s, then 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 60s, 35 circulations, finally extend 10min at 72 DEG C.
Designed Auele Specific Primer OpF/OpR is put back to the Primer-Blast comparison in GeneBank, only have the rRNA gene of Dutch elm disease bacterium can produce the fragment of 304bp, describe this to the specificity of primer in current GeneBank.In addition, adopt this to primer pair for examination 4 strain Dutch elm disease bacterium, 4 kinds of common Ceratocystis fimbriata Strainses, and 2 kinds of common Ceratocystis fimbriata bacterium carry out nest-type PRC, result as shown in Figure 1, all Dutch elm disease bacterium amplifications are all positive, and obtaining size is the amplified production of 304bp, and other reference strain amplifications are negative.Adopt fungi ITS universal primer to carry out PCR to all for examination fungal DNA, all obtain the product of about 750bp size.Result shows that this can only carry out specific amplification to Dutch elm disease bacterium to nest-type PRC primer, and other fungal DNAs can not obtain corresponding amplified production.
In order to confirm the specificity of PCR primer further, with TaKaRa DNA Fragment Purification Kit(purchased from the precious biotech firm in Dalian) nested PCR electrophoresis product is carried out cutting glue recovery, connect pMD19-T carrier, transform JM109, select positive colony and serve the order-checking of Hai Meiji company after Insert Fragment checking, after order-checking and aim sequence comparison confirm the exactness of amplified production.Nested PCR product gel reclaims sequencing result and shows, the amplified production of 304bp size mates completely with one section of sequence in Dutch elm disease bacterium ITS district.Result shows, primer OpF/OpR can amplify the fragment of Dutch elm disease bacterium ITS district 304bp specifically, and does not have amplified production to other fungies.The sequence of the amplified production of 304bp size is as shown in SEQ ID NO.1.
Embodiment 3
In order to assess the sensitivity of nest-type PRC, being diluted by Dutch elm disease bacterium DNA is 10 gradients, respectively containing DNA profiling 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, 100ag in 20uL PCR reaction system, Standard PCR and nest-type PRC is carried out respectively with Auele Specific Primer, Standard PCR with Auele Specific Primer OpF/OpR for primer, Dutch elm disease bacterium DNA is template, and reaction system and reaction conditions take turns reaction with nest-type PRC in embodiment 2 second.As shown in Figure 1, Standard PCR can only detect the DNA of 10pg to result, and as shown in Figure 2, the most low energy of nest-type PRC detects the DNA of 1pg, and nest-type PRC detection sensitivity is 10 times of Standard PCR.
Embodiment 4
In order to study the Detection results of the designed Auele Specific Primer of this test further, carry out nest-type PRC detection to the 2 years potted plant elm seedlings of life using Dutch elm disease bacterium, nest-type PRC reaction system and reaction conditions are with embodiment 3.As shown in Figure 3, use the elm seedling detected result of Dutch elm disease bacterium for positive, the elm seedling not using Dutch elm disease bacterium does not have amplified production to result.
Embodiment 5
In order to assess the specificity of nest-type PRC, nest-type PRC is carried out respectively with the multipair Auele Specific Primer of design, respectively with the Auele Specific Primer OpF/OpR of embodiment 2 for test primer, the two pairs of Auele Specific Primers designed in conventional manner are contrast, with Dutch elm disease bacterium DNA for template, reaction system and reaction conditions and method are with embodiment 2, result as shown in Figure 4 and Figure 5, only have Auele Specific Primer OpF/OpR can amplify the clear band of specificity 304bp in Dutch elm disease bacterium, other primers all cannot amplify respective strap in Dutch elm disease bacterium, visible primer OpF/OpR is to the specificity of Dutch elm disease bacterium.Wherein, the sequence of two couples of Auele Specific Primers Op1/Op2, Op3/Op4 is respectively: Op1 sequence is: 5'-ATTTCGCTGCGTTCTTCA-3', Op2 sequence is: 5'-TTACTGCTTTGGCGTGGT-3', and amplified production size is 159bp; Auele Specific Primer Op3/Op4, Op3 sequence is: 5'-TCCTCCGCTTATTGATAT-3', Op4 sequence is: 5'-TTACTGCTTTGGCGTGGT-3'.
SEQUENCE LISTING
<110> Nanjing Forestry University
The molecular detecting method of a <120> Dutch elm disease bacterium
<130> 100
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 304
<212> DNA
<213> Ophiostoma ulmi (Buisman) Nannf.
<400> 1
gcccgccact ggttttgagg gccctgccgc cacagagggc ggaggagccc caacgccagc 60
gcacccaaaa gggcatgctg aggggggaaa tgacgctcgg acaggcatgc ccgccagaat 120
gctggcgggc gcaatgtgcg ttcaaagatt cgatgactcg ctgaattctg caattcgcat 180
tacgtatcgc atttcgctgc gttcttcatc gatgccagag ccaagagatc cgttgttgaa 240
agttttggct gtttttgttt gtttctcaga cgtttcgtta ctggtttgga aaaggttcgg 300
gcgg 304
<210> 2
<211> 22
<212> DNA
<213> Artificial
<220>
The sequence of <223> ITS1-F
<400> 2
cttggtcatt tagaggaagt aa 22
<210> 3
<211> 23
<212> DNA
<213> Artificial
<220>
The sequence of <223> ITS4-B
<400> 3
caggagactt gtacacggtc cag 23
<210> 4
<211> 18
<212> DNA
<213> Artificial
<220>
<223> OpF sequence
<400> 4
gcccgccact ggttttga 18
<210> 5
<211> 18
<212> DNA
<213> Artificial
<220>
<223> OpR sequence
<400> 5
ccgcccgaac cttttcca 18
Claims (3)
1. the molecular detecting method of a Dutch elm disease bacterium, it is characterized in that: with testing sample DNA for template, the first round reaction of nest-type PRC is carried out with universal primer ITS1-F/ITS4-B, with first round reaction product for template, with primer OpF and primer OpR for Auele Specific Primer, carry out nest-type PRC second takes turns reaction, and reaction product carries out electrophoresis detection, occur in electrophorogram that 304bp band is and detect Dutch elm disease bacterium, otherwise do not detect; Wherein, primer OpF sequence is: 5'-GCCCGCCACTGGTTTTGA-3'; Primer OpR sequence is: 5'-CCGCCCGAACCTTTTCCA-3'.
2. the molecular detecting method of Dutch elm disease bacterium according to claim 1, it is characterized in that, concrete steps are as follows:
(1) mycelia of the freezing mistake of 0.5g is got, be put in the flat centrifuge tube of 2mL, adding 2 diameters is 3mm steel ball, closing upper tube cap is placed on beveller, 30f/min, grinding 3min, take out steel ball, add 500 μ L CTAB, freezing 2min in liquid nitrogen, be put in again in 75 DEG C of water-baths and melt 2 minutes, repeat twice, 30min is melted for the last time in 75 DEG C of water-baths, then the phenol of the 25:24:1 of 500 μ L is added: chloroform: primary isoamyl alcohol, to turn upside down mixing, the centrifugal 10min of 12000rpm, get supernatant, add the precooling dehydrated alcohol precipitation 1h of 2 times of volumes, the centrifugal 10min of 12000rpm, abandon supernatant, precipitation uses 70% washing with alcohol, natural air drying, 50 μ L ddH
2o dissolving DNA ,-20 DEG C save backup,
(2) nest-type PRC reaction, the primer that nest-type PRC first round PCR reacts is universal primer ITS1-F/ITS4-B, PCR reaction cumulative volume is 25 μ L, comprise 1 μ L genomic dna, the each 1 μ L of upstream and downstream primer of 10 μMs, 2.5 μ L 10 × PCR buffer, the MgCl of 2 μ L 25mM
2, 2.5 μ L dNTP, 1.25U Taq archaeal dna polymerase, 14.75 μ L ddH
2o; PCR response procedures is: pre-amplification 95 DEG C of 180s, then 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 60s, and 30 circulations, finally extend 10min at 72 DEG C; Nest-type PRC second takes turns reaction with Auele Specific Primer OpF/OpR for primer, nest-type PRC first round PCR primer dilutes 50 times, get 2uL and take turns reaction template as second, PCR reaction system is identical with the first round, and PCR reaction conditions is: pre-amplification 95 DEG C of 180s, then 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 60s, 30 circulations, finally extend 10min at 72 DEG C;
(3) get nested PCR product and carry out agargel electrophoresis detection, occur in electrophorogram that 304bp band is and detect Dutch elm disease bacterium.
3. the molecular detecting method of Dutch elm disease bacterium according to claim 1, is characterized in that: in step (1), when carrying out mycelia DNA extraction, after DNA is dissolved in 100uL TE solution, again adds 200uL CTAB and repeats to extract once.
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| CN108315392A (en) * | 2018-05-15 | 2018-07-24 | 北京市园林科学研究院 | A kind of nest type PCR detection reagent and its application in the detection of smoke tree wilt |
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| US5539095A (en) * | 1994-08-04 | 1996-07-23 | Board Of Trustees Operating Michigan State University | Chitinase cDNA clone from a disease resistant American elm tree |
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| US5539095A (en) * | 1994-08-04 | 1996-07-23 | Board Of Trustees Operating Michigan State University | Chitinase cDNA clone from a disease resistant American elm tree |
Non-Patent Citations (2)
| Title |
|---|
| N.D.PIPE, ET AL.: "Evolutionary Relationships of the Dutch Elm Disease Fungus Ophiostoma novoulmi to Other Ophiostoma Species Investigated by Restriction Fragment Length Polymorphism Analysis of the rDNA Region", 《J. PHYTOPATHOLOGY》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104911262A (en) * | 2015-05-28 | 2015-09-16 | 中南林业科技大学 | Phyllachora dalbergiicola molecular detection primer and fast detecting method for dalbergia odorifera |
| CN104911262B (en) * | 2015-05-28 | 2018-01-02 | 海南科大林业有限公司 | A kind of dalbergia odorifera tar spot bacterium molecule detection primer and quick determination method |
| CN108315392A (en) * | 2018-05-15 | 2018-07-24 | 北京市园林科学研究院 | A kind of nest type PCR detection reagent and its application in the detection of smoke tree wilt |
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Application publication date: 20150520 Assignee: Nanjing Forest Protection Bird Biotechnology Co., Ltd. Assignor: Nanjing Forestry University Contract record no.: 2018320000399 Denomination of invention: Molecular detection method for elm blight pathogen Granted publication date: 20170118 License type: Common License Record date: 20181218 Application publication date: 20150520 Assignee: Nanjing biological pest control technology, Limited by Share Ltd Assignor: Nanjing Forestry University Contract record no.: 2018320000400 Denomination of invention: Molecular detection method for elm blight pathogen Granted publication date: 20170118 License type: Common License Record date: 20181218 Application publication date: 20150520 Assignee: Guizhou Linxin Ecological Agriculture Technology Co., Ltd. Assignor: Nanjing Forestry University Contract record no.: 2018320000401 Denomination of invention: Molecular detection method for elm blight pathogen Granted publication date: 20170118 License type: Common License Record date: 20181218 |