CN101698882A - Method for checking cucumber fusarium axysporum molecules - Google Patents
Method for checking cucumber fusarium axysporum molecules Download PDFInfo
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- CN101698882A CN101698882A CN200910073197A CN200910073197A CN101698882A CN 101698882 A CN101698882 A CN 101698882A CN 200910073197 A CN200910073197 A CN 200910073197A CN 200910073197 A CN200910073197 A CN 200910073197A CN 101698882 A CN101698882 A CN 101698882A
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Abstract
The invention relates to a method for checking cucumber fusarium axysporum molecules. The invention solves the problems that the specificity of fungi general primers is not strong and the method is complicated by using the PCR-RFLP method and the nested PCR method needs two steps for amplification and is easy to pollute environment in the existing methods for checking the cucumber fusarium axysporum moleculars. The method for checking the cucumber fusarium axysporum moleculars comprises the following steps: (1) primers Fc-1 and Fc-2; (2) extracting ailing sample genome DNA to perform the PCR amplification; and (3) performing electrophoresis for a PCR amplification product and analyzing the product by a gel imaging system, wherein if a special strip appears at the position of 315bp, the ailing sample is provided with the cucumber fusarium axysporum, therefore the checking step is finished. The primers Fc-1 and Fc-2 have high specificity and sensitivity; and the checking result can be obtained in 2 hours with only one step, and the method is fast, simple and not easy to pollute environment.
Description
Technical field
The present invention relates to a kind of cucumber fusarium axysporum detection method.
Background technology
Cucumber fusarium axysporum is by cucumber fusarium axysporum (Fusarium oxysporum, be also referred to as Fusarium oxysporum) parasitogenic a kind of worldwide silborne fungal diseases, infect extensively, pathogenic bacteria is invaded from root, cause vascular bundle diseases, cause plant withered, all can take place, especially becoming the morbidity of strain phase serious in the time of infertility of plant.
Cucumber fusarium axysporum is particularly serious, in case morbidity, be difficult to eradicate, and along with the increase in continuous cropping time, sickness rate raises, and is a kind of soil-borne disease of stubbornness, the sick general underproduction 20%～30% in field, serious field piece can reach 50%～60%, even total crop failure, is to cause cucumber to produce the major cause that drops in production over a large area at present.Therefore, early the wilt initial stage of infecting is made and detecting and diagnosis, set up the diagnostic method of blight early warning fast and accurately and have important theory and using value, significant to propagation and its harm of the popular and timely and effective control of disease of timely and effective control pathogenic bacteria.
At present, the research of method for checking cucumber fusarium axysporum molecules, people such as Chen Wei adopt PCR-RFLP and nested PCR method, can detect the existence of pathogenic bacteria from the susceptible cucumber seedling of artificial inoculation, PCR-RFLP method the primer is the fungi universal primer, and specificity is not strong, and needs the PCR product is carried out restricted enzyme cutting analysis, method is loaded down with trivial details, and specificity is not strong; Nested PCR method needs 2 step pcr amplifications, has increased opportunities for contamination, and the susceptibility of two kinds of methods has not been analyzed.
Summary of the invention
The present invention seeks to that to adopt the PCR-RFLP method to exist fungi universal primer specificity not reach method by force in detecting loaded down with trivial details in order to solve existing cucumber fusarium axysporum molecules, and adopt nested PCR method to need 2 step pcr amplifications, easy pollution problems, and a kind of method for checking cucumber fusarium axysporum molecules is provided.
Method for checking cucumber fusarium axysporum molecules is realized according to the following steps: one, according to the characteristic of rrna internal transcribed spacer district (ITS) dna sequence dna of cucumber fusarium axysporum, design is to a pair of primers F c-1 and the Fc-2 of the effect of wilt tool specific amplified; Two, extract the sample gene group DNA that catches an illness, adopt primers F c-1 and Fc-2 that genomic dna is carried out pcr amplification; Three, pcr amplification product is carried out electrophoresis with 1 * TAE damping fluid containing on 1.0% sepharose of GelRed staining agent, electrophoresis finishes the back and analyzes with gel imaging system, specific band occurs at the 315bp place, the sample of then catching an illness has cucumber fusarium axysporum, promptly finishes detection; Wherein Fc-1 is 5`-CATACCACTTGTTGCCTC-3` in the step 1, and Fc-2 is 5`-ATTAACGCGAGTCCCACC-3`; Pcr amplification reaction system in the step 2: 25 μ l reaction systems, by 10 * PCR damping fluid, 2.5 μ l (200mM Tris-HCl pH 8.4,200mM KCl, 100mM (NH
4)
28O
4, 20mM MgSO
4), 2.5mM dNTP 2 μ l, 10 μ M primers F c-11 μ l, 10 μ M primers F c-21 μ l, 10ng/ μ l genomic dna 1 μ l, Easy Tag DNAPolymerase 1U and surplus aseptic deionized water are formed; The pcr amplification condition is in the step 2: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s then, 60 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 40 circulations, last 72 ℃ are extended 10min polishing ends, and amplification is finished; Electrophoretic voltage is 4～5V/cm in the step 3.
In the method for checking cucumber fusarium axysporum molecules of the present invention, choose rrna internal transcribed spacer district dna sequence dna with specific cucumber fusarium axysporum, the primers F c-1 and the Fc-2 of design have high degree of specificity, show as this primer only to cucumber fusarium axysporum tool specific amplified product, other 33 kinds of pathogenic fungies and bacterium are not all had amplified production.The susceptibility height of PCR method among the present invention, showing as this PCR method is 10fg to the limit of identification of wilt genomic dna, is 1000 spores/gram soil to the detected level of germ spore in the soil.The rapidity of PCR method shows as only need be reflected in the 2h in 1 step and just can obtain detected result among the present invention, fast, simply, and difficult the pollution.PCR can detect pathogenic bacteria among the present invention from asymptomatic early stage disease plant, and repeatability and stability are high, can be used as cucumber fusarium axysporum early warning diagnostic method and carry out practical application.
Description of drawings
Fig. 1 is to the electrophorogram of primers F c-1 and Fc-2 specific detection in the embodiment one, wherein 1 swimming lane is 100-bp DNA ladder marker (a 100-bp dna molecular amount standard), the 2-5 swimming lane is F.oxysporum f.sp.cucumerineum isolates (cucumber fusarium axysporum), 6 swimming lanes are F.avenaceum (root rotof flax bacterium), 7 swimming lanes are F.proliferatum (fusarium moniliforme), 8 swimming lanes are F.solani (pathogen of soybean root rot), 9 swimming lanes are F.poae (root rotof flax bacterium), 10 swimming lanes are Botrytiscinerea (botrytis cinerea), 11 swimming lanes are Alternaria solani (tomato early blight bacterium), 12 swimming lanes are Ascochyta citrallina (cucumber didymella bryoniae), 13 swimming lanes are Septoria lycopersici (spotted wilt of tomato), 14 swimming lanes are Pythium aphanidermatum (cucumber da mping-off fungi), 15 swimming lanes are Fusarium spp. (pathogen of soybean root rot), the 16-17 swimming lane is Rhizoctonia solani (a cucumber rhizoctonia rot bacterium), 18 swimming lanes are Cladosporium fulvum (leaf muld of tomato bacterium), 19 swimming lanes are Sclerotinia sclerotiorum (soybean sclerotinia crown rot bacterium), 20 swimming lanes are F.graminearum (fusarium graminearum), 21 swimming lanes are Verticilliumdahliae (sunflower verticillium wilt pathogen), and 22 swimming lanes are negative control (negative control);
Fig. 2 is to the electrophorogram of primers F c-1 and Fc-2 specific detection in the embodiment one, wherein 1 swimming lane is 100-bp DNA ladder marker (a 100-bp dna molecular amount standard), the 2-3 swimming lane is F.oxysporum f.sp.cucumerineum isolates (cucumber fusarium axysporum), 4 swimming lanes are F.moniliforme (fusarium moniliforme), 5 swimming lanes are F.solani (Kidney bean pine root fungus), 6 swimming lanes are F.equiseti (Kidney bean leaf mold germ), 7 swimming lanes are F.vasinfectum (stem of Bush Redpepper rot bacterium), 8 swimming lanes are F.oxysporum f.sp.soybean (pathogen of soybean root rot), 9 swimming lanes are Phytophthora melonis (muskmelon parasitica), 10 swimming lanes are Bacillus subtilus B29 (subtilis), 11 swimming lanes are E.coliJM109 (intestinal bacteria), 12 swimming lanes are Bacillus amyloliquefaciens TF28 (bacillus amyloliquefaciens), the 13-14 swimming lane is F.nivale (a Herba Hordei Vulgaris rot bacterium), 15 swimming lanes are Colletortichumlindemuthianum (bean anthrax bacteria), 16 swimming lanes are Sphacelotheca reiliana (Sporisorium reilianum), 17 swimming lanes are Pyricularia oryzae (rice blast fungus), 18 swimming lanes are Botrytis cinerea (botrytis cinerea pers), 19 swimming lanes are Alternaria cucumerina (cucumber alternaria), 20 swimming lanes are Alternaria dauci (black rot of carrot bacterium), 21 swimming lanes are Colletotrichum lagenarium (cucumber anthracnose), the 22-23 swimming lane is Pythium spp. (pathogen of soybean root rot), and 24 swimming lanes are negativecontrol (negative control);
Fig. 3 is that PCR method detects electrophorogram to the susceptibility of the limit of identification of wilt genomic dna in the embodiment one, wherein 1 swimming lane is 100-bp DNA ladder marker (a 100-bp dna molecular amount standard), the 2-9 swimming lane is PCR products using DNA at concentrations of 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, (the 2-9 swimming lane is that template DAN concentration is respectively 10ng to 1fg, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, the pcr amplification product of 1fg), 10 swimming lanes are negative control (negative control);
Fig. 4 is that PCR method is to the susceptibility detection electrophorogram of the detected level of germ spore in the soil in the embodiment one, and wherein 1 swimming lane is negative control (negative control), and the 2-6 swimming lane is numbers ofspores were 10,10
2, 10
3, 10
4And 10
5, (it is 10,10 that the 2-6 swimming lane is respectively spore count to respectively
2, 10
3, 10
4With 10
5Pcr amplification product), 7 swimming lanes are positive control (positive control), 8 swimming lanes are 100-bp DNA ladder marker (100-bp dna molecular amount standard);
Fig. 5 is that the pcr amplification to the cucumber fusarium axysporum of susceptible naturally cucumber root detects figure in the embodiment four, wherein 1 swimming lane is 100-bp DNA ladder marker (a 100-bp dna molecular amount standard), 2 swimming lanes are positive control (positive control), 3 swimming lanes are negative control (negative control), 4 swimming lanes are healthy cucumber root (healthy cucumber root), 5 swimming lanes are healthy cucumber stem (healthy cucumber stem), 6 swimming lanes are infected cucumber stem (Chengxi) (the susceptible cucumber in west of a city village stem), 7 swimming lanes are infected cucumber root (Chengxi) (the susceptible cucumber root in west of a city village), 8 swimming lanes are infected cucumber stem (Xuejia) (susceptible cucumber stem of Xue family), 9 swimming lanes are infectedcucumber root (Xuejia) (the susceptible cucumber root of Xue family), 10 swimming lanes are infected cucumber stem (Jianguo) (the susceptible cucumber in foundation village stem), and 11 swimming lanes are infected cucumber root (Jianguo) (the susceptible cucumber root in foundation village);
Fig. 6 is that the pcr amplification to the cucumber fusarium axysporum of susceptible soil naturally detects figure in the embodiment four, wherein 1 swimming lane is 100-bp DNA ladder marker (a 100-bp dna molecular amount standard), 2 swimming lanes are positive control (positive control), 3 swimming lanes are rhizosphere soil of infectedcucumber (Chengxi) (west of a city village susceptible cucumber root sick soil), 4 swimming lanes are rhizosphere soil ofinfected cucumber (Xuejia) (Xue family's susceptible cucumber root sick soil), 5 swimming lanes are rhizosphere soilof infected cucumber (Jianguo) (foundation village susceptible cucumber root sick soil), 6 swimming lanes are rhizosphere soil of healthy cucumber (Xuejia) (the healthy cucumber root soil of Xue family), and 7 swimming lanes are negative control (negative control);
Fig. 7 is that the pcr amplification to artificial inoculation different times sample cucumber root wilt detects figure in the embodiment four, wherein 1 swimming lane is 100-bp DNA ladder marker (a 100-bp dna molecular amount standard), 2 swimming lanes are positive control (positive control), 3 swimming lanes are root 1 days afterinoculation (inoculating 1 day root sample), 4 swimming lanes are root 3 days after inoculation (connecing 3 days root sample), 5 swimming lanes are root 5 days after inoculation (inoculating 5 days root sample), 6 swimming lanes are root 7 days after inoculation (inoculating 7 days root sample), 7 swimming lanes are root 9days after inoculation (inoculating 9 days root sample), 8 swimming lanes are root 15 days afterinoculation (inoculating 15 days root sample), 9 swimming lanes are root 21 days after inoculation (inoculating 21 days root sample), 10 swimming lanes are root 30 days after inoculation (inoculating 30 days root sample), 11 swimming lanes are healthy root (healthy root), and 12 swimming lanes are negative control (negative control).
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the present embodiment method for checking cucumber fusarium axysporum molecules is realized according to the following steps: one, according to the characteristic of rrna internal transcribed spacer district (ITS) dna sequence dna of cucumber fusarium axysporum, design is to a pair of primers F c-1 and the Fc-2 of the effect of wilt tool specific amplified; Two, extract the sample gene group DNA that catches an illness, adopt primers F c-1 and Fc-2 that genomic dna is carried out pcr amplification; Three, pcr amplification product is carried out electrophoresis with 1 * TAE damping fluid containing on 1.0% sepharose of GelRed staining agent, electrophoresis finishes the back and analyzes with gel imaging system, specific band occurs at the 315bp place, the sample of then catching an illness has cucumber fusarium axysporum, promptly finishes detection; Wherein Fc-1 is 5`-CATACCACTTGTTGCCTC-3` in the step 1, and Fc-2 is 5`-ATTAACGCGAGTCCCACC-3`; Pcr amplification reaction system in the step 2: 25 μ l reaction systems, by 10 * PCR damping fluid, 2.5 μ l (200mM Tris-HCl pH 8.4,200mM KCl, 100mM (NH
4)
2SO
4, 20mM MgSO
4), 2.5mMdNTP 2 μ l, 10 μ M primers F c-11 μ l, 10 μ M primers F c-21 μ l, 10ng/ μ l genomic dna 1 μ l, Easy Tag DNA Polymerase 1U and surplus aseptic deionized water are formed; The pcr amplification condition is in the step 2: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s then, 60 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 40 circulations, last 72 ℃ are extended 10min polishing ends, and amplification is finished; Electrophoretic voltage is 4～5V/cm in the step 3.
Primers F c-1 in the present embodiment and Fc-2 are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
The specific detection result of primers F c-1 and Fc-2 as depicted in figs. 1 and 2 in the present embodiment, primers F c-1 and Fc-2 have high degree of specificity, this primer does not all have amplified production only to cucumber fusarium axysporum tool specific amplified product to other 33 kinds of pathogenic fungies and bacterium.
The sample of catching an illness in the present embodiment step 2 is the root of the susceptible cucumber of nature, artificial inoculation different times sample cucumber root or susceptible naturally soil.
1.0% sepharose in the present embodiment step 3, promptly the mass concentration of agarose is 1.0% in the gel.
Carry out pcr amplification in the present embodiment, its susceptibility detected result as shown in Figure 3 and Figure 4, the susceptibility height of PCR method in the present embodiment, this PCR method is 10fg (Fig. 3) to the limit of identification of wilt genomic dna, is 1000 spores/gram soil (Fig. 4) to the detected level of germ spore in the soil.
Embodiment two: present embodiment and embodiment one are different is that rrna internal transcribed spacer district (ITS) dna sequence dna of cucumber fusarium axysporum in the step 1 is:
cataccactt?gttgcctcgg?cggatcagcc?cgctcccggt?aaaacgggac?ggcccgccag
aggaccccta?aactctgttt?ctatatgtaa?cttctgagta?aaaccataaa?taaatcaaaa
ctttcaacaa?cggatctctt?ggttctggca?tcgatgaaga?acgcagcaaa?atgcgataag
taatgtgaat?tgcagaattc?agtgaatcat?cgaatctttg?aacgcacatt?gcgcccgcca
gtattctggc?gggcatgcct?gttcgagcgt?catttcaacc?ctcaagcaca?gcttggtgtt
gggactcgcg?ttaat。Other step and parameter are identical with embodiment one.
Embodiment three: present embodiment is different with embodiment one or two is to extract the sample gene group DNA that catches an illness in the step 2 20mg sample of catching an illness is placed-70 ℃ of freezing 3h, putting into the sterilization mortar after the taking-up grinds, TES damping fluid (the 100mM Tris that adds 500 μ l, pH8.0,10mMEDTA, 2%SDS, 100 μ g Proteinase Ks), then solution is changed over to and put into 60 ℃ of water-bath incubation 1h in the centrifuge tube, take out the 5M NaCl of back adding 140 μ l and the 10%CTAB of 65 μ l and put into 65 ℃ of water-bath incubation 10min afterwards, add 700 μ l chloroforms again and place 30min at 0 ℃, take out centrifugal 10min under back 4 ℃, upper solution is moved on in the centrifuge tube of new sterilization, add RNA enzyme 5 μ l, add 510 μ l Virahols behind 37 ℃ of incubation 1h and place 30min deposit D NA on ice, then at 4 ℃, centrifugal 10min under the condition of 12000r/min, collecting precipitation, with mass concentration is 70% ice washing with alcohol 1 time, be dissolved in after drying up in the aseptic deionized water, the dilution DNA final concentration is 10ng/ μ l.Other step and parameter are identical with embodiment one or two.
Embodiment four: the present embodiment method for checking cucumber fusarium axysporum molecules is realized according to the following steps: one, according to the characteristic of rrna internal transcribed spacer district (ITS) dna sequence dna of cucumber fusarium axysporum, design is to a pair of primers F c-1 and the Fc-2 of the effect of wilt tool specific amplified; Two, extract the sample gene group DNA that catches an illness, adopt primers F c-1 and Fc-2 that cucumber root genomic dna is carried out pcr amplification then; Three, pcr amplification product is carried out electrophoresis with 1 * TAE damping fluid containing on 1.0% sepharose of GelRed staining agent, electrophoresis finishes the back and analyzes with gel imaging system, specific band occurs at the 315bp place, the sample of then catching an illness has cucumber fusarium axysporum, promptly finishes detection; Wherein Fc-1 is 5`-CATACCACTTGTTGCCTC-3` in the step 1, and Fc-2 is 5`-ATTAACGCGAGTCCCACC-3`; Pcr amplification reaction system in the step 2: 25 μ l reaction systems add 10 * PCR damping fluid, 2.5 μ l (200mM Tris-HCl pH 8.4,200mM KCl, 100mM (NH successively
4)
2SO
4, 20mM MgSO
4), 2.5mM dNTP 2 μ l, each 1 μ l of 10 μ M primers F c-1 and Fc-2,10ng/ μ l genomic dna 1 μ l, Easy Tag DNA Polymerase 1U uses aseptic deionized water polishing to 25 μ l then; The pcr amplification condition is in the step 2: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30sec then, 60 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 40 circulations, last circulation, 72 ℃ are extended 10min polishing ends, and amplification is finished; Electrophoretic voltage is 5V/cm in the step 3.
The sample of catching an illness in the present embodiment is for extracting the susceptible cucumber root of nature; The result that cucumber fusarium axysporum molecules detects specific band occurs at the 315bp place as shown in Figure 5, and the root of susceptible cucumber has wilt naturally.
The sample of catching an illness in the present embodiment is the susceptible soil of nature; The result that cucumber fusarium axysporum molecules detects specific band occurs at the 315bp place as shown in Figure 6, and susceptible naturally soil has wilt.
The sample of catching an illness in the present embodiment is an artificial inoculation different times sample cucumber root; The result that cucumber fusarium axysporum molecules detects specific band occurs at the 315bp place as shown in Figure 7, and artificial inoculation different times sample cucumber root has wilt.
Sequence table
＜110〉Institute of Microbiology, Heilongjiang Academy of Sciences
＜120〉a kind of method for checking cucumber fusarium axysporum molecules
<160>3
<210>1
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the sequence of wilt tool specific amplified effect primers F c-1.
<400>1
cataccactt?gttgcctc 18
<210>2
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the sequence of wilt tool specific amplified effect primers F c-2.
<400>2
attaacgcga?gtcccacc?19
<210>3
<211>315
<212>DNA
＜213〉cucumber fusarium axysporum (Fusarium oxysporum)
<220>
＜223〉rrna internal transcribed spacer district (ITS) dna sequence dna of cucumber fusarium axysporum.
<400>3
cataccactt?gttgcctcgg?cggatcagcc?cgctcccggt?aaaacgggac?ggcccgccag?60
aggaccccta?aactctgttt?ctatatgtaa?cttctgagta?aaaccataaa?taaatcaaaa?120
ctttcaacaa?cggatctctt?ggttctggca?tcgatgaaga?acgcagcaaa?atgcgataag?180
taatgtgaat?tgcagaattc?agtgaatcat?cgaatctttg?aacgcacatt?gcgcccgcca?240
gtattctggc?gggcatgcct?gttcgagcgt?catttcaacc?ctcaagcaca?gcttggtgtt?300
gggactcgcg?ttaat?315
Claims (3)
1. method for checking cucumber fusarium axysporum molecules, it is characterized in that method for checking cucumber fusarium axysporum molecules realizes according to the following steps: one, according to the characteristic of the rrna internal transcribed spacer district dna sequence dna of cucumber fusarium axysporum, design is to a pair of primers F c-1 and the Fc-2 of the effect of wilt tool specific amplified; Two, extract the sample gene group DNA that catches an illness, adopt primers F c-1 and Fc-2 that genomic dna is carried out pcr amplification; Three, pcr amplification product is carried out electrophoresis with 1 * TAE damping fluid containing on 1.0% sepharose of GelRed staining agent, electrophoresis finishes the back and analyzes with gel imaging system, specific band occurs at the 315bp place, the sample of then catching an illness has cucumber fusarium axysporum, promptly finishes detection; Wherein Fc-1 is 5`-CATACCACTTGTTGCCTC-3` in the step 1, and Fc-2 is 5`-ATTAACGCGAGTCCCACC-3`; Pcr amplification reaction system in the step 2: 25 μ l reaction systems, by 10 * PCR damping fluid, 2.5 μ l, 2.5mM dNTP 2 μ l, 10 μ M primers F c-11 μ l, 10 μ M primers F c-21 μ l, 10ng/ μ l genomic dna 1 μ l, Easy Tag DNA Polymerase 1U and surplus aseptic deionized water are formed; The pcr amplification condition is in the step 2: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s then, 60 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 40 circulations, last 72 ℃ are extended 10min polishing ends, and amplification is finished; Electrophoretic voltage is 4～5V/cm in the step 3.
2. a kind of method for checking cucumber fusarium axysporum molecules according to claim 1 is characterized in that the rrna internal transcribed spacer district dna sequence dna of cucumber fusarium axysporum in the step 1 is:
cataccactt?gttgcctcgg?cggatcagcc?cgctcccggt?aaaacgggac?ggcccgccag
aggaccccta?aactctgttt?ctatatgtaa?cttctgagta?aaaccataaa?taaatcaaaa
ctttcaacaa?cggatctctt?ggttctggca?tcgatgaaga?acgcagcaaa?atgcgataag
taatgtgaat?tgcagaattc?agtgaatcat?cgaatctttg?aacgcacatt?gcgcccgcca
gtattctggc?gggcatgcct?gttcgagcgt?catttcaacc?ctcaagcaca?gcttggtgtt
gggactcgcg?ttaat。
3. a kind of method for checking cucumber fusarium axysporum molecules according to claim 1 and 2, it is characterized in that in the step 2 extracting the sample gene group DNA that catches an illness and be the 20mg sample of catching an illness is placed-70 ℃ of freezing 3h, putting into the sterilization mortar after the taking-up grinds, the TES damping fluid that adds 500 μ l, then solution is changed over to and put into 60 ℃ of water-bath incubation 1h in the centrifuge tube, take out the 5M NaCl of back adding 140 μ l and the 10%CTAB of 65 μ l and put into 65 ℃ of water-bath incubation 10min afterwards, add 700 μ l chloroforms again and place 30min at 0 ℃, take out centrifugal 10min under back 4 ℃, upper solution is moved on in the centrifuge tube of new sterilization, add RNA enzyme 5 μ l, add 510 μ l Virahols behind 37 ℃ of incubation 1h and place 30min deposit D NA on ice, then at 4 ℃, centrifugal 10min under the condition of 12000r/min, collecting precipitation, with mass concentration is 70% ice washing with alcohol 1 time, is dissolved in the aseptic deionized water after drying up, and the dilution DNA final concentration is 10ng/ μ l.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181522A (en) * | 2011-03-07 | 2011-09-14 | 中国检验检疫科学研究院 | Primer for detecting cucumber bacterial angular leaf-spot germ |
| 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 |
| CN108841987A (en) * | 2018-07-05 | 2018-11-20 | 南京农业大学 | A kind of rapid detection method of Fusarium graminearum 2-cyano-3-amino-3-phenylancryic acetate resistant strain |
| CN116411125A (en) * | 2023-04-25 | 2023-07-11 | 东北农业大学 | Method for analyzing interaction of cucumber and wheat root system by quantitative PCR method |
-
2009
- 2009-11-12 CN CN200910073197A patent/CN101698882A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181522A (en) * | 2011-03-07 | 2011-09-14 | 中国检验检疫科学研究院 | Primer for detecting cucumber bacterial angular leaf-spot germ |
| CN102181522B (en) * | 2011-03-07 | 2012-11-21 | 中国检验检疫科学研究院 | Primer for detecting cucumber bacterial angular leaf-spot germ |
| 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 |
| CN108841987A (en) * | 2018-07-05 | 2018-11-20 | 南京农业大学 | A kind of rapid detection method of Fusarium graminearum 2-cyano-3-amino-3-phenylancryic acetate resistant strain |
| CN116411125A (en) * | 2023-04-25 | 2023-07-11 | 东北农业大学 | Method for analyzing interaction of cucumber and wheat root system by quantitative PCR method |
| CN116411125B (en) * | 2023-04-25 | 2023-12-01 | 东北农业大学 | Method for analyzing interaction of cucumber and wheat root system by quantitative PCR method |
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