CN101899506B - Detection primer for No.1 and No.4 physiological strains of fusarium oxysporum f. sp cubense and rapid detection method - Google Patents
Detection primer for No.1 and No.4 physiological strains of fusarium oxysporum f. sp cubense and rapid detection method Download PDFInfo
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Abstract
The invention discloses a detection primer for No.1 and No.4 physiological strains of fusarium oxysporum f. sp cubense and a rapid detection method. The invention provides a universal primer for the No.1 and the No.4 physiological strains of the fusarium oxysporum f. sp cubense and fusarium oxysporum, which can realize the specific simultaneous detection of the No.1 and the No.4 physiological strains of the fusarium oxysporum f. sp cubense in one PCR reaction, and lead the detection sensitivity to achieve 0.2 mu g of fresh mycelium. As the invention simultaneously provides the universal detection primer for a group of the fusarium oxysporum, and the universal detection primer can be used as an internal reference for detecting the DNA quality, the detection primer can avoid the appearance of false negative. The detection method can not only accurately and quickly identify the physiological strains of the fusarium oxysporum f. sp cubense on a template of DNA extracted from banana tissues with the diseases, but also detect the fusarium oxysporum f. sp cubense in the soil, thereby having the advantages of saving time, being simple, simultaneously identifying the No.1 and the No.4 physiological strains, avoiding the false negative and the like.
Description
Technical field
The invention belongs to control of plant disease and pathogenic detection technique field, be specifically related to detection primer and the method for quick of No. 1 and No. 4 physiological strain of banana blight bacteria.
Background technology
The vascular bundle diseases of banana blight dependent territory biography property, development is rapid, and control is difficult, and mortality ratio is high, and because this disease morbidity is being taken out flower bud phase to the phase of bearing fruit more, the as easy as rolling off a log big area that causes is lost receipts, is called as " banana cancer ".Take place in Panama as far back as banana blight in 1890, so the descendant is called Panama disease.Afterwards, this disease took place in Hawaii, America in 1904, and this disease took place in Asia Indonesia in 1916; Between nineteen thirty-five to nineteen thirty-nine, the outburst of South America banana blight big area causes 40,000 hectares of bananas to be ruined; And along with the outlet of local banana propagates into all over the world (Sun Shougong, 1996; Ploetz and Pegg, 1999).By 1987, nearly all there was the generation (Stover and Simmonds, 1987) of banana blight in global banana producing region except the country of Papua New Guinea, island, the South Pacific and Mediterranean Sea bank.
In China mainland, (Wang Bisheng etc., 1997) should disease only take place in the some areas in China Guangxi, Guangdong in nineteen sixty, and the later dwarf banana of yet just causing harm over more than 30 year does not see the banana of causing harm.But; Blight has taken place No. 2 in the banana variety Brazil any of several broadleaf plants and the Guangdong in ten thousand hectares of husky towns, Fanyu, Guangzhou in 1996, and banana blight has also taken place in succession on ground such as contiguous afterwards banana producing region Zhong Shan, Zhuhai, Dongguan, and sick garden sickness rate is 10%～40%; Serious reach (Qi Peikun, 2000 more than 90%; Woods Lan Wen etc., 2003); Through seedling, this disease has been transmitted to banana producing region (woods time lag etc., 2000 in Hainan, Fujian at present; Pu Jinji etc., 2003).At present, the generation of banana blight and constantly spread China's banana production has been constituted great threat has become urgent problem in the banana production.
The pathogenic bacteria of banana blight [Fusarium oxysporum f.sp.cubense (E.F.Smith) Snyder et Hansen] is subordinate to Fusarium, sharp sickle spore Cuba specialized form; Be that a kind of soil habit occupies fungi; So far also do not find to have condition (Snyder and Hansen, 1940; Bu Si, 1988).According to cause harm host's difference of this bacterium; Can be divided into 3 physiological strains; No. 1 physiological strain (FOC1) infects cultivar " big honey what " [Grosmichel (AAA)], fossilia dentis mastodi any of several broadleaf plants (MusaAAB) and the short banana [Dwarf cavendish (AAA)] of banana; This physiological strain is worldwide distribution, and the banana blight that China mainland was reported before 1996 is all caused by this physiological strain; No. 2 physiological strains in Central America, Honduras, El Salvador, Puerto Rico, the Dominican Republic and Virgin Islands, infect triploid hybrid rib banana [Bluggoe (AAB)], do not infect " big honey what " (Stover and Waite, 1960); No. 4 physiological strains (FOC4) are new physiological strains of at first reporting in China Taiwan in 1967; Can infect " big honey what ", short banana, wild any of several broadleaf plants (BB) and rib and refer to any of several broadleaf plants; Especially can infect all Cavendish (AAA) strain of anti-No. 1 physiological strain, crushing maximum (Persley and De Langhe, 1987); Banana industry (Su et al., 1986 in Taiwan in about 10 years time, have almost been destroyed; Hwang, 2004).Afterwards, No. 4 physiological strains are found and report (Ploetz etal., 1990) in Australia, South Africa, Canary Islands and Philippines again.In view of the hazardness of banana wilt germina number-four biological strain, the national Ministry of Agriculture classified it as national plant quarantine object in 2005.
Present No. 4 physiological strains are just caused harm in local banana producing region in China mainland, and implementing quarantine control will be the key that prevents its diffusion.But the banana blight invasioning delitescence is long and the appearance unclear symptoms shows, and has caused the difficulty in the quarantine.
Existing authenticate technology to the wilt physiological strain; The methods that adopt traditional pathogenic bacteria separation and Culture, pathogenic evaluation, the biological tests such as colonial morphology observation of bacterial strain on the Komada improved culture medium more; It takes longer, can't adapt to the production actual needs.
Simultaneously; Except planting fragrant bud any of several broadleaf plants (AAA), also plant dwarf banana (ABB) in the banana production; If dwarf banana infects blight; Its cause of disease or No. 4 physiological strains or No. 1 physiological strain or two physiological strains compound, present detection technique mostly is the detection method to banana wilt germina number-four biological strain, for example application number is 200710032118.2 patented claim disclosed " a kind of method for quick of banana wilt germina number-four biological strain ".There are two significant defectives in existing detection technique: (1) can only judge and confirm whether the plant (comprising fragrant bud any of several broadleaf plants and dwarf banana) of doubtful banana blight is that No. 4 physiological strains are caused a disease; If confirm not to be No. 4 physiological strains through detecting, it is just unknown whether to be that No. 1 physiological strain is done; (2) do not detect the technique means of DNA quality, can not avoid occurring false-negative detected result.
Therefore the banana blight bacteria detection technique of quick, accurate and No. 1 and No. 4 physiological strain of complete detection is explored in research; For its spread in china that prevents disease, take Preventing Countermeasures early, guarantee that China's banana safety in production has important significance for theories and using value.
Summary of the invention
An object of the present invention is to the existing banana blight cause of disease of China, (F.oxysporum f.sp.cubense race 1 race4), designs new detection primer for No. 1, sharp sickle spore Cuba specialized form and No. 4 physiological strains.
Another object of the present invention is to set up a kind of detection method, utilizes above-mentioned detection primer, in the hope of in the shortest time, uses the easiest method and identifies that simultaneously the banana blight pathogenic bacteria is No. 1 or No. 4 physiological strains, still haves both at the same time.
The object of the invention is achieved through following technical proposals:
Provide three pairs to detect primer, see shown in the table 1:
No. 1, No. 4 physiological strains of table 1 banana blight bacteria and sharp sickle spore specific detection primer sequence
The present invention provides the method for utilizing No. 1 and/or No. 4 physiological strain of above-mentioned primer rapid detection banana blight bacteria simultaneously, may further comprise the steps:
1, the extracting banana disease is organized total DNA;
2, organizing extractive total DNA with banana disease is template, carries out the triple PCR amplification with No. 1, No. 4 physiological strains of banana blight bacteria and sharp sickle spore confidential reference items primer;
3, amplified production is detected with 1.0% agarose gel electrophoresis.
Gather the diseased tissues that preferably adopts the banana bulb when banana disease is organized, adopt the CTAB method to carry out extracting, specifically may further comprise the steps:
(1) gets the curved shot stem disease and organize 2g, diseased tissues is shredded, place mortar to add liquid nitrogen and grind to form finely powdered with the sterilization scissors; The extraction buffer [2%CTAB, 0.1mol/L Tris-HCl (pH 8.0), 1.4mol/LNaCl, 20mmol/L EDTA (pH 8.0), 1% beta-mercaptoethanol (time spent adds)] that adds 65 ℃ of preheatings of 5～6mL rapidly, gentle mixing; Lapping liquid is changed in the 15mL centrifuge tube, and in 65 ℃ of following water-bath 1h, every 10min shakes once;
(2) add 5mL phenol chloroform, behind the abundant mixing of thermal agitation, under the room temperature 12,000g, centrifugal 15min;
(3) carefully pipette supernatant (every pipe 600 μ L) in new 1.5mL centrifuge tube, add the equal-volume chloroform, behind the abundant mixing of short term oscillation, under the room temperature 12,000g, centrifugal 10min;
(4) carefully pipette supernatant (every pipe 600 μ L) in new 1.5mL centrifuge tube, add 1/10 3mol/L NaAc (pH 5.2) that gets the supernatant volume and with the isopyknic Virahol of getting supernatant, gentle mixing, under the room temperature 12,000g, centrifugal 4min;
(5) abandoning supernatant to the greatest extent, is that 70% ethanol cleans twice with volumetric concentration, room temperature air dried deposition;
(6) deposition of all same samples is dissolved in the ddH of 65 ℃ of preheatings of 600 μ L
2Among the O, add 4 μ L RNase A (final concentration is 20 μ g/mL) simultaneously, mixing, 37 ℃ of water-bath 1h fully clear up RNA;
(7) add 600 μ L chloroforms; Mixing fully vibrates; The centrifugal 10min of 12000g under the chamber draws in the new 2mL centrifuge tube of supernatant to then, adds 1/10 and gets the 3mol/LNaAc of supernatant volume and the precooling absolute ethyl alcohol of 2 times of supernatant volumes of getting; Behind the mixing this mixed solution is moved to DNA column (commercially available DNA extraction agent box provides), the centrifugal 1min of 10000g under the room temperature then gently;
(8) abandon filtrating, add 700 μ L concentration and be 70% ethanol to the DNA column, the centrifugal 1min of 10000g under the room temperature abandons filtrating and repeated washing DNA column, abandons the centrifugal empty DNA column 1min of 10000g under the room temperature of filtrating back, to guarantee to abandon to the greatest extent ethanol;
(9) the DNA column is contained on the new 1.5mL centrifuge tube, the 1 * TE solution that adds 65 ℃ of preheatings of 100 μ L is in the pillar center, room temperature held 2min, and fully behind the dissolving DNA, the centrifugal 1min of 10000g, centrifugal gained is extractive DNA;
(10) get purity and the concentration that the extractive DNA of 1～2 μ L utilizes UV spectrophotometer measuring DNA sample, with OD
260/ OD
280Ratio be 1.8～2.0 o'clock best, the concentration of adjustment DNA is that 10ng/ μ L～50ng/ μ L is advisable; It is subsequent use to place-20 ℃ of refrigerators to preserve then.
The phenol chloroform of using in the extracting DNA process of the present invention is phenol: chloroform: the mixed solution of primary isoamyl alcohol volume ratio=25: 24: 1, chloroform is chloroform: the mixed solution of primary isoamyl alcohol volume ratio=24: 1.
The system of the said triple PCR amplification of step (2) is as shown in table 2:
The reaction system (25 μ L) that table 2 triple PCR detects
Method for quick of the present invention promptly can be applicable to the detection of banana disease tissue DNA, can be applicable on diseased tissues, be separated to the detection of fungal DNA again, also can directly be used for the detection of the existing banana blight bacteria physiological strain of soil.
Beneficial effect of the present invention is mainly reflected in two aspects:
One, institute of the present invention designed primer.They are that the inventor belongs to and analyzes design on the basis of the isolating a large amount of banana wilt germina number-four biological strain pathogenic related gene sequences in research department (Agricultural University Of South China tropical and subtropical zone fungal studies chamber); Three pairs of primers that from No. 4 physiological strain Auele Specific Primers obtaining, No. 1 physiological strain Auele Specific Primer, sharp sickle spore universal primer, filter out through combined authentication; Its stripe size of PCR product and the quantity of every pair of primer also are not quite similar, therefore directly pathogen identification to the physiological strain rank of banana blight bacteria.
Two, the banana blight bacteria triple PCR detection method that the present invention set up, said method has following meliority:
1. the DNA with the banana disease tissue is a template, in a PCR reaction, can detect simultaneously No. 1 and No. 4 physiological strains;
2. the sharp sickle spore universal primer in the while combination of primers can be used as the quality that internal reference detects DNA simultaneously, has avoided false-negative appearance;
3. detection method of the present invention can be used for from falling ill the detection of banana separate tissue fungal DNA, and the fresh mycelia that the sensitivity of detection reaches 0.2 μ g can detect the existence of this pathogenic bacteria;
4. detection architecture of the present invention can also detect the banana blight bacteria in the soil, and this is significant to the pollution whether detection any of several broadleaf plants garden mould earth receives banana blight bacteria or other sharp sickle spore.
Description of drawings
Fig. 1 banana wilt germina number-four biological strain Auele Specific Primer pcr amplification product electrophorogram;
No. 1 physiological strain Auele Specific Primer of Fig. 2 banana blight bacteria pcr amplification product electrophorogram;
Fig. 3 point sickle spore universal primer PCR amplified production electrophorogram;
Fig. 4 triple PCR detection architecture is to the amplification electrophorogram of banana blight bacteria and control strain DNA;
The sensitivity of Fig. 5 triple PCR detection architecture detects electrophorogram;
Fig. 6 triple PCR detection architecture is to the amplification electrophorogram of the banana tissue DNA of falling ill;
The amplification electrophorogram of the pathogenic bacteria DNA that Fig. 7 triple PCR detection architecture obtains separation;
Fig. 8 triple PCR detection architecture is to mixing the amplification electrophorogram of soil bacteria DNA;
Fig. 9 triple PCR detection architecture detects electrophorogram to mixing soil bacteria DNA sensitivity.
Embodiment
Below in conjunction with accompanying drawing and each routine further explain the present invention of practical implementation.
Embodiment 1: the design and the screening of No. 1, banana blight bacteria and No. 4 physiological strain Auele Specific Primers and sharp sickle spore universal primer sequence
Design primer according to this research department at the distinguished sequence that No. 4, banana blight bacteria of clone and No. 1 physiological strain pathogenic related gene are obtained.
Wherein, No. 1 physiological strain specific DNA sequence of banana blight bacteria table is shown in SEQ ID NO:1, and banana wilt germina number-four biological strain specific DNA sequence table is shown in SEQ ID NO:4, and sharp sickle spore bacterium specific DNA sequence is shown in table SEQ ID NO:7.According to dna sequence dna information provided by the invention; Those skilled in the art also can easily obtain the dna sequence dna that is equal to through following method: (1) obtains from the genomic dna of the corresponding physiological strain bacterial strain of this pathogenic bacteria with the method for pcr amplification according to said dna sequence dna information design primer; (2) method with chemosynthesis obtains.
Primer sequence according to the Rule Design different lengths that designs primer; Guarantee its annealing temperature (54 ℃～56 ℃) in a metastable scope; Guaranteeing that the different detection primer can bring into play best detection effect under same annealing temperature, and it is synthetic to transfer to dna primer Synesis Company.Through conventional PCR reaction system screening; The Auele Specific Primer of obtain detecting banana blight bacteria No. 1, No. 4 physiological strains and sharp sickle spore, respectively like table SEQ ID NO:2 with SEQ ID NO:3, table SEQ ID NO:5 and table SEQ ID NO:6, table SEQ ID NO:8 with show shown in the SEQ ID NO:9; Behind the screening experiment of the different amounts of primer, sum up the PCR reaction system that obtains suiting again, see shown in the table 3.Under this system, react the physiological strain that to identify banana blight bacteria through once conventional PCR.
The reaction system (25 μ L) that table 3 Auele Specific Primer PCR detects
Embodiment 2: the separation of banana blight bacteria, the extracting of DNA and Auele Specific Primer screening and evaluation
Gather the banana blight sample from the banana region of disease that China is different, cut the sick strong little block organization of intersection of diseased plant bulb, adopt the conventional organization partition method to separate (Fang Zhongda; 1998), in PDA (potato dextrose agar), 25 ℃ of following dark culturing 2～3d, after waiting to grow bacterium colony; Choose the most advanced and sophisticated mycelia pure culture of colony edge; Carry out the monospore separation after producing conidium, and single-ascospore strain is carried out pathogenic mensuration, it is subsequent use to preserve pathogenic single-ascospore strain.Bacterium source and numbering are seen table 4.The bacterial strain of preserving is carried out activation on the PDA flat board, 25 ℃ of following dark culturing 3～4d, the picking inoculated by hypha block is to YPD nutrient solution (yeast powder peptone glucose culture solution; 2% glucose; 1% yeast extract powder, 2% peptone) in, shaking culture 3d under 25 ℃, 200rpm.Leach mycelia with B, subsequent use through the water thorough washing final vacuum lyophilize collection mycelia of sterilization.
Table 4 supplies No. 1, examination banana blight bacteria and No. 4 physiological strain strain numbers, source and little type
With the DNA of improved SDS method extracting banana blight bacteria, concrete steps are following:
(1) takes by weighing 100～200mg freeze-drying mycelia and put into the mortar of precooling, add liquid nitrogen and be ground to Powderedly, rapidly go to it in 1.5mL centrifuge tube then and add the DNA extraction damping fluid [prescription: 50mmol/L Tris-HCl (pH 7.2) of 65 ℃ of preheatings of 750 μ L; 50mmol/L EDTA; 3%SDS, 1% beta-mercaptoethanol (time spent adds)], abundant mixing; Place 65 ℃ of water-bath water-bath 1h then, every 10min shakes once;
(2) add equal-volume (750 μ L) phenol chloroform, the abundant mixing of short term oscillation, the centrifugal 15min of 12000g under the room temperature;
(3) carefully pipette supernatant and in a new 1.5mL centrifuge tube, (note not drawing the cell debris of layering intersection), add the equal-volume chloroform, behind the vibration mixing, the centrifugal 10min of 12000g under the room temperature;
(4) carefully pipette supernatant in a new 1.5mL centrifuge tube, add 1/10 3mol/L NaAc that gets the supernatant volume and with the isopyknic Virahol of supernatant, in 4 ℃ of refrigerators, place 30min behind the mixing gently, then the centrifugal 10min of 12000g under the room temperature;
(5) carefully outwell supernatant and also wash twice (centrifuge tube several times gently overturn) of deposition, outwell ethanol, blot mouth of pipe drop with sterilization filter paper then with 70% ethanol of precooling, air-dry;
(6) add 300 μ L ddH
2O dissolving DNA deposition adds 2～4 μ L RNase A (final concentration is 20 μ g/mL) simultaneously, mixing, and 37 ℃ of water-bath 1h fully clear up RNA;
(7) add 300 μ L phenol chloroforms; Mixing fully vibrates; Then in the centrifugal 15min of room temperature 12000g, draw in the new 1.5mL centrifuge tube of supernatant to, add 1/10 3mol/L NaAc that gets the supernatant volume and with the isopyknic Virahol of getting supernatant; In 4 ℃ of refrigerators, place 30min behind the mixing gently, then the centrifugal 10min of 12000g under the room temperature;
(8) carefully removing supernatant and use the volume by volume concentration of precooling is twice (centrifuge tube gently overturns) of 70% washing with alcohol deposition, air-dry DNA deposition;
(9) add 100 μ L and be preheated to 65 ℃ ddH
2O or TE dissolving DNA after treating to dissolve fully, are got purity and concentration that 1～2 μ L utilizes UV spectrophotometer measuring DNA sample, with OD
260/ OD
280Ratio be 1.8～2.0 o'clock for best, the concentration of adjustment DNA is that 10ng/ μ L～50ng/ μ L is advisable, it is subsequent use to place-20 ℃ of refrigerators to preserve then.
With through No. 1, the banana blight bacteria identified and No. 4 physiological strains and control strain the genomic dna of totally 20 bacterial strains be template; So that being carried out PCR, No. 1, No. 4 physiological strains and the specific primer of sharp sickle spore detect respectively; Amplification system is with reference to table 3, and establishes the clear water contrast.Amplified production detects with 1.0% agarose gel electrophoresis.The result is shown in accompanying drawing 1～3, in accompanying drawing 1, accompanying drawing 2 and accompanying drawing 3: M is DL2000; 1～6 is FOC4; 7～11 is FOC1; 12 are the clear water contrast; 13 is the balsam pear wilt; 14 are joint cucurbit wilt bacterium; 15 is the wax gourd blight; 16 is cotton-wilt fusarium; 17 is the eggplant wilt; 18 is the tomato wilt bacterium; 19 is cucumber fusarium axysporum; 20 is half-naked sickle spore; 21 is dry thread Pyrenomycetes; 22 positive contrasts, wherein accompanying drawing 1 is that XJZ2, accompanying drawing 2 are XJZ2 for FJZ3, accompanying drawing 3; 23 are the clear water contrast.No. 4 physiological strain Auele Specific Primers, No. 1 physiological strain Auele Specific Primer and the specific primer of sharp sickle spore can amplify specific band respectively, and size is respectively 593bp, 354bp, 729bp, and peripheral bacterial strain does not then have amplified production.
The primer that the explanation of present embodiment experimental result is filtered out has excellent specificity, can identify the physiological strain of pathogenic bacteria respectively.
Embodiment 3: the triple PCR detection architecture is identified the detection of banana blight bacteria DNA and contrast and peripheral bacterial strain DNA
With No. 1, banana blight bacteria and No. 4 physiological strains and control strain the genomic dna of totally 20 bacterial strains be template, triple PCR detects primer (seeing table 1) and carries out pcr amplification, amplification system is seen table 2, and establishes the clear water contrast.Amplified production detects with 1.0% agarose gel electrophoresis.The result is shown in accompanying drawing 4, and in the accompanying drawing 4, M is DL2000; 1～6 is FOC4; 7～11 is FOC1; 12～13 is FOC4+FOC1; 14 are the clear water contrast; 15 is the balsam pear wilt; 16 are joint cucurbit wilt bacterium; 17 is the wax gourd wilt; 18 is cotton-wilt fusarium; 19 is the eggplant wilt; 20 is the tomato wilt bacterium; 21 is cucumber fusarium axysporum; 22 is half-naked sickle spore; 23 is dry thread Pyrenomycetes; 24 positive contrasts (XJZ2); 25 positive contrasts (FJZ3); 26 are the clear water contrast.It is 593bp and 729bp that No. 4 physiological strain bacterial strains can get two stripe size; No. 1 the physiological strain bacterial strain also has two bands, and size is 354bp and 729bp, and the bacterial strain of sharp sickle spore has a band simultaneously; Size is 729bp, and the half-naked sickle spore of clear water and peripheral bacterial strain, dry thread Pyrenomycetes then do not have amplified production.
Visible by experimental result, use triple PCR detection primer and system and can in same reaction, detect banana blight bacteria simultaneously No. 1 and No. 4 physiological strains; Simultaneously, because the sharp sickle spore Auele Specific Primer that can be used as internal reference is arranged, thus can check the quality of the DNA that puies forward again, thus the appearance of false negative result avoided.
Embodiment 4: the sensitivity of triple PCR detection architecture detects to be identified
Be dissolved in the water of 50 μ L after getting the fresh mycelia 100mg extracting DNA of No. 4 physiological strain strain X JZ2, detect with carrying out triple PCR as template behind 10 times of concentration gradient doubling dilutions, and establish the clear water contrast.Amplified production detects with 1.0% agarose gel electrophoresis.Triple PCR detection architecture sensitivity detected result is seen accompanying drawing 5, and the DNA extract is in dilution 10
4Still can amplify specific band doubly, show that this system can detect the minimum existence that is equivalent to the fresh mycelia of 0.2 μ g.In the accompanying drawing 5, M is DL2000; 1～7 is the genomic dna dilution gradient (10 of XJZ2
0～10
-6); 8 are the clear water contrast.
Embodiment 5: the triple PCR detection architecture is identified the detection of morbidity banana tissue DNA
Banana disease sample is gathered in the banana blight region of disease from Guangdong Province; Carry out the extracting of the total DNA of banana method diseased tissues by detection of the present invention, and be template, use clear water simultaneously; The bacterial strain DNA of known No. 1 and No. 4 physiological strain does contrast, carries out triple PCR and detects.Amplified production detects with 1.0% agarose gel electrophoresis.Shown in accompanying drawing 6, it is 729bp and 593bp amplified band that banana disease tissue DNA and known No. 4 physiological strain DNA obtain two sizes, and two amplified productions of dwarf banana tissue DNA of falling ill and known No. 1 physiological strain size is for Wei not 729bp and 354bp.Can judge that thus the banana disease sample of being gathered is caused that by No. 4 physiological strains the disease of dwarf banana is then caused by No. 1 physiological strain.In the accompanying drawing 6, M is DL2000; 1 is XJZ2 bacterium DNA; 2 is FJZ1 bacterium DNA; 3 is Zhuhai banana disease appearance; 4 is the sick appearance of Zhuhai dwarf banana; 5 is Dongguan banana disease appearance; 6 is the sick appearance of Dongguan dwarf banana; 7 are the clear water contrast.
Embodiment 6: the triple PCR detection architecture is identified the detection of the pathogenic bacteria DNA that separation obtains
The disease sample that embodiment 5 is gathered carries out the separation and Culture of pathogenic bacteria, and concrete steps are with shown in the embodiment 2, the DNA of extracting pathogenic strains; Method is with embodiment 2; And, do contrast with clear water and known No. 1, No. 4 physiological strain bacterial strain DNA simultaneously as template, carry out triple PCR and detect.Amplified production detects with 1.0% agarose gel electrophoresis.Shown in accompanying drawing 7; By pathogenic bacteria that is separated on the banana of falling ill and known No. 4 physiological strains two sizes that all can increase is 729bp and 593bp amplified band, and pathogenic bacteria that is separated to from dwarf banana and known No. 1 physiological strain then obtain two sizes for Wei not 729bp and the product of 354bp.In the accompanying drawing 7, M is DL2000; 1 is XJZ2 bacterium DNA; 2 is FJZ1 bacterium DNA; 3 is Zhuhai banana disease appearance; 4 is the sick appearance of Zhuhai dwarf banana; 5 is Dongguan banana disease appearance; 6 is the sick appearance of Dongguan dwarf banana; 7 are the clear water contrast.
Experimental result has further been verified the result of embodiment 5, thereby no matter the explanation triple PCR detection architecture that the present invention set up is that the DNA of the pathogenic bacteria that still therefrom is separated to all can obtain reliable qualification result to the banana tissue of falling ill.
Embodiment 7: the triple PCR detection architecture is identified the detection of mixing soil bacteria
The fresh mycelia that in three parts of 200mg sterile soils, adds No. 4 physiological strain strain X of 10mg JZ2, No. 1 physiological strain bacterial strain FJZ3 and balsam pear wilt respectively, mixing separately.Extract the genomic dna of each bacterium in the soil with E.Z.N.ASoil DNA kit (OMEGA) test kit (commercial), concrete steps are following:
(1) sample of adding mixing in the 15mL centrifuge tube; The SLX solution that adds 1 milliliter again; Vortex 3 minutes is broken up until sample under top speed, adds the DS damping fluid of 100 μ L again, the vortex mixing; Place 70 ℃ of water-bath temperature to bathe 10min then, concussion sample 2 times is with abundant mixing sample in the process of hatching;
(2) add the SP2 solution of 200 μ L, the 2 minutes abundant mixing samples that vibrates, leave standstill 5min on ice after, 4 ℃ times 13,000g, centrifugal 5min;
(3) the careful transfer got in supernatant to the new 2mL centrifuge tube, notes not being drawn onto deposition or cell debris, add and the isopyknic Virahol of institute's supernatant of getting, and upset centrifuge tube 5～10 times is with the mixing sample, and under the room temperature 13,000g, centrifugal 10min;
(4) carefully outwell supernatant and guarantee that DNA deposition is not poured out, and is upside down in air-dry DNA sample on the thieving paper with centrifuge tube;
(5) ddH of adding 200 μ L
2O and vortex 10 seconds, 65 ℃ of temperature are bathed 10～20min and are precipitated with complete dissolving DNA, of short durationly centrifugally are bonded at the liquid on the centrifugal tube wall with collection.
(6) add 100 μ L by evenly resuspended HTR solution and after shaking 10 seconds, leave standstill 2min under the room temperature, 13,000g, centrifugal 2min;
(7) shift in supernatant to the new centrifuge tube.Attention:, repeat HTR extraction steps 6 if supernatant still presents the color of attaching most importance to;
(8) add 2 μ L RNase A (25mg/ml) and shake 10s, 37 ℃ down temperature bathe 10min to eliminate the DNA in the sample;
(9) add 300 μ L XP2 solution, vibration mixing sample moves in the HiBind DNA column that the 2ml collection tube is housed, under the room temperature 13, and 000g, centrifugal 1min discards filtrating;
(10) HiBind DNA column is placed original collection tube, add the XP2 solution of 300 μ L, under the room temperature 13,000g, centrifugal 1min discards filtrating;
(11) the DNA column is moved on another collection tube, add under 700 μ L SPW washing lotion (diluting with the absolute ethyl alcohol) room temperatures 13,000g, centrifugal 1min discards filtrating, repeats this step once;
(12) under the room temperature 13,000g, centrifugal empty DNA column 2min is to abandon the SPW washing lotion to the greatest extent;
(13) the DNA column is moved on the new 1.5ml centrifuge tube, add TE (or the ddH of 50 μ L
2O) to the central authorities of pillar, after 65 ℃ of temperature are bathed at least 5min, room temperature 13,000g is centrifugal with eluted dna.
(14) get purity and the concentration that the extractive DNA of 1～2 μ L utilizes UV spectrophotometer measuring DNA sample, with OD
260/ OD
280Ratio be 1.8～2.0 o'clock best, the concentration of adjustment DNA is that 10ng/ μ L～50ng/ μ L is advisable; It is subsequent use to place-20 ℃ of refrigerators to preserve then.
What obtain required detection mixes soil bacteria DNA, and carries out triple PCR as template and detect.Amplified production detects with 1.0% agarose gel electrophoresis.Detected result is shown in accompanying drawing 8, and in the accompanying drawing 8, M is DL2000 Marker; 1 pedotheque for inoculation XJZ2; 2 pedotheques for inoculation FJZ3; 3 pedotheques for inoculation balsam pear wilt; 4 are the contrast of sterilization soil; 5 is the FOC4 positive control; 6 is the FOC1 positive control; 7 is sharp sickle spore positive control; 8 are the clear water contrast.The triple PCR detection architecture can detect No. 1, No. 4 physiological strains of banana blight bacteria and other the sharp sickle spore in the soil.Carry out triple PCR after with 10 times of gradient dilutions and increase by extracting DNA in the soil that is mixed with strain X JZ2, amplification such as accompanying drawing 9, in the accompanying drawing 9, M is DL2000 Marker; 1～6 pedotheque DNA dilution gradient (10 for inoculation XJZ2
0～10
-5); 7 is the FOC4 positive control; 8 are the clear water contrast.Experimental result shows that dna profiling can also effectively detect in the soil that is mixed with XJZ2 after diluting 100 times.Embodiment 7 shows that the triple PCR detection architecture can be applicable to the detection of DNA in the banana blight lesion soil, to identify this growing area whether is arranged existing of cause of disease, for producing plantation convenience and technical guarantee is provided.
Claims (7)
1. No. 1, banana blight bacteria and No. 4 physiological strains detect primer, have following sequence:
No. 1 physiological strain specific detection primer sequence is:
Last primer: 5 ' GTTGAGTCTCGATAAACAGCAAT 3 ';
Following primer: 5 ' GACGAGGGGAGATATGGTC 3 ';
No. 4 physiological strain specific detection primer sequence is:
Last primer: 5 ' GCCGATGTCTTCGTCAGGTA 3 ';
Following primer: 5 ' CTGAGACTCGTGCTGCATGA 3 ';
Point sickle spore universal primer sequence is:
Last primer: 5 ' GCAGTCGTACGTCATCGACC 3 ';
Following primer: 5 ' CCATGGCAGATGGCGAGTCA 3 '.
2. method of utilizing the said primer of claim 1 to detect the banana blight bacteria physiological strain is characterized in that may further comprise the steps:
(1) the extracting banana of falling ill is organized total DNA;
(2) organizing extractive total DNA with the banana of falling ill is template, carries out the triple PCR amplified reaction with the said primer of claim 1;
(3) amplified production is detected with 1.0% agarose gel electrophoresis.
3. method according to claim 2 is characterized in that the said banana of falling ill of step (1) is organized as banana bulb tissue.
4. method according to claim 2 is characterized in that the said triple PCR amplification reaction system of step (2) is:
5. the application of the said method of claim 2 is characterized in that being applied to detect branch physiological strain under the fungal DNA of banana blight strain.
6. the application of the said method of claim 2 is characterized in that being applied to detect contained physiological strain among the total DNA of banana blight strain diseased tissues.
7. the application of the said method of claim 2 is characterized in that being applied to detect existing banana blight bacteria physiological strain in the soil.
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| CN103451298B (en) * | 2013-09-09 | 2015-05-06 | 中国农业科学院蔬菜花卉研究所 | Kit for detecting physiological races of brussels sprouts wilt pathogens I and II and detection method thereof |
| CN103773854A (en) * | 2013-12-30 | 2014-05-07 | 中华人民共和国中山出入境检验检疫局 | LAMP method for simultaneously detecting fusarium oxysporum cubeba specialized type No. 1 and No. 4 microspecies |
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| CN114561483A (en) * | 2020-11-27 | 2022-05-31 | 广东省农业科学院植物保护研究所 | Dual detection kit for fusarium oxysporum f.sp.cubense and application thereof |
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| CN114480700B (en) * | 2021-12-27 | 2024-03-19 | 仲恺农业工程学院 | PCR primer, method and application for detecting and identifying banana fusarium wilt bacteria No.1 and No. 4 physiological race |
| CN117721233A (en) * | 2023-12-12 | 2024-03-19 | 中国热带农业科学院环境与植物保护研究所 | Dual RPA primer combination for banana wilt and bacterial wilt, application and detection method thereof |
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| CN100588716C (en) * | 2006-07-03 | 2010-02-10 | 华南农业大学 | Detection primer for banana wilt germina number-four biological strain and method for detecting same |
| CN101178379B (en) * | 2007-12-05 | 2010-12-08 | 华南农业大学 | Method for detecting No.4 race of Fusarium oxysporum f sp. Cubense |
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