CN102690887A - LAMP (loop-mediated isothermal amplification) detection primers of banana fusarium wilt bacteria No. 4 microspecies and application thereof - Google Patents
LAMP (loop-mediated isothermal amplification) detection primers of banana fusarium wilt bacteria No. 4 microspecies and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biotechnology, and particularly relates to LAMP (loop-mediated isothermal amplification) detection primers of banana fusarium wilt bacteria No. 4 microspecies and an application thereof. The LAMP detection primers of the banana fusarium wilt bacteria No. 4 microspecies in a reaction system are F3, B3, FIP, BIF, F-loop and B-loop, the DNA of the banana fusarium wilt bacteria in an ill plant tissue is extracted, and LAMP isothermal amplification detection is carried out, so that the banana fusarium wilt bacteria can be quickly and specifically detected. The LAMP detection primers of the banana fusarium wilt bacteria No. 4 microspecies disclosed by the invention have the advantages of strong specificity, high sensibility, speediness, high efficiency and easiness in operation, the result of the detection method is reliable, and the primers have high application values in the aspects of on-site rapid detection and early diagnosis of the banana fusarium wilt bacteria.
Description
Technical field
The invention belongs to the molecular Biological Detection field of Plant diseases, the LAMP that is specifically related to No. 4 microspecies of a kind of banana blight bacteria detects primer and application thereof.
Background technology
By sharp sickle spore bacterium Cuba specialized form (
Fusarium oxysporumF.sp.
Cubense) banana blight that causes is a kind of crushing soil-borne disease, also is one of China's importquarantine object.Found this disease in the Hawaii, America reported first in 1904, caused heavy losses in Panama because of this disease in 1910, this disease world that extensively distributes produces banana country at present.China mainland was at first found on the Saigon in Guangxi any of several broadleaf plants in nineteen sixty should disease, and this disease takes place domestic soon each important Jiao Qu in succession, also finds banana blight in recent years in Fujian, and has and continue the situation that expansion spreads.It infects the source mainly is germ-carrying suction bud, the residual body of diseased plant and the soil that carries disease germs.This germ can carry out closely its spread in china through the residual body of diseased plant, farming instrument, carry disease germs soil and lesion irrigation water, rainwater, worm etc., and the suction bud of disease carrying germ, soil and secondary seedling are the modes of long-distance communications.It is reported that banana blight bacteria has 4 physiological strains, wherein production is caused serious harm with No. 1 microspecies and No. 4 microspecies.
In recent years; Along with Agricultural Products Trade and the alternative development of both sides of the Straits agricultural; Kind and the quantity of introducing agricultural-food and seedling from country, banana blight epidemic-stricken area increase sharply; Banana blight bacteria also imports in trade along with these plant prods and plant, and China's agricultural development and Agricultural Products Trade have been constituted serious threat.At present, the method for control banana blight mainly be strengthen to detect, quarantine, be strictly on guard against that seedling gets into no epidemic-stricken area in spite of illness, the anosis tissue cultured seedling of popularizing planting is made regular check on Jiao Yuan, finds that fragmentary diseased plant should in time remove destruction, and spreads fertilizer over the fields lime or urea processing soil; Plant the banana variety of disease-resistant and anti-disease; Cash crop such as other crop of crop rotation such as paddy rice, peanut, sugarcane are then answered in the grave illness district.The quarantine detection technique of this disease is the weak link in the former studies; Therefore; Still do not have the diffusion of highly effective way control banana blight both at home and abroad at present and spread and cause harm, simultaneously, the construction of anosis seedling production base is for no epidemic-stricken area farm crop; Especially the control of perennial crop banana blight such as fruit tree is most important; Therefore in order in time to intercept importing into of external pathogenic bacteria, prevent that banana blight from importing Pest-or disease-free area into from the epidemic-stricken area, set up that a cover is stable, reliable, easy, quick, the sensitive molecular detecting method is to banana seedlings and to plant that regional soil detects and monitor be necessity very.
The quarantine detection technique of traditional pathogenic is because easy, easy row; Still be used at present; But because sensitivity is low and length consuming time, can not satisfy Plant diseases diagnosis under the new situation and disease control requirements of one's work, it is supporting with it to press for novel detection technique.Various countries all are devoted to the molecular Biological Detection Study on Technology in recent years, have obtained very big progress, and the cause of disease detection method that wherein is the basis with polymerase chain reaction (PCR) technology has been successfully applied to the detection and the evaluation of various plants cause of disease.The PCR molecular detection technology of plant pathogeny organism is the developing direction in current this field, and many important causal organisms have all been set up molecular detection technology, and increasing molecular detection kit or is developed.Because odjective cause; Though China is starting late aspect the plant pathogeny organism molecular detection technology; But also developed many technology that plant pathogeny organism detects that are used at present, more especially had the detection method of independent intellectual property right, and it has been put in commercial prodn and the application.
At present existing utilize the molecule means to identify banana blight bacteria, promptly sharp sickle spore bacterium Cuba specialized form (
Fusarium oxysporumF.sp.
Cubense) method, John A. etc. has reported the Molecular Detection of banana blight bacteria based on multiplex PCR; Liao Linfeng etc. have reported with randomly amplified polymorphic DNA (RAPD) technology, from 90 RAPD random primers, filter out primer and carry out the RAPD-PCR amplification, and the design specific primer is as the specific SCAR label of No. 4 microspecies of banana blight bacteria; Liu Jingmei etc. are with the RAPD technology; From 200 random primers, filter out 8 primers and can produce 12 of physiological strain RAPD marks; Design corresponding SCAR primer respectively according to these specific fragment sequences; Through to the check of the pcr amplification of 18 bacterial strains, have 4 RAPD marks successfully to be converted into the SCAR mark, wherein 1 of Race 1-SCAR mark, 2 of Race 4-SCAR marks, can identify 1 of the SCAR mark of 2 microspecies simultaneously; Lv Weicheng etc. analyze the banana blight bacteria genetic diversity, have made up banana blight bacteria physiological strain ITS-SCAR double PCR detection architecture, and this method can detect No. 1, banana blight bacteria and No. 4 physiological strains simultaneously; Ye Jianjuns etc. have made up the banana pathogenic bacteria FOC4 molecular detecting method that combines with the SCAR molecule marker with the ITS order-checking; (design special primer such as the conserved sequence of β-tubulin) detects banana blight bacteria according to ITS sequence, 16SrDNA sequence, 'beta '-tubulin gene for kingdom's sweet smell, Ye Mingzhen, the gorgeous perfume (or spice) of lacquer, Chen Jinghua etc.
PCR specific detection technology still needs expensive professional instrument and molecular biology reagent such as PCR appearance, electrophoresis and gel imaging system at present; And need molecular biology Specialty Experiment personnel operation; More than detect and under laboratory condition, just can carry out, limited PCR detection method applying aborning.
Circulation isothermal amplification technology (Ioop-mediated isothermal amplification is called for short LAMP) is a kind of New Cycle constant temperature nucleic acid amplification technology by people such as the Japanese Rong Yan Notomi of Co., Ltd. exploitation in 2000.LAMP reaction is designed 4 primers to 6 sites of target gene, utilize a kind of chain type substitute activity archaeal dna polymerase (
BstDNA polymerase), (65 ℃ of 60 –) insulation 30 – 90 minutes under constant temperature, can accomplish amplified reaction.Because the high efficiency and the isothermal rapid amplifying of LAMP reaction can be with amplifications 10 in 90 minutes
9– 10
10Doubly.Methods such as optical dye visual observations, agarose gel electrophoresis and turbidity observation are generally adopted in the detection of LAMP amplified production.Because the LAMP reaction is simple, quick, efficient, the economic dispatch characteristic, thereby has very application prospects.LAMP detects and is mainly used in people and animals' pathogen, food safety and sanitary detection at present, and report is few in phytopathogen detects, and the detection of banana blight bacteria does not all appear in the newspapers both at home and abroad.
Summary of the invention
The LAMP that the invention provides No. 4 microspecies of a kind of banana blight bacteria detects primer.
The present invention also provides the LAMP detection method of No. 4 microspecies of a kind of banana blight bacteria.
The present invention provides the LAMP detection kit of No. 4 microspecies of a kind of banana blight bacteria in addition.
It is following that the LAMP of No. 4 microspecies of a kind of banana blight bacteria detects primer:
Outside primers F 3:5 '-AGGACCTCTTCGAATGGCA-3 ',
B3：5’?-GACGCTGCAGCTATGACAA-?3’；
Inboard primers F IP:5 '-GGTGGCTCAATAGCCCAGTGAACCGATACCTGTGAAGTCGC-3 ',
BIP：5’?-CGACATCATCAGCATCTCCGCTAGCTTTGGCTCTTGTGACAG-?3’；
Ring primers F-loop:5 '-GCCTAATTGAACATTCAGTATAAAC-3 ',
B-Loop：5’?-?ACTCCAAGGAACTAGACGACG?-?3’。
The LAMP detection method of No. 4 microspecies of a kind of banana blight bacteria comprises design of primers, LAMP amplification and amplification observation, and the reaction system of said LAMP detection method comprises: primer mixed solution, reaction mixture, 1.0U
BstArchaeal dna polymerase and 25ng dna profiling are supplied 25 uL with the sterilization distilled water; Said primer mixed solution is by the described outside of claim 1 primers F 3, B3; Inboard primers F IP, BIP and ring primer B-loop, F-loop form; Primers F 3 and each 5pmol of B3 wherein, each 40 pmol of inboard primers F IP and BIP, ring primer B-loop, each 20 μ mol of F-loop; Said reaction mixture is prepared as follows: 40mM Tris-HCL, 20mM (NH
4)
2SO
4, 20mM KCL, 16 mM MgSO
4, 0.2% Triton X-100,1.6M trimethyl-glycine and 2.8 mM dNTPs.
The reaction conditions of said LAMP detection method is at 60-65 ℃ of incubation 60 min, 80-85 ℃ of insulation 10min.Said reaction conditions is preferably at 63 ℃ of incubation 60 min, 80 ℃ of insulation 10min.
Said amplification is viewed as optical dye visual observations method or agarose gel electrophoresis method; Said optical dye visual observations method: in the final amplified production of LAMP reaction, add the 1uL developer, said developer is a SYBR green I, and the colour developing result observes green fluorescence and is judged as the positive, the orange feminine gender that is judged as; Said agarose gel electrophoresis method: the final amplified production of getting the 2uLLAMP reaction detects with 2% agarose gel electrophoresis, if the distinctive trapezoid belt of LAMP is judged as the positive, amplified band does not occur and is judged as feminine gender.
No. 4 specific detection kit of microspecies of a kind of banana blight bacteria comprise:
1) primer mixed solution: outside primers F 3 and each 5pmol of B3, each 40 pmol of inboard primers F IP and BIP, each 20 μ mol of ring primer B-loop and F-loop;
2) reaction mixture: 40mM Tris-HCL, 20mM (NH
4)
2SO
4, 20mM KCL, 16 mM MgSO
4, 0.2% Triton X-100,1.6M Betaine, 2.8 mM dNTPs;
3) 1.0U
BstArchaeal dna polymerase.
Said LAMP detection kit also comprises developer, and described developer is a SYBR green I.
Said LAMP detection kit also comprises the extraction reagent that from fall ill banana blight plant tissue or soil, extracts DNA, is phenol/chloroform/primary isoamyl alcohol mixed solution, chloroform, 3M NaAC, 0.4% skim-milk solution, the 7.5 M NH of 25:24:1 by 2% CTAB, 10% SDS, volume ratio
4AC, 10mg/ml Proteinase K, absolute ethyl alcohol, 70% ethanol, 1 * TE form.
Said dna profiling is from the plant tissue of the banana blight of falling ill or soil, to extract.
When being used for plant tissue and having banana blight bacteria, described process for extracting is that the CTAB method is extracted banana blight bacteria DNA (concrete steps are seen embodiment 2) in the plant tissue.
When in pedotheque, existing under the banana blight bacteria condition, adopt the soil DNA extraction method to extract the DNA (concrete steps are seen embodiment 3) of banana blight bacteria in the soil.
Above-mentioned banana blight bacteria No. 4 microspecies LAMP molecular detecting method or test kit are in the banana blight bacteria infection conditions of diagnosis of plant tissue, soil or the application in the discriminating banana blight bacteria.
The present invention solves the problems of the technologies described above the technical scheme of mainly taking and comprises following three parts: 1,4 LAMP primer sequences that are used to detect banana blight bacteria are provided, respectively called after F3, B3, FIP, BIP; Preparation has also been optimized LAMP reaction system and reaction conditions, helps LAMP and with top efficiency sample is increased; 2, multiple LAMP is provided detection method as a result, has mainly contained optical dye visual observations, agarose gel electrophoresis.Comprise that specifically the following step gathers:
1, the banana blight bacteria DNA extraction in plant tissue or the soil of falling ill:
Adopt the CTAB method to extract banana blight bacteria DNA in the plant tissue; Adopt the soil DNA extraction method to extract the DNA of banana blight bacteria in the soil.
2, LAMP design of primers:
1) adopts the CTAB method to extract and supply examination banana blight strain gene group DNA; 2) use random primer and carry out the RAPD Analysis and Screening, screening obtains a RAPD random primer, and sequence is 5
,-TGCCGAGCTG-3
,, use the random primer that filters out once more the strains tested genomic dna to be carried out RAPD and analyze, obtain 1 RAPD fragment that banana blight bacteria is special; 3) use is cut glue recovery test kit and from sepharose, is reclaimed the special RAPD fragment of banana blight bacteria; Be connected on the carrier; Behind the optimal temperature thermal shock, be transformed in the competent cell, treat the bacterium state that restore normal growth; And behind the antibiotics resistance gene of expression plasmid coding, be added with the positive hickie of screening on the LB flat board that contains penbritin Amp, isopropylthio-and 5-bromo-4-chloro-3-indoles-D-galactoside; 4) positive colony of picking transformant hickie adopts the plasmid extraction test kit to extract DNA, uses restriction endonuclease
EcoRI with
HindThe III double digestion detects and confirms that institute's cloned sequence is the specific dna fragmentation of banana blight bacteria really; 5) carry out the order-checking of DNA, obtain the banana blight bacteria special gene sequence; 6) 6 LAMP primers of design from this special gene sequence;
F3：5’?-AGGACCTCTTCGAATGGCA-?3’，
B3：5’?-GACGCTGCAGCTATGACAA-?3’，
FIP：5’?-GGTGGCTCAATAGCCCAGTGAACCGATACCTGTGAAGTCGC-?3’，
BIP：5’?-CGACATCATCAGCATCTCCGCTAGCTTTGGCTCTTGTGACAG-?3’
F-loop：5’?-?GCCTAATTGAACATTCAGTATAAAC-?3’
B-Loop：5’?-?ACTCCAAGGAACTAGACGACG?-?3’
3, the preparation of LAMP reaction system.
4, LAMP reaction amplification condition: at 60-65 ℃ of incubation 60 min, 80-85 ℃ of insulation 10min deactivation
BsT DNA polysaccharase.
5, LAMP result detects: the result can adopt following two kinds of methods to detect: add the developer of 1uL in 1. that above-mentioned reaction is the intact system, gently shake mixing, can observe, the colour developing result observes green fluorescence and is judged as the positive, the orange feminine gender that is judged as.
2. get 2uL amplified production electrophoresis detection in 2% agarose gel electrophoresis,, amplified band do not occur and be judged as feminine gender if the distinctive trapezoid belt of LAMP is judged as the positive.
No. 4 microspecies LAMP of banana blight bacteria provided by the present invention Fast Detection Technique have other detection method incomparable advantage:
1, reliable results: a kind of LAMP specific detection primer that the present invention designed; Only detect to No. 4 microspecies of banana blight bacteria physiology; The banana blight bacteria that comes from ground such as banana blight takes place in China Fujian, Hainan, Guangdong and soil sample, the plant tissue of band banana blight bacteria have been carried out testing authentication, so result reliability has sufficient assurance;
2, high specificity: the primer that is adopted is to design 4 Auele Specific Primers to 6 different zones in the special RAPD gene order of banana blight bacteria, and any zone and primer do not match and all can not carry out nucleic acid amplification in 6 zones, so specificity is high.
3, highly sensitive: the detection sensitivity to banana blight bacteria can reach 10fg on dna level, detects high more than 100 times than conventional PCR.
4, rapidly and efficiently: nucleic acid amplification can be accomplished in 1.5h, will save 2 – 3h than conventional PCR, and product can increase 10
9– 10
10Doubly.
5, simple to operate: the LAMP nucleic acid amplification is under isothermal condition, to carry out, and only needs a water-bath to get final product, and does not need complicated plant and instrument and expensive molecular agents, and naked eyes are directly visible as a result.
Description of drawings
No. 4 microspecies LAMP of the banana blight bacteria specific detection figure that Fig. 1 will detect by the present invention; Wherein A is amplification rear electrophoresis detected result; B is the dyestuff detected result; Among the figure: M is DL 2000 DNA ladder marker, the negative contrast of 1 swimming lane, and 2 swimming lanes are Fusarium oxysporum Cuba specialized form (Race4); 3 swimming lanes are Fusarium oxysporum Cuba specialized form (Race1), and 4 swimming lanes are Fusarium oxysporum cotton specialized form, and 5 swimming lanes are Fusarium oxysporum cucumber specialized form; 6 swimming lanes are Fusarium oxysporum tomato specialized form, and 7 swimming lanes are Fusarium oxysporum watermelon specialized form, and 8 swimming lanes are Fusarium oxysporum sweet potato specialized form; 9 swimming lanes are Fusarium oxysporum watermelon specialized form, and 10 swimming lanes are for intending verticillate sickle-like bacteria, and 11 swimming lanes are the rotten sickle-like bacteria of eggplant; 12 swimming lanes are for intending verticillate sickle-like bacteria, and 13 swimming lanes are Fusarium equiseti, and 14 swimming lanes are Fusarium graminearum; 15 swimming lanes are Fijian ball chamber bacterium, and 16 swimming lanes are bajiao banana ball chamber bacterium, and 17 swimming lanes are the bajiao banana anthrax-bacilus; 18 swimming lanes are leaf of Broadbean shell two born of the same parents bacterium, and 19 swimming lanes are dry thread Pyrenomycetes, and 20 swimming lanes are phytophthora infestans.
Fig. 2 detects figure for No. 4 microspecies LAMP of banana blight bacteria of the present invention susceptibility;
Among the figure: M is DL 2000 DNA ladder marker, and 1 swimming lane is 100 ng, and 2 swimming lanes are 10 ng, and 3 swimming lanes are 1 ng; 4 swimming lanes are 100 pg, and 5 swimming lanes are 10 pg, and 6 swimming lanes are 1 pg, and 7 swimming lanes are 100 fg; 8 swimming lanes are 10 fg, and 9 swimming lanes are 1 fg, and 10 swimming lanes are 0.1 fg.
Fig. 3 is the banana incidence tissue of the present invention and the soil LAMP detected result figure that carries disease germs;
Among the figure: swimming lane 1,2,3 is the banana tissue of morbidity, and swimming lane 6 is morbidity soil, and swimming lane 4,5,7 is healthy banana tissue, swimming lane 8 negative contrasts, and the positive contrast of 9 swimming lanes, M is DL 2000 DNA ladder marker.
Embodiment
Embodiment is described further the present invention below in conjunction with accompanying drawing.
The special detection primer of No. 4 microspecies LAMP of banana blight bacteria:
F3：5’?-AGGACCTCTTCGAATGGCA-?3’，
B3：5’?-GACGCTGCAGCTATGACAA-?3’，
FIP：5’?-GGTGGCTCAATAGCCCAGTGAACCGATACCTGTGAAGTCGC-?3’，
BIP：5’?-CGACATCATCAGCATCTCCGCTAGCTTTGGCTCTTGTGACAG-?3’。
No. 4 microspecies specificitys of banana blight bacteria LAMP detection kit comprises:
1) primer mixed solution: outside primers F 3 and each 5pmol of B3, each 40 pmol of inboard primers F IP and BIP;
2) reaction mixture: 40mM Tris-HCL, 20mM (NH
4)
2SO
4, 20mM KCL, 16 mM MgSO
4, 0.2% Triton X-100,1.6M Betaine, 2.8 mM dNTPs;
3) 1.0U
BstArchaeal dna polymerase.
Test kit also comprises developer, and described developer is a SYBR green I.
Test kit also comprises DNA extraction reagent from fall ill banana blight plant tissue or soil, by 2% CTAB, 10% SDS, phenol/chloroform/primary isoamyl alcohol (volume ratio is 25:24:1), chloroform, 3M NaAC, 0.4% skim-milk solution, 7.5 M NH
4AC, 10mg/ml Proteinase K, absolute ethyl alcohol, 70% ethanol, 1 * TE form.
The LAMP reaction system comprises: primer mixed solution, reaction mixture, 1.0U
BstArchaeal dna polymerase and 25ng dna profiling are supplied 25 uL with the sterilization distilled water, and said primer mixed solution is outside primers F 3 and each 5pmol of B3, each 40 pmol of inboard primers F IP and BIP; Described reaction mixture is 40mM Tris-HCL, 20mM (NH
4)
2SO
4, 20mM KCL, 16 mM MgSO
4, 0.2% Triton X-100,1.6M Betaine, 2.8 mM dNTPs.
Described LAMP reaction conditions is at 63 ℃ of incubation 60 min, 82 ℃ of insulation 10min.
Described developer is a SYBR green I.
The specificity that detects: in the final amplified production of LAMP reaction, add the 1uL developer, except since in the banana blight bacterial strain in provinces such as Hainan, China blight spot, Guangdong and Fujian is the result of template, can be observed the intensive green fluorescence, with
Fusarium oxysporumNon-Cuba specialized form, other sickle-like bacteria
FusariumBacterial strains such as spp and other fungi and bacterium are then not observe among the result of template, still are orange.Getting above-mentioned reaction solution 2uL at 2% agarose gel electrophoresis, is that the distinctive trapezoid belt of LAMP all appears in the result of template with the banana blight bacterial strain, and with the result that other fungi and bacterial isolates is template do not find that amplified production is arranged (table 1, Fig. 1).
The susceptibility that detects: the banana blight bacteria DNA with different concns carries out the susceptibility detection; Pure dna with 10fg is that template still can be observed green fluorescence; The distinctive trapezoid belt of LAMP also appears in the agarose gel electrophoresis result, explains that the minimum verification and measurement ratio of LAMP can reach 10fg (Fig. 2) on dna level.
Table 1 checking primer is to banana blight bacteria specificity bacterial strain uses therefor, host, source and LAMP detected result
+: expression is positive; –: expression is negative
CGMCC is a Chinese common micro-organisms DSMZ
The detection of No. 4 microspecies of banana blight bacteria in the morbidity plant tissue.
1) from incidence tissue, extracts DNA
Adopt the CTAB method to extract the DNA of banana blight bacteria in the tissue.Concrete grammar is following: the plant tissue of will falling ill grinds, and the plant tissue after getting 50mg and grinding is in the 1.5ml centrifuge tube, and (prescription of extracting solution is: 2% CTAB to add 900 μ l, 2% CTAB (cetyl trimethylammonium bromide) extracting solution; 100 m mol/L Tris-HCl (Tri(Hydroxymethyl) Amino Methane Hydrochloride), PH 8.0; 20mmol/L EDTA (EDTA Disodium), pH8.0; 1.4 mol/L NaCl) and 90 μ l, 10% SDS (X 2073) back mixing, in 55～60 ℃ of water-bath 1.5 h, per 10 min vibration mixing once; It is centrifugal behind the water-bath 1.5h that (12,000rpm) 15min gets supernatant and adds and the isopyknic phenol/chloroform of supernatant/primary isoamyl alcohol (volume ratio of phenol, chloroform and primary isoamyl alcohol is 25:24:1); It is centrifugal that (12,000rpm) 5 min get supernatant (water); Once (12,000rpm) centrifugal 5min sucts clearly (350 μ l) with the isopyknic chloroform extracting of supernatant in adding; Add the 3mol/L NaAC solution of 0.1 volume (35 μ l) and the ice absolute ethyl alcohol of 2 volumes (700 μ l), behind-20 ℃ of settle 30min 12, centrifugal 5 min of 000rpm; Remove supernatant lightly, add 700 μ l and ice 70% ethanol and wash (centrifugal slightly, as to incline and fall supernatant); (pH8.0) solution dissolves for 10mmol/L Tris-HCL, 0.1mmol/L EDTA with 1 * TE on Bechtop, to dry alcohol-free flavor back naturally; Obtain dna solution, with UV spectrophotometer measuring DNA concentration and to be diluted to 50ng/ μ l for use.
2) LAMP of banana blight bacteria detects
The LAMP reaction system comprises: primer mixed solution, reaction mixture, 1.0U
BstArchaeal dna polymerase and 25ng dna profiling are supplied 25 uL with the sterilization distilled water, and said primer mixed solution is outside primers F 3 and each 5pmol of B3, each 40 pmol of inboard primers F IP and BIP; Described reaction mixture is 40mM Tris-HCL, 20mM (NH
4)
2SO
4, 20mM KCL, 16 mM MgSO
4, 0.2% Triton X-100,1.6M Betaine, 2.8 mM dNTPs.
The LAMP reaction conditions is at 63 ℃ of incubation 60 min, 82 ℃ of insulation 10min.
The result sees Fig. 3, and the colour developing result can be observed green fluorescence, and the distinctive trapezoid belt of LAMP also appears in the agarose gel electrophoresis result, thereby the incidence tissue of judging banana blight and the artificial inoculation banana of falling ill is organized and all infected banana blight bacteria.
The detection of No. 4 microspecies of banana blight bacteria in the morbidity pedotheque.
1) from the morbidity pedotheque, extracts DNA
Add a small amount of silica sand after getting the freezing 24-48 of the draining h of the soil that sieves, pour liquid nitrogen into and fully grind, the soil fine powder branch after grinding is filled in the 1.5 ml centrifuge tubes, every pipe adds 500 μ l, 0.4% skim-milk solution, vortex mixing.Centrifugal 15 min of 12000 rpm.Get supernatant and add equal-volume Proteinase K damping fluid, adding final concentration is 10 μ g/ml Proteinase Ks, 55 ℃ of water-bath 1-3 h.After water-bath finishes, add 7.5 M NH of 1/2 volume
4AC solution, mixing turns upside down.Centrifugal 15 min of 12000 rpm.Suct and reset and add 2 times of volume absolute ethyl alcohols-20 ℃ deposition (STs 1.5 h).After deposition finishes, centrifugal 15 min of 12000 rpm.Go with the hypsokinesis of 70% washing with alcohol deposition, room temperature is dried.DNA that every duplicate samples is carried dissolves with 10 μ l TE (or aseptic ultrapure water), and-20 ℃ of preservations are subsequent use.
2) LAMP of banana blight bacteria detects
The LAMP reaction system comprises: primer mixed solution, reaction mixture, 1.0U
BstArchaeal dna polymerase and 25ng dna profiling are supplied 25 uL with the sterilization distilled water, and said primer mixed solution is outside primers F 3 and each 5pmol of B3, each 40 pmol of inboard primers F IP and BIP; Described reaction mixture is 40mM Tris-HCL, 20mM (NH
4)
2SO
4, 20mM KCL, 16 mM MgSO
4, 0.2% Triton X-100,1.6M Betaine, 2.8 mM dNTPs.
The LAMP reaction conditions is at 63 ℃ of incubation 60 min, 82 ℃ of insulation 10min.
The result sees Fig. 3, and the colour developing result can be observed green fluorescence, and the distinctive trapezoid belt of LAMP also appears in the agarose gel electrophoresis result, thereby judges that pedotheque infects banana blight bacteria.
The present invention does not carry out the experiment with embodiment 2 and 3 to having the banana plant tissue and the pedotheque that infect banana blight bacteria, and the colour developing result is orange, and the agarose gel electrophoresis result does not find to have amplified production (Fig. 3).
Can know from above result; Banana plant tissue or pedotheque to supply examination carry out total DNA extraction; Use LAMP special primer F3 and B3, FIP and the BIP of banana blight bacteria to carry out pcr amplification; The colour developing result observes green fluorescence, and the distinctive trapezoid belt of LAMP appears in the agarose gel electrophoresis result, can judge that the incidence tissue of banana blight and the banana tissue and the pedotheque of artificial inoculation morbidity all infect banana blight bacteria.
< 110>Inst. of Plant Protection, fujian Academy of Agricultural Science
< 120>LAMP of No. 4 microspecies of banana blight bacteria detects primer and application thereof
<160> 6
<170> PatentIn?version?3.3
<210> 1
<211> 19
<212> DNA
< 213>artificial sequence
<400> 1
aggacctctt?cgaatggca?19
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< 213>artificial sequence
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gacgctgcag?ctatgacaa?19
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<211> 41
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ggtggctcaa?tagcccagtg?aaccgatacc?tgtgaagtcg?c?41
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cgacatcatc?agcatctccg?ctagctttgg?ctcttgtgac?ag?42
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gcctaattga?acattcagta?taaac?25
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actccaagga?actagacgac?g?21
Claims (7)
1. the LAMP of No. 4 microspecies of a banana blight bacteria detects primer, and it is characterized in that: said primer is following:
Outside primers F 3:5 '-AGGACCTCTTCGAATGGCA-3 ',
B3：5’?-GACGCTGCAGCTATGACAA-?3’；
Inboard primers F IP:5 '-GGTGGCTCAATAGCCCAGTGAACCGATACCTGTGAAGTCGC-3 ',
BIP：5’?-CGACATCATCAGCATCTCCGCTAGCTTTGGCTCTTGTGACAG-?3’；
Ring primers F-loop:5 '-GCCTAATTGAACATTCAGTATAAAC-3 ',
B-Loop：5’?-?ACTCCAAGGAACTAGACGACG?-?3’。
2. the LAMP detection method of No. 4 microspecies of a banana blight bacteria comprises design of primers, LAMP amplification and amplification observation, and it is characterized in that: the reaction system of said LAMP detection method comprises: primer mixed solution, reaction mixture, 1.0U
BstArchaeal dna polymerase and 25ng dna profiling are supplied 25 uL with the sterilization distilled water; Said primer mixed solution is by the described outside of claim 1 primers F 3, B3; Inboard primers F IP, BIP and ring primer B-loop, F-loop form; Primers F 3 and each 5pmol of B3 wherein, each 40 pmol of inboard primers F IP and BIP, ring primer B-loop, each 20 μ mol of F-loop; Said reaction mixture is prepared as follows: 40mM Tris-HCL, 20mM (NH
4)
2SO
4, 20mM KCL, 16 mM MgSO
4, 0.2% Triton X-100,1.6M trimethyl-glycine and 2.8 mM dNTPs.
3. the LAMP detection method of No. 4 microspecies of banana blight bacteria according to claim 2 is characterized in that: the reaction conditions of said LAMP detection method is at 60-65 ℃ of incubation 60 min, 80-85 ℃ of insulation 10min.
4. the LAMP detection method of No. 4 microspecies of banana blight bacteria according to claim 2, it is characterized in that: said amplification is viewed as optical dye visual observations method or agarose gel electrophoresis method; Said optical dye visual observations method: in the final amplified production of LAMP reaction, add the 1uL developer, said developer is a SYBR green I, and the colour developing result observes green fluorescence and is judged as the positive, the orange feminine gender that is judged as; Said agarose gel electrophoresis method: the final amplified production of getting the 2uLLAMP reaction detects with 2% agarose gel electrophoresis, if the distinctive trapezoid belt of LAMP is judged as the positive, amplified band does not occur and is judged as feminine gender.
5. No. 4 specific detection kit of microspecies of a banana blight bacteria, it is characterized in that: said LAMP detection kit comprises:
1) primer mixed solution: outside primers F 3 and each 5pmol of B3, each 40 pmol of inboard primers F IP and BIP, each 20 μ mol of ring primer B-loop and F-loop;
2) reaction mixture: 40mM Tris-HCL, 20mM (NH
4)
2SO
4, 20mM KCL, 16 mM MgSO
4, 0.2% Triton X-100,1.6M Betaine, 2.8 mM dNTPs;
3) 1.0U
BstArchaeal dna polymerase.
6. the LAMP detection kit of No. 4 microspecies of banana blight bacteria according to claim 5 is characterized in that: said LAMP detection kit also comprises developer, and described developer is a SYBR green I.
7. the LAMP detection kit of No. 4 microspecies of banana blight bacteria according to claim 5; It is characterized in that: said LAMP detection kit also comprises the extraction reagent that from fall ill banana blight plant tissue or soil, extracts DNA, is phenol/chloroform/primary isoamyl alcohol mixed solution, chloroform, 3M NaAC, 0.4% skim-milk solution, the 7.5 M NH of 25:24:1 by 2% CTAB, 10% SDS, volume ratio
4AC, 10mg/ml Proteinase K, absolute ethyl alcohol, 70% ethanol, 1 * TE form.
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