CN108220204A - A kind of method of quick separating sugarcane ratoon stunting disease pathogenic bacteria Lxx - Google Patents
A kind of method of quick separating sugarcane ratoon stunting disease pathogenic bacteria Lxx Download PDFInfo
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Abstract
The invention discloses a kind of methods of quick separating sugarcane ratoon stunting disease pathogenic bacteria Lxx.Using FeSO4And FeCl3Magnetic nanoparticle is made, adds in 3 aminopropyl triethoxysilanes, adjusts pH value, stirring；Drying is washed, obtains amidized Fe3O4Nano-particle；Glutaraldehyde reaction is added, stirs after adding in Lxx polyclonal antibodies, is collected after washing with magnetite；It is incubated after sugarcane juice after filtering is added thereto mixing, supernatant is abandoned in centrifugation, and Lxx is obtained after being cleaned with PBS buffer solution.The method of the present invention has the advantages that fast separating rate, efficient, favorable repeatability, easy to operate, not need to expensive instrument and equipment, pollution rate low etc., quickly Lxx pathogens can more importantly be separated from sugarcane in the case where not changing bacterial condition, for studying the transcript profile of bacterium and metabolism group under state instantly, solve the problems, such as that in bacterium and pathogen Interaction a large amount of pathogens can not be quickly obtained.
Description
Technical field
The present invention relates to strain separation method more particularly to a kind of quick separating sugarcane ratoon stunting disease pathogenic bacteria Lxx
Method.
Background technology
The in vitro culture of the pathogenic bacteria of sugarcane ratoon stunting disease (Ratoon Stunting Disease, RSD) is very tired
Difficulty, although being found that in nineteen forty-four, until Davis in 1980 is just successfully separately cultured.The related RSD pathogens point in China
Less from the report of culture, Chen Jian texts in 2008 etc. are separately cultured from sugarcane sugarcane juice using the SC culture mediums of improvement to one kind
Bacillus according to the morphological feature of bacterium colony, and combines serology and PCR detection method, confirm it is separated to bacterium be sugarcane
The pathogenic bacteria Lxx of ratoon stunting disease；Zhang little Qiu in 2015 etc. is successfully separated Lxx, the same year Wang Li using the MSC culture mediums of improvement
It has been successfully separated RSD pathogens.
Still also have no idea repeated isolation Lxx in many laboratories at present, and main cause is that the bacterium grows especially in itself
Slowly, bacterium colony could be observed on culture dish by generally requiring the time of 4 weeks or so, due to culture when it is long, once have miscellaneous bacteria
It generates, separation just failure, so control pollution is the key link that Lxx bacterium are separately cultured, it is this for other bacteriums
The repeatability being separately cultured is poor.
Invention content
The purpose of the present invention is to provide a kind of separating rate is fast, efficient, favorable repeatability, it is easy to operate, do not need to
The method of the low quick separating sugarcane ratoon stunting disease pathogenic bacteria Lxx of expensive instrument and equipment, pollution rate.
To achieve the above object, the present invention provides a kind of method of quick separating sugarcane ratoon stunting disease pathogenic bacteria Lxx,
It is characterized in that, includes the following steps：
The preparation of nanometer magnetic bead：Take FeSO4And FeCl3Mixing is poured into three-necked flask and is placed on constant-temperature heating magnetic stirring apparatus,
Dissolving is stirred under conditions of inflated with nitrogen, adjusts pH value most 8-10, solution immediately becomes black, and the reaction was continued, and magnetism is made
Nano particle；It is placed in magnetic field after abandoning supernatant, it is dry after being washed with deionized；
The amination of nanometer magnetic bead：Above-mentioned gained magnetic nanoparticle is taken, is dissolved in absolute ethyl alcohol, ultrasonication is extremely
It is completely dispersed, adds in 3- aminopropyl triethoxysilanes, adjusting pH value is 10-12, is stirred to react in a water bath overnight；After
With being freeze-dried after deionized water repeated washings, amidized Fe is obtained3O4Nano-particle；
Amination nanometer magnetic bead activates：Take the amidized Fe of above-mentioned gained3O4Nano-particle is dissolved in PBS, and ultrasound is disperseed
Property good magnetic bead solution, add in glutaraldehyde, be stirred to react at room temperature；It is collected after PBS washings with magnetite again, constant volume is lived
Amination nanometer magnetic bead after change；
The preparation of Lxx immunomagnetic beads：Lxx polyclonal antibodies are added to the amination nanometer magnetic bead after above-mentioned gained activation
In after stir, then collected to obtain Lxx immunomagnetic beads with magnetite after being washed with PBS；
The separation of Lxx：Sugarcane juice after filtering is added in gained Lxx immunomagnetic beads after mixing and is incubated, low-speed centrifugal is abandoned
Supernatant obtains the isolate of Lxx after being cleaned with PBS buffer solution.
Further, in the preparation process of the nanometer magnetic bead, FeSO4And FeCl3Molar ratio be 2：(2-6)；Preferably,
FeSO4And FeCl3Molar ratio be 2:3.
Optional, temperature when adjusting pH value is 70-80 DEG C；Preferably, temperature is 75 DEG C；
It is optional, the reaction was continued 30min；
Optional, it is washed with deionized and dries afterwards three times.
Further, in the amination step of the nanometer magnetic bead, the magnetic nanoparticle：Absolute ethyl alcohol：3- aminopropyls
The amount ratio of triethoxysilane is (40-60) mg：(80-120)mL：(5-8)mL；Preferably, magnetic nanoparticle：Anhydrous second
Alcohol：The amount ratio of 3- aminopropyl triethoxysilanes is 50mg：100mL：6mL；
Optional, it is 10-12 to adjust pH value with ammonium hydroxide；Preferably, it is 11 to adjust pH value with ammonium hydroxide；
Optional, the temperature of water-bath is 50-70 DEG C；Preferably, the temperature of water-bath is 60 DEG C.
Further, in the amination nanometer magnetic bead activation step, the amidized Fe3O4Nano-particle：PBS：25%
The amount ratio of glutaraldehyde is (8-15) mg：(8-15)mL：(2-3)mL；Preferably, amidized Fe3O4Nano-particle：PBS：
The amount ratio of 25% glutaraldehyde is 10mg：10mL：2.5mL；A concentration of 0.01M of wherein PBS, pH value 7.4；
Optional, the time being stirred to react is 6h；
Optional, the number of washing is 3 times.
Further, in the preparation process of the Lxx immunomagnetic beads, the preparation method of the Lxx polyclonal antibodies is：
It is prepared by Lxx antigens：Sugarcane juice after filtering is coated on the fresh MSC culture mediums containing acidum nalidixicum, light culture, 30-
Observation and picking colony after 50 days；
Lxx Antibody preparations：0.4% formalin-inactivated is added in using the bacterium colony of above-mentioned picking, Freund's adjuvant is added in, directly notes
Rabbit is penetrated, is beaten within two weeks once, extraction serum is made affine using Protein A-Sepharose 4B after being immunized 4 times, two months altogether
Chromatography media purifies to obtain antibody；
Preferably, the sugarcane juice after the filtering is put into super-clean bench to cut sugarcane basal part of stem after carrying out surface clean sterilizing
In dry；Sugarcane stem is cut into suitable size and squeezes juice with tweezers, 4000r/min collects sugarcane juice after centrifuging 10min, uses ordinary filter paper
Be filtered again with 0.45 μm of biofilter after tentatively filtering.
Further, in the preparation process of the Lxx immunomagnetic beads, a concentration of 5-15 μ g/ml of the acidum nalidixicum；It is preferred that
, a concentration of 10 μ g/ml of the acidum nalidixicum；
Optional, the temperature of light culture is 26-30 DEG C, and the time of culture is 35-45 days.
Further, in the preparation process of the Lxx immunomagnetic beads, the Lxx polyclonal antibodies and the amination after activation
The dosage of nanometer magnetic bead is (5-15) ml；Preferably, Lxx polyclonal antibodies and the dosage of the amination nanometer magnetic bead after activation are
5μmol：10ml；
Optional, the condition of the stirring is to stir 3h at 37 DEG C；
Optional, the number of the washing is 3 times.
Further, in the separating step of the Lxx, the sugarcane juice squeezes juice for sugarcane stem is cut into suitable size with tweezers,
It collects and obtains after 4000r/min, centrifugation 10min；
Optional, the filtering using ordinary filter paper carried out with 0.45 μm of biofilter again after tentatively filtering
Filter；
Optional, the sugarcane juice after the filtering：Lxx immunomagnetic beads dosage is 1：(30-50)；Preferably, the sugarcane after filtering
Juice：Lxx immunomagnetic beads dosage is 1：40；
Optional, the condition of the incubation is 37 DEG C of incubation 1h；
Optional, the rotating speed of the low-speed centrifugal is 2000-5000rpm；
Optional, the number of the PBS washings is 2-3 times.
Immuno magnetic cell separation technology (Immunomagnetic separation, IMS) is a kind of with special antigen-antibody
Immunology detection and isolation technics based on reaction.It is using the coated magnetic bead of antibody as carrier, is situated between by antibody with reacting
Specific antigen combines in matter, forms antigen --- antibody complex, this compound is determined under the action of externally-applied magnetic field
To movement, so as to achieve the purpose that detach antigen.
Lxx immunomagnetic beads are prepared in the present invention in sugarcane ratoon stunting disease pathogen separation, with traditional isolated culture method
Compared to separating rate is fast, efficient, favorable repeatability, easy to operate, not need to expensive instrument and equipment, pollution rate low etc.
Advantage, it is often more important that quickly can separate Lxx pathogens from sugarcane in the case where not changing bacterial condition, for studying
Instantly the transcript profile of bacterium and metabolism group under state, a large amount of diseases can not be quickly obtained by solving in bacterium and pathogen Interaction
The problem of opportunistic pathogen.
Description of the drawings
Fig. 1 is tem observation Fe304Magnetic particle structure chart.
Fig. 2 is Lxx immunomagnetic beads Electronic Speculum observation result figure.
Fig. 3 is Lxx isolates Electronic Speculum observation result figure.
Fig. 4 is Lxx isolates Electronic Speculum observation result figure.
Fig. 5 is Lxx isolate PCR result electrophoretograms.
Fig. 6 is the susceptible sugarcane plant separation bacterium colonies of bacterium of RSD and thalli morphology figure.
Specific embodiment
The embodiment of the present invention is described below in detail, the example of the embodiment is shown in the drawings, wherein from beginning to end
Same or similar label represents same or similar element or the element with same or like function.Below with reference to attached
The embodiment of figure description is exemplary, it is intended to for explaining the present invention, and is not considered as limiting the invention.Embodiment
In particular technique or condition person is not specified, according to the described technology of document in the art or condition or according to the description of product
Book carries out.Reagents or instruments used without specified manufacturer, being can be with conventional products that are commercially available.
A kind of method of quick separating sugarcane ratoon stunting disease pathogenic bacteria Lxx, which is characterized in that include the following steps：
The preparation of nanometer magnetic bead：Take FeSO4And FeCl3Mixing is poured into three-necked flask and is placed on constant-temperature heating magnetic stirring apparatus,
Dissolving is stirred under conditions of inflated with nitrogen, adjusts pH value most 8-10, solution immediately becomes black, and the reaction was continued, and magnetism is made
Nano particle；It is placed in magnetic field after abandoning supernatant, it is dry after being washed with deionized；
The amination of nanometer magnetic bead：Above-mentioned gained magnetic nanoparticle is taken, is dissolved in absolute ethyl alcohol, ultrasonication is extremely
It is completely dispersed, adds in 3- aminopropyl triethoxysilanes, adjusting pH value is 10-12, is stirred to react in a water bath overnight；After
With being freeze-dried after deionized water repeated washings, amidized Fe is obtained3O4Nano-particle；
Amination nanometer magnetic bead activates：Take the amidized Fe of above-mentioned gained3O4Nano-particle is dissolved in PBS, and ultrasound is disperseed
Property good magnetic bead solution, add in glutaraldehyde, be stirred to react at room temperature；It is collected after PBS washings with magnetite again, constant volume is lived
Amination nanometer magnetic bead after change；
The preparation of Lxx immunomagnetic beads：Lxx polyclonal antibodies are added to the amination nanometer magnetic bead after above-mentioned gained activation
In after stir, then collected to obtain Lxx immunomagnetic beads with magnetite after being washed with PBS；
The separation of Lxx：Sugarcane juice after filtering is added in gained Lxx immunomagnetic beads after mixing and is incubated, low-speed centrifugal is abandoned
Supernatant obtains the isolate of Lxx after being cleaned with PBS buffer solution.
Further, in the preparation process of the nanometer magnetic bead, FeSO4And FeCl3Molar ratio be 2：(2-6)；Preferably,
FeSO4And FeCl3Molar ratio be 2:3.
Optional, temperature when adjusting pH value is 70-80 DEG C；Preferably, temperature is 75 DEG C；
It is optional, the reaction was continued 30min；
Optional, it is washed with deionized and dries afterwards three times.
Further, in the amination step of the nanometer magnetic bead, the magnetic nanoparticle：Absolute ethyl alcohol：3- aminopropyls
The amount ratio of triethoxysilane is (40-60) mg：(80-120)mL：(5-8)mL；Preferably, magnetic nanoparticle：Anhydrous second
Alcohol：The amount ratio of 3- aminopropyl triethoxysilanes is 50mg：100mL：6mL；
Optional, it is 10-12 to adjust pH value with ammonium hydroxide；Preferably, it is 11 to adjust pH value with ammonium hydroxide；
Optional, the temperature of water-bath is 50-70 DEG C；Preferably, the temperature of water-bath is 60 DEG C.
Further, in the amination nanometer magnetic bead activation step, the amidized Fe3O4Nano-particle：PBS：25%
The amount ratio of glutaraldehyde is (8-15) mg：(8-15)mL：(2-3)mL；Preferably, amidized Fe3O4Nano-particle：PBS：
The amount ratio of 25% glutaraldehyde is 10mg：10mL：2.5mL；A concentration of 0.01M of wherein PBS, pH value 7.4；
Optional, the time being stirred to react is 6h；
Optional, the number of washing is 3 times.
Further, in the preparation process of the Lxx immunomagnetic beads, the preparation method of the Lxx polyclonal antibodies is：
It is prepared by Lxx antigens：Sugarcane juice after filtering is coated on the fresh MSC culture mediums containing acidum nalidixicum, light culture, 30-
Observation and picking colony after 50 days；
Lxx Antibody preparations：0.4% formalin-inactivated is added in using the bacterium colony of above-mentioned picking, Freund's adjuvant is added in, directly notes
Rabbit is penetrated, is beaten within two weeks once, extraction serum is made affine using Protein A-Sepharose 4B after being immunized 4 times, two months altogether
Chromatography media purifies to obtain antibody；
Preferably, the sugarcane juice after the filtering is put into super-clean bench to cut sugarcane basal part of stem after carrying out surface clean sterilizing
In dry；Sugarcane stem is cut into suitable size and squeezes juice with tweezers, 4000r/min collects sugarcane juice after centrifuging 10min, uses ordinary filter paper
Be filtered again with 0.45 μm of biofilter after tentatively filtering.
Further, in the preparation process of the Lxx immunomagnetic beads, a concentration of 5-15 μ g/ml of the acidum nalidixicum；It is preferred that
, a concentration of 10 μ g/ml of the acidum nalidixicum；
Optional, the temperature of light culture is 26-30 DEG C, and the time of culture is 35-45 days.
Further, in the preparation process of the Lxx immunomagnetic beads, the Lxx polyclonal antibodies and the amination after activation
The dosage of nanometer magnetic bead is (5-15) ml；Preferably, Lxx polyclonal antibodies and the dosage of the amination nanometer magnetic bead after activation are
5μmol：10ml；5μmol：10ml；
Optional, the condition of the stirring is to stir 3h at 37 DEG C；
Optional, the number of the washing is 3 times.
Further, in the separating step of the Lxx, the sugarcane juice squeezes juice for sugarcane stem is cut into suitable size with tweezers,
It collects and obtains after 4000r/min, centrifugation 10min；
Optional, the filtering using ordinary filter paper carried out with 0.45 μm of biofilter again after tentatively filtering
Filter；
Optional, the sugarcane juice after the filtering：Lxx immunomagnetic beads dosage is 1：(30-50)；Preferably, the sugarcane after filtering
Juice：Lxx immunomagnetic beads dosage is 1：40；
Optional, the condition of the incubation is 37 DEG C of incubation 1h；
Optional, the rotating speed of the low-speed centrifugal is 2000-5000rpm；
Optional, the number of the PBS washings is 2-3 times.
Embodiment 1：The quick separating of Lxx
The preparation of nanometer magnetic bead：Take 10mL 0.25mol/L FeSO4With 15ml 0.25mol/L FeCl3Mixing pours into three
Mouth flask is placed on constant-temperature heating magnetic stirring apparatus, and dissolving is stirred under conditions of nitrogen charging institute, and temperature is set as 75 DEG C, adds in
NaOH adjusts pH value to 9, and solution immediately becomes black, the reaction was continued 30min, and magnetic nanoparticle is made.It is placed in magnetic field and abandons
After supernatant, it is washed with deionized and dries afterwards three times.
The amination of nanometer magnetic bead：50mg nano particles are taken, are dissolved in 100mL absolute ethyl alcohols, ultrasonication is to complete
Full dispersion, adds in 6mL 3- aminopropyl triethoxysilanes (APTES), pH value is adjusted to 11 with ammonium hydroxide, in 60 stirred in water bath
Reaction is overnight.After with deionized water repeated washings, magnetic Fe that 10mL is taken to synthesize304Suspension is in dry surface plate
Drying to constant weight, and the quality after drying is 0.095g, a concentration of 9.5mg/mL of magnetic bead.After be freeze-dried.Pass through tem observation
Fe304Magnetic particle (see attached drawing 1), it can be seen from the figure that nanoscale Fe304The average grain diameter of magnetic particle 125-
Between 268nm, spherome surface is uniform, there is preferable pattern, favorable dispersibility.
Amination nanometer magnetic bead activates：The amidized Fe of 10mg3O4Nano-particle is dissolved in 10mL 0.01M PH 7.4PBS,
Ultrasound obtains the magnetic bead solution of good dispersion, adds in 25% glutaraldehydes of 2.5mL, is stirred to react 6h at room temperature.PBS washings three
It is collected after secondary with magnetite, is settled to 10mL.
The preparation of Lxx immunomagnetic beads：It adds in the homemade Lxx polyclonal antibodies of 5 μm of ol and 3h, PBS washings 3 is stirred at 37 DEG C
After secondary 4 DEG C of preservations are collected in magnetite.Lxx immunomagnetic beads Electronic Speculum observation result (see Fig. 2), it can be seen from the figure that with magnetism
Fe304Particle is consistent, free from admixture adhesion, also without near-wall air curtain.
The preparation method of wherein Lxx polyclonal antibodies is：
1) prepared by Lxx antigens：Sugarcane basal part of stem is cut, is put into super-clean bench and dries after progress surface clean sterilizing；By sugarcane
Stem is cut into suitable size and squeezes juice with tweezers, and 4000r/min collects sugarcane juice after centrifuging 10min, tentatively filtered with ordinary filter paper
It is filtered again with 0.45 μm of biofilter afterwards.Sugarcane juice after filtering is coated on containing (5-15 μ g/ml；It is preferred that 10 μ g/
Ml the fresh MSC culture mediums of acidum nalidixicum), 26-30 DEG C, the time of culture is light culture, observation and picking bacterium after 30-50 days
It falls；
2) Lxx Antibody preparations：0.4% formalin-inactivated is added in using the bacterium colony of above-mentioned picking, adds in Freund's adjuvant, directly
Rabbit is injected, is beaten within two weeks once, extraction serum is made close using Protein A-Sepharose 4B after being immunized 4 times, two months altogether
It purifies to obtain antibody with chromatography media.
The separation of Lxx：Sugarcane stem is cut into suitable size and squeezes juice with tweezers, 4000r/min collects sugarcane juice after centrifuging 10min,
It is filtered again with 0.45 μm of biofilter after carrying out tentatively filtering with ordinary filter paper, the sugarcane juice after being filtered according to 1 μ L adds in
The amount of 40 μ L Lxx immunomagnetic beads is mixed after adding in Lxx immunomagnetic beads, 37 DEG C of incubation 1h, is abandoned supernatant after low-speed centrifugal, is delayed with PBS
Fliud flushing is cleaned 2 times, obtains the isolate of Lxx.
Lxx isolates Electronic Speculum is observed：As a result Fig. 3 and Fig. 4 are seen, from Fig. 3-4 as can be seen that immunomagnetic beads absorption is in Lxx bacterium
Body surface face, bacterium is more intensive, and corresponding absorption is also more in the immunomagnetic beads of phage surface.
Lxx isolates PCR is verified.Lxx 16S~23S r DNA internal transcribed spacer of the PCR primer reports such as Pan
(ITS) special primer, product 439bp.
Lxx1：5’-CCGAAGTGAGCAGATTGACC-3’、SEQ ID NO:1；
Lxx2：5 '-ACCCTGTGTTGTTTTCAACG-3 ', SEQ ID NO:2；Primer is had by Shanghai English fine horse biotechnology
Limit company synthesizes.
PCR reaction systems：RTaq 0.25 μ L, dNTP 1 μ L, 10 × PCR buffer (Mg2+) 2 μ L, forward primer Lxx1
1 μ L, reverse primer Lxx2 1 μ L, ddH2O 13.75μL.The concentration of forward and reverse primer is 10uM/L.
PCR amplification program is 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 40 cycles；72 DEG C of extension 5min.
PCR result electrophoretograms are shown in Fig. 5, from fig. 5, it can be seen that display isolate can amplify the purpose consistent with positive control
Band determines that the isolate that this experiment is obtained is Lxx.Wherein swimming lane M:DNA markers；Swimming lane 1：Immunomagnetic beads-Lxx
Mixture；Swimming lane P:Positive control；Swimming lane N:Negative control.
Embodiment 2：The quick separating of Lxx
The preparation of nanometer magnetic bead：Take 10mL 0.25mol/LFeSO4With 10ml 0.25mol/L FeCl3Mixing pours into three mouthfuls
Flask is placed on constant-temperature heating magnetic stirring apparatus, and dissolving is stirred under conditions of nitrogen charging institute, and temperature is set as 70 DEG C, adds in NaOH
PH value is adjusted to 9, solution immediately becomes black, the reaction was continued 30min, and magnetic nanoparticle is made.It is placed in magnetic field and abandons supernatant
After liquid, it is washed with deionized and dries afterwards three times.
The amination of nanometer magnetic bead：40mg nano particles are taken, are dissolved in 80mL absolute ethyl alcohols, ultrasonication is to complete
Dispersion adds in 5mL 3- aminopropyl triethoxysilanes (APTES), and pH value is adjusted to 10 with ammonium hydroxide, anti-in 60 stirred in water bath
It should stay overnight.After with deionized water repeated washings, magnetic Fe that 10mL is taken to synthesize304Suspension dries in dry surface plate
It does to constant weight, the quality after drying is 0.095g, a concentration of 9.5mg/mL of magnetic bead.After be freeze-dried.Pass through tem observation Fe304
Magnetic particle (see attached drawing 1), it can be seen from the figure that nanoscale Fe304The average grain diameter of magnetic particle 125-268nm it
Between, spherome surface is uniform, there is preferable pattern, favorable dispersibility.
Amination nanometer magnetic bead activates：The amidized Fe of 8mg3O4Nano-particle is dissolved in 8mL 0.01M PH 7.4PBS, surpasses
Sound obtains the magnetic bead solution of good dispersion, adds in 25% glutaraldehydes of 2mL, is stirred to react 6h at room temperature.After PBS washings three times
It is collected with magnetite, is settled to 10mL.
The preparation of Lxx immunomagnetic beads：The homemade Lxx polyclonal antibodies (method is with embodiment 1) of 10 μm of ol are added in 37 DEG C
Lower stirring 3h, PBS with magnetite are collected in 4 DEG C of preservations after washing 3 times.Lxx immunomagnetic beads Electronic Speculum observation result (see Fig. 2), from figure
In as can be seen that and magnetic Fe304Particle is consistent, free from admixture adhesion, also without near-wall air curtain.
Following steps are with embodiment 1, as a result with embodiment 1.
Embodiment 3：The quick separating of Lxx
The preparation of nanometer magnetic bead：Take 10mL 0.25mol/LFeSO4With 30ml 0.25mol/L FeCl3Mixing pours into three mouthfuls
Flask is placed on constant-temperature heating magnetic stirring apparatus, and dissolving is stirred under conditions of nitrogen charging institute, and temperature is set as 80 DEG C, adds in NaOH
PH value is adjusted to 9, solution immediately becomes black, the reaction was continued 30min, and magnetic nanoparticle is made.It is placed in magnetic field and abandons supernatant
After liquid, it is washed with deionized and dries afterwards three times.
The amination of nanometer magnetic bead：60mg nano particles are taken, are dissolved in 120mL absolute ethyl alcohols, ultrasonication is to complete
Full dispersion, adds in 8mL 3- aminopropyl triethoxysilanes (APTES), pH value is adjusted to 12 with ammonium hydroxide, in 60 stirred in water bath
Reaction is overnight.After with deionized water repeated washings, magnetic Fe that 10mL is taken to synthesize304Suspension is in dry surface plate
Drying to constant weight, and the quality after drying is 0.095g, a concentration of 9.5mg/mL of magnetic bead.After be freeze-dried.Pass through tem observation
Fe304Magnetic particle (see attached drawing 1), it can be seen from the figure that nanoscale Fe304The average grain diameter of magnetic particle 125-
Between 268nm, spherome surface is uniform, there is preferable pattern, favorable dispersibility.
Amination nanometer magnetic bead activates：The amidized Fe of 15mg3O4Nano-particle is dissolved in 15mL 0.01M PH 7.4PBS,
Ultrasound obtains the magnetic bead solution of good dispersion, adds in 25% glutaraldehydes of 3mL, is stirred to react 6h at room temperature.PBS is washed three times
It is collected afterwards with magnetite, is settled to 10mL.
The preparation of Lxx immunomagnetic beads：The homemade Lxx polyclonal antibodies (with embodiment 1) of 7.5 μm of ol are added in stir at 37 DEG C
Mix 3h, PBS with magnetite is collected in 4 DEG C of preservations after washing 3 times.Lxx immunomagnetic beads Electronic Speculum observation result (see Fig. 2), can from figure
To find out, with magnetic Fe304Particle is consistent, free from admixture adhesion, also without near-wall air curtain.
Following steps are with embodiment 1, as a result with embodiment 1.
Embodiment 4：Lxx's is separately cultured
The separation of Lxx：Sugarcane stem is cut into suitable size and squeezes juice with tweezers, 4000r/min collects sugarcane juice after centrifuging 10min,
It is filtered again with 0.45 μm of biofilter after carrying out tentatively filtering with ordinary filter paper, adds in 40 μ L Lxx immunomagnetic beads and mix
, it is incubated 1h for 37 DEG C, abandons supernatant after low-speed centrifugal, cleaned 2 times with PBS buffer solution, obtain Lxx's with 0.45 μm of membrane filtration of φ
Isolate.
The culture of Lxx：Above-mentioned isolate is inoculated on MSC culture mediums, after liquid is waited to be absorbed completely, with sealed membrane handle
Culture medium seals, and is inverted in 28 DEG C of constant incubator and is cultivated, is observed after 40 days.The formula of MSC culture mediums is such as
Table 1：
The formula table of 1 MSC culture mediums of table
The Morphological Identification of pathogen Lxx：Negative staining electron microscope detection is carried out to isolate, 10 μ L bacterium solutions is drawn and drips to film
Copper mesh front, stands 1min, siphons away bacterium solution from edge with filter paper, copper mesh is dried in room temperature, through 1% phosphotungstic acid dyeing 2~
5min is then siphoned away phosphotungstic acid from edge with filter paper, and room temperature, which dries to be placed under transmission electron microscope, to be observed.
As a result it is as follows：The pathogenicbacteria separation of RSD, culture are relatively difficult, long after 3 weeks are cultivated in 28 DEG C of constant incubators
Go out macroscopic colourless, round, dotted size, neat in edge median rise bacterium colony, diameter is about 0.2mm (Fig. 1 and Fig. 6
A).Hereafter its continued growth is allowed again, colony colour no longer changes, and colony shape becomes gradually flat by original intermediate projections
It is flat.Under Electronic Speculum, pathogen is in elongated club-like, and some middle parts or one end are expanded, and inside have mesosome, thalline size is about 0.25 μm
× 2 μm (B of Fig. 1 and Fig. 6), more consistent with the description of other Research Literatures, it is sugarcane RSD that can further infer that the bacterium accordingly
Pathogen Lxx.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, those of ordinary skill in the art are not departing from the principle of the present invention and objective
In the case of can make changes, modifications, substitutions and variations to the above described embodiments within the scope of the invention.
Claims (8)
- A kind of 1. method of quick separating sugarcane ratoon stunting disease pathogenic bacteria Lxx, which is characterized in that include the following steps：The preparation of nanometer magnetic bead：Take FeSO4And FeCl3Mixing is poured into three-necked flask and is placed on constant-temperature heating magnetic stirring apparatus, is filling Dissolving is stirred under conditions of nitrogen, adjusts pH value most 8-10, solution immediately becomes black, and the reaction was continued, and magnetic Nano is made Particle；It is placed in magnetic field after abandoning supernatant, it is dry after being washed with deionized；The amination of nanometer magnetic bead：Above-mentioned gained magnetic nanoparticle is taken, is dissolved in absolute ethyl alcohol, ultrasonication is to complete Dispersion adds in 3- aminopropyl triethoxysilanes, and adjusting pH value is 10-12, is stirred to react in a water bath overnight；After spend It is freeze-dried after ionized water repeated washings, obtains amidized Fe3O4Nano-particle；Amination nanometer magnetic bead activates：Take the amidized Fe of above-mentioned gained3O4Nano-particle is dissolved in PBS, and ultrasound obtains good dispersion Magnetic bead solution, add in glutaraldehyde, be stirred to react at room temperature；It is collected after PBS washings with magnetite again, constant volume, after obtaining activation Amination nanometer magnetic bead；The preparation of Lxx immunomagnetic beads：After Lxx polyclonal antibodies are added in the amination nanometer magnetic bead after above-mentioned gained activation Stirring, then collected to obtain Lxx immunomagnetic beads with magnetite after being washed with PBS；The separation of Lxx：Sugarcane juice after filtering is added in gained Lxx immunomagnetic beads after mixing and is incubated, low-speed centrifugal abandons supernatant, The isolate of Lxx is obtained after being cleaned with PBS buffer solution.
- 2. the method for quick separating sugarcane ratoon stunting disease pathogenic bacteria Lxx as described in claim 1, which is characterized in that described to receive In the preparation process of rice magnetic bead, FeSO4And FeCl3Molar ratio be 2：(2-6)；Preferably, FeSO4And FeCl3Molar ratio be 2:3。Optional, temperature when adjusting pH value is 70-80 DEG C；Preferably, temperature is 75 DEG C；It is optional, the reaction was continued 30min；Optional, it is washed with deionized and dries afterwards three times.
- 3. the method for quick separating sugarcane ratoon stunting disease pathogenic bacteria Lxx as described in claim 1, which is characterized in that described to receive In the amination step of rice magnetic bead, the magnetic nanoparticle：Absolute ethyl alcohol：The amount ratio of 3- aminopropyl triethoxysilanes is (40-60)mg：(80-120)mL：(5-8)mL；Preferably, magnetic nanoparticle：Absolute ethyl alcohol：3- aminopropyl-triethoxy silicon The amount ratio of alkane is 50mg：100mL：6mL；Optional, it is 10-12 to adjust pH value with ammonium hydroxide；Preferably, it is 11 to adjust pH value with ammonium hydroxide；Optional, the temperature of water-bath is 50-70 DEG C；Preferably, the temperature of water-bath is 60 DEG C.
- 4. the method for quick separating sugarcane ratoon stunting disease pathogenic bacteria Lxx as described in claim 1, which is characterized in that the ammonia In base nanometer magnetic bead activation step, the amidized Fe3O4Nano-particle：PBS：The amount ratio of 25% glutaraldehyde is (8- 15)mg：(8-15)mL：(2-3)mL；Preferably, amidized Fe3O4Nano-particle：PBS：The amount ratio of 25% glutaraldehyde is 10mg：10mL：2.5mL；A concentration of 0.01M of wherein PBS, pH value 7.4；Optional, the time being stirred to react is 6h；Optional, the number of washing is 3 times.
- 5. the method for quick separating sugarcane ratoon stunting disease pathogenic bacteria Lxx as described in claim 1, which is characterized in that the Lxx In the preparation process of immunomagnetic beads, the preparation method of the Lxx polyclonal antibodies is：It is prepared by Lxx antigens：Sugarcane juice after filtering is coated on the fresh MSC culture mediums containing acidum nalidixicum, light culture, 30-50 days Observation and picking colony afterwards；Lxx Antibody preparations：0.4% formalin-inactivated is added in using the bacterium colony of above-mentioned picking, adds in Freund's adjuvant, direct injection rabbit Son is beaten once for two weeks, is extracted serum after being immunized 4 times, two months altogether and is made affinity chromatography using Protein A-Sepharose 4B Medium purification obtains antibody；Preferably, the sugarcane juice after the filtering is put into super-clean bench after progress surface clean sterilizing and dries in the air to cut sugarcane basal part of stem It is dry；Sugarcane stem is cut into suitable size and squeezes juice with tweezers, 4000r/min collects sugarcane juice after centrifuging 10min, carried out with ordinary filter paper It is filtered again with 0.45 μm of biofilter after preliminary filtering.
- 6. the method for quick separating sugarcane ratoon stunting disease pathogenic bacteria Lxx as described in claim 1, which is characterized in that the Lxx In the preparation process of immunomagnetic beads, a concentration of 5-15 μ g/ml of the acidum nalidixicum；Preferably, a concentration of 10 μ of the acidum nalidixicum g/ml；Optional, the temperature of light culture is 26-30 DEG C, and the time of culture is 35-45 days.
- 7. the method for quick separating sugarcane ratoon stunting disease pathogenic bacteria Lxx as described in claim 1, which is characterized in that the Lxx In the preparation process of immunomagnetic beads, the dosage of the Lxx polyclonal antibodies and the amination nanometer magnetic bead after activation is 5 μm of ol： (5-15)ml；Preferably, the dosage of Lxx polyclonal antibodies and the amination nanometer magnetic bead after activation is 5 μm of ol：10ml；Optional, the condition of the stirring is to stir 3h at 37 DEG C；Optional, the number of the washing is 3 times.
- 8. the method for quick separating sugarcane ratoon stunting disease pathogenic bacteria Lxx as described in claim 1, which is characterized in that the Lxx Separating step in, the sugarcane juice is that sugarcane stem is cut into suitable size to squeeze juice with tweezers, and 4000r/min is collected after centrifuging 10min It obtains；Optional, the filtering using ordinary filter paper be filtered with 0.45 μm of biofilter again after tentatively filtering；Optional, the sugarcane juice after the filtering：Lxx immunomagnetic beads dosage is 1：(30-50)；Preferably, the sugarcane juice after filtering： Lxx immunomagnetic beads dosage is 1：40；Optional, the condition of the incubation is 37 DEG C of incubation 1h；Optional, the rotating speed of the low-speed centrifugal is 2000-5000rpm；Optional, the number of the PBS washings is 2-3 times.
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