CN108251560A - Method and kit for rapidly identifying ralstonia solanacearum by using ralstonia solanacearum bacteriophage - Google Patents
Method and kit for rapidly identifying ralstonia solanacearum by using ralstonia solanacearum bacteriophage Download PDFInfo
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Abstract
the invention provides a method and a kit for rapidly identifying ralstonia solanacearum by using ralstonia solanacearum phages, belonging to the technical field of ralstonia solanacearum identification, wherein the method comprises the following steps of 1) coating a strain to be detected on a L B agar plate, standing for 15-20 min to obtain a plate to be detected, 2) dropwise adding the ralstonia solanacearum phages to the plate to be detected to obtain an identification system, and 3) standing and culturing the identification system obtained in the step 2) at 25-32 ℃ for 24-48 h, wherein if plaques appear, the strain to be detected is ralstonia solanacearum.
Description
Technical field
The invention belongs to bacterial wilt strain identification technology fields, and in particular to a kind of to use ralstonia solanacearum bacteriophage Rapid identification
The method and kit of ralstonia solanacearum.
Background technology
Ralstonia solanacearum is a kind of destructive native transmissibility pathogen, and host range is wide, from monocotyledon, dicotyledon
To trees and shrub, plant is planted in can infect a section more than 50 more than 200, which infects since the root of host plant, enters
Xylem vessel is invaded, is expanded to the aerial part of plant rapidly by fibrovascular system, a large amount of colonize of germ causes conduit work(
It can lose, and then cause plant wilt, lead to Plant death.Bacterial wilt is difficult to prevent, and crop is once susceptible, it will causes a large amount of
The underproduction.Therefore, it is to prevent the effective means of bacterial wilt generation to the detection in advance of ralstonia solanacearum in plant.
At present, the identification of ralstonia solanacearum is depended on colony characteristics, morphological features, physiological and biochemical property and
16S rDNA sequence analysis methods identify bacterial strain.But since bacterial strain is grown under different growing environment, thalline
Form and the bacterium colony of generation have different degrees of difference, while using method for identifying molecules tend not to accurately determine to be detected
Bacterial strain classification position, therefore, in the prior art often by morphological feature, physiological and biochemical property is combined with method for identifying molecules
Bacterial strain is identified together.To sum up, it is complex to the identification method of ralstonia solanacearum at present, and rely on microbial morphology
Correct conclusion can just be obtained by learning the long-term work experience of expert, it can be seen that, which is unfavorable for quickly detecting, and then limits
Make its extensive use.
Invention content
In view of this, the purpose of the present invention is to provide a kind of sides with ralstonia solanacearum bacteriophage Rapid identification ralstonia solanacearum
Method and kit.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
The present invention provides a kind of method using ralstonia solanacearum bacteriophage Rapid identification ralstonia solanacearum, includes the following steps：
1) by a concentration of 107~109CFU/mL strains to be checked are coated on LB agar plates, stand 15~20min, are obtained
Tablet to be checked；
2) ralstonia solanacearum bacteriophage mixed liquor is added dropwise on the tablet to be checked, obtains identification system；
3) identification system obtained in step 2) is placed in 25~32 DEG C, 24~48h of quiescent culture, observation identification system is such as
Fruit has plaque appearance, and strain idenfication to be checked is ralstonia solanacearum.
Preferably, the body fluid of ralstonia solanacearum phagocytosis described in step 1) includes 15~30 kinds of ralstonia solanacearum bacteriophages.
Preferably, the coating volume of the step 1) strain to be checked is 100~200 μ l；
When the coating volume of the strain to be checked is 100~200 μ l, ralstonia solanacearum bacteriophage drop in the step 2)
It is 20~50 μ l to add volume.
Preferably, the potency of the ralstonia solanacearum bacteriophage described in step 2) is 108~1010PFU/mL。
Preferably, the acquisition methods of the ralstonia solanacearum phagocytosis body fluid include the following steps：1) bacterial wilt that will be separated to
Bacterium bacteriophage obtains culture solution with host strain ralstonia solanacearum mixed culture；2) by culture solution separation of solid and liquid, collecting liquid phase component is
Bacterial wilt phagocytosis body fluid.
Preferably, the method for the separation of solid and liquid is centrifugation.
Preferably, the rotating speed of the centrifugation is 6000~12000rpm, and the time of the centrifugation is 5~20min.
Preferably, the temperature of the centrifugation is 2~10 DEG C.
The present invention also provides a kind of kit of Rapid identification bacterial wilt, including ralstonia solanacearum phagocytosis body fluid.
Preferably, the kit further includes LB agar plates.
Beneficial effects of the present invention
The identification method of ralstonia solanacearum of the present invention infects the specificity of host strain using bacteriophage and cracks original
Reason, ralstonia solanacearum is identified using ralstonia solanacearum bacteriophage, and at low cost, time saving and energy saving, required time is only 24~48h, and
Ralstonia solanacearum can be fast and accurately identified, particularly suitable for the preliminary screening of a large amount of ralstonia solanacearums.
Description of the drawings
Fig. 1 is the photo of ralstonia solanacearum identification system plaque in embodiment 1.
Specific embodiment
The present invention provides a kind of methods with ralstonia solanacearum bacteriophage Rapid identification ralstonia solanacearum, include the following steps：
1) by a concentration of 107~109CFU/mL strains to be checked are coated on LB agar plates, stand 15~20min, are obtained
Tablet to be checked；
2) ralstonia solanacearum bacteriophage drop is added on tablet to be checked, obtains identification system；
3) identification system obtained in step 2) is placed in 25~32 DEG C, 24~48h of quiescent culture, observation identification system is such as
Fruit has plaque appearance, and strain idenfication to be checked is ralstonia solanacearum.
In the present invention, by a concentration of 107~109CFU/mL strains to be checked are coated on LB agar plates, and standing 15~
20min obtains tablet to be checked.The present invention does not limit the source of the strain to be checked, in specific implementation process of the present invention,
The strain to be checked can be detached from environmental sample, such as water sample, soil sample or animal tissue etc..The strain to be checked is preferred
For the pure bacterium after isolating and purifying.
In the present invention, the bacterium to be checked is preferably cultivated before identification, and the incubation time of the bacterium to be checked is preferred
For 8~12h；The method and condition of the culture using this field routine bacterial screening during cultural method and condition be
It can.
The concentration of the bacterium to be checked is preferably 108CFU/mL.In the present invention, it is preferred to bacterium to be checked is coated on LB agar
On tablet, in the present invention, the coating preferably carries out in gnotobasis, can specifically be carried out in aseptic operating platform；
In the present invention, the coating volume of the strain to be checked is 100~200 μ l, more preferably preferably 120~180 μ l, 150 μ l.
After the bacterium to be checked is coated on LB agar plates, 15~20min is stood, the time of the standing is preferably 16~18min.This hair
It is bright that tablet to be checked is obtained after the standing.
The present invention adds to ralstonia solanacearum bacteriophage drop on tablet to be checked after tablet to be checked is obtained, and obtains identification body
System.In the present invention, the ralstonia solanacearum phagocytosis body fluid preferably includes 10 kinds or more ralstonia solanacearum bacteriophages, more preferably including 15
~25 kinds；The potency of the ralstonia solanacearum phagocytosis body fluid is preferably 108~1010PFU/mL, more preferably 109PFU/mL；Institute
The volume for stating bacterial wilt phagocytosis body fluid is preferably 20~50 μ l, more preferably 30~40 μ l.
In the present invention, the ralstonia solanacearum bacteriophage is preferably isolated from soil.The present invention is to the side of the separation
Method is not particularly limited, using bacteriophage separation method known in the art.
In the present invention, preferably culture obtains ralstonia solanacearum phagocytosis body fluid, institute to the ralstonia solanacearum bacteriophage before use
The method for stating culture preferably includes following steps：A the ralstonia solanacearum bacteriophage being separated to) is mixed into training with host strain ralstonia solanacearum
It supports and obtains culture solution；B) by culture solution separation of solid and liquid, collection liquid phase component is ralstonia solanacearum phagocytosis body fluid.
In the present invention, it can also be the bacterium that self-control isolates and purifies that the host strain ralstonia solanacearum, which can be commercially available strain,
Kind；Specifically the host strain ralstonia solanacearum described in implementation process of the present invention is derived from and is suffered from the plant of bacterial wilt.The present invention exists
Ralstonia solanacearum bacteriophage is with before host strain ralstonia solanacearum mixed culture, preferably cultivating host strain ralstonia solanacearum, in this hair
The culture of host strain ralstonia solanacearum described in bright is inoculated with for solid single bacterium colony or liquid inoculation culture.When the host strain blueness is withered
When germ is inoculated with for solid single bacterium colony, preferably aseptically the single bacterium colony inoculation on picking conservation tablet, cultivation temperature are excellent
It is selected as 25~32 DEG C, more preferably 28~30 DEG C；The culture rotating speed is preferably 50~200rpm, more preferably 100~
180rpm；14~18h of incubation time.When the host strain is liquid inoculation culture, preferably aseptically by low temperature
The liquid bacterium solution of storage, which is inoculated in fluid nutrient medium, is cultivated；The inoculum concentration of the liquid bacterium solution is preferably 10~20%
(volume), more preferably 12~18%；The density of the liquid bacterium solution is preferably 106~108CFU/mL, more preferably 107CFU/
mL；The incubation time is preferably 10~16h, more preferably 12~14h；Cultivation temperature during the liquid inoculation culture turns
Consistent when speed is with solid single bacterium colony inoculated and cultured, details are not described herein.The present invention is to the culture medium of the Liquid Culture without spy
It is different to limit, using the ralstonia solanacearum culture medium of this field routine.
The present invention is not special to the ralstonia solanacearum bacteriophage and condition of culture that host strain ralstonia solanacearum is mixed
It limits, it is withered using above-mentioned host strain blueness in specific implementation process using the condition of culture of the ralstonia solanacearum of this field routine
The condition of culture of germ.
In the present invention, the method for the separation of solid and liquid preferably centrifuges；The rotating speed of the centrifugation is preferably 1000~
8000rpm, more preferably 3000~6000rpm；The time of the centrifugation is preferably 5~20min, more preferably 10~15min；
The temperature of the centrifugation is preferably 2~10 DEG C, more preferably 4~6 DEG C.
The present invention is placed in 25~32 DEG C, 24~48h of quiescent culture after identification system is obtained, by the identification system of acquisition,
Identification system is observed, if there is plaque occurs, strain idenfication to be checked is ralstonia solanacearum.The identification system is quiet in the present invention
It is preferably 28~30 DEG C to put cultivation temperature；The time of the quiescent culture is preferably 24~36h, more preferably 32h.The present invention exists
After quiescent culture, identification system is observed, if there is plaque appearance in identification system, strain idenfication to be checked is bacterial wilt
Bacterium.
The present invention also provides a kind of kit of Rapid identification ralstonia solanacearum, including ralstonia solanacearum phagocytosis body fluid.It is described
Ralstonia solanacearum phagocytosis body fluid preferably includes 10 kinds or more ralstonia solanacearum bacteriophages.The ralstonia solanacearum phagocytosis body fluid preferably includes 15
~20 kinds；The potency of the ralstonia solanacearum bacteriophage mixed liquor is preferably 108~1010PFU/mL, more preferably 109PFU/
mL.The kit further preferably includes LB agar plates.The present invention does not have special limit to the preparation method of the LB agar plates
System, using preparation scheme well-known to those skilled in the art.
The application method of heretofore described kit uses ralstonia solanacearum bacteriophage Rapid identification ralstonia solanacearum with reference to above-mentioned
Method, details are not described herein.
With reference to embodiment to provided by the invention a kind of using ralstonia solanacearum bacteriophage Rapid identification ralstonia solanacearum
Method is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
The present embodiment is included the following steps using the method for ralstonia solanacearum bacteriophage Rapid identification ralstonia solanacearum：Step A：It is green
Blight bacterium isolates and purifies, if the present invention is detached from the vega soil that bacterial wilt is suffered from the area such as Guangdong, Fujian, Shandong, Henan
It is dry to obtain 40 plants of ralstonia solanacearum；Step B：The separation identification of bacteriophage, carries out bacteriophage primary dcreening operation by the bacterium being separated to, obtains phagocytosis
The bacterium of body carries out biochemical identification and 16SrDNA identifications again, so that it is determined that separation obtains 24 plants of virulent phages.By using bilayer
Plate method carries out purifying conservation to bacteriophage.Step D：Bacteriophage spreads cultivation, and spreads cultivation to the bacteriophage separated；Step E：It will
The culture solution of gained, 8000rpm centrifuges 5min at 4 DEG C so that host strain falls to bottom, and upper strata is taken to clarify part, adjustment
Its potency is 108PFU/mL；Step F：Be separated to 24 plants of phagocytosis body fluid equal proportions are mixed, as ralstonia solanacearum identification liquid.
Using drop method, by 40 plants be incubated overnight a concentration of 107The various ralstonia solanacearums of CFU/mL use liquid relief similar to bacterium
Rifle draws 100 μ l and drops in tablet center, then is spread evenly across LB agar plates with spreading rod, stands 20 minutes, treats that liquid parches,
The ralstonia solanacearum bacteriophage mixed liquor of 20 μ l is added dropwise, keeps 32 DEG C of environment temperature and quiescent culture is for 24 hours, observation, which whether there is plaque, to go out
It is existing；If so, it is proved to be ralstonia solanacearum.
As a result, it has been found that：For 40 plants of ralstonia solanacearums similar in bacterium, 35 plants there is plaque (Fig. 1), and biochemical and 16s is carried out to it
RDNA is accredited as ralstonia solanacearum, rate of accuracy reached to 100%.
Embodiment 2
The present embodiment is included the following steps using the method for ralstonia solanacearum bacteriophage Rapid identification ralstonia solanacearum：Step A：It is green
Blight bacterium isolates and purifies, and the present invention suffers from separation in bacterial wilt vega soil from Guangxi, Shandong, Hebei Province and obtains ralstonia solanacearum
Similar 50 plants of bacterium, purifies conservation.Step B：LB fluid nutrient mediums are prepared, respectively on aseptically picking conservation tablet
Single bacterium colony is inoculated with, and cultivation temperature makes its concentration reach 10 for 30 DEG C, rotating speed 150rpm, incubation time 16h7More than.Step C：
Using drop method, by 50 plants be incubated overnight a concentration of 107The various ralstonia solanacearums of CFU/mL are drawn similar to bacterium with liquid-transfering gun
100 μ l drop in tablet center, then are spread evenly across LB agar plates with spreading rod, stand 20 minutes, the bacterial wilt of 20 μ l is added dropwise
Bacterium bacteriophage mixed liquor, keeps 30 DEG C of environment temperature and quiescent culture 48h, and observation whether there is plaque appearance；If so, it proves
It is ralstonia solanacearum.
As a result, it has been found that：For 50 plants of ralstonia solanacearums similar in bacterium, 32 plants there is plaque, and biochemical and 16srDNA mirror are carried out to it
It is ralstonia solanacearum to be set to 32 plants, rate of accuracy reached to 100%.
Embodiment 3
The present embodiment is included the following steps using the method for ralstonia solanacearum bacteriophage Rapid identification ralstonia solanacearum：Step A：Place
Main bacterium isolates and purifies, and ralstonia solanacearum of the invention suffers from separation in bacterial wilt vega soil from different regions and obtains doubtful bacterial wilt
100 plants of bacterial strain.Step B：By 121 DEG C of high pressure sterilization 20min of LB fluid nutrient mediums, it is cooled to room temperature.In aseptic condition, pass through bacterium
Liquid is inoculated with, and 100 plants of bacterium is shaken respectively logarithmic phase is spare, and condition is that temperature is 28 DEG C, rotating speed 200rpm, incubation time
10h；Step C：Using drop method, by 100 plants of ralstonia solanacearums in logarithmic phase similar to bacterium solution, 150 μ l drops are drawn with liquid-transfering gun
LB agar plates are spread evenly across in tablet center, then with spreading rod, stand 15min, the ralstonia solanacearum that 20 μ l are added dropwise respectively is bitten
Thalline mixed liquor, keeps 28 DEG C of environment temperature and quiescent culture 36h, and observation whether there is plaque appearance；If so, it is proved to be green
Blight bacterium.
The results show that 100 plants of ralstonia solanacearums, similar in bacterium, 78 plants there is plaque, through further identify be determined as it is green withered
Germ, rate of accuracy reached to 100%.
Embodiment 4
The present embodiment is included the following steps using the method for ralstonia solanacearum bacteriophage Rapid identification ralstonia solanacearum：Step A：Place
Main bacterium isolates and purifies, and ralstonia solanacearum of the invention obtains doubtful bacterial wilt bacterial strain 200 from separation in bacterial wilt vega soil is suffered from
Strain.Step B：By 121 DEG C of high pressure sterilization 20min of LB fluid nutrient mediums, it is cooled to room temperature, in aseptic condition, is inoculated with by bacterium solution,
200 plants of bacterium are shaken respectively logarithmic phase is spare, and condition is that temperature is 25 DEG C, rotating speed 120rpm, incubation time 14h；Step
C：Using drop method, by 200 plants of ralstonia solanacearums in logarithmic phase similar to bacterium solution, draw 200 μ l with liquid-transfering gun and drop in tablet
Centre, then LB agar plates are spread evenly across with spreading rod, 15min is stood, the ralstonia solanacearum bacteriophage mixing of 20 μ l is added dropwise respectively
Liquid, keeps 28 DEG C of environment temperature and quiescent culture 48h, and observation whether there is plaque appearance；If so, it is proved to be ralstonia solanacearum.
The results show that 200 plants of ralstonia solanacearums, similar in bacterium, 144 plants there is plaque, through being further determined as bacterial wilt
Bacterium, rate of accuracy reached to 100%.
Embodiment 5
A kind of kit of Rapid identification ralstonia solanacearum, puts down including 20 kinds of ralstonia solanacearum bacteriophage mixed liquors and LB agar
Plate.The potency of the ralstonia solanacearum bacteriophage mixed liquor is 109PFU/mL。
The application method of the kit：
Using drop method, by 8 plants be incubated overnight a concentration of 107The similar bacterium of the various ralstonia solanacearums of CFU/mL, with shifting
Liquid rifle draws 200 μ l and drops in the LB agar plates center in kit, then is spread evenly across LB agar plates with spreading rod, stands
The ralstonia solanacearum bacteriophage mixed liquor in 20 μ l kits is added dropwise in 20min, keeps 28 DEG C of environment temperature and quiescent culture 28h, sees
It examines and whether there is plaque appearance；If so, it is proved to be ralstonia solanacearum.
As a result, it has been found that：In the similar bacterium for identifying 8 plants of ralstonia solanacearums, 6 kinds there is plaque, are accredited as ralstonia solanacearum, accurately
Rate reaches 100%.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of method using ralstonia solanacearum bacteriophage Rapid identification ralstonia solanacearum includes the following steps：
1) by a concentration of 107~109CFU/mL strains to be checked are coated on LB agar plates, stand 15~20min, are obtained to be checked
Tablet；
2) ralstonia solanacearum bacteriophage drop is added on the tablet to be checked, obtains identification system；
3) identification system obtained in the step 2) is placed in 25~32 DEG C, 24~48h of quiescent culture, observes identification system,
If there is plaque occurs, then strain idenfication to be checked is ralstonia solanacearum.
2. according to the method described in claim 1, it is characterized in that, the body fluid of ralstonia solanacearum phagocytosis described in step 2) includes 15
~30 kinds of ralstonia solanacearum bacteriophages.
3. according to the method described in claim 1, it is characterized in that, the coating volume of strain to be checked is 100 in the step 1)
~200 μ l；
When the coating volume of the strain to be checked is 100~200 μ l, ralstonia solanacearum bacteriophage drop adds body in the step 2)
Product is 20~50 μ l.
4. method according to claim 1 or 2, which is characterized in that the effect of ralstonia solanacearum phagocytosis body fluid in the step 2)
Valency is 108~1010PFU/mL。
5. according to the method described in claim 1, it is characterized in that, the acquisition methods of the ralstonia solanacearum phagocytosis body fluid include with
Lower step：
A) ralstonia solanacearum bacteriophage and host strain ralstonia solanacearum are mixed, obtain culture solution；
B) by the culture solution separation of solid and liquid, collection liquid phase component is ralstonia solanacearum phagocytosis body fluid.
6. according to the method described in claim 5, it is characterized in that, the method for the separation of solid and liquid is centrifugation.
7. according to the method described in claim 6, it is characterized in that, the rotating speed of the centrifugation be 6000~12000rpm, it is described
The time of centrifugation is 5~20min.
8. the method described according to claim 6 or 7, which is characterized in that the temperature of the centrifugation is 2~10 DEG C.
9. a kind of kit of Rapid identification ralstonia solanacearum, which is characterized in that including ralstonia solanacearum phagocytosis body fluid.
10. kit according to claim 9, which is characterized in that the kit further includes LB agar plates.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109321462A (en) * | 2018-10-17 | 2019-02-12 | 中国烟草总公司郑州烟草研究院 | A kind of method that separation ralstonia solanacearum is combined using resistant panel screening and test strips identification in field |
| CN109406778A (en) * | 2018-10-15 | 2019-03-01 | 国家烟草质量监督检验中心 | The time-resolved fluorescence quantitative test paper item and its preparation method and application of ralstonia solanacearum in a kind of detection tobacco leaf |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109406778A (en) * | 2018-10-15 | 2019-03-01 | 国家烟草质量监督检验中心 | The time-resolved fluorescence quantitative test paper item and its preparation method and application of ralstonia solanacearum in a kind of detection tobacco leaf |
| CN109406778B (en) * | 2018-10-15 | 2021-06-29 | 国家烟草质量监督检验中心 | Time-resolved fluorescence quantitative test strip for detecting ralstonia solanacearum in tobacco leaves and preparation method and application thereof |
| CN109321462A (en) * | 2018-10-17 | 2019-02-12 | 中国烟草总公司郑州烟草研究院 | A kind of method that separation ralstonia solanacearum is combined using resistant panel screening and test strips identification in field |
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