CN109609666A - A kind of sweet potato potato seasonal febrile diseases bacterium molecule detection primer and its application - Google Patents
A kind of sweet potato potato seasonal febrile diseases bacterium molecule detection primer and its application Download PDFInfo
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Abstract
The present invention provides a kind of sweet potato potato seasonal febrile diseases bacterium molecule detection primer and its application, which is SPRS-F and SPRS-R, and gene order, as shown in SEQ ID NO.1 and SEQ ID NO.2, the rapid molecular which is applied to sweet potato potato seasonal febrile diseases bacterium detects；The present invention establishes corresponding polymerase chain reaction PCR amplification system simultaneously, rapid molecular detection for sweet potato with potato pest bacterium sample, by PCR amplification and agarose gel electrophoresis, the specific amplified product that fragment length is 1641bp can be amplified in sweet potato potato seasonal febrile diseases bacterium pure dna, the disease plant to carry disease germs tissue.Result of the present invention is reliable, easy to operation, high sensitivity, high specificity, the detection to sweet potato potato seasonal febrile diseases bacterium in invalid body, disease plant and seedling can be passed through, long term monitoring for prediction, Prevalent district before plant disease epidemic in production practices, and control approach and method are instructed, it is of great significance for sweet potato potato seasonal febrile diseases prevention and treatment in production.
Description
[technical field]
The present invention relates to a kind of sweet potato potato seasonal febrile diseases bacterium molecule detection primer and its applications.
[background technique]
1, potato pest bacterium endangers sweet potato serious, but endangers the later period and be difficult to the differentiation of other pathogenic bacteria of same sweet potato, needs accurately
Molecular labeling is identified to be prevented and treated with Instructing manufacture.
Sweet potato (Ipomoea batatas (L.) Lam.) is one of world food, the energy and insutrial crop, and China is
Maximum sweet potato plants state in the whole world.The cause of disease of sweet potato potato seasonal febrile diseases is ot Pseudomonas solanacearum in Sweet Potatoes (Ralstonia solanacearum),
Belong to Evolution Type I type, No. 1 biological strain, No. 15 sequence mutation in Ralstonia solanacearum.Sweet potato potato seasonal febrile diseases can make southern sweet potato producing region
At destructive strike, field General Loss 20%~30% of falling ill, severe one is up to 60%~70%, or even full field total crop failure, because
This disease has been cited as sweet potato quarantine disease.Ralstonia solanacearum to the infection processs of plant be commonly from plant roots stem wound or from
Right aperture intrusion, first colonizes in cortex, space between cells etc., then the mass propagation in flora conduit and tissue around, and point
It secretes exocellular polysaccharide obstruction conduit and destroys surrounding tissue, finally make plant wilt dead, classical symptom is exactly plant in wilting
Dark green color is still kept under state.But in the morbidity middle and later periods, sweet potato stalk becomes brown, dark brown, and plant is wilted, with
Other sweet potato Disease symptoms are similar, such as black rot, stem rot, and microbe species are various in plant tissue.Therefore, in production
It was found that symptom too time-consuming work consuming if be separately cultured with traditional symptom identification, microorganism and identified one by one again, if there is
Whether specific molecular marker is just being detected at the first time, can at least illustrate in disease sample containing sweet potato potato seasonal febrile diseases bacterium, can be with
Quickly take measures to be prevented and treated.
2, there has been no specific molecular markers for sweet potato potato seasonal febrile diseases bacterium
Ralstonia solanacearum (Ralstonia solanacearum) belonging to sweet potato potato seasonal febrile diseases bacterium can pass through specific molecular mark
It remembers row into fast and accurately to identify, molecular labeling is typically based on the intergenic spacer region 16S-23S rRNA (ITS), c1 cytochromes
The specific sequences such as signal peptide.But Ralstonia solanacearum is a complicated monoid, is embodied in genetic diversity abundant and (plants interior DNA
Homology is lower than general standard 70%), (54 sections, more than 450 plant for the highly variable of genome and extensive host range
Object).At this stage, molecular marker identification method is 2005 by Fegan and Prior under the most accurate Ralstonia solanacearum kind of international endorsement
The Evolution Type frame (Phylotype classification) proposed jointly, is considered as species complex (Species for Ralstonia solanacearum
Complex), Ralstonia solanacearum group is divided by kind of (Species), Evolution Type according to the phylogenetic affiliation of Ralstonia solanacearum
(Phylotype), sequence mutation (Sequevar) and clone (Clone), lose its significance lies in that being able to reflect in the kind of Ralstonia solanacearum
Diversity and geographic origin are passed, but does not have specific correlation with the effect host of Ralstonia solanacearum.And Ralstonia solanacearum host is related
Discrimination method be generally to be identified with biological strain (Race) partitioning, but more host's discrimination methods of this artificial infection
Lack accuracy to take time and effort very much again, there has been no identify molecular labeling, sweet potato potato pest under the relevant Ralstonia solanacearum kind of host specificity
Germ does not also have specific molecular marker.In other words, for sweet potato disease sample, the prior art can identify whether invaded by Ralstonia solanacearum
Dye, but potato pest bacterium or other Ralstonia solanacearums can not just judge.
3, pathogen specific gene sequences can be found to develop numerator detection mark by genomic information
Since carrying out genome sequencing to Ralstonia solanacearum GMI1000 with Salanoubat in 2002 et al., at this stage
Had the bacterial strain of 71 Ralstonia solanacearums carried out gene order-checking (according to the website NCBI Genome search result, comprising complete figure with
Frame diagram), due to the changeability of Ralstonia solanacearum genome, with the difference of genome between different Isolates of Pseudomonas Solanacearum Smith, ratio can be passed through
Exploitation compared with the specific gene that the method for genomics excavates independent Ralstonia solanacearum as molecular labeling.Such as Yang Jiaodi et al. base
Tobacco Ralstonia solanacearum specific detector molecules label is had developed in tobacco Ralstonia solanacearum genome specificity sequence.
Early-stage study basis of the present invention be based on carrying out gene order-checkings to 2 plants of sweet potato potato pest bacterium, and with other 66 plants of blueness
Withered bacterium genome is compared genome analysis, obtains potato pest bacterium specific gene.And by the gene in NCBI BLAST
Comparison result on website, choose can not compared with any reference sequences region design specific primer SPRS-F and
SPRS-R.It is compared detection with the cause of disease Ralstonia solanacearum of other 6 kinds of crops, it is found that the primer has sweet potato potato pest bacterium specificity.
Primer SPRS-F and SPRS-R and corresponding sweet potato potato seasonal febrile diseases bacterium PCR detection method can be provided to production application more to be accelerated
Speed, reliable detection, quickly to determine that field has whether symptom sample is that epiphytotic pathogen causes to take corresponding measure
Carry out prevention and control.
[summary of the invention]
One of the technical problem to be solved in the present invention is to provide a kind of sweet potato potato seasonal febrile diseases bacterium molecule detection primer, can
It realizes potato pest bacterium specific molecular marker and quickly detection, operation, high sensitivity, high specificity is as a result reliable, easy to, for life
The prevention and treatment of sweet potato potato seasonal febrile diseases is of great significance in production.
The present invention is realized in one of above-mentioned technical problem:
A kind of sweet potato potato seasonal febrile diseases bacterium molecule detection primer, the sequence of the sweet potato potato seasonal febrile diseases bacterium molecule detection primer are as follows:
SPRS-F:5 '-GACACATCAGGGCCATTTTCACC-3 '
SPRS-R:5 '-GCCTCTGCCAGTGAGATCATC-3 '.
The second technical problem to be solved by the present invention is to provide a kind of answering for sweet potato potato seasonal febrile diseases bacterium molecule detection primer
With, can be realized potato pest bacterium specific molecular marker and quickly detection, be as a result reliable, easy to operation, high sensitivity, specificity
By force, it is of great significance for sweet potato potato seasonal febrile diseases prevention and treatment in production.
The present invention is realized in the twos' of above-mentioned technical problem:
A kind of application of sweet potato potato seasonal febrile diseases bacterium molecule detection primer, the molecular detection primer are applied to sweet potato potato seasonal febrile diseases bacterium
Rapid molecular detection, detection method is as follows:
(1) DNA or (2) are extracted from the doubtful plant infected by sweet potato potato seasonal febrile diseases bacterium from doubtful by sweet potato potato seasonal febrile diseases bacterium sense
DNA is extracted in the bacteria suspension of dye plant preparation；The above step DNA is logical for template a pair of molecular detection primer SPRS-F and SPRS-R
Polymerase chain reaction PCR amplification is crossed, PCR amplified production is separated with agarose electrophoresis, the Yu Ning after SYBR Green I dyeing
It is observed under glue imaging analysis instrument ultraviolet lamp, if amplified production is 1641bp, that is, can determine whether that there are sweet potato potatos in the sample
Seasonal febrile diseases bacterium；
The gene order of the molecular detection primer SPRS-F is as shown in SEQ ID NO.1, the molecular detection primer
The gene order of SPRS-R is as shown in SEQ ID NO.2, and obtained amplified production gene order is as shown in SEQ ID NO.3.
Further, in the detection method (1) from it is doubtful by sweet potato potato seasonal febrile diseases bacterium infect plant in extract DNA tool
Body method are as follows: take 50-300mg sweet potato diseased tissues, ground after liquid nitrogen frozen, 300 μ l ddH2O are added and mix；Solution is set into boiling water
In boil 10min；Solution takes 1 μ l to participate in PCR reaction as DNA profiling after using ddH2O to dilute 100 times.
Further, in the detection method (2) from it is doubtful by sweet potato potato seasonal febrile diseases bacterium infected plant prepare bacteria suspension in
Extract DNA's method particularly includes: site of pathological change is taken, on clean slide, covered after clear water to be added containing vascular tissue
And tissue is squeezed from coverslip, later by slide to whether generated around light or micro- sem observation vascular bundle spray bacterium phenomenon；If
Spray bacterium phenomenon is generated, then the bacteria suspension exhaustion on glass slide around tissue is added in centrifuge tube；Bacteria suspension is set in boiling water
Boil 10min；Solution takes 1 μ l to participate in PCR reaction as DNA profiling after using ddH2O to dilute 100 times.
Further, PCR reaction system is 20 μ l, including 10 μ l 2 × EasyTaq PCR SuperMix hybrid reactions
Solution, each 0.2 μm of ol/l of primer SPRS-F and SPRS-R, 10-100ng template DNA, ddH2O supply 20 μ l.
Further, PCR reaction system is 20 μ l, including 10 μ l 2 × EasyTaq PCR SuperMix hybrid reactions
Solution, each 0.2 μm of ol/l of primer SPRS-F and SPRS-R, 10-100ng template DNA, 0.2 μ l DMSO dimethyl sulfoxide,
DdH2O supplies 20 μ l.
Further, PCR reaction condition are as follows: 94 DEG C of initial denaturation 4min, 94 DEG C of denaturation 30s, 52~64 DEG C of annealing 30s, 72
DEG C extend 90s, recycle 30 times, 72 DEG C of extension 5min.
The present invention has the advantage that
1, high specificity, accurate and reliable: the primer SPRS-F and SPRS-R of the present invention is to utilize sweet potato potato seasonal febrile diseases bacterium base
Because of the specific gene orf428 design in group.The gene has not yet been sequenced in 66 Isolates of Pseudomonas Solanacearum Smith genomes at other
It finds, while comparison result of the gene in the website NCBI BLAST shows that the gene specific is very high (withered with immediate blueness
Sequence similarity in bacterium allied species Ralstonia insidiosa be only 71%), and the design position of primer not with it is any
On reference sequences compare.The primer of the present invention and method are in Fujian different regions and Zhanjiang area potato pest bacterium
Resource is verified on 6 kinds of other crop ralstonia solanacearums in addition to potato pest bacterium, and determination can expand shaping in all potato pest bacterium resources
Band, and all do not have in other 6 kinds of Ralstonia solanacearums, that is, illustrate the technology of the present invention high specificity, as a result accurately and reliably.
2, strong operability, fast and convenient: the method for the present invention does not need that microorganism in sick sample is separately cultured and is given birth to
Object identification；DNA extraction method is also more easier than traditional extracting method, does not need removal sample contamination precipitation purification DNA,
DNA extraction can be completed in 30 minutes；It can be judged by banding pattern after the direct agarose electrophoresis of PCR result, not need to be sequenced；Entire inspection
3-4 hour of survey process can complete.
3, practical, high sensitivity: found in production sick sample without complex process can DNA rapid extraction tested
Card, for potato pest bacterium DNA content demand in sample down to 10pg.
In short, the present invention at this stage there has been no potato pest bacterium specific molecular marker and rapid detection method, pass through
Potato pest bacterium genome and other Ralstonia solanacearum genome comparisons find specific gene orf428, and sequence design goes out a pair of special accordingly
Specific primer SPRS-F and SPRS-R, and corresponding polymerase chain reaction PCR amplification system is established, for sweet potato band potato pest
The rapid molecular of bacterium sample detects.This technical result is reliable, easy to operation, high sensitivity, high specificity, can be applied to disease stream
The long term monitoring of prediction, Prevalent district before row, and control approach and method are instructed, for sweet potato potato seasonal febrile diseases in production
Prevention and treatment is of great significance.
[Detailed description of the invention]
The present invention is further illustrated in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 is the specific detection PCR amplification result electrophoretogram of 1 sweet potato potato seasonal febrile diseases bacterium of the embodiment of the present invention.
Fig. 2 is that the sensitivity of 2 sweet potato potato seasonal febrile diseases bacterium of the embodiment of the present invention detects amplification electrophoretogram.
Fig. 3 is the potato pest bacterial examination survey amplification electrophoretogram that 3 Zhong Ge sweet potato incidence tissue of the embodiment of the present invention extracts DNA.
Fig. 4 is the potato pest bacterial examination survey amplification that DNA is extracted in 4 Zhong Ge sweet potato incidence tissue bacteria suspension of the embodiment of the present invention
Electrophoretogram.
Fig. 5 is that sweet potato incidence tissue prepares the micro- sem observation of spray bacterium phenomenon during bacteria suspension in the embodiment of the present invention 4
Figure.
[specific embodiment]
Refering to fig. 1-5, the present invention relates to a kind of sweet potato potato seasonal febrile diseases bacterium molecule detection primer, the sweet potato potato seasonal febrile diseases bacterium molecules
The sequence of detection primer are as follows:
SPRS-F:5 '-GACACATCAGGGCCATTTTCACC-3 '
SPRS-R:5 '-GCCTCTGCCAGTGAGATCATC-3 '.
The product of 1641bp can be gone out from specific amplified on sweet potato potato seasonal febrile diseases bacterium using the primer.
The specific primer sequences preparation method of sweet potato potato seasonal febrile diseases bacterium molecule detection primer are as follows:
According to different pathological form sweet potato potato seasonal febrile diseases bacterium (Ralstonia solanacearum) bacterial strain genome sequencing knots
Fruit, and genomics analysis is compared with 66 Ralstonia solanacearum genome sequencing results in ncbi database, that is, it uses
The amino acid and nucleotide sequence of all species for participating in analysis is compared in OrthoMCL v2.0.3 software, selected threshold
(BLASTP E value is not more than 1e-5, MCL_INFLATION=1.5) carries out similitude cluster, obtains homologous gene list, and
Count the species distribution situation of each albumen cluster CLUSTER.As a result, it has been found that different pathological form potato seasonal febrile diseases bacterium all contain one
The specific gene of 948 amino acid of codified, i.e. specific gene orf428, and the gene is in the website NCBI BLAST
Comparison result shows the very high (sequence belonged in Ralstonia insidiosa approximate with immediate Ralstonia solanacearum of the gene specific
71%) column similitude is only.Sweet potato potato seasonal febrile diseases bacterium specific primer SPRS-F and SPRS- are designed according to the gene specific region
R。
The invention further relates to a kind of application of sweet potato potato seasonal febrile diseases bacterium molecule detection primer, the molecular detection primer is applied to
The rapid molecular of sweet potato potato seasonal febrile diseases bacterium detects, and detection method is as follows:
(1) DNA or (2) are extracted from the doubtful plant infected by sweet potato potato seasonal febrile diseases bacterium from doubtful by sweet potato potato seasonal febrile diseases bacterium sense
DNA is extracted in the bacteria suspension of dye plant preparation；The above step DNA is logical for template a pair of molecular detection primer SPRS-F and SPRS-R
Polymerase chain reaction PCR amplification is crossed, PCR amplified production is separated with agarose electrophoresis, the Yu Ning after SYBR Green I dyeing
It is observed under glue imaging analysis instrument ultraviolet lamp, if amplified production is 1641bp, that is, can determine whether that there are sweet potato potatos in the sample
Seasonal febrile diseases bacterium；
The gene order of the molecular detection primer SPRS-F is as shown in SEQ ID NO.1, the molecular detection primer
The gene order of SPRS-R is as shown in SEQ ID NO.2, and obtained amplified production gene order is as shown in SEQ ID NO.3.
(1) extracts the specific method of DNA from the doubtful plant infected by sweet potato potato seasonal febrile diseases bacterium in the detection method
Are as follows: 50-300mg sweet potato diseased tissues is taken, is ground after liquid nitrogen frozen, 300 μ l ddH2O are added and mix；Solution is set in boiling water and is boiled
10min；Solution takes 1 μ l to participate in PCR reaction as DNA profiling after using ddH2O to dilute 100 times.
(2) extract DNA's from the doubtful bacteria suspension prepared by sweet potato potato seasonal febrile diseases bacterium infected plant in the detection method
Method particularly includes: it takes site of pathological change containing vascular tissue on clean slide, covered is added after clear water and from lid glass
On piece squeeze tissue, later by slide to whether generated around light or micro- sem observation vascular bundle spray bacterium phenomenon；If generating spray bacterium
Bacteria suspension exhaustion on glass slide around tissue is then added in centrifuge tube by phenomenon；Bacteria suspension is set and boils 10min in boiling water；
Solution takes 1 μ l to participate in PCR reaction as DNA profiling after using ddH2O to dilute 100 times.
PCR reaction system is 20 μ l, including 10 μ l 2 × EasyTaq PCR SuperMix mixed reaction solutions, primer
Each 0.2 μm of ol/l of SPRS-F and SPRS-R, 10-100ng template DNA, ddH2O supply 20 μ l.
Or PCR reaction system is 20 μ l, including 10 μ l 2 × EasyTaq PCR SuperMix mixed reaction solutions, primer
Each 0.2 μm of ol/l of SPRS-F and SPRS-R, 10-100ng template DNA, 0.2 μ l DMSO dimethyl sulfoxide, ddH2O supply 20 μ l.
PCR reaction condition are as follows: 94 DEG C of initial denaturation 4min, 94 DEG C of denaturation 30s, 52~64 DEG C of annealing 30s, 72 DEG C extend
90s is recycled 30 times, 72 DEG C of extension 5min.
Below in conjunction with specific embodiment, the present invention is further explained:
Embodiment 1:
Specific amplification of the detection primer to sweet potato potato seasonal febrile diseases bacterium
1. the specific detection of sweet potato potato seasonal febrile diseases bacterium
With sweet potato potato seasonal febrile diseases bacterium (Ralstonia solanacearum) and belong to other 6 kinds of crop Ralstonia solanacearums (tomatoes, flower
Life, capsicum, eggplant, ginger, potato Ralstonia solanacearum) it is material to be tested, using Beijing Quanshijin Biotechnology Co., Ltd's production
EasyPure Bacteria Genomic DNA Kit bacterial genomes extracts kit extracts each strains tested genomic DNA,
The specific method is as follows: the bacterium 1ml, 12000 × g that are incubated overnight being taken to be centrifuged 1 minute, as far as possible abandoning supernatant；100 μ l LB11 are added
With 20 μ l Proteinase K, oscillation thoroughly suspends to thallus；55 DEG C are incubated for 15 minutes up to the limpid shape of solution, every 5 minutes
It shakes up primary；20 μ l RNase A are added and mix standing 2 minutes；400 μ l BB11 are added to be vortexed 30 seconds；Whole solution is added
Enter in centrifugal column, 1000g is centrifuged 30 seconds, abandons efflux；500 μ l CB11,12000 × g is added centrifugation 30 seconds, abandons efflux；Weight
Multiple upper step is primary；500 μ l WB11,12000 × g is added centrifugation 30 seconds, abandons efflux；It is walked in repetition primary；12000 × g from
The heart 2 minutes, thoroughly remove remaining WB11；Centrifugal column is placed in clean centrifuge tube, 100 μ l 60- are added in column center
The EB solution of 70 DEG C of preheatings, is stored at room temperature 2 minutes, and 12000 × g is centrifuged 1 minute, eluted dna；Dilution DNA concentration is to 50ng/ μ
L, be stored in -20 DEG C it is spare.
With specific primer SPRS-F and SPRS-R to sweet potato potato seasonal febrile diseases bacterium and belong to other 6 kinds of crop Ralstonia solanacearums (tomato,
Peanut, capsicum, eggplant, ginger, potato Ralstonia solanacearum) carry out PCR amplification verifying specific reaction system: 20 μ l of PCR reaction system,
Including 10 μ l 2 × EasyTaq PCR SuperMix mixed reaction solutions, (Beijing Quanshijin Biotechnology Co., Ltd is raw
Produce), each 0.2 μm of ol/l and 30ng template DNA of primer SPRS-F and SPRS-R, ddH2O supplies 20 μ l, PCR reaction conditions are as follows:
94 DEG C of initial denaturations 4min, 94 DEG C of denaturation 30s, 52~64 DEG C of annealing 30s, 72 DEG C of extension 90s are recycled 30 times, 72 DEG C of extensions
5min。
2. testing result
Specific detection result is referring to Fig. 1: wherein each swimming lane is expressed as follows: M is DL 2000bp DNA molecular weight standard,
1-13 be different regions source sweet potato potato seasonal febrile diseases bacterium, 14-16 be Strain of Pseudomonas Solanacearum different strains, 17-19 be peanut Ralstonia solanacearum not
Same bacterial strain, 20-22 are capsicum Ralstonia solanacearum different strains, and 23-25 is eggplant Ralstonia solanacearum different strains, and 26 be ginger Ralstonia solanacearum, and 27 are
Potato Ralstonia solanacearum, 28 is using ddH2O as the negative control of template；As shown in Figure 1, in addition to the sweet potato potato in different regions source
Seasonal febrile diseases bacterium can specifically amplify outside the product of 1641bp, and 3 plants of Strain of Pseudomonas Solanacearum detected, 3 plants of peanut Ralstonia solanacearums, 3 plants peppery
Green pepper Ralstonia solanacearum, 3 plants of eggplant Ralstonia solanacearum different strains, the bacterial strains DNA such as 1 plant of ginger Ralstonia solanacearum and 1 plant of potato Ralstonia solanacearum fail to expand
Increase corresponding product out, illustrates that primer SPRS-F and SPRS-R has very strong specificity.
Embodiment 2: detection primer detects the sensitivity of sweet potato potato seasonal febrile diseases bacterium DNA
1. the DNA of detection bacterium is extracted and dilution
DNA extraction method is by the EasyPure Bacteria Genomic DNA Kit bacterium base in above-described embodiment 1
Because group extracts kit carries out, sweet potato potato seasonal febrile diseases bacterium DNA solution through gradient dilution at 100 ng/ μ l, 10ng/ μ l, 1ng/ μ l,
Then 100pg/ μ l, 10pg/ μ l, 1pg/ μ l, 100fg/ μ l, 10fg/ μ l draw 1 μ l and carry out PCR detection.
2. the sensitivity of sweet potato potato seasonal febrile diseases bacterium DNA detects
20 μ l of PCR reaction system, including 10 μ l 2 × EasyTaq PCR SuperMix mixed reaction solution (full formulas in Beijing
Golden Bioisystech Co., Ltd's production), each 0.2 μm of ol/l and 30ng template DNA of primer SPRS-F and SPRS-R, ddH2O is supplied
20μl；PCR reaction condition are as follows: 94 DEG C of initial denaturation 4min, 94 DEG C of denaturation 30s, 52~64 DEG C of annealing 30s, 72 DEG C of extension 90s are followed
Ring 30 times, 72 DEG C of extension 5min.Agar electrophoresis detects amplified production, and electrophoresis applied sample amount is 6 μ l.
3. testing result is referring to fig. 2: wherein each swimming lane is expressed as follows: M is DL 2000bp DNA molecular amount standard, and 1 is
The sweet potato potato seasonal febrile diseases bacterium DNA of 100ng, 2 for 10ng sweet potato potato seasonal febrile diseases bacterium DNA, 3 be the sweet potato potato seasonal febrile diseases bacterium DNA of 1ng, and 4 are
The sweet potato potato seasonal febrile diseases bacterium DNA of 100pg, 5 for 10pg sweet potato potato seasonal febrile diseases bacterium DNA, 6 be the sweet potato potato seasonal febrile diseases bacterium DNA of 1pg, and 7 are
The sweet potato potato seasonal febrile diseases bacterium DNA of 100 fg, 8 for 10fg sweet potato potato seasonal febrile diseases bacterium DNA, 9 is using ddH2O as the negative control of template.
As can be seen from Figure 2, swimming lane 1-6 has obvious amplified band, i.e., in 20 μ l reaction systems, there is the sweet potato potato seasonal febrile diseases bacterium base of 1pg or more
Because a group DNA can be obtained obvious amplified band.
Embodiment 3: the detection of sweet potato potato seasonal febrile diseases bacterium in morbidity plant tissue
1. sample acquires: five groups of morbidity sweet potato Plant tissue samples of acquisition, label A, B, C, D, E, morbidity sweet potato are planted respectively
Object tissue sample picks up from Foochow planting base Xin Dian.
2.DNA is extracted and detection
50-300mg sweet potato diseased tissues is taken, is ground after liquid nitrogen frozen, 300 μ l ddH are added2O is mixed；Solution is set into boiling water
In boil 10min；Solution ddH2O takes 1 μ l to participate in PCR reaction as DNA profiling after diluting 100 times.PCR reaction system is 20 μ
L, including (Beijing Quanshijin Biotechnology Co., Ltd is raw for 10 μ l 2 × EasyTaq PCR SuperMix mixed reaction solutions
Produce), each 0.2 μm of ol/l of primer SPRS-F and SPRS-R, 10-100ng template DNA, 0.2 μ l DMSO dimethyl sulfoxide is (optional
Add), ddH2O supplies 20 μ l.PCR reaction condition are as follows: 94 DEG C of initial denaturations 4min, 94 DEG C of denaturation 30s, 52~64 DEG C of annealing 30s,
72 DEG C of extension 90s are recycled 30 times, 72 DEG C of extension 5min.Agar electrophoresis detects amplified production, and electrophoresis applied sample amount is 6 μ l.
3. testing result is referring to Fig. 3: wherein each swimming lane is expressed as follows: M is DL 2000bp DNA molecular amount standard, and 1-5 divides
Not Wei sample A-E (PCR reaction system is not added with DMSO dimethyl sulfoxide), 6 is using ddH2O as the negative control of template；7-12
The DMSO of 0.2 μ l is wherein added to relative to 1-6 for 20 μ l systems of PCR amplification, wherein 7-11 is respectively sample A-E, and 12 are
Using ddH2O as the negative control of template；As can be known from Fig. 3, in A-E sweet potato diseased tissues sample, A-D sample can be amplified
1641bp potato pest bacterium specific band illustrates to contain potato pest bacterium in A-D sample.And it is added in 20 μ l PCR reaction systems
Amplification efficiency can be improved in the DMSO of 0.2 μ l, can allow band brighter (the swimming lane 7- in Fig. 3 while having not been changed qualitative results
11)。
Embodiment 4: the detection of sweet potato potato seasonal febrile diseases bacterium in morbidity plant tissue bacteria suspension
1. sample acquires: five groups of morbidity sweet potato Plant tissue samples of acquisition, label F, G, H, I, J, morbidity sweet potato are planted respectively
Object tissue sample picks up from Foochow planting base Xin Dian.
2.DNA is extracted and detection
It takes site of pathological change containing vascular tissue on clean slide, covered is added after clear water and from coverslip
Tissue is squeezed, later by slide to spray bacterium phenomenon whether is generated around light or micro- sem observation vascular bundle, referring in particular to Fig. 5, Fig. 5
In: A is sweet potato health tissues；B is sweet potato incidence tissue；If generating spray bacterium phenomenon, the bacterium on glass slide around tissue is hanged
Liquid exhaustion is added in centrifuge tube；Bacteria suspension is set and boils 10min in boiling water；Solution ddH2O takes 1 μ l conduct after diluting 100 times
DNA profiling participates in PCR reaction.PCR reaction system is 20 μ l, including 10 μ l 2 × EasyTaq PCR SuperMix mixing is instead
It answers solution (Beijing Quanshijin Biotechnology Co., Ltd's production), primer SPRS-F and SPRS-R each 0.2 μm of ol/l, 10-
100ng template DNA, 0.2 μ l DMSO dimethyl sulfoxide (optional to add), ddH2O supplies 20 μ l.PCR reaction condition are as follows: 94 DEG C pre-
It is denaturalized 4min, 94 DEG C of denaturation 30s, 52~64 DEG C of annealing 30s, 72 DEG C of extension 90s, is recycled 30 times, 72 DEG C of extension 5min.Agar
Electrophoresis detection amplified production, electrophoresis applied sample amount are 6 μ l.
3. testing result is referring to Fig. 4: wherein each swimming lane is expressed as follows: M is DL 2000bp DNA molecular amount standard, and 1-5 divides
Not Wei sample F-J (PCR reaction system is not added with DMSO dimethyl sulfoxide), 6 is using ddH2O as the negative control of template；7-12
The DMSO of 0.2 μ l is wherein added to relative to 1-6 for 20 μ l systems of PCR amplification, wherein 7-11 is respectively sample F-J, and 12 are
Using ddH2O as the negative control of template；As can be known from Fig. 4, in F-J sweet potato diseased tissues sample, F-I sample can be amplified
1641bp potato pest bacterium specific band illustrates to contain potato pest bacterium in F-I sample.And it is added in 20 μ l PCR reaction systems
Amplification efficiency can be improved in the DMSO of 0.2 μ l, can allow band brighter (the swimming lane 7- in Fig. 4 while having not been changed qualitative results
11)。
To sum up, the present invention at this stage there has been no potato pest bacterium specific molecular marker and rapid detection method, pass through
Potato pest bacterium genome and other Ralstonia solanacearum genome comparisons find specific gene orf428, and sequence design goes out a pair of special accordingly
Specific primer SPRS-F and SPRS-R, and corresponding polymerase chain reaction PCR amplification system is established, for sweet potato band potato pest
The rapid molecular of bacterium sample detects.This technical result is reliable, easy to operation, high sensitivity, high specificity, can be applied to disease stream
The long term monitoring of prediction, Prevalent district before row, and control approach and method are instructed, for sweet potato potato seasonal febrile diseases in production
Prevention and treatment is of great significance.
Compared with prior art, the present invention having following remarkable advantage:
1, high specificity, accurate and reliable: the primer SPRS-F and SPRS-R of the present invention is to utilize sweet potato potato seasonal febrile diseases bacterium base
Because of the specific gene orf428 design in group.The gene has not yet been sequenced in 66 Isolates of Pseudomonas Solanacearum Smith genomes at other
It finds, while comparison result of the gene in the website NCBI BLAST shows that the gene specific is very high (withered with immediate blueness
Sequence similarity in bacterium allied species Ralstonia insidiosa be only 71%), and the design position of primer not with it is any
On reference sequences compare.The primer of the present invention and method are in Fujian different regions and Zhanjiang area potato pest bacterium
Resource is verified on 6 kinds of other crop ralstonia solanacearums in addition to potato pest bacterium, and determination can expand shaping in all potato pest bacterium resources
Band, and all do not have in other 6 kinds of Ralstonia solanacearums, that is, illustrate the technology of the present invention high specificity, as a result accurately and reliably.
2, strong operability, fast and convenient: the method for the present invention does not need that microorganism in sick sample is separately cultured and is given birth to
Object identification；DNA extraction method is also more easier than traditional extracting method, does not need removal sample contamination precipitation purification DNA,
DNA extraction can be completed in 30 minutes；It can be judged by banding pattern after the direct agarose electrophoresis of PCR result, not need to be sequenced；Entire inspection
3-4 hour of survey process can complete.
3, practical, high sensitivity: found in production sick sample without complex process can DNA rapid extraction tested
Card, for potato pest bacterium DNA content demand in sample down to 10pg.
Although specific embodiments of the present invention have been described above, those familiar with the art should be managed
Solution, we are merely exemplary described specific embodiment, rather than for the restriction to the scope of the present invention, it is familiar with this
The technical staff in field should be covered of the invention according to modification and variation equivalent made by spirit of the invention
In scope of the claimed protection.
Sequence table
<110>Fujian Academy of Agricultural Sciences's crop investigations institute
<120>a kind of sweet potato potato seasonal febrile diseases bacterium molecule detection primer and its application
<130> 100
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>(artificial sequence)
<400> 1
gacacatcag ggccattttc acc 23
<210> 2
<211> 21
<212> DNA
<213>(artificial sequence)
<400> 2
gcctctgcca gtgagatcat c 21
<210> 3
<211> 1641
<212> DNA
<213>(Ralstonia solanacearum)
<400> 3
gacacatcag ggccattttc accagttgct gatgatgtca gtgtaaaagt cataaaccaa 60
atctttggta gtttggtcaa tggtggcaat gatgctttcg gctctgcaat atccacgttc 120
aatgcagcca tcctaatagt aggcggtatt ctcgccacct atacccttct tgctggaact 180
cttggcacag cccatgatgg ggagatgcta ggcaagaaat tttcttctgt ctggattccc 240
attcgctatg cattaggaac agcattggta cttcctgtgc ttgctaatgg ctactgcatc 300
atgcagcaaa tggtcatttg gcttgcaatg caaggaatcg ggttggctga ctctgtgtgg 360
gatgcctata tgcagacacc tggcatagca gccaacatga gtatcagcaa aaacactgaa 420
gacaaggccc gtgctcttgc tgaaaacatc ttcatggctg aagtctgtat tgaggccaat 480
aagtatgcag tttcaaagac agacaacatt ttgaccatca agagtagata tgactacaac 540
atggtctacg acgacaaaac cagaacctac agcttcggtg atcagaaagg cattgtctca 600
ggttggacta agaatgactg tggtgaggtg agctttggtg agaagatcca gaattcaaca 660
gttgcttcag gtccttcaac ttccaatgtg ggctaccttg gcccattgga caacttgttc 720
gctccaacag atgtacagcc tatcaaccag gcacatgaga cagccaccaa ggcattgata 780
actgctgctc aaactgctgc acaagcgatt gtgcaatcag gtggtgatat tgatgcagct 840
acagcagcca cctactatca aggaattgat gcagccacta ctgcctacct gactgcgatc 900
aaagcagcag ccaatgcagt tgccatgtca cctagtgaca ctaccaagac tgcccacaag 960
tatggctggt tcatggcagg tgcctacttc atgaacactg ttgtgaccaa caacaagata 1020
actaatgcga ttgctgcggt tcctgacagt aagttctcta agtctgcttt caatgaccag 1080
gcggaagcat tgcaagcaac tggcttgaaa attcttgctg ctggaaatcc tgtctatgga 1140
tcagcagcca atgcaatcaa tcagaagcaa gccaatgaaa gtgcaaagga aacagaggaa 1200
ttaggctttg gtggaagaat catgaatgca atcactcgtg gattgacttc tattgatctt 1260
taccagttaa agaatgaccc aagacatcca gtgattgttg tcaatgaaat gggtaacaga 1320
cttcaagcct actggacagg catggtcgtt acattgatcg ctgttactgg tgcggcaggc 1380
attgcagcat tgttgaagtc ttcaatcgca acaaccgtca ccaacgtttt ggtgatcatt 1440
gttggattcc ttggattgcc aatcatggct ttggcagcta cgtcatttac tgccagctat 1500
ctgattccaa tgatgccatt tatgatgtgg ctaggtattc ttggaggttg gttgattgct 1560
cttgtaatag gagtgatcgc tgctccactt tgggccatca tgcacttgca tccaaatgga 1620
gatgatctca ctggcagagg c 1641
Claims (7)
1. a kind of sweet potato potato seasonal febrile diseases bacterium molecule detection primer, it is characterised in that: the sweet potato potato seasonal febrile diseases bacterium molecule detection primer
Sequence are as follows:
SPRS-F:5 '-GACACATCAGGGCCATTTTCACC-3 '
SPRS-R:5 '-GCCTCTGCCAGTGAGATCATC-3 '.
2. a kind of application of sweet potato potato seasonal febrile diseases bacterium molecule detection primer, it is characterised in that: the molecular detection primer is applied to sweet
The rapid molecular of potato potato seasonal febrile diseases bacterium detects, and detection method is as follows:
(1) DNA or (2) are extracted from the doubtful plant infected by sweet potato potato seasonal febrile diseases bacterium to plant from doubtful infected by sweet potato potato seasonal febrile diseases bacterium
DNA is extracted in the bacteria suspension of strain preparation；The above step DNA is template a pair of molecular detection primer SPRS-F and SPRS-R by gathering
Polymerase chain reacts PCR amplification, and pcr amplification product separates with agarose electrophoresis, after SYBR Green I dyeing in gel at
As observing under analyzer ultraviolet lamp, if amplified production is 1641bp, that is, it can determine whether that there are sweet potato potato seasonal febrile diseases in the sample
Bacterium；
The gene order of the molecular detection primer SPRS-F is as shown in SEQ ID NO.1, the molecular detection primer SPRS-R
Gene order as shown in SEQ ID NO.2, obtained amplified production gene order is as shown in SEQ ID NO.3.
3. a kind of application of sweet potato potato seasonal febrile diseases bacterium molecule detection primer according to claim 2, it is characterised in that: the inspection
(1) extracts DNA's from the doubtful plant infected by sweet potato potato seasonal febrile diseases bacterium in survey method method particularly includes: takes 50-300mg sweet potato
Diseased tissues is ground after liquid nitrogen frozen, and 300 μ l ddH2O are added and mix；Solution is set and boils 10min in boiling water；Solution is dilute with ddH2O
1 μ l is taken to participate in PCR reaction as DNA profiling after releasing 100 times.
4. a kind of application of sweet potato potato seasonal febrile diseases bacterium molecule detection primer according to claim 2, it is characterised in that: the inspection
(2) extract DNA's from the doubtful bacteria suspension prepared by sweet potato potato seasonal febrile diseases bacterium infected plant in survey method method particularly includes: take hair
Sick position on clean slide, is added covered after clear water and squeezes tissue from coverslip containing vascular tissue, it
Afterwards by slide to whether generated around light or micro- sem observation vascular bundle spray bacterium phenomenon；If spray bacterium phenomenon is generated, by glass slide
Bacteria suspension exhaustion around upper tissue is added in centrifuge tube；Bacteria suspension is set and boils 10min in boiling water；Solution is diluted with ddH2O
1 μ l is taken to participate in PCR reaction as DNA profiling after 100 times.
5. a kind of application of sweet potato potato seasonal febrile diseases bacterium molecule detection primer according to claim 2, it is characterised in that: PCR is anti-
Answering system is 20 μ l, including 10 μ l 2 × EasyTaq PCR SuperMix mixed reaction solutions, primer SPRS-F and SPRS-R
Each 0.2 μm of ol/l, 10-100ng template DNA, ddH2O supply 20 μ l.
6. a kind of application of sweet potato potato seasonal febrile diseases bacterium molecule detection primer according to claim 2, it is characterised in that: PCR is anti-
Answering system is 20 μ l, including 10 μ l 2 × EasyTaq PCR SuperMix mixed reaction solutions, primer SPRS-F and SPRS-R
Each 0.2 μm of ol/l, 10-100ng template DNA, 0.2 μ l DMSO dimethyl sulfoxide, ddH2O supply 20 μ l.
7. a kind of application of sweet potato potato seasonal febrile diseases bacterium molecule detection primer according to claim 2, it is characterised in that: PCR is anti-
Answer condition are as follows: 94 DEG C of initial denaturations 4min, 94 DEG C of denaturation 30s, 52~64 DEG C of annealing 30s, 72 DEG C of extension 90s are recycled 30 times, 72 DEG C
Extend 5min.
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| CN110923342A (en) * | 2019-12-12 | 2020-03-27 | 福建省农业科学院作物研究所 | RPA detection primer, kit and detection method for sweet potato blast bacteria |
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