CN106222291B - A kind of kit of Molecular Detection strain different with identification sugarcane red streak bacterium - Google Patents
A kind of kit of Molecular Detection strain different with identification sugarcane red streak bacterium Download PDFInfo
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- CN106222291B CN106222291B CN201610686714.1A CN201610686714A CN106222291B CN 106222291 B CN106222291 B CN 106222291B CN 201610686714 A CN201610686714 A CN 201610686714A CN 106222291 B CN106222291 B CN 106222291B
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Abstract
The present invention relates to a kind of Molecular Detections and identification sugarcane red streak bacteriumAcidovorax avenae subsp.avenae(Aaa) different strain kits.Outside primer is included at least in kit of the invention to 16S-F4/23S-R4 and inner primer to ITS-F1/ITS-R1, in kit there are also PCR reaction solution I and II, restriction enzyme reaction liquid, Ex Taq enzyme liquid,HinDIII restriction endonuclease,EcoRI restriction endonuclease, positive sample DNA, negative sample DNA and nuclease free sterile water.The advantages that high sensitivity and restriction enzyme slitting strip length polymorphism and uniqueness of set of the present invention nested PCR detection method, provides a kind of simplicity, fast and accurately method for sugarcane red streak bacterium detection and identification.The reagent provided using kit of the present invention can determine that it is sugarcane red streak bacterium and its strain type through detection.
Description
Technical field
The present invention relates to a kind of kits for detecting plant disease, and in particular to a kind of Molecular Detection and the identification red item of sugarcane
GermAcidovorax avenae subsp. avenae(Aaa) kit of different strains, platymiscium Defect inspection technology neck
Domain.
Background technique
Sugarcane red streak (red stripe disease) is a kind of bacterial disease generally occurred, in Major Sugarcane kind
Planting country has generation, seriously affects the quality and yield of sugarcane.It is reported that the disease becomes Argentinian sugarcane sugarcane district in recent years
Important disease, cause about 30% economic loss.Sugarcane red streak symptom has 2 seed type of leaf streak and top rot, can individually send out
It is raw, it can also occur simultaneously.The disease is considered as by Pseudomonas rubrilineansPseudomonas rubrilineans Stapp draws
It rises, Gram's staining reaction is feminine gender, and no gemma is in the shape of a rod, and size is 0.7 ~ 1.6 μm, has single polar flagella, there is thin-walled pod
Film (Martin & Wismer, 1989)；Then renamed for oat lactic acid bacteria oat subspecies (Acidovorax avenae
subsp. avenae,Aaa) (Willems etc., 1992；Schaad etc., 2008).AaaThe natural host of germ is in addition to sugarcane
Outside, there are also some gramineae plants such as rice, corn, oat, broomcorn millet, foxtail, or even can also infect the prestige of Ravenala madagascariensis University of Science and Technology crane
The plants such as orchid.
Phytopathogen detection method has biological assay, electron microscopy, Enzyme-multiplied immune technique and molecular biology skill
Art etc..AaaGerm identification and detection method existing research on the gramineae plants such as rice, this spy grass, broomcorn millet, sugarcane.Song etc.
(2004) culture of germ biology is established and BIO-PCR method that nest-type PRC combines, to rice paddy seedAaaGerm carries out
Detection.But many plant diseases do not show typical Disease Characters, and germ separation, training under the natural conditions of field
Biological assays time and effort consuming and the complex steps such as feeding and its Pathogenicity.Molecular biosciences means are a kind of sensitivity
High, high specificity Fast Detection Technique, is widely used to the detection and identification of microorganism, mainly there is molecule hybridization and nucleosides
Acid sequencing, as Southern hybridization, polymerase chain reaction (PCR), real time PCR amplification, biochip, universal label technique with
And ring mediated isothermal amplification.Pathogen ribosomes 16S ~ 23S Transcribed Spacer sequence is because both ends are by highly conserved rRNA packet
It encloses, which has not only guarded but also relative variable, and segment is small, reproducible, may not only be applied to the identification between strain, may further be used to point
It distinguishes the strain and plant interior bacterial strain that 16S rRNA gene can not identify, is that 16S rRNA genetic analysis means are supplemented well.
Therefore, the invention patent early period a large amount of sugarcane red streak bacterium separation, purifying and identification working foundation on, according toAaa
16S ~ 23S rDNA gene and its spacer region design the specificity inside of germ, Outside primer, by having gathered nest-type PRC detection
The advantages that high sensitivity and restriction enzyme slitting strip length polymorphism and uniqueness of method, has developed a kind of quickly inspection
Survey identification sugarcane red streak bacterium kit and its detection method, to for China sugarcane introduce a fine variety inspection and quarantine, evaluation of resistance and
Seedling quality monitoring provides technical support.
Summary of the invention
The object of the present invention is to provide a kind of kits of Molecular Detection identification sugarcane red streak bacterium difference strain, to overcome
Shortcoming in the prior art.
The purpose of the present invention is what is be achieved through the following technical solutions.
A kind of kit of Molecular Detection of the invention strain different with identification sugarcane red streak bacterium, it is characterised in that the examination
Agent box consists of the following reagents:
(1) PCR reaction solution I: each PCR reaction solution includes: 10 × PCR buffer, 2.5 μ L, 2.5 mmol/L dNTPs
2.0 μ L and concentration are each 1.0 μ L of Outside primer 16S-F4 and 23S-R4 of 10 μm of ol/L, totally 6.5 μ L, 50 reactions
System amounts to 325 μ L, and -20 DEG C save backup；The Outside primer 16S-F4:5'-CGTGAAGTCGGAATCGCTA-3'；
23S-R4:5'-TCGGAATCTCGGTTGATG-3'；
(2) PCR reaction solution II: each PCR reaction solution includes: 10 × PCR buffer, 5.0 μ L, 2.5 mmol/L dNTPs
4.0 μ L and concentration are each 2.0 μ L of inner primer ITS-F1 and ITS-R1 of 10 μm of ol/L, totally 13.0 μ L, 50 reactions
System amounts to 650 μ L, and -20 DEG C save backup；The inner primer ITS-F1:5'-AGACCCACCAAATCTTCCG-3'；
ITS-R1:5'-GACATCTCCGCTTTCTTTCAAG-3'；
(3) restriction enzyme enzyme buffer liquid: each endonuclease reaction liquid includes 10 × enzyme cutting buffering liquid, 2.0 μ L, 50 reactions
System amounts to 100 μ L, and -20 DEG C save backup；
(4) Ex Taq enzyme: each PCR reaction system includes 5 U/ μ L Ex Taq enzyme, 0.75 μ L, and 50 reaction systems are total
37.5 μ L are counted, -20 DEG C save backup；
(5)HinDIII restriction endonuclease: each restriction enzyme reaction system includes 15 U/ μ LHinDIII restriction endonuclease 1.0
μ L, 50 reaction systems amount to 50 μ L, and -20 DEG C save backup；
(6)EcoRI restriction endonuclease: each restriction enzyme reaction system includes 15 U/ μ LEco1.0 μ L of RI restriction endonuclease,
50 reaction systems amount to 50 μ L, and -20 DEG C save backup；
(7) DNA: four kinds of positive sample Aaa germ different sugarcane red streak bacteriumAcidovorax avenae subsp.avenae(Aaa) strain HE, H, E1, E2 germ original DNA is as standard sample, 1 ng/ μ L, 50 L/ bottles of μ, -20 DEG C of preservations
It is spare；
(8) negative sample RNA: the Sugarcane Leaves total DNA of no Aaa infection is negative control, 100 ng/ μ L, 50 μ L/
Bottle, -20 DEG C save backup；
(9) nuclease free sterile water: 2 × 10 mL/ bottles.
It is an advantage of the invention that overcoming the shortcomings of the survey of conventional sugarcane red streak bacterial examination and identification method, a kind of molecule is provided
The kit of Testing and appraisal sugarcane red streak bacterium difference strain, the present invention have high sensitivity, high specificity, at low cost, easy
Quick advantage can be used for field early diagnosis and extensive quick detection and identification.
Detailed description of the invention:
Fig. 1 is the nested PCR product restriction enzyme band polymorphic detection electrophoresis of different sugarcane red streak bacterial strain systems
Figure, wherein M is 20 bp DNA standard relative molecular masses, and 1-12 is the Sugarcane Leaves sample for infecting red streak bacterium, and 13 be strong
Health Sugarcane Leaves sample, 14 be nuclease free sterile water.
There is PCR product digestion band simultaneously in 330 bp, 68 bp and 56 positions bp in sample 1-3, indicates in the sample
Contain Aaa-HE strain；There is PCR product digestion band simultaneously in 330 bp and 124 positions bp in sample 4-6, indicates the sample
In contain Aaa-H strain；There is PCR product digestion band simultaneously in 386 bp and 68 positions bp in sample 7-9, indicates the sample
In contain Aaa-E1 strain；There is PCR product digestion band in 369 bp and 68 positions bp in sample 10-12, indicates these samples
In contain Aaa-E2 strain.13(negative control in Fig. 1) do not occur PCR product digestion band, indicate the sample without Aaa.
14(nuclease free sterile water in Fig. 1) do not occur PCR product digestion band, indicate that experiment is not polluted.
Specific embodiment:
In order to which the present invention is furture elucidated rather than the limitation present invention, it is illustrated with reference to embodiments.
A kind of method of embodiment 1, Molecular Detection and identification sugarcane red streak bacterium difference strain, comprising the following steps:
1, Sugarcane Leaves Genome DNA extraction
(1) 100 mg of blade is taken, with the rapid grind into powder of liquid nitrogen, is set in 2 mL centrifuge tubes.
(2) dehydrated alcohol of 900 μ 2 × CTAB of L buffers and 100 μ L is added, is vortexed to abundant dissolution.
(3) centrifuge tube is placed in 65 DEG C of 60 min of water-bath, is mixed by inversion every 5-10 min, makes the abundant lytic cell of CTAB,
12000 rpm after taking-up is centrifuged 5 min.
(4) it takes supernatant in 2 new mL centrifuge tubes, and isometric phenol: chloroform: isoamyl alcohol 25:24:1 is added；
It mixes, 12000 rpm are centrifuged 10 min.
(5) it takes supernatant in 1.5 new mL centrifuge tubes, and isometric chloroform: isoamyl alcohol 24:1 is added；It mixes,
12000 rpm are centrifuged 10 min.
(6) supernatant is taken, is set in 1.5 new mL centrifuge tubes, and 3 mol/L NaAC(pH of addition 1/10 volume pre-cooling=
5.2) it precipitates DNA sufficiently, mixes, add the isopropanol of isometric -20 DEG C of pre-coolings, rolling is even, is placed in -20 DEG C of 30 min of placement.
(7) 4 DEG C of 10000 rpm are centrifuged 5 min, go liquid phase, add 1000 μ L that 75 % ethyl alcohol are pre-chilled and wash precipitating twice, then use
Dehydrated alcohol is washed once, and centrifuge tube, drying at room temperature DNA are inverted.
(8) 40 μ L ddH are added2O, and keep the temperature in 37 DEG C until DNA is completely dissolved.
(9) it is detected with spectrophotometer spare in DNA mass and 4 DEG C of DNA concentration postposition, or sets -20 DEG C of long-term preservations.
2, design of primers
Early period a large amount of sugarcane red streak bacterium separation, purifying and identification working foundation on, according to sugarcane red streak
16S ~ 23S rDNA gene of bacterium Aaa and its 2 pairs of specific primers of spacer region be respectively Outside primer 16S-F4/23S-R4 and
The purpose nucleic acid band size of inner primer ITS-F1/ ITS-R1, nested PCR amplification are 454 bp.The Outside primer 16S-
F4 has the nucleotide sequence as shown in SEQ ID NO:1 in sequence table；23S-R4 has such as SEQ ID NO:2 in sequence table
Shown in nucleotide sequence；The inner primer ITS-F1 has the nucleotides sequence as shown in SEQ ID NO:3 in sequence table
Column；ITS-R1 has the nucleotide sequence as shown in SEQ ID NO:4 in sequence table.
3, the 1st wheel PCR amplification
Following reagent is added in PCR pipe:
It mixes, 8000 rpm are centrifuged 94 DEG C of 3 min of initial denaturation, 94 DEG C of 45 S of denaturation, 58 DEG C of annealing 30 S, 72 after 5 S
DEG C extend 45 S, totally 35 circulation, last 72 DEG C re-extend 10 min.
4, the 2nd wheel PCR amplification
After 1st wheel PCR product is diluted 100 times, following reagent is added in PCR pipe:
Mix, 8000 rpm be centrifuged 5 S after 94 DEG C of 3 min of initial denaturation, 94 DEG C of 45 S of denaturation, 58 DEG C of 30 S of annealing,
72 DEG C of 60 S of extension, totally 35 recycle, and last 72 DEG C re-extend 10 min.
5, PCR product purifies
(1) it is settled to after 100 μ L with 50 μ L nuclease free sterile waters and isometric phenol/chloroform/isoamyl alcohol (25 is added
: 24: 1) mixing.
(2) room temperature, 13,500 rpm, 5 min centrifugation, takes supernatant.
(3) isometric chloroform/isoamyl alcohol (24: 1) is added to mix.
(4) room temperature, 13,500 rpm, 5 min centrifugation, takes supernatant.
(5) 10 μ L, 3 mol/L sodium acetate, the 250 cold ethyl alcohol of μ L, -20 DEG C of 30 min of placement are added.
(6) 4 DEG C, supernatant is abandoned in 13,500 rpm, 15 min centrifugation.
(7) the cold ethyl alcohol of 500 μ L 70% is added to clean, 4 DEG C, supernatant is abandoned in 13,500 rpm, 5 min centrifugation.
(8) drying is precipitated.
(9) dissolution of 100 μ L nuclease free sterile waters is added.
6, endonuclease reaction
It is provided using this kitHinDIII andEcoRI restriction enzyme and its buffer carry out endonuclease reaction,
Following reagent is added in PCR pipe:
It mixes, 37 DEG C digestion 2 hours, 65 DEG C of 5 min of processing, terminate endonuclease reaction, drop after 8000 rpm centrifugation, 5 s
To room temperature.
7, electrophoresis detection
5 μ L reaction solutions are taken to carry out the low melting-point agarose gel electricity that mass volume ratio is 4.0 % after endonuclease reaction
Swimming, in 0.5 × electrophoretic buffer environment, then 100 V pressure stabilizing electrophoresis, 1.5 h is observed and is taken pictures with gel imaging system, in
Occur PCR product digestion band in electrophoresis detection figure, indicates the sample containing Aaa；Do not occur PCR product digestion band, indicating should
Sample is free of Aaa.Different Aaa strains is distinguished according to the banding pattern of appearance.
Sugarcane red streak bacterium difference strain restriction enzyme digestion sites and band length polymorphism are listed in table 1, from table 1
As it can be seen that the position of nested PCR product digestion band is different, indicate that the strain of contained Aaa in sample is different.In 330 bp, 68 bp
There is PCR product digestion band simultaneously with 56 positions bp, indicates to contain Aaa-HE strain in the sample.In 330 bp and 124
There is PCR product digestion band simultaneously in the position bp, indicates to contain Aaa-H strain in the sample.It is same in 386 bp and 68 positions bp
When there is PCR product digestion band, indicate in the sample containing Aaa-E1 strain.There is PCR production in 369 bp and 68 positions bp
Object digestion band indicates to contain Aaa-E2 strain in these samples.
1 sugarcane red streak bacterium difference strain restriction enzyme digestion sites of table and band length polymorphism
The present embodiment has detected 72 samples with red streak evil classical symptom collected from China Provinces (regions) sugarcane field
Product, representative sample electrophoresis detection result is as shown in Figure 1,1 ~ 12 expression sample 1 ~ 12 is through detection containing Aaa in Fig. 1.Wherein, sample
There is PCR product digestion band simultaneously in 330 bp, 68 bp and 56 positions bp in 1-3, indicates to contain Aaa-HE plants in the sample
System；There is PCR product digestion band simultaneously in 330 bp and 124 positions bp in sample 4-6, indicates to contain Aaa-H plants in the sample
System；There is PCR product digestion band simultaneously in 386 bp and 68 positions bp in sample 7-9, indicates to contain Aaa-E1 plants in the sample
System；There is PCR product digestion band in 369 bp and 68 positions bp in sample 10-12, indicates to contain Aaa-E2 plants in these samples
System.13(negative control in Fig. 1) do not occur PCR product digestion band, indicate the sample without Aaa；14(in Fig. 1 is seedless
Sour enzyme sterile water) do not occur PCR product digestion band, indicate that experiment is not polluted.M in Fig. 1 is that 20 bp DNA standards are opposite
Molecular mass.The above 62 sample identification results with red streak evil classical symptom are that 31 parts of samples have infected Aaa-HE plants
System, 20 parts of samples have infected Aaa-H strain, and 18 parts of samples have infected Aaa-E1 strain, and 3 parts of samples have infected Aaa-E2 strain；
It is completely the same with the experimental result by nested PCR product purifying, clone, sequencing and sequence analysis, further demonstrate experiment
Accuracy and reliability.
The main agents that the present invention uses are as follows: (all chemical reagent are that analysis is pure)
1, CTAB Extraction buffer: 100 mmol/L Tris-HCl (pH8.0), 20 mmol/L EDTA-Na2, 1.4
Mol/LNaCl, 2 % CTAB of mass volume ratio use preceding 0.1 % beta -mercaptoethanol of addition volume ratio；
2, sample-loading buffer: 0.1 % bromophenol blue, 40 % sucrose；
3,0.5 × tbe buffer liquid: 44.5 mmol/L Tris, 50 mmol/L HBO3, 1 mmol/L EDTA；
4, PCR amplification reagent is purchased from Dalian treasured biotech firm.
Instrument used in the present invention is mainly as follows:
1, PCR amplification instrument: German 5331 PCR amplification instrument of Eppendorf；
2, electrophoresis apparatus: Liuyi Instruments Plant, Beijing DYCZ-20A DNA sequence analysis electrophoresis apparatus；
3, centrifuge: German Eppendorf 5810/5810R multifunctional table-type centrifuge；
4, gel imaging system: 2000 gel imaging system of U.S. BIO-RAD Gel Doc；
5, spectrophotometer: BioTek company, U.S. SynergyTMH1 ultramicrospectrophotometer.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
SEQUENCE LISTING
Sequence table
<110>University Of Agriculture and Forestry In Fujian
<120>kit of a kind of Molecular Detection strain different with identification sugarcane red streak bacterium
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>artificial sequence 16S-F4
<400> 1
cgtgaagtcg gaatcgcta 19
<210> 2
<211> 18
<212> DNA
<213>artificial sequence 23S-R4
<400> 2
tcggaatctc ggttgatg 18
<210> 3
<211> 19
<212> DNA
<213>artificial sequence ITS-F1
<400> 3
agacccacca aatcttccg 19
<210> 4
<211> 22
<212> DNA
<213>artificial sequence ITS-R1
<400> 4
gacatctccg ctttctttca ag 22
Claims (1)
1. a kind of kit of Molecular Detection strain different with identification sugarcane red streak bacterium, it is characterised in that the kit is by following
Reagent composition:
(1) PCR reaction solution I: each PCR reaction solution includes: 10 × PCR buffer, 2.5 μ L, 2.5 mmol/L dNTPs 2.0
μ L and concentration are each 1.0 μ L of Outside primer 16S-F4 and 23S-R4 of 10 μm of ol/L, totally 6.5 μ L, 50 reaction systems
Total 325 μ L, -20 DEG C save backup；The Outside primer 16S-F4:5'-CGTGAAGTCGGAATCGCTA-3'；23S-
R4:5'-TCGGAATCTCGGTTGATG-3'；
(2) PCR reaction solution II: each PCR reaction solution includes: 10 × PCR buffer, 5.0 μ L, 2.5 mmol/L dNTPs 4.0
μ L and concentration are each 2.0 μ L of inner primer ITS-F1 and ITS-R1 of 10 μm of ol/L, totally 13.0 μ L, 50 reaction systems
Total 650 μ L, -20 DEG C save backup；The inner primer ITS-F1:5'-AGACCCACCAAATCTTCCG-3'；ITS-
R1:5'-GACATCTCCGCTTTCTTTCAAG-3'；
(3) restriction enzyme enzyme buffer liquid: each endonuclease reaction liquid includes 10 × enzyme cutting buffering liquid, 2.0 μ L, 50 reaction systems
Total 100 μ L, -20 DEG C save backup；
(4) Ex Taq enzyme: each PCR reaction system includes 5 U/ μ L Ex Taq enzyme, 0.75 μ L, and 50 reaction systems are total
37.5 μ L, -20 DEG C save backup；
(5)HinDIII restriction endonuclease: each restriction enzyme reaction system includes 15 U/ μ LHin1.0 μ L of dIII restriction endonuclease,
50 reaction systems amount to 50 μ L, and -20 DEG C save backup；
(6)EcoRI restriction endonuclease: each restriction enzyme reaction system includes 15 U/ μ LEcoRI restriction endonuclease 1.0 μ L, 50
Reaction system amounts to 50 μ L, and -20 DEG C save backup；
(7) DNA: four kinds of positive sample Aaa germ different sugarcane red streak bacteriumAcidovorax avenae subsp. avenae
(Aaa) strain HE, H, E1, E2 germ original DNA is as standard sample, and 1 ng/ μ L, 50 L/ bottles of μ, -20 DEG C save backup；
(8) negative sample DNA: the Sugarcane Leaves total DNA of no Aaa infection is negative control, 100 ng/ μ L, 50 L/ bottles of μ, -20
It DEG C saves backup；
(9) nuclease free sterile water: 2 × 10 mL/ bottles.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102864244A (en) * | 2012-10-16 | 2013-01-09 | 福建农林大学 | Nested polymerase chain reaction (PCR) detection method of acidovorax avenae subsp. avenae |
| CN103555844A (en) * | 2013-11-07 | 2014-02-05 | 福建农林大学 | Fluorescent quantitative PCR (polymerase chain reaction) kit for detecting sugarcane red stripe germ |
| CN104313188A (en) * | 2014-11-13 | 2015-01-28 | 福建农林大学 | Kit for detecting and judging genotype of sugarcane yellow leaf virus |
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2016
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102864244A (en) * | 2012-10-16 | 2013-01-09 | 福建农林大学 | Nested polymerase chain reaction (PCR) detection method of acidovorax avenae subsp. avenae |
| CN103555844A (en) * | 2013-11-07 | 2014-02-05 | 福建农林大学 | Fluorescent quantitative PCR (polymerase chain reaction) kit for detecting sugarcane red stripe germ |
| CN104313188A (en) * | 2014-11-13 | 2015-01-28 | 福建农林大学 | Kit for detecting and judging genotype of sugarcane yellow leaf virus |
Non-Patent Citations (2)
| Title |
|---|
| Genetic Relationships among Xanthomonas Species and Pathovars Based on RFLP Analyses of PCR-amplified 16S, 23S rDNA and rDNA Internal Transcribed Spacer;OCHIAI,H.等;《日植病报》;19991231;第63卷;437-446 |
| 甘蔗赤条病病原菌分离与鉴定;李晓燕等;《中国植物病理学会2016年学术年会论文集》;20160805;330 |
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Effective date of registration: 20231227 Address after: 513055 Lin Wu, Xianjiao Keng Village, Qiaotou Town, Yingde City, Qingyuan City, Guangdong Province Patentee after: Yingde Fenglin Agricultural Professional Cooperative Address before: No. 15, Cangshan District, Fujian, Fuzhou, Fujian Patentee before: FUJIAN AGRICULTURE AND FORESTRY University |