CN106222291A - A kind of Molecular Detection and the test kit identifying the Caulis Sacchari sinensis different strain of red streak bacterium - Google Patents
A kind of Molecular Detection and the test kit identifying the Caulis Sacchari sinensis different strain of red streak bacterium Download PDFInfo
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Abstract
The present invention relates to a kind of Molecular Detection and identify Caulis Sacchari sinensis red streak bacteriumAcidovorax avenae subsp. avenae(Aaa) different strain test kits.The test kit of the present invention at least includes Outside primer to 16S F4/23S R4 and inner primer to also have in ITS F1/ITS R1, test kit PCR reactant liquor I and II, restriction enzyme reaction liquid, Ex Taq enzyme liquid,HinDIII restriction endonuclease,EcoRI restriction endonuclease, positive DNA, negative sample DNA and nuclease free sterilized water.The advantages such as the high sensitivity of set of the present invention nested PCR detection method and restricted enzyme cutting strip length polymorphism and uniqueness, provide a kind of easy, method fast and accurately for the detection of Caulis Sacchari sinensis red streak bacterium and qualification.The reagent using test kit of the present invention to provide, can determine that it is Caulis Sacchari sinensis red streak bacterium and strain type thereof after testing.
Description
Technical field
The present invention relates to a kind of test kit detecting plant disease, be specifically related to a kind of Molecular Detection and identify the red bar of Caulis Sacchari sinensis
Pathogenic bacteriaAcidovorax avenae subsp. avenae(Aaa) test kit of different strains, platymiscium
Defect inspection technical field.
Background technology
Caulis Sacchari sinensis red streak (red stripe disease) is a kind of bacterial disease generally occurred, in Major Sugarcane kind
Plant country and all have generation, have a strong impact on the quality and yield of Caulis Sacchari sinensis.It is reported, this disease becomes Caulis Sacchari sinensis sugarcane district of Argentina in recent years
Important disease, cause the economic loss of about 30%.Caulis Sacchari sinensis red streak symptom has leaf streak and top rot 2 type, all can individually send out
Raw, it is possible to occur simultaneously.This disease is considered as by Pseudomonas rubrilineansPseudomonas rubrilineans
Stapp causes, and Gram’s staining reaction is feminine gender, and without spore, in shaft-like, size is 0.7 ~ 1.6 m, has and single extremely gives birth to whip
Hair, has thin-walled pod film (Martin & Wismer, 1989)；Renamed subsequently as Herba bromi japonici lactic acid bacteria Herba bromi japonici subspecies
(Acidovorax avenae subsp.avenae,Aaa) (Willems etc., 1992；Schaad etc.,
2008).AaaThe natural host of pathogenic bacteria in addition to Caulis Sacchari sinensis, also some grass family such as Oryza sativa L., Semen Maydis, Herba bromi japonici, broomcorn millet, foxtail
Plant, even can also infect the plants such as Streliziaceae Strelitzia augusta.
Phytopathogen detection method has biological assay, electron microscopy, Enzyme-multiplied immune technique and molecular biology skill
Art etc..AaaPathogenic bacteria is identified and detection method has been studied on the grasses such as Oryza sativa L., this spy grass, broomcorn millet, Caulis Sacchari sinensis.Song
The pathogenic bacteria BIO-PCR method that biology, cultivation combined is established with nest-type PRC, to rice paddy seed Deng (2004)AaaPathogenic bacteria
Detect.But, many plant diseases do not show typical Disease Characters under the natural conditions of field, and pathogenic bacteria is divided
From, cultivate and Biological assay time and effort consuming and the complex steps such as Pathogenicity.Molecular biosciences means are a kind of spirits
Sensitivity is high, the Fast Detection Technique of high specificity, is widely used to detection and the qualification of microorganism, mainly have molecule hybridization and
Nucleotide sequencing, such as Southern hybridization, polymerase chain reaction (PCR), real time PCR amplification, biochip, universal tag skill
Art and ring mediated isothermal amplification.Pathogen ribosome 16S ~ 23S Transcribed Spacer sequence is because two ends are by the rRNA of high conservative
Being surrounded, this sequence is the most conservative but also relative variable, and fragment is little, reproducible, may not only be applied to the discriminating between strain, can also be used with
Differentiate strain and the interior bacterial strain of kind that 16S rRNA gene cannot identify, be that 16S rRNA gene analysis means are well mended
Fill.Therefore, patent of the present invention on the working foundation of separation, purification and the qualification of early stage substantial amounts of Caulis Sacchari sinensis red streak bacterium, according toAaa16S ~ 23S rDNA gene of pathogenic bacteria and spacer thereof are designed inside specificity, Outside primer, by having gathered nido
The high sensitivity of PCR detection method and the advantage such as restricted enzyme cutting strip length polymorphism and uniqueness, have developed one
Plant test kit and the detection method thereof of quick Testing and appraisal Caulis Sacchari sinensis red streak bacterium, to introducing a fine variety inspection and quarantine for China Caulis Sacchari sinensis, resist
Property evaluate and seedling quality monitoring provide technical support.
Summary of the invention
It is an object of the invention to provide a kind of Molecular Detection and identify the test kit of Caulis Sacchari sinensis red streak bacterium difference strain, to overcome
Weak point of the prior art.
It is an object of the invention to be achieved through the following technical solutions.
A kind of Molecular Detection of the present invention and the test kit identifying the Caulis Sacchari sinensis different strain of red streak bacterium, it is characterised in that this examination
Agent box is made up of following reagent:
(1) PCR reactant liquor I: each PCR reactant liquor includes: 10 × PCR buffer 2.5 L, 2.5 mmol/L dNTPs 2.0
L and Outside primer 16S-F4 and 23S-R4 each 1.0 L, totally 6.5 L that concentration is 10 mol/L, 50 reaction systems
Amounting to 325 L ,-20 DEG C save backup；Described Outside primer 16S-F4:5'-CGTGAAGTCGGAATCGCTA-3'；23S-
R4:5'-TCGGAATCTCGGTTGATG-3'；
(2) PCR reactant liquor II: each PCR reactant liquor includes: 10 × PCR buffer 5.0 L, 2.5 mmol/L dNTPs 4.0
L and inner primer ITS-F1 and ITS-R1 each 2.0 L, totally 13.0 L that concentration is 10 mol/L, 50 reaction systems
Amounting to 650 L ,-20 DEG C save backup；Described inner primer ITS-F1:5'-AGACCCACCAAATCTTCCG-3'；ITS-
R1:5'-GACATCTCCGCTTTCTTTCAAG-3'；
(3) restricted enzyme buffer: each endonuclease reaction liquid comprises 10 × enzyme cutting buffering liquid 2.0 L, 50 reaction systems
Amounting to 100 L ,-20 DEG C save backup；
(4) Ex Taq enzyme: each PCR reaction system includes that 5 U/ L Ex Taq enzyme 0.75 L, 50 reaction systems amount to
37.5 L ,-20 DEG C save backup；
(5)HinDIII restriction endonuclease: each restriction enzyme reaction system includes 15 U/ LHinDIII restriction endonuclease 1.0
L, 50 reaction systems amount to 50 L, and-20 DEG C save backup；
(6)EcoRI restriction endonuclease: each restriction enzyme reaction system includes 15 U/ LEcoRI restriction endonuclease 1.0 L,
50 reaction systems amount to 50 L, and-20 DEG C save backup；
(7) positive Aaa pathogenic bacteria DNA: four kinds different Caulis Sacchari sinensis red streak bacteriumAcidovorax avenae
subsp.avenae(Aaa) strain HE, the former DNA of H, E1, E2 pathogenic bacteria are as standard sample, 1 ng/ L, 50 L/
Bottle ,-20 DEG C save backup；
(8) negative sample RNA: the Sugarcane Leaves STb gene infected without Aaa is negative control, 100 ng/ L, 50 L/ bottles ,-20
DEG C save backup；
(9) nuclease free sterilized water: 2 × 10 mL/ bottles.
It is an advantage of the invention that and overcome the detection of conventional sugarcane red streak bacterium and the deficiency of authentication method, it is provided that a kind of molecule
The test kit of Testing and appraisal Caulis Sacchari sinensis red streak bacterium difference strain, the present invention has highly sensitive, high specificity, low cost, simplicity
Quickly advantage, can be used for field early diagnosis and extensive quickly detection and identifies.
Accompanying drawing illustrates:
Fig. 1 is the nested PCR product restricted enzyme band polymorphic detection electrophoretogram of different Caulis Sacchari sinensis red streak bacterial strain system, its
In, M is 20 bp DNA standard relative molecular masses, and 1-12 is the Sugarcane Leaves sample infecting red streak bacterium, and 13 is health cane
Leaf sample, 14 is nuclease free sterilized water.
There is PCR primer enzyme action band at 330 bp, 68 bp and 56 bp positions simultaneously in sample 1-3, represents in this sample
Containing Aaa-HE strain；There is PCR primer enzyme action band at 330 bp and 124 bp positions simultaneously in sample 4-6, represents this sample
In containing Aaa-H strain；There is PCR primer enzyme action band at 386 bp and 68 bp positions simultaneously in sample 7-9, represents this sample
In containing Aaa-E1 strain；There is PCR primer enzyme action band at 369 bp and 68 bp positions in sample 10-12, represents these samples
In containing Aaa-E2 strain.13(negative control in Fig. 1) PCR primer enzyme action band does not occurs, represent that this sample is without Aaa.
14(nuclease free sterilized water in Fig. 1) PCR primer enzyme action band does not occurs, represent that experiment is not polluted.
Detailed description of the invention:
In order to be further elucidated with the present invention rather than limit the present invention, it is illustrated below in conjunction with embodiment.
Embodiment 1, a kind of Molecular Detection and the method identifying the Caulis Sacchari sinensis different strain of red streak bacterium, comprise the following steps:
1, Sugarcane Leaves total DNA extraction
(1) take blade 100 mg, with the rapid grind into powder of liquid nitrogen, put in 2 mL centrifuge tubes.
(2) adding 900 L 2 × CTAB buffer and the dehydrated alcohol of 100 L, vortex is to fully dissolving.
(3) centrifuge tube is placed in 65 DEG C of water-bath 60 min, every the reverse mixing of 5-10 min, makes the abundant cell lysis of CTAB,
12000 rpm after taking-up, centrifugal 5 min.
(4) take supernatant in 2 new mL centrifuge tubes, and add isopyknic phenol: chloroform: isoamyl alcohol 25:24:1；
Mixing, 12000 rpm are centrifuged 10 min.
(5) take supernatant in 1.5 new mL centrifuge tubes, and add isopyknic chloroform: isoamyl alcohol 24:1；Mixing,
12000 rpm are centrifuged 10 min.
(6) take supernatant, put in 1.5 new mL centrifuge tubes, and add 3 mol/L NaAC(pH=of 1/10 volume pre-cooling
5.2) DNA is made fully to precipitate, mixing, add the isopropanol of-20 DEG C of pre-coolings of equal-volume, shake even, be placed in-20 DEG C and place 30 min.
(7) 4 DEG C of 10000 rpm are centrifuged 5 min, go liquid phase, add 1000 L pre-cooling 75 % ethanol and wash precipitation twice, then use
Dehydrated alcohol is washed once, is inverted centrifuge tube, drying at room temperature DNA.
(8) 40 L ddH are added2O, and be incubated until DNA is completely dissolved in 37 DEG C.
(9) with standby in spectrophotometer detection DNA mass and rearmounted 4 DEG C of DNA concentration, or-20 DEG C long-term preservations are put.
2, design of primers
On the working foundation of separation, purification and the qualification of early stage substantial amounts of Caulis Sacchari sinensis red streak bacterium, according to Caulis Sacchari sinensis red streak bacterium Aaa
16S ~ 23S rDNA gene and 2 pairs of specific primers of spacer be respectively Outside primer 16S-F4/23S-R4 and inner side
Primer I TS-F1/ ITS-R1, the purpose nucleic acid belt size of nested PCR amplification is 454 bp.Described Outside primer 16S-F4, tool
Just like the nucleotide sequence shown in SEQ ID NO:1 in sequence table；23S-R4, has as shown in SEQ ID NO:2 in sequence table
Nucleotide sequence；Described inner primer ITS-F1, has the nucleotide sequence as shown in SEQ ID NO:3 in sequence table；
ITS-R1, has the nucleotide sequence as shown in SEQ ID NO:4 in sequence table.
3, the 1st PCR amplification is taken turns
Following reagent is added in PCR pipe:
Mixing, 8000 rpm be centrifuged after 5 S 94 DEG C of denaturation 3 min, 94 DEG C of degeneration 45 S, 58 DEG C of annealing 30 S, 72 DEG C
Extending 45 S, totally 35 circulations, last 72 DEG C re-extend 10 min.
4, the 2nd PCR amplification is taken turns
Take turns PCR primer to dilute the 1st after 100 times, PCR pipe add following reagent:
Mixing, 8000 rpm be centrifuged after 5 S 94 DEG C of denaturation 3 min, 94 DEG C of degeneration 45 S, 58 DEG C of annealing 30 S, 72 DEG C
Extending 60 S, totally 35 circulations, last 72 DEG C re-extend 10 min.
5, PCR primer purification
(1) add after being settled to 100 μ L with 50 μ L nuclease free sterilized water isopyknic phenol/chloroform/isoamyl alcohol (25: 24:
1) mixing.
(2) room temperature, 13,500 rpm 5 min are centrifuged, take supernatant.
(3) mixing of isopyknic chloroform/isoamyl alcohol (24: 1) is added.
(4) room temperature, 13,500 rpm 5 min are centrifuged, take supernatant.
(5) add 10 μ L 3 mol/L sodium acetates, the 250 cold ethanol of μ L, place 30 min for-20 DEG C.
(6) 4 DEG C, 13,500 rpm 15 min are centrifuged, and abandon supernatant.
(7) adding the 500 cold ethanol of μ L 70% to clean, 4 DEG C, 13,500 rpm 5 min are centrifuged, and abandon supernatant.
(8) precipitation is dried.
(9) add 100 μ L nuclease free sterilized water to dissolve.
6, endonuclease reaction
This test kit is applied to provideHinDIII andEcoRI restricted enzyme and buffer thereof carry out endonuclease reaction,
Following reagent is added in PCR pipe:
Mixing, 8000 rpm are centrifuged after 5 s 37 DEG C of enzyme action 2 hours, and 65 DEG C process 5 min, terminate endonuclease reaction, are often down to
Temperature.
7, electrophoresis detection
Endonuclease reaction takes 5 L reactant liquors and carries out the low melting-point agarose gel electrophoresis that mass volume ratio is 4.0 % after terminating,
In 0.5 × electrophoretic buffer environment, 100 V voltage stabilizing electrophoresis 1.5 h, then observe with gel imaging system and take pictures, in electrophoresis
Detection figure occurs PCR primer enzyme action band, represents that this sample is containing Aaa；Occur without PCR primer enzyme action band, represent this sample
Without Aaa.Different Aaa strains is distinguished according to the banding pattern occurred.
Caulis Sacchari sinensis red streak bacterium difference strain restriction enzyme digestion sites and band length polymorphism are listed in table 1, from table 1
Visible, the position of nested PCR product enzyme action band is different, represents that in sample, the strain of contained Aaa is different.At 330 bp, 68 bp
With 56 bp positions, PCR primer enzyme action band occurs simultaneously, represent in this sample containing Aaa-HE strain.At 330 bp and 124
There is PCR primer enzyme action band simultaneously in bp position, represents in this sample containing Aaa-H strain.Same at 386 bp and 68 bp positions
Time PCR primer enzyme action band occurs, represent in this sample containing Aaa-E1 strain.Occur that PCR produces at 369 bp and 68 bp positions
Thing enzyme action band, represents in these samples containing Aaa-E2 strain.
Table 1 Caulis Sacchari sinensis red streak bacterium difference strain restriction enzyme digestion sites and band length polymorphism
The present embodiment have detected 72 samples with red streak evil classical symptom collected from China's Provinces (regions) sugarcane field, generation
Table sample electrophoresis testing result is as it is shown in figure 1,1 ~ 12 expression sample 1 ~ 12 is after testing containing Aaa in Fig. 1.Wherein, sample 1-3 exists
There is PCR primer enzyme action band simultaneously in 330 bp, 68 bp and 56 bp positions, represent in this sample containing Aaa-HE strain；Sample
There is PCR primer enzyme action band at 330 bp and 124 bp positions simultaneously in product 4-6, represents in this sample containing Aaa-H strain；Sample
There is PCR primer enzyme action band at 386 bp and 68 bp positions simultaneously in product 7-9, represents in this sample containing Aaa-E1 strain；Sample
There is PCR primer enzyme action band at 369 bp and 68 bp positions in product 10-12, represents in these samples containing Aaa-E2 strain.Figure
13(negative control in 1) PCR primer enzyme action band does not occurs, represent that this sample is without Aaa；14(nuclease free in Fig. 1 without
Bacterium water) PCR primer enzyme action band does not occurs, represent that experiment is not polluted.M in Fig. 1 is 20 bp DNA standard average molecular matter
Amount.Above 62 sample identification results with red streak evil classical symptom are that 31 parts of samples have infected Aaa-HE strain, 20 parts
Sample has infected Aaa-H strain, and 18 parts of samples have infected Aaa-E1 strain, and 3 parts of samples have infected Aaa-E2 strain；Pass through nido
PCR primer purification, clone, check order and sequence analysis, completely the same with this experimental result, demonstrate the accuracy of experiment further
And reliability.
The main agents that the present invention uses is as follows: (all chemical reagent are analytical pure)
1, CTAB Extraction buffer: 100 mmol/L Tris-HCl (pH8.0), 20 mmol/L EDTA-Na2, 1.4 mol/
LNaCl, mass volume ratio 2 % CTAB, add volume ratio 0.1 % beta-mercaptoethanol before using；
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 amplifing reagent is purchased from Dalian treasured biotech firm.
Instrument used in the present invention is as follows:
1, PCR amplification instrument: Germany's Eppendorf 5331 PCR amplification instrument；
2, electrophresis apparatus: Liuyi Instruments Plant, Beijing DYCZ-20A DNA sequence analysis electrophresis apparatus；
3, centrifuge: Germany's Eppendorf 5810/5810R multifunctional table-type centrifuge；
4, gel imaging system: U.S.'s BIO-RAD Gel Doc 2000 gel imaging system；
5, spectrophotometer: BioTek company of U.S. SynergyTMH1 ultramicrospectrophotometer.
The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with
Modify, all should belong to the covering scope of the present invention.
SEQUENCE LISTING
Sequence table
<110>University Of Agriculture and Forestry In Fujian
<120>a kind of Molecular Detection and the test kit identifying the Caulis Sacchari sinensis different strain of 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 Molecular Detection and the test kit identifying the Caulis Sacchari sinensis different strain of red streak bacterium, it is characterised in that this test kit is by following
Reagent forms:
(1) PCR reactant liquor I: each PCR reactant liquor includes: 10 × PCR buffer 2.5 L, 2.5 mmol/L dNTPs 2.0
L and Outside primer 16S-F4 and 23S-R4 each 1.0 L, totally 6.5 L that concentration is 10 mol/L, 50 reaction systems
Amounting to 325 L ,-20 DEG C save backup；Described Outside primer 16S-F4:5'-CGTGAAGTCGGAATCGCTA-3'；23S-
R4:5'-TCGGAATCTCGGTTGATG-3'；
(2) PCR reactant liquor II: each PCR reactant liquor includes: 10 × PCR buffer 5.0 L, 2.5 mmol/L dNTPs 4.0
L and inner primer ITS-F1 and ITS-R1 each 2.0 L, totally 13.0 L that concentration is 10 mol/L, 50 reaction systems
Amounting to 650 L ,-20 DEG C save backup；Described inner primer ITS-F1:5'-AGACCCACCAAATCTTCCG-3'；ITS-
R1:5'-GACATCTCCGCTTTCTTTCAAG-3'；
(3) restricted enzyme buffer: each endonuclease reaction liquid comprises 10 × enzyme cutting buffering liquid 2.0 L, 50 reaction systems
Amounting to 100 L ,-20 DEG C save backup；
(4) Ex Taq enzyme: each PCR reaction system includes that 5 U/ L Ex Taq enzyme 0.75 L, 50 reaction systems amount to
37.5 L ,-20 DEG C save backup；
(5)HinDIII restriction endonuclease: each restriction enzyme reaction system includes 15 U/ LHinDIII restriction endonuclease 1.0
L, 50 reaction systems amount to 50 L, and-20 DEG C save backup；
(6)EcoRI restriction endonuclease: each restriction enzyme reaction system includes 15 U/ LEcoRI restriction endonuclease 1.0 L,
50 reaction systems amount to 50 L, and-20 DEG C save backup；
(7) positive Aaa pathogenic bacteria DNA: four kinds different Caulis Sacchari sinensis red streak bacteriumAcidovorax avenae
subsp.avenae(Aaa) strain HE, the former DNA of H, E1, E2 pathogenic bacteria are as standard sample, 1 ng/ L, 50 L/
Bottle ,-20 DEG C save backup；
(8) negative sample RNA: the Sugarcane Leaves STb gene infected without Aaa is negative control, 100 ng/ L, 50 L/ bottles ,-20
DEG C save backup；
(9) nuclease free sterilized water: 2 × 10 mL/ bottles.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107034284A (en) * | 2017-05-15 | 2017-08-11 | 华南农业大学 | Sang Sheng ball pin shells ITS sequence and its application in Sang Sheng ball pin shell Molecular Detections |
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 |
-
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 |
|---|
| OCHIAI,H.等: "Genetic Relationships among Xanthomonas Species and Pathovars Based on RFLP Analyses of PCR-amplified 16S, 23S rDNA and rDNA Internal Transcribed Spacer", 《日植病报》 * |
| 李晓燕等: "甘蔗赤条病病原菌分离与鉴定", 《中国植物病理学会2016年学术年会论文集》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107034284A (en) * | 2017-05-15 | 2017-08-11 | 华南农业大学 | Sang Sheng ball pin shells ITS sequence and its application in Sang Sheng ball pin shell Molecular Detections |
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