CN106967823A - The authentication method of pleurotus eryngii Pathogen of Fusarium Wilt - Google Patents
The authentication method of pleurotus eryngii Pathogen of Fusarium Wilt Download PDFInfo
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- CN106967823A CN106967823A CN201710325362.1A CN201710325362A CN106967823A CN 106967823 A CN106967823 A CN 106967823A CN 201710325362 A CN201710325362 A CN 201710325362A CN 106967823 A CN106967823 A CN 106967823A
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Abstract
The invention discloses the authentication method of pleurotus eryngii Pathogen of Fusarium Wilt.In order to identify pleurotus eryngii Pathogen of Fusarium Wilt, the invention provides general bacterium special primer of picking up the ears to K3143FR, it is made up of primer K3143F and primer K3143R；Primer K3143F is the single strand dna shown in SEQ ID No.1；Primer K3143R is the single strand dna shown in SEQ ID No.2.General bacterium special primer of picking up the ears only in the genomic DNA for picking up the ears general bacterium has amplified specific band to K3143FR, and amplified production is not obtained in other bacterial strains, and general bacterium special primer of picking up the ears is picked up the ears general bacterium to K3143FR available for Rapid identification.The present invention picks up the ears the infection sources of general microbial pleurotus eryngii droop for early prevention and in time cut-out and route of transmission provides technical support, achievable to general microbial pleurotus eryngii droop progress early prevention and the control of picking up the ears.
Description
Technical field
The present invention relates to the authentication method of pleurotus eryngii Pathogen of Fusarium Wilt in biological technical field.
Background technology
China's pleurotus eryngii large-scale planting since last century the nineties.General Pseudomonas includes being used as plant growth-promoting bacteria, life
Fungi-proofing or phytopathogen several kinds of (Pantoea:insights into a highly versatile and diverse
genus within the Enterobacteriaceae,FEMS Microbiology Reviews(2015)39:968-
984).By general microbial pleurotus eryngii bacterialo wilt disease be in recent years at home and abroad Pleurotus eryngii industrial production in occur it is tight
Grave illness does harm to, and the pathogen for being reported in the pleurotus eryngii droop of Beijing area is accredited and named as the general bacterium (Pantoea that picks up the ears
pleuroti sp.nov.,isolated from the fruiting bodies of Pleurotus eryngii,Ma et
al.Current Microbiology,(2016)72(2)207-212).Pleurotus eryngii bacterialo wilt disease has a strong impact on fructification
Quality, be unfavorable for factorial praluction.The exploitation for general bacterium quick diagnosis technology of picking up the ears, effective prevention and control to pleurotus eryngii droop have
It is significant.
The content of the invention
The technical problems to be solved by the invention are how to identify or aid in identification to pick up the ears general bacterium (Pantoea
pleuroti)。
In order to solve the above technical problems, the invention provides general bacterium specific PCR primers pair of picking up the ears.
General bacterium specific PCR primers provided by the present invention of picking up the ears are to entitled K3143FR, by primer K3143F and primer
K3143R is constituted；The primer K3143F is the single strand dna shown in SEQ ID No.1；The primer K3143R is SEQ
Single strand dna shown in ID No.2.
General bacterium of picking up the ears is identified present invention also offers the identification or auxiliary including general bacterium special primer of picking up the ears to K3143FR
Reagent or kit.
Above-mentioned identification or auxiliary identify that the reagent or kit of general bacterium of picking up the ears also include removing primer needed for reacting into performing PCR
Outer other reagents.
Present invention also offers preparation method of the general bacterium special primer to K3143FR of picking up the ears, identification or auxiliary identification are picked up the ears
The reagent of general bacterium or the preparation method of kit.
General bacterium special primer provided by the present invention of picking up the ears specifically draws to K3143FR preparation method including the general bacterium that will pick up the ears
The step of thing is individually packed to K3143FR two single strand dnas.
Identification provided by the present invention or auxiliary identification pick up the ears general bacterium reagent or kit preparation method, including by side
The step of general bacterium special primer of ear is individually packed to K3143FR two single strand dnas.
Present invention also offers the DNA molecular shown in SEQ ID No.3.
DNA molecular shown in SEQ ID No.3 is the specific DNA fragment of general bacterium of picking up the ears.
General bacterium special primer of picking up the ears is identifying or aided in identification to pick up the ears application in general bacterium or to identify preparing K3143FR
Or auxiliary identification pick up the ears application in general bacterium product (as identified or auxiliary identification is picked up the ears the reagent or kit of general bacterium), identification or
Auxiliary identifies the application and SEQID No.3 institutes of the reagent or kit of general bacterium of picking up the ears in general bacterium is picked up the ears in identification or auxiliary identification
The DNA molecular shown picks up the ears application in general bacterium or preparing identification or auxiliary identification is picked up the ears general bacterium product identifying or aid in identification
Application in (as identification or auxiliary identify the reagent or kit for general bacterium of picking up the ears) falls within protection scope of the present invention.
Above-mentioned application does not include the diagnostic method and/or treatment method of disease.
Present invention also offers the method that identification or auxiliary identify general bacterium of picking up the ears.
The method that identification provided by the present invention or auxiliary identify general bacterium of picking up the ears, including with the genome of biological sample to be measured
DNA is template, and entering performing PCR amplification to K3143FR with general bacterium special primer of picking up the ears obtains PCR primer, detects the PCR primer
Size, if the PCR primer contain 500-700bp DNA fragmentation (or the PCR primer be 500-700bp DNA pieces
Section), the biological sample to be measured is general bacterium or general to pick up the ears containing general bacterium of picking up the ears, or the biological sample candidate to be measured of picking up the ears
Bacterium or candidate contain general bacterium of picking up the ears；If the PCR primer does not contain 500-700bp DNA fragmentation, (or the PCR primer is not
For 500-700bp DNA fragmentation), the biological sample to be measured general bacterium or does not contain general bacterium of picking up the ears to pick up the ears, or described treats
Biological sample candidate is surveyed to pick up the ears general bacterium or candidate does not contain general bacterium of picking up the ears.
Above-mentioned identification or auxiliary identify in the method for general bacterium of picking up the ears that the DNA fragmentation of the 500-700bp is concretely
660bp DNA fragmentation.
It is demonstrated experimentally that general bacterium special primer of picking up the ears only has amplified spy to K3143FR in the genomic DNA for picking up the ears general bacterium
Different band, amplified production is not obtained in other bacterial strains, and general bacterium special primer of picking up the ears can be used for Rapid identification to K3143FR
Pick up the ears general bacterium.The present invention is early prevention and cuts off general microbial pleurotus eryngii droop (pleurotus eryngii bacterium of picking up the ears in time
Property droop) the infection sources and route of transmission provide technical support, can be achieved to general microbial pleurotus eryngii droop of picking up the ears
Carry out early prevention and control.
Brief description of the drawings
Fig. 1 is agarose gel electrophoresis of the primer pair 16sp1-16sp2 to the 16s rDNA of general bacterium pcr amplification product
Figure.
Fig. 2 is Ago-Gel of the general bacterium special primer to the pcr amplification product of the general bacterium of K3143FR specific detections of picking up the ears
Electrophoretogram.
Fig. 3 is agarose gel electrophoresis figure of the general bacterium special primer to the pcr amplification product of the general bacterium of K6FR detections of picking up the ears.
Fig. 1 is into Fig. 3, and M is 1kb plus DNA ladder (Tiangeng)；1 is general bacterium (the Pantoea pleuroti that pick up the ears
sp.nov.)JZB2120015T；2 be Pantoea vagans LMG24199；3 be Pantoea stewartii
subsp.stewartii LMG2715；4 be Pantoea stewartii subsp.Indologenes LMG2632；5 are
Pantoea wallisii LMG26277；6 be Pantoea allii LMG24248；7 be Pantoea ananatis
LMG2665；8 be Pantoea deleyi LMG24200；9 be Pantoea brenneri LMG5343；10 be Pantoea
rodasii LMG 26273；11 be Pantoea eucalypti 24197；12 be Pantoea agglomerans
JCM1236；13 be the general bacterium in Beijing (Pantoea beijingensis sp.nov.) JZB2120001T；14 be Pantoea
gaviniae DSM22758；15 be Pantoea anthophila DSM23080；16 be Pantoea dispersa DSM
30073。
Embodiment
The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the scope being not intended to be limiting of the invention.
Experimental method in following embodiments, is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
The general bacterium in Beijing (Pantoea beijingensis sp.nov.) JZB2120001 in following embodimentsT(i.e.
LMG27579TOr KCTC 32406T)(Pantoea beijingensis sp.nov.,isolated from the
fruiting body of Pleurotus eryngii.Liu et al.Antonie van Leeuwenhoek(2013)
104:1039-1047) public can be from Beijing City Academy Of Agriculture and Forest Sciences INST OF PLANT PROT ＆ ENVIRONME or Belgium BCCM/LMG
DSMZ or KCTC DSMZs of South Korea obtain the bacterial strain.
General bacterium of picking up the ears (Pantoea pleuroti sp.nov.) JZB2120015 in following embodimentsT(Pantoea
pleuroti sp.nov.,Isolated from the Fruiting Bodies of Pleurotus eryngii.Ma et
Al.Current Microbiology.19November 2015) public can be from Beijing City Agriculture and Forestry Institute plant protection environment
Protective strategy institute or BCCM/LMG DSMZs of Belgium or CGMCC DSMZs of China obtain the bacterial strain.
Pantoea gaviniae DSM22758, Pantoea anthophila DSM23080 in following embodiments and
Pantoea dispersa DSM 30073 are the bacterial strain of German DSM DSMZs.
Pantoea agglomerans JCM1236 in following embodiments are the bacterial strain of JCM DSMZs of Japan.
Pantoea vagans LMG24199, Pantoea stewartii subsp.Stewartii LMG2715,
Pantoea stewartii subsp.Indologenes LMG 2632, Pantoea wallisii LMG 26277,
Pantoea allii LMG24248, Pantoea ananatis LMG2665, Pantoea deleyi LMG24200,
Pantoea brenneri LMG5343, Pantoea rodasii LMG26273, Pantoea eucalypti LMG24197
For the bacterial strain of Belgian LMG DSMZs.
Embodiment 1, general bacterium of being picked up the ears using PCR primer to K3143FR identifications
1st, the specific PCR reagent for general bacterium of picking up the ears is identified
Pick up the ears general bacterium (Pantoea pleuroti sp.nov.) JZB2120015TGenomic DNA in contain SEQ ID
Section of DNA sequence shown in No.3, is compared on NCBI without obvious sequence similarity, with this sequences Design special primer pair,
It is named as K3143FR.
The identification of the present embodiment pick up the ears general bacterium PCR reagent by general bacterium special primer of picking up the ears to K3143FR, 2 × Taq PCR
Mix (Bioeasy) and ddH2O is constituted.
Wherein, pick up the ears general bacterium special primer to K3143FR by K3143F (sense primer) and K3143R (anti-sense primer) group
Into K3143F nucleotide sequence is 5'-CGGCGAATCAACCACCAAAAAC-3'(SEQ ID No.1), K3143R nucleosides
Acid sequence is 5'-GAATGAGGTCAGGTAGCGGAAC-3'(SEQ ID No.2).
2nd, general bacterium of picking up the ears is identified
2.1st, the genomic DNA of bacterial strain is extracted
From 16 strains (bacterial strain) (general bacterium in Beijing (Pantoea beijingensis sp.nov.) of -80 DEG C of preservations
JZB2120001T；Pick up the ears general bacterium (Pantoea pleuroti sp.nov.) JZB2120015T；Pantoea gaviniae
DSM22758；Pantoea anthophila DSM23080；Pantoea dispersa DSM 30073；Pantoea
agglomerans JCM1236；Pantoea vagans LMG24199；Pantoea stewartii subsp.Stewartii
LMG2715；Pantoea stewartii subsp.Indologenes LMG 2632；Pantoea wallisii
LMG26277；Pantoea allii LMG 24248；Pantoea ananatis LMG2665；Pantoea deleyi
LMG24200；Pantoea brenneri LMG5343；Pantoea rodasii LMG26273 and Pantoea eucalypti
LMG24197) rule respectively in solid LB media, 28 DEG C of culture 20h, with each bacterium bacterial strain single bacterium colony of oese picking
It is connected in the test tube equipped with LB culture mediums (3mL), with 28 DEG C of shaken cultivation 18h of 200rpm.By each strains tested DNeasy
Blood＆Tissue Kit (QIAGEN) extract STb gene as pcr template, are operated according to the specification of kit, specific steps
It is as follows：
1) 2ml inoculums are collected, 12000rpm centrifugation 1min remove supernatant.
2) 180 μ l Buffer ATL of addition, 20 μ l Proteinase Ks, 4 μ l RNaseA, 56 DEG C are handled 1 hour.
3) 15s is vibrated, 200 μ l buffer AL is added and mixes.
4) 200 μ l absolute ethyl alcohols, 8000rpm centrifugations 3min are added.
5) supernatant is collected into centrifugal column, 8000rpm centrifugations 1min.
6) 500 μ l Buffer AW1,8000rpm centrifugations 1min are added in centrifugal column.
7) 500 μ l Buffer AW2,8000rpm centrifugations 1min are added in centrifugal column.
8) centrifugal column is put on a new 1.5ml centrifuge tube, adds 50 μ l Buffer AE, room temperature places 1min,
8000rpm centrifuges 1min, and gained is the genomic DNA of each strain of above-mentioned 16 strains.
The genomic DNA using above-mentioned each strain expands each bacterial strain as template using primer pair 16sp1-16sp2PCR respectively
16s rDNA fragments.Primer pair 16sp1-16sp2 by 16sp1 (sequence is 5 '-
AGAGTTTGATCCTGGCTCAGAACGAACGCT-3 ') and 16sp2 (sequence is 5 '-
TACGGCTACCTTGTTACGACTTCACCCC-3 ') composition.PCR reaction systems (20 μ L)：10μmol·L-1Upstream and downstream draw
Each μ L of 1 μ L, 2 × Taq PCR Mix (Bioeasy) 10 of thing, template DNA 0.5 μ L, ddH2O 7.5μL.PCR reaction conditions：95
DEG C, 5min；95 DEG C, 30s, 55 DEG C, 30s, 72 DEG C of 2min, 30 circulations；72 DEG C of extension 10min.1% agarose gel electrophoresis is examined
Survey.
As a result show that primer pair 16sp1-16sp2 can amplify purpose band in 16 bacterial strains for examination, illustrate to supply
The DNA extraction effects for trying bacterial strain are good (Fig. 1).
2.2nd, performing PCR amplification is entered using primer pair K3143FR
The genomic DNA using the 2.1 each bacterial strains extracted utilizes the general bacterium special primer pair of picking up the ears of step 1 as template respectively
K3143FR enters performing PCR.PCR reaction systems (20 μ L)：10μmol·L-1Each 1 μ L, 2 × Taq PCR Mix of upstream and downstream primer
(Bioeasy) 10 μ L, template DNA 0.5 μ L, ddH2O 7.5μL.PCR reaction conditions：95 DEG C, 5min；95 DEG C, 30s, 60 DEG C,
30s, 72 DEG C of 1min, 30 circulations；72 DEG C of extension 10min.1% agarose gel electrophoresis is detected.
As a result the general bacterium special primer that shows to pick up the ears can only be picked up the ears general bacterium (Pantoea to K3143FR with No. 1 bacterial strain
pleuroti sp.nov.)JZB2120015TGenomic DNA be that to go out size be 500-700bp bands to template amplification, pick up the ears general
Bacterium special primer does not amplify band (Fig. 2) to K3143FR in other 15 bacterial strains.Recovery is picked up the ears general bacterium (Pantoea
pleuroti sp.nov.)JZB2120015T500-700bp bands, be sequenced, as a result show the 500-700bp bands
Size be 660bp, its sequence be SEQ ID No.3.The general bacterium special primer that illustrates to pick up the ears is the spy of general bacterium of picking up the ears to K3143FR
Different primer pair, picks up the ears general bacterium available for Rapid identification.
Comparative example 1, PCR primer can not identify general bacterium of picking up the ears to K6FR
Pick up the ears general bacterium (Pantoea pleuroti sp.nov.) JZB2120015TGenomic DNA in contain other one
Segment DNA sequence (entitled K6), is compared on NCBI without obvious sequence similarity, with this sequences Design special primer to K6FR
By K6F (sequence is 5 '-CCATTACACAGTTTCGGTTCC-3 ') and K6R, (sequence is 5 '-GCTTGCCCAATAAATTCCTTC-
3 ') constitute, enter performing PCR respectively using the genomic DNA of 16 strains of embodiment 1 as template, as a result show special primer pair
K6FR's is specific bad, except No. 1 bacterial strain is picked up the ears general bacterium (Pantoea pleuroti sp.nov.) JZB2120015TIt can expand
Increase and outside specific band, No. 9 bacterial strain Pantoea brenneri LMG5343 can also amplify purpose band (Fig. 3), illustrate side
The general bacterium special primer of ear is unable to unique identification to K6FR and picked up the ears general bacterium.Wherein, PCR reaction systems be the same as Example 1, PCR reaction bars
Part：95 DEG C, 5min；95 DEG C, 30s, 60 DEG C, 30s, 72 DEG C of 1min, 30 circulations；72 DEG C of extension 10min.1% Ago-Gel
Electrophoresis detection.
<110>Beijing City Agriculture and Forestry Institute
<120>The authentication method of pleurotus eryngii Pathogen of Fusarium Wilt
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 1
cggcgaatca accaccaaaa ac 22
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 2
gaatgaggtc aggtagcgga ac 22
<210> 3
<211> 660
<212> DNA
<213>Pick up the ears general bacterium（Pantoea pleuroti）
<400> 3
cggcgaatca accaccaaaa acggtatcgc tgccgcttct ggagaaaccg gtcagatggc 60
ccgcagatgt gcgtcagcct gaccgcttct tgcccgccag cagtcttgaa ctttcgcaac 120
aatggaaact ttatcgcgct actgaggata agcagctcca tgtcggtgac gccattgagc 180
gcgtcgtgac attgcgtgcg accgatgtga ttgccgtaca aatcccgcag ctgctttacg 240
ccattcccgg ttccggtgcg caacgtttgc cgccggtaaa cagcaacctg acgcagggac 300
gcgatgaaat catcggcgca cagcgagaag agcggctgcg atatctgccc tctcagtctg 360
gtgaactggt gataccaccg ttgcgtttac gctggtggga tacacgccag catcagtggc 420
aactggccga attacccggt gcgcaatacg ctattgccgc tgcgcgtccg gcgggcagtg 480
aacaggcgtt gaaagcccac gtcccggtca actggacgtc gttgttgatg ggcccggcca 540
tcctgctgac gttgctgctg ttgagcttct tttttcgaca cgcccttaaa aaaagcctgc 600
aaagagtagt gcagcgcaca cgggaatact ggcggatcgt tccgctacct gacctcattc 660
<210> 4
<211> 611
<212> DNA
<213>Pick up the ears general bacterium（Pantoea pleuroti）
<400> 4
ccattacaca gtttcggttc cctataaaaa tgaaaagcag catgttacag agttactttc 60
tttgataagt aaaagcattg gcgttaaagc gacaggcaat ttctcttaca gtcttcattt 120
tcctgatgtt gtgcttgatg aggtgaggca ctttcttagt ccgtgggaaa ataagaaaaa 180
aattgttatt aatgcttttg ctggcacgcc ggaaaggaac ttctcacaac agcaattgct 240
ggaaattatt aatatgatta ataataacag cagagaggtg aaagtcatca ttctggatca 300
tcgtaatgaa atagcggtgc cgcttcctgg aaatgttgtg aaaaatccct tttatacttt 360
gcatcatgtg atggcattaa ttaaagaatc ggacgtggtg ataacgccag atacatcaat 420
tgtacatatc tctgctgcgt ggaaaaaacc tcttatcgcc gtttacaaaa atgcgcctaa 480
taataatatg tgggcgccag gctatgaaaa cgcaagccac atcattgttc atgatgataa 540
aacatccgat gctgagaata tccctgagcg tatttttaaa gagataatcc gaaggaattt 600
attgggcaag c 611
Claims (10)
1. general bacterium special primer pair of picking up the ears, is made up of primer K3143F and primer K3143R；The primer K3143F is SEQ ID
Single strand dna shown in No.1；The primer K3143R is the single strand dna shown in SEQ ID No.2.
The reagent or kit of general bacterium 2. identification or auxiliary identification are picked up the ears, including general bacterium of picking up the ears described in claim 1 are specifically drawn
Thing pair.
3. the preparation method of the general bacterium special primer pair of picking up the ears described in claim 1, including by the general bacterium special primer of picking up the ears
To two single strand dnas individually pack the step of.
4. the preparation method of the reagent or kit described in claim 2, including by the institute of the general bacterium special primer pair of picking up the ears
State the step of two single strand dnas are individually packed.
5. the general bacterium special primer of picking up the ears described in claim 1 is to identifying or aiding in identification to pick up the ears application in general bacterium or in system
Standby identification or auxiliary identify the application in general bacterium product of picking up the ears.
6. the application of reagent or kit in identifying or aiding in identifying general bacterium of picking up the ears described in claim 2.
The method of general bacterium 7. identification or auxiliary identification are picked up the ears, including using the genomic DNA of biological sample to be measured as template, use right
It is required that the general bacterium special primer of picking up the ears described in 1 obtains PCR primer to entering performing PCR amplification, the size of the PCR primer is detected, such as
Really described PCR primer contains 500-700bp DNA fragmentation, and the biological sample to be measured is the general bacterium or containing general bacterium of picking up the ears of picking up the ears,
Or the biological sample candidate to be measured is picks up the ears general bacterium or candidate contains general bacterium of picking up the ears；If the PCR primer is not contained
500-700bp DNA fragmentation, the biological sample to be measured general bacterium or does not contain general bacterium of picking up the ears to pick up the ears, or described to be measured
Biological sample candidate is to pick up the ears general bacterium or candidate does not contain general bacterium of picking up the ears.
8. method according to claim 7, it is characterised in that：The DNA fragmentation of the 500-700bp is 660bp DNA pieces
Section.
DNA molecular shown in 9.SEQ ID No.3.
DNA molecular shown in 10.SEQ ID No.3 identify or aid in identification pick up the ears application in general bacterium or prepare identification or
Auxiliary identifies the application in general bacterium product of picking up the ears.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105463105A (en) * | 2016-01-04 | 2016-04-06 | 北京市农林科学院 | Method for authenticating Beijing Pantoea and paired DNA molecules applied by method |
| CN105463107A (en) * | 2016-01-04 | 2016-04-06 | 北京市农林科学院 | Primer pair AWF-AWR for identifying pantoea beijingensis |
| CN105483260A (en) * | 2016-01-04 | 2016-04-13 | 北京市农林科学院 | Pantoea beijingensis specific primer and application thereof |
| CN105506117A (en) * | 2016-01-04 | 2016-04-20 | 北京市农林科学院 | Method for authenticating Peking pantoea through primer pair AHF-AHR |
-
2017
- 2017-05-10 CN CN201710325362.1A patent/CN106967823B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105463105A (en) * | 2016-01-04 | 2016-04-06 | 北京市农林科学院 | Method for authenticating Beijing Pantoea and paired DNA molecules applied by method |
| CN105463107A (en) * | 2016-01-04 | 2016-04-06 | 北京市农林科学院 | Primer pair AWF-AWR for identifying pantoea beijingensis |
| CN105483260A (en) * | 2016-01-04 | 2016-04-13 | 北京市农林科学院 | Pantoea beijingensis specific primer and application thereof |
| CN105506117A (en) * | 2016-01-04 | 2016-04-20 | 北京市农林科学院 | Method for authenticating Peking pantoea through primer pair AHF-AHR |
Non-Patent Citations (1)
| Title |
|---|
| MA Y ET AL: "Pantoea pleuroti sp. nov., Isolated from the Fruiting Bodies of Pleurotus eryngii", 《CURR MICROBIOL》 * |
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