CN102816835A - Clavibacter michiganensis subsp.nebraskensis(Cmn) PCR detection method, and detection primer - Google Patents
Clavibacter michiganensis subsp.nebraskensis(Cmn) PCR detection method, and detection primer Download PDFInfo
- Publication number
- CN102816835A CN102816835A CN2012101378028A CN201210137802A CN102816835A CN 102816835 A CN102816835 A CN 102816835A CN 2012101378028 A CN2012101378028 A CN 2012101378028A CN 201210137802 A CN201210137802 A CN 201210137802A CN 102816835 A CN102816835 A CN 102816835A
- Authority
- CN
- China
- Prior art keywords
- corn
- primer
- pcr
- cmn
- state
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a Cmn PCR detection method and a detection primer. The Cmn PCR detection method is established through designing the specific primer PSM1(5'- cctttccgtcgtcctttc-3')/CM3(5'- gcatccaccgtttgctct-3') according to the 16S-23S difference between different subspecies of the Cmn. The detection sensitivity of the method is 4pg of a thallus DNA or 680CFU of a target bacterium, and detection tests of entrance corn samples show that the PCR method can be used for the rapid detection of the Cmn in the entrance corn samples.
Description
Technical field
The present invention relates to agricultural and Plant Quarantine technical field, be specifically related to the detection method of state wilting germ in a kind of corn, relate in particular to the PCR detection method of state wilting germ in a kind of corn.In addition, the invention still further relates to the primer that detects state wilting germ in the corn.
Background technology
Clavate Bacillaceae Clavibacter only comprises a kind of phytopathogen Clavibacter michiganensis (Cm); Difference according to its host's specialization; Be divided into 5 subspecies; Comprise state wilting germ C.m.subsp.nebraskensis (Cmn), tomato bacterial canker germ C.m.subsp.Michiganenis (Cmm) in the corn; Bacterial ring rot o potato bacterium C.m.subsp.sepedonicus (Cms), state wilting germ Cmn and wheat mosaic bacterium C.m.subsp.tessellarius (Cmt) (Eichenlaub et al.2006) in the clover wilting germ C.m.subsp.insidiouss (Cmi), corn.They all can cause important Plant diseases, and EPPO classifies Cmm, Cms and Cmi as quarantine harmful organisms, and in the quarantine harmful organisms register is listed Cmn, Cmm, Cms and Cmi simultaneously by China.
State wilting germ Cmn has another name called clavate bacillus Nebraska, Michigan subspecies in the corn, is under the jurisdiction of Firmicutes Firmicutes, heavy wall Gammaproteobacteria Firmibacteria, excellent type Bacillaceae Clavibacter.1969, United States Nebraska was found state wilt (Goss's bacterial wilt and leaf blight of corn) (Vidaver and Mandel, 1974) in this germ corn first.To 1972, this disease extended to contiguous 5 states.In recent years along with the popularization of transformed variety; Disease spreads each corn planting district to the Middle West; Comprise the Nebraska State, the Kansas State, the South Dakota State, the state of Colorado, the Wyoming State, the state of Michigan, Wei Sikang good fortune state, Iowa, Illinois, Indiana (Ruhl et al.2009), the Minnesota State (Malvick et al.2010), Deco Sa Si state (Korus et al.2011) and Ontario, Canada, Manitoba province (Agarkova et al.2011), become the serious plant disease on the north America region Maize Production.
State wilting germ Cmn can endanger dent corn, sweet corn, flint corn, quick-fried corn, Herba Setariae Viridis, barnyard grass grass and sugarcane in the corn.Cmn may vary seed dispersal (Biddle? Et? Al.1990), but most of our corn-growing areas suitable for the occurrence of disease (Chen Shi, etc. .2009).In a single day germ imports into surely and grows, and break out easily and cause disease popular, and the disease control difficulty is big.China's corn yield and cultivated area all occupy the second in the world, in national economy, occupy critical role.2011, only the annual import volume of fodder maize China reached millions of tons.Therefore, it is huge that germ is imported the potential risk that China and diffusion propagate into.At present, the domestic report of not seeing that as yet Cmn detects in the corn sample that enters the territory, accurately practical fast micro-germ detection method has crucial potential using value.
Summary of the invention
The technical problem that the present invention will solve is to provide the PCR detection method of state wilting germ in a kind of corn; For this reason, the present invention also is provided for the detection primer of aforesaid method.
In order to solve the problems of the technologies described above, the present invention realizes through following technical scheme:
In one aspect of the invention, the PCR detection method of state wilting germ in a kind of corn is provided, comprises the steps:
(1) design primer, the sequence of this primer is:
PSM 1:5 '-cctttccgtcgtcctttc-3 ' (SEQ ID NO.1) or its complementary strand;
CM3:5 '-gcatccaccgtttgctct-3 ' (SEQ ID NO.2) or its complementary strand;
(2) DNA of bacteria extracts in the corn sample (or corn seed sample);
(3) adopting step (1) designed primer to carry out PCR detects.
In the step (2), DNA of bacteria extracts and is specially in the said corn sample: put into PBS damping fluid (PH7.2) behind the corn sample mixing, 4 ℃ of soaked overnight; Filter, the centrifugal supernatant of abandoning of filtrating, deposition is extracted DNA with test kit.
In the step (3), the reaction system that said PCR detects is 25 μ L, comprises 2.5 μ L, 10 * PCRbuffer (Mg
2+Plus), 2.5 μ L dNTP (each 250 μ M), each 1 μ L (100pM) of step (1) upstream and downstream primer, 2 μ L template DNAs, 1.5U Taq polysaccharase (5U/ μ L) add water and mend to 25 μ L.
In the step (3), the response procedures that said PCR detects is: 94 ℃ of preparatory sex change 3min; Then with 94 ℃ of 30s, 63 ℃ of 30s, 35 circulations of 72 ℃ of 30s amplifications; Last 72 ℃ are extended 5min.
In another aspect of this invention, a kind of primer that is used to detect state wilting germ in the corn is provided, its sequence is:
PSM1:5 '-cctttccgtcgtcctttc-3 ' (SEQ ID NO.1) or its complementary strand;
CM3:5 '-gcatccaccgtttgctct-3 ' (SEQ ID NO.2) or its complementary strand.
For Cmn in the aimed detection corn sample, the present invention designs Cmn in the primer PSM1/CM3 specific detection corn sample according to 16S-23S sequence difference between Cmn among the GenBank and relevant kind the thereof, has set up the PCR detection method of Cmn.Test-results shows, but primer PSM1/CM3 specific detection Cmn, and the sensitivity that detects is very high, and can the increase 4 strain Cmn bacterial strains that supply examination of this primer obtain the expection product of 208bp, and 36 strains that supply the to try bacterial strain of being correlated with all can not amplify the expection band.DNA and bacteria suspension to different series extent of dilution Cmn carry out the test of PCR detection sensitivity, and the result shows that the detection sensitivity of primer PSM1/CM3 is DNA or the 680CFU target bacteria of 4pg; The detection test of corn sample shows that this PCR method can be used for the rapid detection of Cmn in the inward corn sample.
Description of drawings
Fig. 1 is the detection sensitivity synoptic diagram as a result of PCR in the embodiment of the invention; Wherein, Figure 1A is the state germ bacteria suspension detection sensitivity electrophorogram that here withers in the corn, and Figure 1B is that here wither germ DNA detection sensitivity electrophorogram in the state in the corn.
Fig. 2 is the PCR detection electrophorogram that primer PSM1/CM3 detects corn sample in the embodiment of the invention.
Embodiment
Following examples only be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions among the embodiment usually by normal condition, or is pressed the condition that manufacturer advises.
1 material method
1.1 supply examination corn sample and bacterial strain
Supplying the examination corn sample is U.S.'s import fodder maize, numbering 1058, and the inward time is in July, 2011.
Strains tested amounts to 40 strains; Comprise 4 strain Cmn; Bacterial strain removes and derives from ATCC (American type culture collection; U.S. typical case DSMZ) and NCPPB (The national collection of plant pathogenic bacteria; Britain plant pathogenetic bacteria preservation center) outside, units such as Ningbo, Gansu and Tianjin Entry-Exit Inspection and Quarantine Bureau, Yunnan Prov Agriculture University, China Inst. of Quarantine Inspection Sciences and academy of agricultural sciences, Shanxi Province provide the part test bacterial strain, and other has 10 strains isolating Gram-positive bacteria strain from the fodder maize sample that enters the territory.Table 1 is seen in strains tested and source thereof for details.
Table 1 strains tested and PCR detected result
Table?1?Isolates?used?in?the?test?and?the?result?of?PCR?reaction
Continuous table 1
| Isolates | Collection?no. | PCR?reaction |
| Rhodococcus?sp. | 1057-6 g | - |
| Microbacterium?arborescens | 1057-4 g | - |
| Microbacterium?testaceum | 1057-922 g | － * |
| Bacillus?sp. | 1057-12 g | - |
Annotate: in the table 1, a: Zhang Huili, Ningbo Entry-Exit Inspection and Quarantine Bureau; B: Liu Qing, Gansu Entry-Exit Inspection and Quarantine Bureau; C: Zhao Wenjun, China Inst. of Quarantine Inspection Sciences; D: Wang Ruixia, the academy of agricultural sciences, Shanxi Province; E: Ji Guanghai, Yunnan Prov Agriculture University; F: Liu Peng, Tianjin Entry-Exit Inspection and Quarantine Bureau; G: isolate on the corn sample that enters the territory.
+: positive reaction;-: negative reaction.
*: non-specific amplification.
1.2 supply the examination primer
According to Cmn among the GenBank and sibling species 16S-23S sequence difference thereof; Designs C mn primer PSM1 (5 '-cctttccgtcgtcctttc-3 '; SEQ ID NO.1)/and CM3 (5 '-gcatccaccgtttgct ct-3 ', SEQ ID NO.2), amplified production is 208bp.
1.3 microbial culture and DNA extraction
Strains tested is rule on nutrient agar medium (NA) flat board, cultivates 48 ~ 72h, sterilized water wash-out for 28 ℃; Get the centrifugal 5min of bacterium liquid 12000r/min; Collect thalline, extract test kit with plant genome DNA and extract DNA (TIANGEN DP305), measure DNA concentration with the nucleic acid determination appearance.
1.4 primer specificity detects
Increasing respectively with primer PSM1/CM3 supplies 40 bacterial strain DNA of examination, tests its specificity.
PCR reaction system 25 μ L comprise 2.5 μ L, 10 * PCR buffer (Mg
2+Plus), 2.5 μ L dNTP (each 250 μ M), 1 μ L upstream and downstream primer (PSM1/CM3) (100pM), 2 μ L template DNAs, 1.5U Taq polysaccharase (5U/ μ L) adds water to 25 μ L.
Response procedures is: 94 ℃ of 3min; 94 ℃ of 30s, 63 ℃ of 30s, 72 ℃ of 30s circulate 35 times; Last 72 ℃ of 5min.
Amplified production is with 1.5% sepharose, 1 * TAE damping fluid electrophoresis, and EB dyeing back is analyzed with gel imaging system.
1.5 primer sensitivity test
Get DNA and the bacteria suspension of Cmn strains A TCC 27822, adjustment DNA concentration is to 20ng/ μ L, and gradient dilution is 2ng/ μ L, 200pg/ μ L, 20pg/ μ L, 2pg/ μ L; Adjustment bacteria suspension concentration to 3.4 * 10
8CFU/mL, gradient dilution are 3.4 * 10
7CFU/mL, 3.4 * 10
6CFU/mL, 3.4 * 10
5CFU/mL, 3.4 * 10
4CFU/mL.Get 2 μ L DNA and bacteria suspension respectively as the PCR reaction template.Primer PSM1/CM3 carries out pcr amplification to serial dilution DNA and bacteria suspension respectively, reaction system, and program, electrophoresis and imaging analysis are with 1.4.
DNA of bacteria extracts in the corn sample 1.6 enter the territory
Get 100g behind the corn sample mixing and be used for test, take by weighing 100g and put into 100mL PBS damping fluid (PH7.2), 4 ℃ of soaked overnight; Filtered through gauze, the centrifugal 20min of filtrating 10000r/min, 1mL PBS suspend to precipitate and change EP over to and manage the centrifugal 5min of 12000r/min, and deposition is extracted test kit with plant genome DNA and is extracted DNA (TIANGEN DP305).
Cmn detects in the corn sample 1.7 enter the territory
Every duplicate samples is extracted DNA of bacteria according to the method in 1.6, and get 2 μ L DNA respectively and detect with PCR, reaction system, program, electrophoresis and imaging analysis repeat 2 times with 1.4 and 1.5.
1.8PCR product sequential analysis
Cmn is examined among splicing back and the GenBank in the two-way order-checking of pcr amplification product, sequence and relevant kind of sequence compares, and the corresponding sequence of trying 4 strain Cmn reference cultures with confession simultaneously compares.
2 results and analysis
2.1 primer specific property testing
Primer PSM1/CM3 carries out PCR to 40 strain strains tested DNA respectively and detects.Supply 4 strain Cmn bacterial strains of examination that specific amplification is all arranged, obtain the re-set target fragment of 208bp; Other four subspecies and other strains tested all do not have expection amplified production (seeing table 1) under the Cm kind; The have an appointment non-specific amplification of 500bp of the strain separated 922-3 in the corn sample of only entering the territory varies in size with the expection amplified production.Test-results shows that primer PSM1/CM3 can be used for the specific detection of Cmn.
2.2 primer sensitivity test
Increase the respectively DNA of bacterial strain 27822 of serial dilution of primer PSM1/CM3; Amplification shows; Primer PSM1/CM3 can both amplify the intended purposes fragment of 208bp to the DNA of 4ng, 400pg, 40pg, 4pg; And the DNA cloning of 400fg does not obtain expecting band (seeing Figure 1A), and the sensitivity that effectively detects DNA is 4pg.Among Figure 1A, M is Markers DL2000 (Takara) 100,250,500,750,1000,2000bp; 1 ~ 5 expression is numbered 4ng, 400pg, 40pg, 4pg, the 400fg DNA of Cmn bacterial strain 27822; 6 expression blanks.
Amplification to bacterial strain 27822 bacteria suspensions of serial dilution shows that primer PSM1/CM3 is to 6.8 * 10
5CFU, 6.8 * 10
4CFU, 6.8 * 10
3The bacteria suspension of CFU, 680CFU can both increase and obtain the 208bp title product, and the bacteria suspension of 68CFU is no amplified production (seeing Figure 1B) after increasing, and the sensitivity that effectively detects bacteria suspension is 680CFU.Among Figure 1B, M is Markers DL2000 (Takara) 100,250,500,750,1000,2000bp; 1 ~ 5 expression is numbered 6.8 * 10 of Cmn bacterial strain 27822
5CFU, 6.8 * 10
4CFU, 6.8 * 10
3CFU, 6.8 * 10
2CFU, 68CFU bacteria suspension; 6 expression blanks.
The detection of Cmn in the american corn sample 2.3 enter the territory
Corn sample 1058-1,1058-3,1058-4,1058-6 are through the PBS soaked overnight; The centrifuging and taking deposition is extracted DNA, with 35 circulations of primer PSM1/CM3 amplification; Only positive amplification appears in sample 1058-6, and sample 1058-1,1058-3,1058-4 all do not have the amplified production (see figure 2).Among Fig. 2, M is Markers DL2000 (Takara) 100,250,500,750,1000,2000bp; 1 ~ 4 expression sample 1058-6, sample 1058-1, sample 1058-3, sample 1058-4; 5 expression positive controls; 6 expression blanks.
2.4PCR product sequential analysis
Choose PCR and detect male corn sample 1058-6, obtain the sequence of 208bp after the two-way order-checking of pcr amplification product.BLAST analyzes among the GenBank, and Cmn bacterial strain KACC20788 (JN613835) 16s-23s region sequence similarity is 100% among sample 1058-6 sequence and the GenBank.Compare with supplying examination 4 strain Cmn bacterial strain 16S-23S sequences, the 16s-23s region sequence similarity of sample 1058-6 order-checking gained 208bp sequence and ATCC27822, ATCC27794, ATCC27795, NCPPB2578 is 100%; And with GenBank in 15 Cmm bacterial strain 16s-23s region sequence similaritys be 97% ~ 99%, sequence difference is 3bp ~ 7bp; With 3 Cmi bacterial strain 16S-23S region sequence similaritys among the GenBank be 97% ~ 98%, sequence difference 4bp ~ 7b; With 4 Cms bacterial strain 16S-23S region sequence similaritys among the GenBank be 97% ~ 98%, sequence difference is 5bp; Differ greatly with 1 Cmt bacterial strain KACC2080016S-23S region sequence among the GenBank, sequence similarity is merely 60%.The product sequencing result has been verified the specificity that detects primer, also shows in the corn sample that enters the territory to have Cmn.
3 discuss
Detection method to Cmn mainly comprises separation and Culture, serology detection, molecular Biological Detection etc. both at home and abroad.Gross and Vidaver have invented the half selectivity culture medium C orynebacterium nebraskense selective (CNS) of suitable separation of C mn in 1979, can be from fresh corn tissue, exsiccant invalid body and soil separation of C mn.But the lithium chloride in the CNS substratum has restraining effect (Gross and Vidaver for the growth of Cmn; 1979); Therefore be removed from CNS this component in 1986, Shepherd further is improved to the sCNS substratum with the CNS substratum, has improved the recovery to Cmn; And Cmn is formed the time that can distinguish bacterium colony shortened 24h (Smidt and Vidaver, 1986) on substratum.But growing, Cmn still needs 4-7 days time on the sCNS substratum; Consuming time longer; Colony colour, the form of different Cmn bacterial strains on substratum has certain difference (Smidt and Vidaver; 1987); And some other Gram-positive coryneform bacteria also can be grown on sCNS like Curtobacterium flaccumfaciens pv.betae, C.f.pv.oortii, C.f.pv.flaccumfaciens etc., and we find also that when carrying out the separating experiment of corn sample some bacterial strain colony colours, form and the Cmn of Curtobacterium sp. is closely similar; In actual detected, easily the result is caused interference, but do not see have the scholar to report that other is more suitable for the selective medium of separation of C mn at present always.
Molecular Biological Detection has fast, sensitive, easy and simple to handle characteristics, makes it in the Preliminary detection of phytopathogen, bring into play more and more important effect.Louws etc. have passed through based on the Rep-PCR technical Analysis of the short-and-medium Tumor-necrosis factor glycoproteins repetitive of bacterial genomes sequence the genome fingerprinting map spectrum of the different subspecies of Cm; Different subspecies form unique finger printing; And the finger printing between different strains is stablized and consistent (Louws et al.1998) in the same subspecies, so this method can be used for the discriminating of the different subspecies of Cm.Pastrik etc. utilize the 16S-23S sequence difference of 5 subspecies of Cm to design different primers respectively; Can from the genomic dna of Cmn, amplify a certain size fragment specifically, Cmn and other subspecies distinguished (Pastrik and Rainey 1999).Bach etc. have designed a pair of universal primer and five kinds of subspecies specificity T aqMan probes according to the 16S-23S sequence difference of following 5 subspecies of Cm kind, have set up the real-time fluorescence detection method (Bach et al.2003) to following 5 the subspecies plant pathogenetic bacterias of Cm kind.But these researchs focus mostly in the detection to the pure culture bacterium, and less to the detection method report of Cmn in the actual corn seed sample.
The different subspecies 16S-23S of Cm sequence difference among this experimental evidence GenBank; Design specific primers PSM1/CM3; Can be in the 4 strain corns that supply examination the specific band of a 208bp of amplification the wilting germ bacterial strain of state, supply that the target amplification product does not all appear in other 4 subspecies and the approximate bacterial strain of 27 strains under the Cm kind of examination, only to the have an appointment non-specific amplification of 500bp of strain separated 922-3 in the corn sample; But the title product that does not have 208bp; 922-3 is accredited as Microbacterium testaceum with it after 16S rDNA order-checking, it is not a phytopathogen, possibly be the endogenetic bacteria on the corn seed sample; Behind the amplified production electrophoresis band a little less than, explain that primer PSM1/CM3 is not high to its amplification efficiency, and amplified production and title product differ greatly, therefore very little to the detection influence of actual sample.Sensitivity test to state wilting germ DNA and bacteria suspension in the different series extent of dilution corn shows that primer PSM1/CM3 can detect the object bacteria of DNA or the 680CFU of 4pg; When the corn seed sample is detected; We find that corn seed sample soaked overnight can obtain more bacterium amount; To the centrifugal impurity such as starch of can removing with low-speed centrifugal earlier when concentrated of corn seed sample soak solution, effectively reduce the restraining effect that other composition in the corn seed sample possibly cause PCR, the target stripe of 208bp all appears after to the corn sample DNA cloning in PCR; There is not other assorted band influence; Therefore the PCR method set up of the present invention can be used for the detection of corn seed sample, the PCR product is carried out two-way order-checking after, Cmn bacterial strain and the accurate bacterial strain 16S-23S of 4 strains confession test-object sequence height are similar among its sequence and the GeneBank; Similarity is 100%, explains in the corn sample that enters the territory to have Cmn.
Claims (5)
1. the PCR detection method of the interior state of a corn wilting germ is characterized in that, comprises the steps:
(1) design primer, the sequence of this primer is:
PSM1:5 '-cctttccgtcgtcctttc-3 ' (SEQ ID NO.1) or its complementary strand;
CM3:5 '-gcatccaccgtttgctct-3 ' (SEQ ID NO.2) or its complementary strand;
(2) DNA of bacteria extracts in the corn sample;
(3) adopting step (1) designed primer to carry out PCR detects.
2. the PCR detection method of state wilting germ is characterized in that in the corn as claimed in claim 1,
In the step (2), DNA of bacteria extracts and is specially in the said corn sample: put into the PBS damping fluid behind the corn sample mixing, 4 ℃ of soaked overnight; Filter, the centrifugal supernatant of abandoning of filtrating, deposition is extracted DNA with test kit.
3. the PCR detection method of state wilting germ in the corn as claimed in claim 1; It is characterized in that in the step (3), the reaction system that said PCR detects is 25 μ L; Comprise 2.5 μ L, 10 * PCR buffer; 2.5 μ LdNTP, each 1 μ L of step (1) upstream and downstream primer, 2 μ L template DNAs, 1.5U Taq polysaccharase add water and mend to 25 μ L.
4. like the PCR detection method of state wilting germ in claim 1 or the 3 described corns, it is characterized in that in the step (3), the response procedures that said PCR detects is: 94 ℃ of preparatory sex change 3min; Then with 94 ℃ of 30s, 63 ℃ of 30s, 35 circulations of 72 ℃ of 30s amplifications; Last 72 ℃ are extended 5min.
5. primer that is used to detect state wilting germ in the corn is characterized in that its sequence is:
PSM1:5 '-cctttccgtcgtcctttc-3 ' (SEQ ID NO.1) or its complementary strand;
CM3:5 '-gcatccaccgtttgctct-3 ' (SEQ ID NO.2) or its complementary strand.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210137802.8A CN102816835B (en) | 2012-05-04 | 2012-05-04 | Clavibacter michiganensis subsp.nebraskensis(Cmn) PCR detection method, and detection primer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210137802.8A CN102816835B (en) | 2012-05-04 | 2012-05-04 | Clavibacter michiganensis subsp.nebraskensis(Cmn) PCR detection method, and detection primer |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102816835A true CN102816835A (en) | 2012-12-12 |
| CN102816835B CN102816835B (en) | 2014-06-18 |
Family
ID=47301278
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210137802.8A Expired - Fee Related CN102816835B (en) | 2012-05-04 | 2012-05-04 | Clavibacter michiganensis subsp.nebraskensis(Cmn) PCR detection method, and detection primer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102816835B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962610A (en) * | 2015-06-01 | 2015-10-07 | 舟山出入境检验检疫局综合技术服务中心 | LAMP rapid detecting method for Clavibacter michiganensis subsp.nebraskensis(Cmn) |
| CN105200130A (en) * | 2015-09-22 | 2015-12-30 | 中国检验检疫科学研究院 | Barcode identification method for identifying different subspecies of clavibacter michiganensis and primer specially adopted in method |
| CN105525011A (en) * | 2016-01-28 | 2016-04-27 | 河南农业大学 | Rapid detection method for pathogens of bipolaris carbonum Wilson |
| CN113528679A (en) * | 2021-04-07 | 2021-10-22 | 兰州百源基因技术有限公司 | Specific primer and probe for detecting wilting disease in corn and real-time fluorescent quantitative PCR detection kit |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1584054A (en) * | 2004-06-16 | 2005-02-23 | 南京农业大学 | Detecting gene for tomato ulcer germs and detecting reagent case thereof |
-
2012
- 2012-05-04 CN CN201210137802.8A patent/CN102816835B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1584054A (en) * | 2004-06-16 | 2005-02-23 | 南京农业大学 | Detecting gene for tomato ulcer germs and detecting reagent case thereof |
Non-Patent Citations (3)
| Title |
|---|
| K.-H.PASTRIK等: "Identification and Differentiation of Clavibacter michiganensis Subspecies by Polymerase Chain Reaction-based Techniques", 《J.PHYTOPATHOLOGY》, vol. 147, 31 December 1999 (1999-12-31) * |
| XIANG LI等: "Selection of Polymerase Chain Reaction Primers from an RNA Intergenic Spacer Region for Specific Detection of Clavibacter michiganensis subsp. sepedonicus", 《DISEASE DETECTION AND LOSSES》, vol. 85, no. 8, 31 December 1995 (1995-12-31), pages 837 - 842 * |
| 周琦等: "PCR技术快速检测玉米内州萎蔫病菌研究", 《植物检疫》, vol. 24, no. 2, 31 December 2010 (2010-12-31) * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962610A (en) * | 2015-06-01 | 2015-10-07 | 舟山出入境检验检疫局综合技术服务中心 | LAMP rapid detecting method for Clavibacter michiganensis subsp.nebraskensis(Cmn) |
| CN104962610B (en) * | 2015-06-01 | 2017-12-01 | 舟山出入境检验检疫局综合技术服务中心 | Wilting germ LAMP quick determination methods in state in a kind of corn |
| CN105200130A (en) * | 2015-09-22 | 2015-12-30 | 中国检验检疫科学研究院 | Barcode identification method for identifying different subspecies of clavibacter michiganensis and primer specially adopted in method |
| CN105200130B (en) * | 2015-09-22 | 2018-09-28 | 中国检验检疫科学研究院 | The bar code identification method and its primer special of Michigan clavibacter difference subspecies |
| CN105525011A (en) * | 2016-01-28 | 2016-04-27 | 河南农业大学 | Rapid detection method for pathogens of bipolaris carbonum Wilson |
| CN113528679A (en) * | 2021-04-07 | 2021-10-22 | 兰州百源基因技术有限公司 | Specific primer and probe for detecting wilting disease in corn and real-time fluorescent quantitative PCR detection kit |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102816835B (en) | 2014-06-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Xiao et al. | Effects of cultivation ages and modes on microbial diversity in the rhizosphere soil of Panax ginseng | |
| Xu et al. | Bacterial communities in soybean rhizosphere in response to soil type, soybean genotype, and their growth stage | |
| Beneduzi et al. | Genetic and phenotypic diversity of plant-growth-promoting bacilli isolated from wheat fields in southern Brazil | |
| Tian et al. | Community structures of the rhizomicrobiomes of cultivated and wild soybeans in their continuous cropping | |
| Maarastawi et al. | Temporal dynamics and compartment specific rice straw degradation in bulk soil and the rhizosphere of maize | |
| Wang et al. | Effect of soil type and soybean genotype on fungal community in soybean rhizosphere during reproductive growth stages | |
| CN108587974B (en) | Bacillus belgii, application and preparation method of fermentation liquor of bacillus belgii | |
| CN101805793B (en) | PCR detection method of L.maculans and primer used for detection | |
| CN102816835B (en) | Clavibacter michiganensis subsp.nebraskensis(Cmn) PCR detection method, and detection primer | |
| CN102230015B (en) | Nucleotide sequence and kit for detecting Burkholderia gladioli | |
| CN105441543B (en) | It is a kind of identification Fusarium oxysporum f. sp. niveum biological strain primer and its application | |
| CN108588249B (en) | Primer pair for detecting sweet potato stem rot bacteria and detection method thereof | |
| CN103725782A (en) | Method, kit and primer for detecting pathogenic bacteria of lotus fungal rot disease carried by lotus seeds | |
| CN102433387B (en) | Molecular detection method and primers for detecting fusarium oxysporum | |
| CN104232782A (en) | PCR (polymerase chain reaction) primer for detecting tobacco soil-borne fungal pathogens as well as application and method of PCR primer | |
| CN104726557A (en) | Double-PCR (polymerase chain reaction) molecular detection primer for tobacco phytophthora parasitica and thielaviopsis basicola and detection method | |
| CN104004842A (en) | Multiplex PCR primer set and detection method for simultaneously detecting three pathogenic bacteria causing sepsis of aquatic animals | |
| CN104099408A (en) | Qualitative detection method for potato scab germs | |
| CN114686609B (en) | Specific primer for identifying tomato bacterial wilt and application thereof | |
| CN114277166B (en) | RPA detection primer, probe and detection method for melon bacterial fruit blotch | |
| CN104212896B (en) | The Molecular Identification primer of a kind of Tobacco Angular Leaf Spot Disease bacterium and authentication method | |
| CN102936621B (en) | Bacillus cereus detection method and kit | |
| CN109136394A (en) | One kind proposing Dick's Salmonella TaqMan fluorescent quantitative PCR detection method and application until dawn | |
| Osdaghi | Dickeya solani (black leg disease of potato) | |
| Zhang et al. | Genetically modified WYMV-resistant wheat exerts little influence on rhizosphere microbial communities |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140618 Termination date: 20170504 |