CN102816835B - 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
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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 corn.
Background technology
Clavibacter belongs to Clavibacter and only comprises a kind of phytopathogen Clavibacter michiganensis (Cm), according to the difference of its host preference, be divided into 5 subspecies, comprise state wilting germ C.m.subsp.nebraskensis(Cmn in corn), Tomato Caused by Clavibacter michiganensis subsp. michiganensis bacterium C.m.subsp.Michiganenis (Cmm), Potato Ring Rot C.m.subsp.sepedonicus (Cms), clover wilting germ C.m.subsp.insidiouss (Cmi), state wilting germ Cmn and wheat mosaic bacterium C.m.subsp.tessellarius (Cmt) (Eichenlaub et al.2006) in corn.They all can cause important Plant diseases, and EPPO classifies Cmm, Cms and Cmi as quarantine harmful organisms, and in quarantine harmful organisms register is listed Cmn, Cmm, Cms and Cmi simultaneously by China.
In corn, state wilting germ Cmn has another name called clavibacter Nebraska, Michigan subspecies, is under the jurisdiction of Firmicutes Firmicutes, heavy wall Gammaproteobacteria Firmibacteria, excellent type Bacillaceae Clavibacter.1969, United States Nebraska was found state wilt disease (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-growing regions to 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 in north America region Maize Production.
In corn, state wilting germ Cmn can endanger dent corn, sweet corn, flint corn, quick-fried corn, Herba Setariae Viridis, barnyard grass and sugarcane.Cmn can be with seed dispersal (Biddle et al.1990), and the most of corn-growing regions of China is applicable to disease .2009 such as () old Shi occurs.Grow once germ is imported into surely, easily break out and cause disease popular, and disease control difficulty is large.China's corn yield and cultivated area all occupy the second in the world, occupy critical role in national economy.2011, only the annual import volume of fodder maize China reached millions of tons.Therefore it is huge that, germ is imported China's potential risk that also diffusion is propagated into.At present, the domestic report that there is not yet Cmn detection in the corn sample that enters the territory, accurately the micro-Pathogen detection method of Fast Practical has very important potential using value.
Summary of the invention
The technical problem to be solved in the present invention is to provide the PCR detection method of state wilting germ in a kind of corn; For this reason, the present invention is also provided for the detection primer of aforesaid method.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
In one aspect of the invention, provide the PCR detection method of state wilting germ in a kind of corn, comprise 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) in corn sample (or corn seed sample), DNA of bacteria is extracted;
(3) adopt the primer of step (1) design to carry out PCR detection.
In step (2), in described corn sample, DNA of bacteria is extracted and is specially: after corn sample mixes, put into PBS damping fluid (PH7.2), 4 DEG C of soaked overnight; Filter, the centrifugal supernatant of abandoning of filtrate, precipitation is extracted DNA with test kit.
In step (3), the reaction system that described PCR detects is 25 μ L, comprises 2.5 μ L 10 × PCRbuffer (Mg
2+plus), each 250 μ M of 2.5 μ L dNTP(), the 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 step (3), the response procedures that described PCR detects is: 94 DEG C of denaturation 3min; Then with 94 DEG C of 30s, 63 DEG C of 30s, 35 circulations of 72 DEG C of 30s amplifications; Last 72 DEG C are extended 5min.
In another aspect of this invention, provide a kind of primer for detection of state wilting germ in corn, 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 aimed detection corn sample, the present invention, according to 16S-23S sequence difference between Cmn in GenBank and relevant kind thereof, designs Cmn in primer PSM1/CM3 specific detection corn sample, has set up the PCR detection method of Cmn.Test-results shows, primer PSM1/CM3 can specific detection Cmn, and the sensitivity detecting is very high, and this primer can increase and obtain the expection product of 208bp for 4 strain Cmn bacterial strains of examination, all can not amplify expection band for 36 strains that the try bacterial strain of being correlated with.DNA to different series extent of dilution Cmn and bacteria suspension carry out the test of PCR detection sensitivity, and result shows DNA or the 680CFU target bacteria that the detection sensitivity of primer PSM1/CM3 is 4pg; The detection test of corn sample shows that this PCR method can be used for the rapid detection of Cmn in inward corn sample.
Brief description of the drawings
Fig. 1 is the detection sensitivity result schematic diagram of PCR in the embodiment of the present invention; Wherein, Figure 1A is the state germ bacteria suspension detection sensitivity electrophorogram that here withers in corn, and Figure 1B is that in corn here wither germ DNA detection sensitivity electrophorogram in state.
Fig. 2 is the PCR detection electrophorogram that in the embodiment of the present invention, primer PSM1/CM3 detects corn sample.
Embodiment
Following examples are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in embodiment, condition routinely conventionally, or by the condition of manufacturer's suggestion.
1 material method
1.1 for examination corn sample and bacterial strain
Be imported from America fodder maize for examination corn sample, numbering 1058, the inward time is in July, 2011.
Strains tested amounts to 40 strains, comprise 4 strain Cmn, bacterial strain is except deriving from ATCC(American type culture collection, typical case DSMZ of the U.S.) and NCPPB (The national collection of plant pathogenic bacteria, Britain's plant pathogenetic bacteria preservation center) outside, Ningbo, Gansu and Tianjin Entry-Exit Inspection and Quarantine Bureau, Yunnan Prov Agriculture University, the units such as China Inst. of Quarantine Inspection Sciences and academy of agricultural sciences of Shanxi Province provide part test bacterial strain, the Gram-positive bacteria strain that separately has 10 strains to separate from the fodder maize sample that enters the territory.Table 1 is referred in strains tested and source thereof.
Table 1 strains tested and PCR detected result
Table 1 Isolates used in the test and the result of PCR reaction
Continued 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 | - |
Note: in 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, academy of agricultural sciences of 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 for examination primer
According to Cmn in GenBank and sibling species 16S-23S sequence difference thereof, design Cmn primer PSM1(5 '-cctttccgtcgtcctttc-3 ', SEQ ID NO.1)/CM3(5 '-gcatccaccgtttgct ct-3 ', SEQ ID NO.2), amplified production is 208bp.
1.3 microbial culture and DNA extraction
Strains tested is at the flat lining out of nutrient agar medium (NA), cultivate 48 ~ 72h for 28 DEG C, sterilized water wash-out, get the centrifugal 5min of bacterium liquid 12000r/min, collect thalline, extract test kit with plant genome DNA and extract DNA (TIANGEN DP305), by nucleic acid determination instrument mensuration DNA concentration.
1.4 primer specificity detect
Increase respectively and supply 40 bacterial strain DNA of examination with primer PSM1/CM3, test its specificity.
PCR reaction system 25 μ L, comprise 2.5 μ L 10 × PCR buffer (Mg
2+plus), each 250 μ M of 2.5 μ L dNTP(), 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 DEG C of 3min; 94 DEG C of 30s, 63 DEG C of 30s, 72 DEG C of 30s, circulate 35 times; Last 72 DEG C of 5min.
1.5% sepharose 1 × TAE damping fluid electrophoresis for amplified production, analyzes with gel imaging system after EB dyeing.
1.5 primer sensitivity tests
Get DNA and the bacteria suspension of Cmn strains A TCC 27822, adjust DNA concentration to 20ng/ μ L, gradient dilution is 2ng/ μ L, 200pg/ μ L, 20pg/ μ L, 2pg/ μ L; Adjust bacteria suspension concentration to 3.4 × 10
8cFU/mL, gradient dilution is 3.4 × 10
7cFU/mL, 3.4 × 10
6cFU/mL, 3.4 × 10
5cFU/mL, 3.4 × 10
4cFU/mL.Get respectively 2 μ L DNA and bacteria suspension as 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.
In 1.6 inward corn samples, DNA of bacteria is extracted
After corn sample mixes, get 100g for test, take 100g and put into 100mL PBS damping fluid (PH7.2), 4 DEG C of soaked overnight; Filtered through gauze, the centrifugal 20min of filtrate 10000r/min, 1mL PBS suspends and precipitates and proceed to the centrifugal 5min of EP pipe 12000r/min, and precipitation is extracted test kit with plant genome DNA and is extracted DNA (TIANGEN DP305).
In 1.7 inward corn samples, Cmn detects
Every duplicate samples is extracted DNA of bacteria according to the method in 1.6, and get respectively 2 μ L DNA PCR and detect, reaction system, program, electrophoresis and imaging analysis, with 1.4 and 1.5, repeat 2 times.
The sequential analysis of 1.8PCR product
The two-way order-checking of pcr amplification product, Cmn and a relevant kind of sequence thereof compare after sequence is examined splicing and in GenBank, compare simultaneously and for the corresponding sequence of trying 4 strain Cmn reference cultures.
2 results and analysis
2.1 primer specificity tests
Primer PSM1/CM3 carries out PCR detection to 40 strain strains tested DNA respectively.The 4 strain Cmn bacterial strains for examination all have specific amplification, obtain the re-set target fragment of 208bp; Under Cm kind, other four subspecies and other strains tested are all without expection amplified production (in table 1); The have an appointment non-specific amplification of 500bp of the bacterial strain 922-3 that separates in corn sample of only entering the territory, varies in size with expection amplified production.Test-results shows that primer PSM1/CM3 can be used for the specific detection of Cmn.
2.2 primer sensitivity tests
Increase the respectively DNA of bacterial strain 27822 of serial dilution of primer PSM1/CM3, amplification shows, primer PSM1/CM3 can amplify the expection object 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), the sensitivity that effectively detects DNA is 4pg.In Figure 1A, M is Markers DL2000(Takara) 100,250,500,750,1000,2000bp; 1 ~ 5 represents to be numbered 4ng, 400pg, 40pg, 4pg, the 400fg DNA of Cmn bacterial strain 27822; 6 represent blank.
The amplification of bacterial strain 27822 bacteria suspensions to serial dilution shows, primer PSM1/CM3 is to 6.8 × 10
5cFU, 6.8 × 10
4cFU, 6.8 × 10
3the bacteria suspension of CFU, 680CFU can increase and obtain 208bp target product, and the bacteria suspension of 68CFU is without amplified production (being shown in Figure 1B) after amplification, and the sensitivity that effectively detects bacteria suspension is 680CFU.In Figure 1B, M is Markers DL2000(Takara) 100,250,500,750,1000,2000bp; 1 ~ 5 represents to be 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 represent blank.
The detection of Cmn in 2.3 black spot from America corn samples
Corn sample 1058-1,1058-3,1058-4,1058-6 are through PBS soaked overnight, centrifuging and taking precipitation, extract DNA, with 35 circulations of primer PSM1/CM3 amplification, there is positive amplification in sample 1058-6 only, and sample 1058-1,1058-3,1058-4 are all without amplified production (being shown in Fig. 2).In Fig. 2, M is Markers DL2000(Takara) 100,250,500,750,1000,2000bp; 1 ~ 4 represents sample 1058-6, sample 1058-1, sample 1058-3, sample 1058-4; 5 represent positive control; 6 represent blank.
The sequential analysis of 2.4PCR product
Choose PCR and detect positive corn sample 1058-6, after the two-way order-checking of pcr amplification product, obtain the sequence of 208bp.In GenBank, BLAST analyzes, Cmn bacterial strain KACC20788(JN613835 in sample 1058-6 sequence and GenBank) 16s-23s region sequence similarity is 100%.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 in GenBank be 97% ~ 98%, sequence difference 4bp ~ 7b; With 4 Cms bacterial strain 16S-23S region sequence similaritys in GenBank be 97% ~ 98%, sequence difference is 5bp; Differ greatly with 1 Cmt bacterial strain KACC2080016S-23S region sequence in GenBank, sequence similarity is only 60%.Product sequencing result has been verified the specificity that detects primer, in the corn sample that also shows to enter the territory, has Cmn.
3 discuss
The detection method of Cmn is mainly comprised to 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 applicable separation of C mn in 1979, can be from fresh corn tissue, dry invalid body and soil separation of C mn.But the lithium chloride in CNS substratum has restraining effect (Gross and Vidaver for the growth of Cmn, 1979), therefore be removed from CNS this component in 1986, CNS substratum is further improved to sCNS substratum by Shepherd, improve the rate of recovery to Cmn, and Cmn is formed on substratum can distinguish bacterium colony time shorten 24h(Smidt and Vidaver, 1986).But Cmn grows and still needs the time of 4-7 days on sCNS substratum, consuming time longer, the colony colour of different Cmn bacterial strains on substratum, form has certain difference (Smidt and Vidaver, 1987), and some other Gram-positive coryneform bacteria, as Curtobacterium flaccumfaciens pv.betae, C.f.pv.oortii, C.f.pv.flaccumfaciens etc. also can grow on sCNS, we also find some bacterial strain colony colours of Curtobacterium sp. in the time carrying out the separating experiment of corn sample, form and Cmn are closely similar, in detecting, reality easily result is caused to interference, but report that there are no scholar other is more suitable for the selective medium of separation of C mn at present always.
Molecular Biological Detection has feature quick, sensitive, easy and simple to handle, makes it in the Preliminary detection of phytopathogen, bring into play more and more important effect.The Rep-PCR technical Analysis by based on short tandem repeat repetitive sequence in bacterial genomes such as Louws the genomic collection of illustrative plates 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 same subspecies, therefore the method can be for the discriminating of the different subspecies of Cm.Pastrik etc. utilize the 16S-23S sequence difference of 5 subspecies of Cm to design respectively different primers, can from the genomic dna of Cmn, amplify specifically a certain size fragment, Cmn and other subspecies be distinguished (Pastrik and Rainey 1999).Bach etc. are according to the 16S-23S sequence difference of lower 5 subspecies of Cm kind, design a pair of universal primer and five kinds of subspecies specificity T aqMan probes, set up the real-time fluorescence detection method (Bach et al.2003) to lower 5 the subspecies plant pathogenetic bacterias of Cm kind.But these researchs focus mostly in the detection to pure culture bacterium, and less to the detection method report of Cmn in actual corn seed sample.
The different subspecies 16S-23S of Cm sequence difference in this experimental evidence GenBank, design Auele Specific Primer PSM1/CM3, the specific band of a 208bp can increase the wilting germ bacterial strain of state in the 4 strain corns for examination, all there is not target amplification product for the approximate bacterial strain of other 4 subspecies and 27 strains under the Cm kind of examination, only to the have an appointment non-specific amplification of 500bp of the bacterial strain 922-3 separating in corn sample, but without the target product of 208bp, 922-3 is accredited as Microbacterium testaceum after 16S rDNA order-checking, it is not phytopathogen, it may be the endogenetic bacteria on corn seed sample, after amplified production electrophoresis band a little less than, illustrate that primer PSM1/CM3 is not high to its amplification efficiency, and amplified production and target product differ greatly, therefore very little on the detection impact of actual sample.Sensitivity test to state wilting germ DNA and bacteria suspension in different series extent of dilution corn shows, primer PSM1/CM3 can detect the object bacteria of DNA or the 680CFU of 4pg, in the time that corn seed sample is detected, we find that corn seed sample soaked overnight can obtain more bacterium amount, during to corn seed sample soak solution centrifugal concentrating, first can remove the impurity such as starch with low-speed centrifugal, effectively reduce the restraining effect that in corn seed sample, other composition may cause PCR, all there is the target stripe of 208bp after to corn sample DNA cloning in PCR, without other assorted band impact, therefore the PCR method that the present invention sets up can be used for the detection of corn seed sample, PCR product is carried out after two-way order-checking, its sequence is similar for examination reference culture 16S-23S sequence height to Cmn bacterial strain in GeneBank and 4 strains, similarity is 100%, illustrate in the corn sample that enters the territory and have Cmn.
Claims (2)
1. a PCR detection method for state wilting germ in corn, is characterized in that, comprises the steps:
(1) design primer, the sequence of this primer is:
PSM1:5 '-cctttccgtcgtcctttc-3 ', as shown in SEQ ID NO.1;
CM3:5 '-gcatccaccgtttgctct-3 ', as shown in SEQ ID NO.2;
(2) in corn sample, DNA of bacteria is extracted: after corn sample mixes, put into PBS damping fluid, 4 DEG C of soaked overnight; Filter, the centrifugal supernatant of abandoning of filtrate, precipitation is extracted DNA with test kit;
(3) adopt the primer of step (1) design to carry out PCR detection: the reaction system that described PCR detects is 25 μ L, comprise 2.5 μ L10 × PCR buffer, 2.5 μ L dNTP, the each 1 μ L of step (1) PSM1 and CM3 primer, 2 μ L template DNAs, 1.5U Taq polysaccharase, add water and mend to 25 μ L; The response procedures that described PCR detects is: 94 DEG C of denaturation 3min; Then with 94 DEG C of 30s, 63 DEG C of 30s, 35 circulations of 72 DEG C of 30s amplifications; Last 72 DEG C are extended 5min.
2. for detection of a primer for state wilting germ in corn, it is characterized in that, its sequence is:
PSM1:5 '-cctttccgtcgtcctttc-3 ', as shown in SEQ ID NO.1;
CM3:5 '-gcatccaccgtttgctct-3 ', as shown in SEQ ID NO.2.
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| 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 |
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| 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 |
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