CN104232782B - A kind of detect tobacco soil-borne fungus pathogen PCR primer and application and method - Google Patents
A kind of detect tobacco soil-borne fungus pathogen PCR primer and application and method Download PDFInfo
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Abstract
The invention discloses a kind of detect tobacco soil-borne fungus pathogen PCR primer and application and method, the PCR primer of described detection tobacco soil-borne fungus pathogen is made up of tobacco rhizoctonia solani primer, black shank bacterium primer, root black rot primer and da mping-off fungi primer; The PCR primer being applied as described detection tobacco soil-borne fungus pathogen detects the application in tobacco 4 kinds of soil-borne fungus pathogens at the same time, and 4 kinds of described soil-borne fungus are tobacco rhizoctonia solani, black shank bacterium, root black rot and da mping-off fungi; Method comprises pre-treatment, PCR reaction, electrophoresis detection step; The present invention detects easy to be quick, highly sensitive, accurate: within 4 hours, complete sample preparation and detection, detect 4 kinds of pathogens such as tobacco rhizoctonia solani, black shank bacterium, root black rot, da mping-off fungi simultaneously, can detect the pathogen DNA sample of 0.01ng, detected result accuracy is higher than traditional isolation identification and symptom authentication method.
Description
Technical field
The invention belongs to Plant diseases molecular diagnostic techniques field, be specifically related to a kind of detect tobacco soil-borne fungus pathogen PCR primer and application and method.
Background technology
Tobacco soil-borne fungus diseases comprises balck shank, root Black Rotten, samping off and damping-off etc., and these diseases are a kind of important diseases in tobacco leaf production, cause serious threat to the production of tobacco.In recent years; due to the change of tobacco environment; these diseases occur complicated; main Minor diseases is various in partial area change; and often mixedly to occur, the symptom caused is difficult to distinguish, and adopts the diagnostic means such as isolation identification of traditional classical symptom, pathogenic bacteria; there is the interference that length consuming time, sensitivity are low, be subject to the factors such as artificial and environment, quick and precisely cannot obtain result.The early molecule diagnostic techniques mainly individual event Defect inspection technology of development recent years, as tobacco black shank bacterium, black rot detection (Gao Qiaofen etc. the rapid molecular of Phytophthora nicotianae detects. Yunnan Prov Agriculture University's journal, 2012,27 (2): 156-159; Zhao Yongqiang. the Molecular Detection of black root of tobacco bacterium and fungicide screening. Shandong: Shandong Agricultural University, 2009).These individual events detect and adopt exclusive method, detect one by one single germ, more consuming time, easily miss farming season, are difficult to work out prophylactico-therapeutic measures accurately, incur loss through delay control approach, can not meet the actual needs of production.Multiplex PCR is improved on the basis of regular-PCR, in a PCR reaction system, add multipair Auele Specific Primer, and the different zones for multiple DNA profiling or same template increases the round pcr of multiple object fragment.But up to the present, there is not yet the technology of the multiplex PCR simultaneously detecting black shank, root Black Rotten, samping off and damping-off.
Summary of the invention
The first object of the present invention is to provide a kind of PCR primer detecting tobacco soil-borne fungus pathogen; Second object is to provide the application of the PCR primer of described detection tobacco soil-borne fungus pathogen; 3rd object is to provide the PCR primer of described detection tobacco soil-borne fungus pathogen to detect the method for soil-borne fungus pathogen in tobacco 4 simultaneously.
The first object of the present invention is achieved in that the PCR primer of described detection tobacco soil-borne fungus pathogen is made up of tobacco rhizoctonia solani primer, black shank bacterium primer, root black rot primer and da mping-off fungi primer;
The DNA sequence dna of described tobacco rhizoctonia solani primer is:
Upstream primer P1:5 '-GCACACTCTTCTCTTTCATCCA-3 ';
Downstream primer P2:5 '-TTAGAAGCGGTTCGTCTGCA-3 ';
The DNA sequence dna of described black shank bacterium primer is:
Upstream primer P3:5 '-CGAAGCCAACCATACCACGAA-3 ';
Downstream primer P4:5 '-ATGAAGAACGCTGCGAACTGC-3 ';
The DNA sequence dna of described root black rot primer is:
Upstream primer P5:5 '-AACGTACCTTTTCTAGCTGCTTTG-3 ';
Downstream primer P6:5 '-TGAGGGTTTTTCGGCATGTTA-3 ';
The DNA sequence dna of described da mping-off fungi primer is:
Upstream primer P7:5 '-TGGCTCCCTTTTCGGAGGAGA-3 ';
Downstream primer P8:5 '-TTGCCCAGACCATTGCCTCA-3 '.
The second object of the present invention realizes like this, the PCR primer of described detection tobacco soil-borne fungus pathogen detects the application in tobacco 4 kinds of soil-borne fungus pathogens at the same time, and 4 kinds of described soil-borne fungus are tobacco rhizoctonia solani, black shank bacterium, root black rot and da mping-off fungi.
The third object of the present invention is achieved in that and comprises pre-treatment, PCR reaction, electrophoresis detection step:
A, the germ DNA extracted in tobacco sample to be detected, aseptic ultrapure water dissolving DNA, regulates DNA concentration to be 200ng/ μ L, prepares sample DNA;
B, the PCR primer pair sample DNA described in claim 1 is utilized to carry out multiplexed PCR amplification;
After C, PCR reaction terminates, get product and do agarose gel electrophoresis detection.
The present invention, relative to prior art, has the following advantages and effect:
1, the multiplex PCR detection system of the present invention's foundation, can detect 4 kinds of pathogenic fungies such as tobacco rhizoctonia solani, black shank bacterium, root black rot, da mping-off fungi simultaneously.
2, amplified production clip size of the present invention is consistent with expected results, and 4 pairs of primers are only special to target, and specific band produces nothing but, thus well can distinguish 4 kinds of pathogenic bacterias.
3, the present invention detects easy to be quick, highly sensitive, accurate: within 4 hours, complete sample preparation and detection, detect 4 kinds of pathogens such as tobacco rhizoctonia solani, black shank bacterium, root black rot, da mping-off fungi simultaneously, can detect the pathogen DNA sample of 0.01ng, detected result accuracy is higher than traditional isolation identification and symptom authentication method.
Accompanying drawing explanation
Fig. 14 to grow tobacco silborne fungal diseases pathogen substance pcr amplification product, swimming lane: M.250bpDNALadder; 1. da mping-off fungi (
pythiumaphanidermatum); 2. black shank bacterium (
phytophthoranicotianae); 3. a black rot (
thielavioisbasicola); 4. rhizoctonia solani (
rhizoctoniasolani);
Fig. 2 is the result that multiplex PCR detects different concns tobacco black shank bacterium, da mping-off fungi, root black rot and rhizoctonia solani 4 kinds of disease pathogens simultaneously, swimming lane: M.DL2000DNAMaker; 1-6. pathogen DNA profiling dilutes 10 successively
0-10
5amplification doubly.
Embodiment
Below in conjunction with accompanying drawing, the present invention is further illustrated, but limited the present invention never in any form, and any conversion done based on training centre of the present invention or replacement, all belong to protection scope of the present invention.
The PCR primer of detection tobacco soil-borne fungus pathogen of the present invention, is made up of tobacco rhizoctonia solani primer, black shank bacterium primer, root black rot primer and da mping-off fungi primer;
The DNA sequence dna of described tobacco rhizoctonia solani primer is:
Upstream primer P1:5 '-GCACACTCTTCTCTTTCATCCA-3 ';
Downstream primer P2:5 '-TTAGAAGCGGTTCGTCTGCA-3 ';
The DNA sequence dna of described black shank bacterium primer is:
Upstream primer P3:5 '-CGAAGCCAACCATACCACGAA-3 ';
Downstream primer P4:5 '-ATGAAGAACGCTGCGAACTGC-3 ';
The DNA sequence dna of described root black rot primer is:
Upstream primer P5:5 '-AACGTACCTTTTCTAGCTGCTTTG-3 ';
Downstream primer P6:5 '-TGAGGGTTTTTCGGCATGTTA-3 ';
The DNA sequence dna of described da mping-off fungi primer is:
Upstream primer P7:5 '-TGGCTCCCTTTTCGGAGGAGA-3 ';
Downstream primer P8:5 '-TTGCCCAGACCATTGCCTCA-3 '.
The application of the PCR primer of detection tobacco soil-borne fungus pathogen of the present invention is the application that the PCR primer of described detection tobacco soil-borne fungus pathogen detects in tobacco 4 kinds of soil-borne fungus pathogens at the same time, and 4 kinds of described soil-borne fungus are tobacco rhizoctonia solani, black shank bacterium, root black rot and da mping-off fungi.
The PCR primer of described detection tobacco soil-borne fungus pathogen is carried disease germs in qualification tobacco seedling and is monitored and qualification field tobacco disease early diagnosis and the application be separated in germ.
The PCR primer of detection tobacco soil-borne fungus pathogen of the present invention detects the method for soil-borne fungus pathogen in tobacco 4 simultaneously, it is characterized in that comprising pre-treatment, PCR reaction, electrophoresis detection step:
A, the germ DNA extracted in tobacco sample to be detected, aseptic ultrapure water dissolving DNA, regulates DNA concentration to be 200ng/ μ L, prepares sample DNA;
B, the PCR primer pair sample DNA described in claim 1 is utilized to carry out multiplexed PCR amplification;
After C, PCR reaction terminates, get product and do agarose gel electrophoresis detection.
Extraction described in step A is by 50 ~ 100mg mycelia liquid nitrogen grinding powdered, joins in 1.5ml centrifuge tube.Add 400 μ LBufferDigestion4 μ L beta-mercaptoethanols, concussion mixing.65 DEG C of water-bath 1h are to the complete cracking of cell.Add 200 μ LBufferPF, fully put upside down mixing, 5min placed by-20 DEG C of refrigerators.The centrifugal 5min of room temperature 10,000rpm, transfers to supernatant liquor (500-550 μ L) in new 1.5ml centrifuge tube.Add isopyknic Virahol, put upside down and make it abundant mixing for 5-8 time, room temperature places 2-3min.The centrifugal 5min of room temperature 10,000rpm, abandons supernatant.Add 1ml75% ethanol, put upside down rinsing 1-3min, the centrifugal 2min of 10,000rpm, abandons supernatant.Again be inverted 5-10min by room temperature of uncapping after ethanol rinse one time to volatilize completely to residual ethanol.The aseptic ultrapure water of the DNA obtained dissolves.Utilize ultramicrospectrophotometer to measure DNA concentration, and with aseptic ultrapure water, the adjustment of sample DNA concentration is about 200ng/ μ L, the DNA sample of extraction can carry out next step experiment or-20 DEG C of preservations immediately.
The reaction system of the multiplexed PCR amplification described in step B is: 0.2 μ L concentration is the TaKaRaLaTaq of 5U/ μ L; 2.5 μ L10 × LAPCRBufferII(Mg
2+free); 4.0 μ L concentration are the MgCl of 25mM
2; 4.5 μ L concentration are respectively the dNTPMixture of 2.5mM; Concentration is each 0.5 μ L of upstream and downstream primer P1 ~ P8 of 20 μMs; The DNA profiling of 1 μ L; Add ddH
2o to 25mL.
Multiplexed PCR amplification described in step B reaction conditions be: 94 DEG C of denaturation 4min, 94 DEG C of sex change 40s, 56 DEG C annealing 40s, 72 DEG C extend 45s, 30 circulations, last 72 DEG C extension 7min.
Concrete operations of the present invention are as follows:
1. materials and methods
1.1 material
1.1.1 strains tested
For examination black shank pathogen (
phytophthoranicotianae), root black rot pathogen (
thielavioisbasicola), samping off pathogen (
pythiumaphanidermatum) and damping-off pathogen (
rhizoctoniasolani) preserve by Yunnan Academy of Tobacco Agricultural Science laboratory.
1.1.2 key instrument and reagent is tested
Fungal genomic DNA Rapid extraction test kit is purchased from Shanghai Sheng Gong biotechnology company limited; Whizzer, purchased from German Eppendorf company; ABIVeriti grads PCR instrument (U.S.); Ultramicrospectrophotometer (U.S. ThermoNanoDropND-2000C); TaKaRaLaTaqTM, 250bpDNAMarker, DL2000DNAMarker are purchased from TaKaRa(Dalian) company; Oat, potato, deionized water, agarose, EB(ethidium bromide) dyestuff etc.
1.2 for the extraction trying the mycelial collection of pathogen and genomic dna
Bacterial strain for examination balck shank and samping off pathogen is gone to NA solid medium dull and stereotyped, the bacterial strain of root Black Rotten and damping-off pathogen goes on PDA flat board, small colonies block is cut at colony edge after 28 DEG C of dark culturing 3d, root Black Rotten and damping-off pathogen go in PDB, other bacterial strain goes in tomato juice liquid nutrient medium, after 7d is cultivated in 28 DEG C of concussions, collecting by filtration mycelia.All all adopts fungal genomic DNA Rapid extraction test kit to extract for examination pathogen genomic dna.
The design of 1.3 primers and screening
According to the conserved regions (GenBank accession number is JQ313811.1) of dry thread Pyrenomycetes, application Primier5.0 primer-design software designs a pair Auele Specific Primer RsF/RsR, the Auele Specific Primer TBF/TBR(Zhao Yong that black root of tobacco pathogen adopts is strong. the Molecular Detection of black root of tobacco bacterium and fungicide screening. Shandong: Shandong Agricultural University, 2009), black shank and samping off pathogen adopt Zhang Lifang according to Phytophthora nicotianae Breda (JX978447.1) respectively; The Auele Specific Primer of the gene order design of tobacco scraping and printing unit (KC438411.1) (Zhang Lifang etc. the multiplex PCR of tobacco bacterial wilt, balck shank and samping off detects. North China agronomy reports .2013,28 (Z): 22-26.).Primer is synthesized by precious biotechnology (Dalian) company limited (TaKaRa), and primer sequence is in table 1.
Table 1 is for the Auele Specific Primer of multiplexed PCR amplification
The foundation of 1.4PCR detection method and condition optimizing
1.4.1 individual event PCR method detects
Respectively using the DNA of 4 pathogenic bacterias as template, carry out individual event pcr amplification with corresponding primer.PCR reaction system (25 μ L): TaKaRaLaTaq(5U/ μ L): 0.2 μ L; 10 × LAPCRBufferII(Mg
2+free): 2.5 μ L; MgCl
2(25mM): 2.5 μ L; The each 2.5mM of dNTPMixture(): 4 μ L; The each 0.5 μ L of upstream and downstream primer (20 μMs); DNA profiling: 1 μ L; Add ddH2O to 25 μ L.Reaction conditions is: 94 DEG C of denaturation 4min, 94 DEG C of sex change 40s, 59 DEG C of annealing 40s, and 72 DEG C extend 45s, 30 circulations, and last 72 DEG C extend 7min.Get 10 μ LPCR products through 1.5% agarose gel electrophoresis com-parison and analysis.
1.4.2 the optimization of multi-PRC reaction condition
According to repeatedly revision test and experience, and By consulting literatures (yellow honeysuckle flower, Hu Xiaoxiang, Gao Yu etc. affect the factor of the Efficiency of Mutiplex PCR. heredity 2003.25 (1): 65-68.) concentration etc. of the factor that finds to affect multi-PRC reaction result mainly annealing temperature, dNTPMixure, Mg2+ addition and Taq DNA polymerase, therefore emphasis is optimized above reaction conditions by this experiment, and then screens optimal reaction system and the program of multiplexed PCR amplification.
1.4.2.1 multiplex PCR Mg
2+(25mM) optimization of addition
When other condition is constant, in 25 μ L systems, the addition of Mg2+ is chosen as respectively: 2 μ L, 2.5 μ L, 3 μ L, 3.5 μ L, 4 μ L, 4.5 μ L six process.
1.4.2.2dNTPMixture(each 2.5mM) optimization of addition
In 25 μ L systems, the addition of dNTPs is chosen as 2.5 μ L, 3 μ L, 3.5 μ L, 4 μ L, 4.5 μ L, 5 μ L six process respectively.
1.4.2.3LaTaq the optimization of the addition of enzyme (5U/ μ L)
In 25 μ L systems, the addition of LaTaq enzyme is chosen as 0.2 μ L, 0.25 μ L, 0.3 μ L, 0.35 μ L, 0.4 μ L, 0.45 μ L six process respectively.
1.4.2.4 the optimization of multiplex PCR annealing temperature
In multiplex PCR system, annealing temperature is set as 53 DEG C, 54 DEG C, 55 DEG C, 56 DEG C, 57 DEG C, 58 DEG C, 59 DEG C, 60 DEG C, 61 DEG C, 62 DEG C, 63 DEG C according to 1 DEG C of gradient.Amplification relatively under differing temps, thus the optimum annealing temperature of screening multi-PRC reaction.
1.4.3 multi-PRC reaction specificity
With tobacco black shank bacterium; Root black rot; The genomic dna random combine of da mping-off fungi and rhizoctonia solani, as template, increases by the reaction system optimized, the specificity of checking multi-PRC reaction, the specificity of test multi-PRC reaction system.
1.4.4 the being quick on the draw property of multiplex PCR
The DNA profiling concentration recording black shank, samping off, root Black Rotten and damping-off pathogen through ultramicrospectrophotometer is respectively 227ng/ μ L, 179ng/ μ L, 190ng/ μ L and 254ng/ μ L, successively by 10 from starting point concentration
0-10
5gradient carries out doubling dilution.Increase according to the multiplex PCR condition optimized, measure the susceptibility of multiplex PCR.
2. results and analysis
2.1 substance PCR electrophoresis detection results
Using the template that 4 kinds of fungal DNAs such as black shank, samping off, root Black Rotten, damping-off react as PCR, obtain 4 specific bands that size is 265,364,400 and 541bp respectively, as Fig. 1 through pcr amplification.
2.2 the suitableeest Mg
2+(25mM) selection of addition
In multiplex PCR system, work as Mg
2+addition all can amplify 4 band when being 2 μ L ~ 4.5 μ L, works as Mg
2+when addition is 4 μ L, the band of 4 entries is all comparatively clear, and this experiment finally selects 4 μ L as optimal Mg2+ addition.
The each 2.5mM of 2.3 the suitableeest dNTPMixture() selection of addition
When dNTPMixture addition is at 4 μ L, 4.5 μ L, 5 μ L, all can amplifies four specific bands clearly, but when dNTPMixture addition is less than 4 μ L, just can only amplify part band.Composite factor is considered, 4.5 μ L are defined as the most applicable dNTPMixture addition by this experiment.
The selection of the addition of 2.4 the suitableeest LaTaq enzymes (5U/ μ L)
When the addition of Taq enzyme is between 0.2 μ L-0.45 μ L, 4 specific bands clearly all can be amplified.When Taq enzyme addition is 0.2 μ L, just have the band of gem-pure 4 entries to occur, and conditions of streaking is relatively weak.So composite factor is considered, this experiment the most at last 0.2 μ L is defined as the suitableeest LaTaq enzyme addition.
The selection of 2.5 the suitableeest annealing temperatures
Having there is the band of 4 entries in each temperature of multiplex PCR procedure, can react under 53 ~ 64 ° of C temperature are described.When annealing temperature is 56 DEG C, the band of 4 entries is all comparatively homogeneous, so this experiment is decided to be the suitableeest annealing temperature by 56 DEG C.
2.6 multi-PRC reaction specific detection
4 kinds of random equivalent combinations of germ are carried out multi-PRC reaction as template, and result display multiplex PCR detection system has good stability, and atopic is stronger.
The being quick on the draw property of 2.7 multiplex PCRs
In the 4 heavy PCR system of 25 μ L, the concentration of black shank, samping off, root Black Rotten and damping-off pathogen DNA profiling is respectively 227ng/ μ L, 179ng/ μ L, 190ng/ μ L and 254ng/ μ L.As shown in Figure 2, the sensitivity simultaneously detecting 4 kinds of pathogenic bacterias can reach 10
-2ng/ μ L genomic dna.
Below in conjunction with embodiment, the present invention will be further described.
Embodiment 1 prepares DNA masterplate
The masterplate that the DNA extracting 4 kinds of pathogen samples reacts as PCR, detailed process is as follows:
1, the collection of mycelia
Bacterial strain for examination balck shank and samping off pathogen is gone to NA solid medium dull and stereotyped, the bacterial strain of root Black Rotten and damping-off pathogen goes on PDA flat board, small colonies block is cut at colony edge after 28 DEG C of dark culturing 3d, root Black Rotten and damping-off pathogen go in PDB, other bacterial strain goes in tomato juice liquid nutrient medium, after 7d is cultivated in 28 DEG C of concussions, collecting by filtration mycelia.
2, the extraction of sample DNA
By 50-100mg mycelia liquid nitrogen grinding powdered, join in 1.5ml centrifuge tube.Add 400 μ LBufferDigestion4 μ L beta-mercaptoethanols, concussion mixing.65 DEG C of water-bath 1h are to the complete cracking of cell.Add 200 μ LBufferPF, fully put upside down mixing, 5min placed by-20 DEG C of refrigerators.The centrifugal 5min of room temperature 10,000rpm, transfers to supernatant liquor (500-550 μ L) in new 1.5ml centrifuge tube.Add isopyknic Virahol, put upside down and make it abundant mixing for 5-8 time, room temperature places 2-3min.The centrifugal 5min of room temperature 10,000rpm, abandons supernatant.Add 1ml75% ethanol, put upside down rinsing 1-3min, the centrifugal 2min of 10,000rpm, abandons supernatant.Again be inverted 5-10min by room temperature of uncapping after ethanol rinse one time to volatilize completely to residual ethanol.The aseptic ultrapure water of the DNA obtained dissolves.Utilize ultramicrospectrophotometer to measure DNA concentration, and with aseptic ultrapure water, the adjustment of sample DNA concentration is about 200ng/ μ L, the DNA sample of extraction can carry out next step experiment or-20 DEG C of preservations immediately.
Embodiment 2 sets up PCR reaction system
1,4 kinds of pathogen substance PCR reaction systems are set up
The mixed solution cumulative volume of PCR reaction is 25 μ L, comprises the TaKaRaLaTaq that 0.2 μ L concentration is 5U/ μ L; 2.5 μ L10 × LAPCRBufferII(Mg
2+free); 2.5 μ L concentration are the MgCl of 25mM
2; 4 μ L concentration are respectively the dNTPMixture of 2.5mM; The concentration that 4 kinds of pathogens are corresponding is each 0.5 μ L of upstream and downstream primer of 20 μMs; The DNA profiling of 1 μ L; Volume is supplied with sterilized water.So reagent is all purchased from TaKaR company, ABIVeriti grads PCR instrument carries out amplified reaction.Response procedures is 94 DEG C of denaturation 4min, 94 DEG C of sex change 40s, 56 DEG C of annealing 40s, and 72 DEG C extend 45s, 30 circulations, and last 72 DEG C extend 7min.Get 10 μ LPCR products electrophoresis in 1.5% sepharose, gel imaging system detects and takes pictures, if there are 4 the clear DNA bands (Fig. 1) being respectively 265,364,400 and 541bp, then demonstrate four pairs of primers.
2,4 heavy PCR reaction systems are set up
The mixed solution cumulative volume of PCR reaction is 25 μ L, comprises each 0.5 μ L of upstream and downstream primer P1 ~ P8 that concentration is 20 μMs; 0.2 μ L concentration is the TaKaRaLaTaq of 5U/ μ L; 2.5 μ L10 × LAPCRBufferII(Mg
2+free); 4.0 μ L concentration are the MgCl of 25mM
2; 4.5 μ L concentration are respectively the dNTPMixture of 2.5mM; Supply with sterilized water.Response procedures is with above-mentioned substance PCR system.Get 10 μ LPCR products electrophoresis in 1.5% sepharose, gel imaging system detects and takes pictures, if having 265,364,400 and 541bp totally 4 specific bands, then prove to detect 4 kinds of pathogenic bacterias in a PCR system simultaneously.
The specificity that embodiment 3 multiplex PCR detects and susceptibility
1, specific detection
The genomic dna random combine of 4 kinds of pathogens, as template, adopts Auele Specific Primer P1 ~ P8 that the present invention is designed and screen, increases by the reaction system optimized, and the pathogen of the specific detection random combine of energy, demonstrates the specificity of multiplex PCR.
2,4 heavy PCR sensitivity technique
The sensitivity of 4 set up heavy PCR detection system is measured in 25 μ L reaction systems.Concentration is respectively 227ng/ μ L, 179ng/ μ L, 190ng/ μ L and 254ng/ μ L the DNA sample of 4 kinds of pathogens by 10
0-10
5gradient carries out doubling dilution, and adopts 4 heavy PCR reaction system and the response procedures designed in the present invention, amplifies 4 specific bands (Fig. 2) from 0.01ng/ μ L genomic templates Absorbable organic halogens.
Embodiment 4 Multiple detection system is to the detection of early stage doubtful diseased plant illing tissue
In June, 2014 gathers doubtful diseased plant sample in Zhi Yan district, Yuxi, cleans with tap water rhizome portion tissue of catching an illness, and gets the strong intersection tissue of disease, is cut into 4mm
2fritter, 70% alcohol disinfecting 30s, gets the sterile disease of 200-300mg and is good for the method extraction sample DNA of tissue by embodiment 1.The sample DNA in health tissues is extracted with method.With the tissue sample DNA extracted for template, black shank, samping off, root Black Rotten and damping-off pathogen DNA are positive control, and sterilized water DNA is negative control, adopt embodiment 2 reaction system electroresis appraisal.Result shows, 4 positive controls all present corresponding specific band, positive control, health tissues sample without amplification, the specific band having 16 to amplify 364bp in 20 doubtful diseased plant samples, 2 specific bands amplifying 400bp, 1 specific band amplifying 265bp, 1 specific band amplifying 364bp and 400bp.These results show, there is balck shank in the doubtful diseased plant sample gathered, root Black Rotten, samping off are accidental, there is extremely individually black shank, root Black Rotten Combined Infection, have no damping-off diseased plant.
SEQUENCELISTING
<110> Yunnan Academy of Tobacco Agricultural Science
<120> mono-kind detects the PCR primer of tobacco soil-borne fungus pathogen and application and method
<130>2014
<160>8
<170>PatentInversion3.3
<210>1
<211>22
<212>DNA
<213> tobacco rhizoctonia solani upstream primer
<400>1
gcacactcttctctttcatcca22
<210>2
<211>20
<212>DNA
<213> tobacco rhizoctonia solani downstream primer
<400>2
ttagaagcggttcgtctgca20
<210>3
<211>21
<212>DNA
<213> black shank bacterium upstream primer
<400>3
cgaagccaaccataccacgaa21
<210>4
<211>21
<212>DNA
<213> black shank bacterium downstream primer
<400>4
atgaagaacgctgcgaactgc21
<210>5
<211>24
<212>DNA
<213> root black rot upstream primer
<400>5
aacgtaccttttctagctgctttg24
<210>6
<211>21
<212>DNA
<213> root black rot downstream primer
<400>6
tgagggtttttcggcatgtta21
<210>7
<211>21
<212>DNA
<213> da mping-off fungi upstream primer
<400>7
tggctcccttttcggaggaga21
<210>8
<211>20
<212>DNA
<213> da mping-off fungi downstream primer
<400>8
ttgcccagaccattgcctca20
Claims (3)
1. one kind is detected the method for tobacco 4 kinds of soil-borne fungus pathogens simultaneously, it is characterized in that described 4 kinds of soil-borne fungus are tobacco rhizoctonia solani, black shank bacterium, root black rot and da mping-off fungi, the described tobacco 4 kinds of soil-borne fungus pathogen methods that simultaneously detect comprise pre-treatment, PCR reaction, electrophoresis detection step:
A, pre-treatment: extract the germ DNA in tobacco sample to be detected, aseptic ultrapure water dissolving DNA, regulates DNA concentration to be 200ng/ μ L, prepares sample DNA;
B, PCR react: carry out multiplexed PCR amplification with PCR primer pair sample DNA;
Described PCR primer is made up of tobacco rhizoctonia solani primer, black shank bacterium primer, root black rot primer and da mping-off fungi primer;
The DNA sequence dna of described tobacco rhizoctonia solani primer is:
Upstream primer P1:5 '-GCACACTCTTCTCTTTCATCCA-3 ';
Downstream primer P2:5 '-TTAGAAGCGGTTCGTCTGCA-3 ';
The DNA sequence dna of described black shank bacterium primer is:
Upstream primer P3:5 '-CGAAGCCAACCATACCACGAA-3 ';
Downstream primer P4:5 '-ATGAAGAACGCTGCGAACTGC-3 ';
The DNA sequence dna of described root black rot primer is:
Upstream primer P5:5 '-AACGTACCTTTTCTAGCTGCTTTG-3 ';
Downstream primer P6:5 '-TGAGGGTTTTTCGGCATGTTA-3 ';
The DNA sequence dna of described da mping-off fungi primer is:
Upstream primer P7:5 '-TGGCTCCCTTTTCGGAGGAGA-3 ';
Downstream primer P8:5 '-TTGCCCAGACCATTGCCTCA-3 ';
The reaction system of described multiplexed PCR amplification is: 0.2 μ L concentration is the TaKaRaLaTaq of 5U/ μ L; 2.5 μ L10 × LAPCRBufferIIMg
2+free; 4.0 μ L concentration are the MgCl of 25mM
2; 4.5 μ L concentration are the dNTPMixture of 2.5mM; Concentration is each 0.5 μ L of upstream and downstream primer P1 ~ P8 of 20 μMs; The DNA profiling of 1 μ L; Add ddH
2o to 25 μ L; Reaction conditions is: 94 DEG C of denaturation 4min, 94 DEG C of sex change 40s, 56 DEG C of annealing 40s, and 72 DEG C extend 45s, 30 circulations, and last 72 DEG C extend 7min;
After C, electrophoresis detection: PCR reaction terminates, get product and do agarose gel electrophoresis detection, primer P1, P2 specific band is 541bp, primer P3, P4 specific band is 364bp, primer P5, P6 specific band is 400bp, primer P7, P8 specific band is 265bp, and the sensitivity simultaneously detecting 4 kinds of pathogenic bacterias reaches 10
-2ng/ μ L genomic dna.
2. the method simultaneously detecting tobacco 4 kinds of soil-borne fungus pathogens according to claim 1 to be carried disease germs monitoring and qualification field tobacco disease early diagnosis and the application be separated in germ in qualification tobacco seedling.
3. detect the method for tobacco 4 kinds of soil-borne fungus pathogens according to claim 1 simultaneously, it is characterized in that the extraction described in step A is by 50-100mg mycelia liquid nitrogen grinding powdered, join in 1.5ml centrifuge tube, add 400 μ LBufferDigestion4 μ L beta-mercaptoethanols, concussion mixing, 65 DEG C of water-bath 1h are to the complete cracking of cell, add 200 μ LBufferPF, fully put upside down mixing, 5min placed by-20 DEG C of refrigerators, the centrifugal 5min of room temperature 10000rpm, supernatant liquor 500-550 μ L is transferred in new 1.5ml centrifuge tube, add isopyknic Virahol, put upside down 5 ~ 8 times and make it abundant mixing, room temperature places 2-3min, the centrifugal 5min of room temperature 10000rpm, abandon supernatant, add 1ml75% ethanol, put upside down rinsing 1 ~ 3min, 10, the centrifugal 2min of 000rpm, abandon supernatant, again be inverted 5 ~ 10min by room temperature of uncapping after ethanol rinse one time to volatilize completely to residual ethanol, the aseptic ultrapure water of the DNA obtained dissolves, ultramicrospectrophotometer is utilized to measure DNA concentration, and with aseptic ultrapure water, sample DNA concentration is adjusted to 200ng/ μ L, the DNA sample extracted carries out next step experiment or-20 DEG C of preservations immediately.
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