CN101250580A - Primer, probe and real-time fluorescent PCR reagent case for detecting tobacco ralstonia solanacearum - Google Patents
Primer, probe and real-time fluorescent PCR reagent case for detecting tobacco ralstonia solanacearum Download PDFInfo
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Abstract
A primer which is used to detect pseudomonas solanacearum, a probe and a real-time fluorescence PCR reagent kit thereof, wherein the primer comprises forward primer R. so11 and reverse primer R. so 12, wherein the base sequence thereof respectively are that R. so 11:5'-CCG ACA CCA CCC TGA A-3', and R so 12:5'-GCG GAC GGA TAG ATG TAG TTG C-3'. The base sequence of the probe is any one ribonucleotide sequence in the region of shifting two bases towards to upstream on the two ends of 5'-ACC TGG CAT ACC TTG GCG ACA CC-3', or shifting three bases towards to downstream, fluorescence reporting group is marked on 5' end in the ribonucleotide sequence, fluorescence quenching group is marked on 3' end, thereby forming fluorescence marking probe. The relative reagent kit is prepared with the primer and the probe. The invention can fast and accurately detect pseudomonas solanacearum in field sick soil, thereby providing accurate foundation for real-timely taking measurements to cut off the infection source for plant diseases, can fast and accurately detect diseases on plant, and has extreme important significances.
Description
Technical field
The present invention relates to a kind of biotechnology that departments such as agriculture production, plant protection use that is suitable for, particularly a kind of great crushing soil-borne disease---primer, probe and real-time fluorescent PCR reagent case of tobacco bacterial wilt pathogenic bacteria Ralstonia solanacearum that is used for detecting tobacco production.
Background technology
Tobacco bacterial wilt is the great destructive disease of the global tobacco industry development of influence, takes place comparatively general in China various places.Plant root, stem, Ye Junke are injured, and its most typical symptom is withered.Because plant is susceptible withered very fast, and blade withers to hang down and still is dark green, so the title bacterial wilt.This disease is in case generation can cause complete stool death, and especially in seedling stage, it is withered in flakes that this disease often causes seedling, causes in a large number and be short of seedling, very big to output and the quality influence of tobacco.This disease is based on the soil biography, and wind and rain, flowing water, farming operation etc. all are its routes of transmission.
Detection method for tobacco ralstonia solanacearum has conventional sense methods such as classical symptom appearance method, tissue pathological slice method, pathogenic assay method.The advantage of ordinary method is directly perceived, simple and easy to do, but its shortcoming is that sense cycle is long, the person's of lacking experience False Rate height, and can not detect at the cause of disease initial stage of infecting.In addition, the detection of tobacco ralstonia solanacearum is also had enzyme-linked immunosorbent assay, spot immune absorption method etc., but exist process complexity, sensitivity low, detect problems such as cost height, cycle be long.
After conventional PCR detection technique, emerging real-time fluorescence quantitative PCR technology relies on that it is accurately quantitative, highly sensitive to the original template amount, high specificity, stopped pipe operation, advantage such as simple, convenient and rapid, become the mainstream technology in the domestic and international molecular biology research, it has equally also obtained being extensive use of in the detection of phytopathogen and diagnosis gradually.Begin successively both at home and abroad in recent years the real-time fluorescence PCR technology is applied to the Plant diseases diagnostic detection.
Real-time quantitative fluorescence PCR is to utilize fluorescent signal to be accompanied by the increase of PCR product and the enhanced principle, in the pcr amplification process, and the continuously variation of fluorescent signal in the detection reaction system.According to the mean value of fluorescent signal baseline and average standard deviation, when 99.7% degree of confidence, calculate fluorescent value threshold value, the PCR cycle index (Ct value) when collecting fluorescent signal then and being strengthened to predetermined threshold greater than mean value.Strict linear relationship is arranged between the logarithmic value of initiate dna template amount in this parameter and the PCR reaction system.Utilize the Ct value of positive gradient dilution standard substance to be depicted as typical curve, just can measure the starting template copy number of target pathogenic bacteria again according to the Ct value of test sample exactly.Real-time fluorescence PCR is divided into two class methods of using probe and not using probe according to its principle that produces fluorescence.Because probe and pathogenic bacteria dna sequence dna to be detected have very high specificity, and can effectively avoid the false positive problem of conventional PCR, so use fluorescence labeling probe method comparatively generally at present.
Real-time fluorescence PCR detection method is used for the detection of phytopathogen, at home and abroad all still is at the early-stage.At present, in the real-time fluorescence PCR of tobacco ralstonia solanacearum detects, be primer, classify probe as with any nucleotides sequence in 3 base zones of 2 bases or downstream displacement that upstream are shifted at 5 '-ACC TGG CAT ACC TTG GCG ACA CC-3 ' two ends and be prepared into corresponding real-time fluorescent PCR reagent case still do not have relevant report with 5 '-CCGACA CCA CGA CCC TGA A-3 ' and 5 '-GCG GAC GGA TAG ATG TAG TTG C-3 '.
Summary of the invention
One of purpose of the present invention just provides a kind of primer that is used to detect tobacco ralstonia solanacearum, and it can be in the PCR testing process, qualitative detection tobacco ralstonia solanacearum, and high specificity.
The primer that is used to detect tobacco ralstonia solanacearum provided by the present invention comprises that the primer that forward primer R.sol1 and reverse primer R.sol2 form is right, the nucleotide sequence of described forward primer R.sol1 is seen shown in the sequence 1 in the sequence table, R.sol1:5 '-CCG ACA CCA CGA CCC TGA A-3 '; Described reverse primer R.sol2 nucleotide sequence is seen shown in the sequence 2 in the sequence table, R.sol2:5 '-GCG GAC GGATAG ATG TAG TTG C-3 '.
The applicant is through long-term a large amount of experiment, filters out the above-mentioned primer of being made up of forward primer R.sol1 and reverse primer R.sol2 from numerous PCR primers that are used for detecting tobacco ralstonia solanacearum, and its amplified fragments size is 331bp.The characteristics of this primer are tobacco ralstonia solanacearum to be had the conservative property of height, and do not have significant homology between other nearly edge biological species, carry out pcr amplification with this primer, realize the accurate qualitative detection of tobacco ralstonia solanacearum, and high specificity.
Two of purpose of the present invention just provides a kind of probe that is used to detect tobacco ralstonia solanacearum, and it can be in the real-time fluorescence PCR testing process, and the detection by quantitative tobacco ralstonia solanacearum improves detection sensitivity significantly.The base sequence of this probe is any nucleotide sequence in 5 '-ACC TGG CAT ACC TTG GCG ACACC-3 ' two ends upstream are shifted 2 bases or 3 base zones of downstream displacement, described probe one end is connected with the fluorescent quenching group, and the other end is connected with the fluorescence report group.
Described fluorescence probe reporter group is marked at 5 ' end, and the fluorescent quenching group is marked at 3 ' end, constitutes fluorescence labeling probe.
Described probe is at upstream be shifted the nucleotide sequence 5 '-CCA CCT GGC ATA CCT TGG CGA CAC C-3 ' of 2 bases of 5 '-ACC TGG CAT ACC TTG GCG ACA CC-3 ', sees shown in the sequence 4 in the sequence table.
The nucleotide sequence of described probe is the sequence shown in the sequence 3 in the sequence table: 5 '-ACC TGGCAT ACC TTG GCG ACA CC-3 '.
Described probe is at be shifted downstream the nucleotide sequence 5 '-ACC TGG CAT ACC TTG GCG ACA CCG CC-3 ' of 3 bases of 5 '-ACC TGG CAT ACC TTG GCG ACA CC-3 ', sees shown in the sequence 5 in the sequence table.
Three of purpose of the present invention just provides a kind of test kit that utilizes the detection tobacco ralstonia solanacearum that primer in the purpose one and the probe in the purpose two make, can be in the real-time fluorescence PCR testing process, in the detection by quantitative soil and the tobacco ralstonia solanacearum on the plant, and reliable and stable, highly sensitive quickly and accurately.Described test kit comprises that also testing sample extracts reagent, quantitative criterion product, real-time fluorescence PCR reaction solution and detects articles for use, and described real-time fluorescence PCR reaction solution includes following component and corresponding final concentration thereof: PCR damping fluid: 10%; MgCl
2: 2.5mmol/L; DNTPs:0.2mmol/L; Taq archaeal dna polymerase: 1U/25 μ L; Forward primer R.sol1:0.3 μ mol/L; Reverse primer R.sol2:0.3 μ mol/L; Fluorescence labeling probe: 0.2 μ mol/L; Adopt aseptic double-distilled water to adjust final concentration.
According to practical situation, what each component adopted in the real-time fluorescence PCR reaction solution is: the PCR damping fluid is 10 * PCR damping fluid; MgCl
2Initial concentration be 25mmol/L; The initial concentration of dNTPs is 5mmol/L; The initial concentration of Taq archaeal dna polymerase is 1U/ μ L; The initial concentration of forward primer R.sol1 is 5 μ mol/L; The initial concentration of reverse primer R.sol2 is 5 μ mol/L; The initial concentration of fluorescence labeling probe is 5 μ mol/L; PCR damping fluid, MgCl
2, dNTPs, Taq archaeal dna polymerase, forward primer R.sol1, reverse primer R.sol2, fluorescence labeling probe volume ratio be: 5: 5: 2: 2: 3: 3: 2.
In the real-time fluorescence PCR reaction solution, what each component adopted is: the PCR damping fluid is 10 * PCR damping fluid; MgCl
2Initial concentration be 25mmol/L; The initial concentration of dNTPs is 10mmol/L; The initial concentration of Taq archaeal dna polymerase is 2.5U/ μ L; The initial concentration of forward primer R.sol1 is 10 μ mol/L; The initial concentration of reverse primer R.sol2 is 10 μ mol/L; The initial concentration of fluorescence labeling probe is 5 μ mol/L; PCR damping fluid, MgCl
2, dNTPs, Taq archaeal dna polymerase, forward primer R.sol1, reverse primer R.sol2, fluorescence labeling probe volume ratio be: 50: 50: 10: 8: 15: 15: 20.
Adopt the test kit among the present invention, on the one hand, in the testing sample preprocessing process, the tobacco ralstonia solanacearum DNA that testing sample extraction reagent can directly extract in pedotheque or the tobacco leaf is used as the pcr amplification template, has saved the time; On the other hand, in the real-time fluorescence PCR amplification procedure of testing sample, directly the real-time fluorescence PCR reaction solution that will prepare by best component and volume ratio thereof adds in eight connecting legs, both saved a large amount of time, and reliable and stable, thus reached detect quickly and accurately in the soil and plant on tobacco ralstonia solanacearum.
Owing to adopted technique scheme, the present invention to have following advantage:
(1) high specificity: primer and probe all design according to the tobacco ralstonia solanacearum specific sequence among the present invention, and compare with other biological species, do not have remarkable homology, and detection specificity is strong;
(2) highly sensitive: utilize when real-time fluorescent PCR reagent case detects tobacco ralstonia solanacearum among the present invention, can be accurately quantitative, object bacteria DNA has good linear relationship in 10ng/ μ L-1fg/ μ L concentration range, highly sensitive;
(3) practicality is good: real-time fluorescent PCR reagent case has unlatching promptly with advantages such as, standard are unified, simple and convenient among the present invention, can detect quickly and accurately in the field soil with plant on tobacco ralstonia solanacearum, practicality is good;
(4) applied range: can detection by quantitative soil and plant on tobacco ralstonia solanacearum, be applicable to dynamic monitoring of tobacco ralstonia solanacearum field and disease screening, can be used for the rate of propagation of tobacco ralstonia solanacearum in tobacco plant and the detection by quantitative of seed-borne fungi simultaneously.
In sum, adopt the present invention, can be in the field in spite of illness in the soil and detect tobacco ralstonia solanacearum quickly and accurately on the plant, thus the source of infection that cuts off disease in time taking measures provides accurate foundation, also can detect the disease on the plant quickly and accurately, its meaning is very great.
Embodiment
The invention will be further described below in conjunction with embodiment:
In the present invention, the primer that is used to detect tobacco ralstonia solanacearum comprises forward primer R.sol1 and reverse primer R.sol2, and its base sequence is respectively:
R.sol1:5 '-CCG ACA CCA CGA CCC TGA A-3 ' sees shown in the sequence 1 in the sequence table;
R.sol2:5 '-GCG GAC GGA TAG ATG TAG TTG C-3 ' sees shown in the sequence 2 in the sequence table.
In the present invention, be used to detect the probe of tobacco ralstonia solanacearum, its base sequence is any nucleotide sequence in 5 '-ACCTGG CAT ACC TTG GCG ACA CC-3 ' two ends upstream are shifted 2 bases or 3 base zones of downstream displacement, list at this nucleotides sequence, the fluorescence report group is marked at 5 ' end, the fluorescent quenching group is marked at 3 ' end, to constitute fluorescence labeling probe.
In the amplification region of the primer of forming by forward primer R.sol1 and reverse primer R.sol2, principle of design and method of design according to probe, design the probe that is used for the detection of tobacco ralstonia solanacearum real-time fluorescence PCR, its base sequence is any nucleotide sequence in 5 '-ACC TGG CAT ACC TTG GCG ACA CC-3 ' two ends upstream are shifted 2 bases or 3 base zones of downstream displacement; Carry out the real-time fluorescence PCR amplification with all designed probes, designed probe is screened; Result according to the real-time fluorescence PCR amplification filters out best probe at last.
At upstream the be shifted nucleotide sequence of 2 bases of 5 '-ACC TGG CAT ACC TTG GCG ACA CC-3 ' can be: 5 '-CCA CCT GGC ATA CCT TGG CGA CAC C-3 ', see shown in the sequence 4 in the sequence table
The nucleotide sequence of fluorescence labeling probe also can be: 5 '-ACC TGG CAT ACC TTG GCG ACACC-3 ', see that it is a preferred forms shown in the sequence 3 in the sequence table.
At be shifted the downstream nucleotide sequence of 3 bases of 5 '-ACC TGG CAT ACC TTG GCG ACA CC-3 ' can also be: 5 '-ACC TGG CAT ACC TTG GCG ACA CCG CC-3 ', see shown in the sequence 5 in the sequence table.
5 ' end at fluorescence labeling probe indicates note reporter group FAM, and 3 ' end is marked with fluorescent quenching group TAMRA.When probe was complete, reporter group institute emitted fluorescence energy was absorbed by quenching group, and instrument detecting is less than signal.Along with PCR carries out, the Taq archaeal dna polymerase runs in the chain extension process and template bonded probe, its 5 ' → 3 ' exonuclease activity will cut off probe, reporter group is away from quenching group, and its energy can not be absorbed, and promptly produces fluorescent signal, the fluorescence that reporter group discharged can be detected by the photofluorometer in the detection by quantitative instrument, template is whenever duplicated once, just has a probe to be cut off, and follows the release of a fluorescent signal.Because d/d fluorophor number and PCR product quantity are man-to-man relations, so the accumulation volume of the increase of fluorescence volume and PCR product is proportionlity.The Ct value is the cycle number that the accumulation of fluorescence volume in the PCR process surpasses the substrate fluorescence volume.Utilize the Ct value of positive gradient standard form to make typical curve, again according to the Ct value of testing sample can quantitative exactly detected sample in the concentration of tobacco ralstonia solanacearum, thereby the detection sensitivity of significantly improving.
The test kit of detection tobacco ralstonia solanacearum of the present invention, it includes testing sample and extracts reagent, quantitative criterion product, real-time fluorescence PCR reaction solution and detect articles for use, and wherein the real-time fluorescence PCR reaction solution includes following component and corresponding final concentration thereof: PCR damping fluid: 10%; MgCl
2: 2.5mmol/L; DNTPs:0.3mmol/L; Taq archaeal dna polymerase: 1U/25 μ L; Forward primer R.sol1:0.4 μ mol/L; Reverse primer R.sol2:0.4 μ mol/L; Fluorescence labeling probe: 0.2 μ mol/L; Adopt aseptic double-distilled water to adjust concentration.
Testing sample extracts reagent to be made up of TES damping fluid and TE damping fluid, and TES damping fluid and TE damping fluid all are conventional reagent, is formulated according to methods involving in " fine works molecular biology experiment guide (the 4th edition) ".
The quantitative criterion product comprise standard positive control template, no tobacco ralstonia solanacearum soil sample and the healthy plant negative control template based on the peculiar nucleotide sequence design of tobacco ralstonia solanacearum.The standard positive control template is prepared from by containing the pMD18-T carrier (available from Takara company) that inserts target fragment.Preparation process is: adopt primer R.sol1 and R.sol2 amplification tobacco ralstonia solanacearum genomic dna, cut glue behind the PCR product electrophoresis and reclaim target fragment, be connected under 16 ℃ with the pMD18-T carrier and spend the night, connect product transformed into escherichia coli JM109 competent cell, converted product is coated on the penbritin flat board that contains X-gal and IPTG and cultivates, the picking hickie shakes and adopts behind the bacterium SDS alkaline hydrolysis method to extract plasmid, by the enzyme evaluation positive colony of cutting and check order.The plasmid DNA of positive colony is quantitative through ultraviolet spectrophotometer A260, is 10ng-1fg/ μ L through 10 * gradient dilution, is stored in-20 ℃.
The real-time fluorescence PCR reaction solution includes following component and corresponding final concentration thereof: PCR damping fluid: 10%; MgCl
2: 2.5mmol/L; DNTPs:0.2mmol/L; Taq archaeal dna polymerase: 1U/25 μ L; Forward primer R.sol1:0.3 μ mol/L; Reverse primer R.sol2:0.3 μ mol/L; Fluorescence labeling probe: 0.2 μ mol/L; Adopt aseptic double-distilled water to adjust concentration.Wherein, PCR damping fluid, MgCl
2, dNTPs, Taq archaeal dna polymerase be the conventional reagent of PCR, all available from Beijing ancient cooking vessel state biotechnology responsibility company limited, primer is synthetic by match Parkson, Beijing gene engineering company limited, fluorescence labeling probe is synthetic in Takara company.
Detect articles for use and include self-sealing plastics bag, compound membrane filter and 0.2mL eight connecting legs.
On the basis of the best probe in primer in utilizing purpose one and the purpose two, optimizing reaction system and reaction conditions are determined best tobacco ralstonia solanacearum real-time fluorescence PCR detection architecture.
The real-time fluorescence PCR reaction system cumulative volume of optimization of the present invention is 25 μ L, 10 * PCR damping fluid, 2.5 μ L wherein, 25mmol/L MgCl
22.5 μ L, 5mmol/L dNTPs 1 μ L, 1U/ μ L TaqDNA polysaccharase 1 μ L, each 1.5 μ L of 5 μ mol/L forward primer R.sol1 and reverse primer R.sol2,5 μ mol/L fluorescence labeling probes, 1 μ L, aseptic double-distilled water 13 μ L, dna profiling 1 μ L.Real-time fluorescence PCR adopts two-step approach, and the amplified reaction program is 95 ℃, 4min; 42 circulations then, each circulation is 95 ℃, 10s, 63 ℃, 30s collects fluorescence in each round-robin annealing/extension stage (63 ℃).
Utilize the real-time fluorescent PCR reagent case among the present invention that tobacco ralstonia solanacearum is detected, comprise the real-time fluorescence PCR reaction of testing sample pre-treatment and testing sample.
The preparation of testing sample pcr amplification template and the reaction of the real-time fluorescence PCR of testing sample.
The preparation of testing sample pcr amplification template comprises the preparation of pedotheque pcr amplification template and the preparation of plant sample P CR amplification template:
1. the preparation of pedotheque pcr amplification template:
(1) take by weighing 0.5 gram pedotheque in the 5ml centrifuge tube, add 0.2g polyvinylpolypyrrolidone (PVPP), vortex 30s adds 3mL DNA extraction buffer again and mixes;
(2) place-196 ℃ of freezing 2min of liquid nitrogen rapidly, take out the back and melt (about 3min, the attention time can not be oversize, melts the back and take out at once), 2-3 cracking cell so repeatedly in 65 ℃ of water-baths;
(3) add 20 μ l 10mg/ml Proteinase Ks, 0.5ml 50mg/ml N,O-Diacetylmuramidase, the mixing that turns upside down places the 30min (225r/min) that vibrates on 37 ℃ of shaking tables;
(4) add 600 μ l 100g/LSDS, 65 ℃ of water-bath 30min put upside down mixing gently every 5-10min therebetween;
(5) centrifugal (12,000r/min) 10min collects supernatant liquor to room temperature, transfers in another new 5mL centrifuge tube;
(6) soil precipitation adds 0.5ml extracting solution and 100 μ l 100g/L SDS again, vortex 30s, and 65 ℃ of water-bath 10min, room temperature is centrifugal, and (12,000r/min) 10min collects supernatant liquor and merges with supernatant liquor last time;
(7) add 0.05g PVPP in the supernatant liquor, room temperature in conjunction with 20min after, room temperature is centrifugal, and (12,000r/min) 10min collects supernatant liquor, transfers in another new 5ml centrifuge tube;
(8) supernatant liquor is with isopyknic chloroform/primary isoamyl alcohol (24: 1) extracting twice, centrifugal (12,000r/min, 10min) back is drawn water and is transferred in another 5mL centrifuge tube;
(9) with 0.6 times of volume Virahol precipitation at room temperature 30min of precooling, room temperature is centrifugal, and (12,000r/min) 10min collects the nucleic acid precipitation;
(10) with cold 700mL/L washing with alcohol precipitation twice, dry up naturally, be dissolved in the ultrapure water of 100 μ L sterilization.
2. the preparation of plant sample P CR amplification template:
(1) with 70% alcohol wipe worktable, to guarantee under sterile state, operating whole process;
(2) choose tobacco leaf randomly, shred the back and add and to be equipped with in the plastics bag of 10mL TES damping fluid, soaks 15 minutes (during vibration 2-3 time), the 50mL centrifuge tube of packing into then, the centrifugal 1min of 5000r/min is then with compound membrane filter filtration;
(3) wash thalline from filter membrane, filter membrane is immersed 100 μ L TE damping fluids, thermal agitation, the bacterium liquid of wash-out is follow-up PCR template to be measured.
The real-time fluorescence PCR reactions steps of testing sample is:
(1) be provided with the start of real-time fluorescence PCR instrument standby;
(2) in 0.2mL eight connecting legs, add 24 μ L real-time fluorescence PCR reaction solutions;
(3) add 1 μ L analyte sample fluid, no tobacco ralstonia solanacearum soil sample or health tobacco plant negative control that test kit is provided add in the negative control pipe, the positive control pipe adds 10 * gradient dilution liquid of positive control standard substance, and the consumption of control sample is every pipe 1 μ L;
(4) can carry out the PCR reaction after mixing;
(5) treat that pcr amplification finishes, the analysis software that adopts instrument to carry, analysing amplified result, the production standard curve calculates the DNA amount of tobacco ralstonia solanacearum in the testing sample reaction system according to typical curve.
Adopt the test kit among the present invention, on the one hand, in the testing sample preprocessing process, the tobacco ralstonia solanacearum DNA that testing sample extraction reagent can directly extract in pedotheque or the tobacco leaf is used as the pcr amplification template, has saved the time; On the other hand, in the real-time fluorescence PCR amplification procedure of testing sample, directly the real-time fluorescence PCR reaction solution that will prepare by best component and volume ratio thereof adds in eight connecting legs, both saved a large amount of time, and reliable and stable, thus reached detect quickly and accurately in the soil and plant on tobacco ralstonia solanacearum.
In above-mentioned real-time fluorescence PCR reaction solution, what each component adopted is: the PCR damping fluid is 10 * PCR damping fluid; MgCl
2Initial concentration be 25mmol/L; The initial concentration of dNTPs is 5mmol/L; The initial concentration of Taq archaeal dna polymerase is 1U/ μ L; The initial concentration of forward primer R.sol1 is 5 μ mol/L; The initial concentration of reverse primer R.sol2 is 5 μ mol/L; The initial concentration of fluorescence labeling probe is 5 μ mol/L; PCR damping fluid, MgCl
2, dNTPs, Taq archaeal dna polymerase, forward primer R.sol1, reverse primer R.sol2, fluorescence labeling probe volume ratio be: 5: 5: 2: 2: 3: 3: 2.
In above-mentioned real-time fluorescence PCR reaction solution, what each component adopted is: the PCR damping fluid is 10 * PCR damping fluid; MgCl
2Initial concentration be 25mmol/L; The initial concentration of dNTPs is 10mmol/L; The initial concentration of Taq archaeal dna polymerase is 2.5U/ μ L; The initial concentration of forward primer R.sol1 is 10 μ mol/L; The initial concentration of reverse primer R.sol2 is 10 μ mol/L; The initial concentration of fluorescence labeling probe is 5 μ mol/L; PCR damping fluid, MgCl
2, dNTPs, Taq archaeal dna polymerase, forward primer R.sol1, reverse primer R.sol2, fluorescence labeling probe volume ratio be: 50: 50: 10: 8: 15: 15: 20.
Sequence table
Claims (9)
1. primer that is used to detect tobacco ralstonia solanacearum, it comprises that the primer of forward primer R.sol1 and reverse primer R.sol2 composition is right, the nucleotide sequence of described forward primer R.sol1 is seen shown in the sequence 1 in the sequence table, R.sol1:5 '-CCG ACA CCA CGA CCC TGA A-3 '; Described reverse primer R.sol2 nucleotide sequence is seen shown in the sequence 2 in the sequence table, R.sol2:5 '-GCG GAC GGATAG ATG TAG TTG C-3 '.
2. probe that is used to detect tobacco ralstonia solanacearum, its base sequence is any nucleotide sequence in 5 '-ACC TGG CATACC TTG GCG ACA CC-3 ' two ends upstream are shifted 2 bases or 3 base zones of downstream displacement, the fluorescence report group is marked at 5 ' end, the fluorescent quenching group is marked at 3 ' end, constitutes fluorescence labeling probe.
3. the probe that is used to detect tobacco ralstonia solanacearum as claimed in claim 2, it is characterized in that described probe is at upstream be shifted the nucleotide sequence 5 '-CCA CCT GGC ATA CCT TGG CGA CAC C-3 ' of 2 bases of 5 '-ACC TGG CAT ACC TTG GCG ACA CC-3 ', sees shown in the sequence 4 in the sequence table.
4. the probe that is used to detect tobacco ralstonia solanacearum as claimed in claim 2, the nucleotide sequence that it is characterized in that described probe are the sequences shown in the sequence 3 in the sequence table: 5 '-ACC TGG CAT ACCTTG GCG ACA CC-3 '.
5. the probe that is used to detect tobacco ralstonia solanacearum as claimed in claim 2, it is characterized in that described probe is at be shifted downstream the nucleotide sequence 5 '-ACC TGG CAT ACC TTG GCG ACA CCG CC-3 ' of 3 bases of 5 '-ACC TGG CAT ACC TTG GCG ACA CC-3 ', sees shown in the sequence 5 in the sequence table.
6. a test kit that detects tobacco ralstonia solanacearum comprises described primer of claim 1 and claim 3,4 or 5 described probes.
7. test kit according to claim 6, it is characterized in that: described test kit comprises that also testing sample extracts reagent, quantitative criterion product, real-time fluorescence PCR reaction solution and detects articles for use, and described real-time fluorescence PCR reaction solution includes following component and corresponding final concentration thereof: PCR damping fluid: 10%; MgCl
2: 2.5mmol/L; DNTPs:0.2mmol/L; Taq archaeal dna polymerase: 1U/25 μ L; Forward primer R.sol1:0.3 μ mol/L; Reverse primer R.sol2:0.3 μ mol/L; Fluorescence labeling probe: 0.2 μ mol/L; Adopt aseptic double-distilled water to adjust final concentration.
8. the test kit of detection tobacco ralstonia solanacearum according to claim 6 is characterized in that in the real-time fluorescence PCR reaction solution, and what each component adopted is: the PCR damping fluid is 10 * PCR damping fluid; MgCl
2Initial concentration be 25mmol/L; The initial concentration of dNTPs is 5mmol/L; The initial concentration of Taq archaeal dna polymerase is 1U/ μ L; The initial concentration of forward primer R.sol1 is 5 μ mol/L; The initial concentration of reverse primer R.sol2 is 5 μ mol/L; The initial concentration of fluorescence labeling probe is 5 μ mol/L; PCR damping fluid, MgCl
2, dNTPs, Taq archaeal dna polymerase, forward primer R.sol1, reverse primer R.sol2, fluorescence labeling probe volume ratio be: 5: 5: 2: 2: 3: 3: 2.
9. the test kit of detection tobacco ralstonia solanacearum according to claim 6 is characterized in that: in the real-time fluorescence PCR reaction solution, what each component adopted is: the PCR damping fluid is 10 * PCR damping fluid; MgCl
2Initial concentration be 25mmol/L; The initial concentration of dNTPs is 10mmol/L; The initial concentration of Taq archaeal dna polymerase is 2.5U/ μ L; The initial concentration of forward primer R.sol1 is 10 μ mol/L; The initial concentration of reverse primer R.sol2 is 10 μ mol/L; The initial concentration of fluorescence labeling probe is 5 μ mol/L; PCR damping fluid, MgCl
2, dNTPs, Taq archaeal dna polymerase, forward primer R.sol1, reverse primer R.sol2, fluorescence labeling probe volume ratio be: 50: 50: 10: 8: 15: 15: 20.
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| CN103614474A (en) * | 2013-11-27 | 2014-03-05 | 福建省农业科学院植物保护研究所 | Loop-mediated isothermal amplification detection primer for ralstonia solanacearu and detection method for ralstonia solanacearu |
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| CN104250665B (en) * | 2014-08-20 | 2016-08-17 | 江苏科技大学 | No. 5 microspecies RTQ-PCR detection primers of Ralstonia solanacearum and method |
| CN105112555A (en) * | 2015-10-08 | 2015-12-02 | 中国烟草总公司郑州烟草研究院 | Tobacco bacterial wilt real-time fluorescence quantitative PCR detection kit and detection method |
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| CN113584194A (en) * | 2021-07-09 | 2021-11-02 | 西南大学 | Method for detecting composite infection of ralstonia solanacearum mixed flora |
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