CN103789417A - TaqMan probe real-time fluorescent primer for detecting fusarium oxysporum cubeba specialized No. 4 physiological race and application thereof - Google Patents
TaqMan probe real-time fluorescent primer for detecting fusarium oxysporum cubeba specialized No. 4 physiological race and application thereof Download PDFInfo
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Abstract
The method establishes a real-time fluorescent PCR qualitative detection method based on the specific conservative primer and TaqMan probe of the pathogenic fusarium oxysporum cubeba special type 4 physiological race, can simultaneously and efficiently perform qualitative and quantitative detection on the fusarium oxysporum cubeba special type 1 physiological race within 1.5 hours, has high specificity and high sensitivity, improves the detection sensitivity by 1-2 orders of magnitude compared with the prior common PCR detection research, can perform quantitative detection and multiple detection, and breaks through the limitation that the SYBREN GREEN fluorescent dye method cannot perform double or multiple detection. The real-time fluorescent PCR qualitative detection method is accurate and efficient, and can be used for enhancing the disease prevention, control, detection and quarantine work.
Description
Technical field
The invention belongs to biological technical field, particularly TaqMan probe for real-time fluorescence primer and the detection method thereof of a kind of Rapid identification and No. 4 physiological strains of detection by quantitative Fusarium oxysporum Cuba specialized form.
Background technology
By No. 1 (Fusarium oxysporum f.sp.cubense race1 of Fusarium oxysporum, FOC1) and No. 4 physiological strain (F.oxysporum f.sp.cubense race4, FOC4) research of fusarium wilt disesase of banana causing, be the destructive disease of banana in the world, bring huge harm to the banana industry in the world.Banana mainly concentrates on Africa, South America and Asia, and nearly 100,000,000 tons of annual production is second-biggest-in-the-world fruit crop, is one of main food of nearly 500,000,000 populations simultaneously, is the fourth-largest food crop after paddy rice, wheat and maize.Have 120 countries and regions a few days ago and produce banana.China's banana production development is in recent years swift and violent, occupies the whole world second.Oneself becomes the mainstay industry of China's torrid areas agricultural Banana Industry.But along with banana planting area increases year by year, the harm of banana disease is also day by day serious, especially banana blight brings great threat to banana production.1876, Joseph Bancroft in Australian reported first banana blight.Within 1904, also find this disease at Hawaii, America, 1910, Panama, because of the large generation of banana blight, cause heavy loss, thereby banana blight was called again banana Panama disease.In the same year, be separated to this sick pathogenic bacteria from banana disease tissue in Cuba by Erwin F.Smith, and by its called after Fusarium oxysporum f.cubenseE.F.Smith.Within 1913, Ashby has done detailed description first to this disease and pathogenic bacteria thereof, Brandes has confirmed the pathogenic of this germ first in 1919,1940 is F.oxysporum Schlecht.f.sp.cubense (E.F.Smith) Snyder et Hansen by Snyder and Hansen suggestion by this germ definite designation.{Moore,1999#67}
At present, this disease has been distributed widely in the banana producing region in Asia, Africa, Australia, the South Pacific and tropical America.This disease (No. 1 microspecies) infects in Central and South American countries outburst in 1940-1950, and the large honey of international trade banana variety in the Shi Fengmi world is breathed out (Gros Michel) and stepped down from the stage of history.There is banana blight in Taiwan Province of China in 1967, popular after 20 century 70s, makes the banana area in Taiwan from climax 50000 hectares of 5000 hectares of being down in recent years in period, and half wherein is still subject to the impact of blight.The also local blight that occurs in succession of the banana of the countries and regions such as Philippines, Australia, Malaysia, Africa.
In China in five sixties of 19th-century in Guangdong, Guangxi, Hainan finds that this disease infects the dwarf banana of causing harm, there is blight in the Brazil in Wan Qingsha town, Fanyu, Guangdong in 1996 and No. 2, Guangdong banana, the ground such as middle mountain, Zhuhai, Dongguan, Zhaoqing, Xinyi, Gaozhou county and Zhanjiang (Lezhou Peninsula) have now been diffused to by the route of infection such as seedling and flowing water, this disease is propagated with 30-50 kilometers of speed every year, make many any of several broadleaf plants garden can not plant banana, existing more than 1000 hectare of any of several broadleaf plants garden is no longer cultivated at present.All there is distribution in existing Guangdong, Guangxi (as Nanning, Baise), Fujian (as ZhangZhou), Hainan (as Wenchang, Wanning, Danzhou, stable, Chengmai), Yunnan (as river mouth, Jinghong) and Taiwan, and China's banana planting industry is constituted a serious threat.
This bacterium endangers the banana of different varieties because physiological strain is different, 3 physiological strains are reported in the world, the wherein No. 1 worldwide distribution of microspecies, infects the large honey of Cultivar of dwarf banana, banana and breathes out the banana strains such as (AAA) and Apple (AAB), Silk (AAB), Taiwan Latundan (AAB) and IC2 (AAAA); No. 2 microspecies distribute in Central America Honduras, El Salvador, Puerto Rico, Dominica and Virgin Islands, only infect Triploid induction Bluggoe (ABB) and nearly edge kind and some Jamaica tetraploid (AAAA) any of several broadleaf plants; No. 4 microspecies are mainly distributed in the part Asian countries such as Australia, Africa and Philippines (OK a karaoke club thunder archipelago), can infect the fragrant tooth any of several broadleaf plants (AAA) that large honey is breathed out all bananas and the plantain kinds such as (AAA), TaiwanLatundan (AAB), Pisang Lilin (AA), wild any of several broadleaf plants (BB) and other microspecies had to resistance, therefore dangerous and crushing maximum, cause showing great attention to of each banana planting country of the world.China is the most serious with No. 4, microspecies and No. 1 harm at present.
The at present not good prophylactico-therapeutic measures of this disease, and the region of harm is in continuous expansion, and more there is the phenomenon of Combined Infection for dwarf banana.Dwarf banana economic worth is high, and along with the increase of cultivated area, disease occurs to be also on the rise, and for the detection of dwarf banana seedling and the early detection of their early stage, needs more sensitive and detection method accurately and effectively.The detection of banana blight and cause of disease thereof is identified and monitor epidemic preventing working to be prerequisite and the basis that solves banana blight problem, and the foundation of detection method and application are for stoping disease spread and have great importance.
Domestic detection identification research, wait according to banana blight bacteria ITS sequences Design synthetic three primer: FusF1 (5 ' AACCCCTGTGAACATACCACTTG3 '), FusR1 (5 ' GAGGAACGCGAATTAACGCGAC3 ') and FusR2 (5 ' GACGATTACCAGTAGCGAGGGT3 ') and form primer pair A (FusF1/R1) and B (FusF1/R2) as kingdom is fragrant, Fusarium oxysporum Cuba specialized form germ has been carried out to effective PCR specific amplified test.
Detect evaluation aspect for physiological strain and mainly concentrate on physiological strain No. 4, detect primer as Taiwan Lin Yinghong etc. has set up for the regular-PCR of FOC4 specificity 404bp sequence, and apply SYBR GREEN fluorescence dye and carried out real-time fluorescence PCR detection.Fujian Li etc. has set up for the LAMP method of FOC4 specificity 404bp sequence and has detected No. 4 physiological strains.
In recent years, studies have reported that the regular-PCR evaluation problem that has solved two physiological strains.As the trials such as Liu Jingmei divide physiological strain with RAPD technical area, filter out 4 RAPD marks and be successfully converted into SCAR mark through random primer, wherein 1 of Race1-SCAR mark, 2 of Race4-SCAR marks, can identify 1 of the SCAR mark of 2 microspecies simultaneously.Apply these 4 SCAR marks and 9 germ isolates that pick up from field are detected simultaneously, can identify exactly Fusarium oxysporum Cuba specialized form Racel and the Race4 of Guangdong Jiao Qu.Li Minhui in 2012 etc. have set up the regular-PCR Molecular Detection system that can distinguish two physiological strains.But field sample germ content is low, interfering substance is many, in endogenetic fungus, bacterium and soil, actinomycetes etc. are widely distributed, the sensitivity and the specificity that detect have all been formed to great interference, cause existing detection method and often present the unsettled phenomenon of detected result because of sensitivity and specific restriction, the early detection of disease has been brought to a huge obstacle.
Real-time fluorescence PCR technology is the method that uses real-time fluorescence amplification device Real-Time Monitoring pcr amplification product and analyze.The method is more sensitive reliable compared with regular-PCR method, more stable compared with loop-mediated isothermal amplification method, and be difficult for polluting, and can realize Multiple detection, be now current sensitive and reliable detection method, be widely used.Especially probe method real-time fluorescence PCR technology because its specificity is high, can Multiple detection, resolve virus, detection of pathogens therefore be usually used in SNP.But not yet there is probe method real-time fluorescence PCR to detect the report of Fusarium oxysporum at present, more lack the real-time fluorescence PCR detection method for No. 4 physiological strains of Fusarium oxysporum Cuba specialized form specially.
Present method has been set up the TaqMan probe for real-time fluorescence PCR detection method of can precise and high efficiency distinguishing No. 4 physiological strains of Fusarium oxysporum Cuba specialized form.Wish by the foundation of the method, to solve the difficult problem that content is low, interference is many; Meanwhile, set up a kind of method that can No. 4 physiological strains of detection by quantitative Fusarium oxysporum Cuba specialized form to be applied to the comparative study of bacterial content.
Present method can be used for real-time fluorescence PCR method simultaneously and detect No. 4 physiological strains of Fusarium oxysporum Cuba specialized form, and more stable, and sensitivity is higher, meets the detection general requirement of " fast, accurate and stable, province ".As more stable in LAMP method with respect to other sensitiveer methods, be difficult for polluting, normal PCR method and inoculated identification physiological strain method are more sensitive and quick relatively, can detect at Seedling Stage, for significant in banana production.Because banana belongs to triploid crop, can not breed by traditional cross-breeding, in production, adopt tissue cultured seedling to do field planting, so for the control of field banana blight mainly in the monitoring of seedling, and bacteria containing amount in seedling is very low, need the higher detection method of sensitivity, and the method that this research is set up can address this problem preferably, application future is extensive.And because FOC4 is day by day serious in harm within Chinese territory, field investigation finds that the harm of FOC4 has the trend of further expansion.Present method has been set up TaqMan probe for real-time fluorescence detection method for FOC4 first, can be used for coordinating other physiological strain TaqMan fluoroscopic examination primer to carry out bacterial content (copy number) dual or multiple fluorescence PCR detection by quantitative point fusarium Cuba specialized form simultaneously.For monitoring and prevent further spreading and be of great importance of this physiological strain.
Special conservative primer and the TaqMan probe of Fusarium oxysporum Cuba specialized form No. 4 physiological strains of present method based on this cause of disease have been set up a kind of real-time fluorescence PCR qualitative checking method, can in 1.5 hours, carry out respectively the quantitative and qualitative analysis detection of precise and high efficiency to No. 1 physiological strain of Fusarium oxysporum Cuba specialized form simultaneously, there is high specific and highly sensitive, regular-PCR detection research has improved detection sensitivity and has reached 1-2 the order of magnitude, and can carry out detection by quantitative and Multiple detection, break through SYBRGREEN fluorescence dye method and can not carry out the restriction of double or Multiple detection.The capable precise and high efficiency of this real-time fluorescence PCR qualitative checking method, can be used in and strengthen this disease prevention and control detection and quarantine.
Summary of the invention
Primary and foremost purpose of the present invention is shortcoming and the weak point that overcomes prior art, and a kind of quick and precisely TaqMan probe primer and the detection method thereof of sensitive No. 4 physiological strains of detection Fusarium oxysporum Cuba specialized form are provided.
Another object of the present invention is to provide the application of described detection method.
The present invention adopts following scheme to realize: the TaqMan probe in detecting method of No. 4 physiological strains of a kind of rapid detection Fusarium oxysporum Cuba's specialized form, comprises following steps:
1. primer and probe
Primer adopts PrimerExpress3.0 software design, synthetic by Dalian TAKARA company.
FOC4 the primer and probe are as follows:
Foc4-0422F25’GGCTTCCAGACCGACAAGATAT3’
Foc4-0422R25’TGCTTGGCCTTGATTCTGACT3’
Foc4-0422P25’FAM-ATAATCGAACAGTTTGCG-BHQ13’
2.DNA extracts and measurement of concetration
Sample DNA extracts and adopts CTAB two-step approach, before extracting, fully grinds with liquid nitrogen, after extracting, measures nucleic acid concentration with NanoDrop1000 type nucleic acid microdetermination instrument.Concrete extraction step is as follows:
Take 1g sample (DNA content per sample can be adjusted sample size), join in the centrifuge tube of 50mL; Add 5mLCTAB extracting solution and 20 μ L Proteinase K solution, fully mix.Hatch 30min for 65 ℃, frequently vibration; 65 ℃ spend the night after, the centrifugal 10min of 8000r/min room temperature, gets 1ml supernatant liquor and enters in 2ml centrifuge tube; After adding 700 μ L chloroforms, use forced oscillation, the centrifugal 10min of 13000 × g, shifts supernatant liquor 600 μ L in new 2ml centrifuge tube; Add the CTAB precipitation solution of 2 times of volumes to put upside down the standing 60min of for several times rear room temperature, the centrifugal 10min of 13000 × g, supernatant discarded, add 350 μ LNaCl solution that precipitation is suspended, add 350 μ L chloroforms, vortex vibration mixes, the centrifugal 10min of 13000 × g again, shift and add after supernatant the Virahol of 0.8 times of volume to be used for precipitate nucleic acids, room temperature is placed 20min, the centrifugal 10min of 13000 × g, supernatant discarded, add 500 μ L70% ethanolic soln washing precipitations, be dissolved in 50 μ LTE solution.
3. real-time fluorescence PCR detection method specificity checking
Use fluorescent probe Foc4-0422P2 and primers F oc4-0422F2/Foc4-0422R2 to carry out real-time fluorescence PCR, jointly detect allied species and the outer Penicillium of Fusarium in the Fusariums such as banana fusarium moniliforme, wax gourd wilt, bitter gourd wilt bacterium and Muskmelon Fusarium wilt bacterium, Hylocereus undatus wilt.Amplification kit adopts ABI test kit 2 × TaqMan Universal PCR Master Mix II.
Real-time fluorescence PCR reaction system is as follows:
| Reagent name | Volume |
| TE damping fluid | 8.25μL |
| Primer (upstream) | 0.75μL |
| Primer (downstream) | 0.75μL |
| Probe | 0.25μL |
| TaqMan?Universal?PCR?Master?Mix | 12.5μL |
| DNA profiling (10-100ng/ μ L) | 2.5μL |
| Cumulative volume | 25μL |
Instrument adopts ABI7500 fluorescent PCR instrument, and the reaction parameter of real-time fluorescence quantitative PCR is: 50 ℃, and 5min; 94 ℃ of denaturations, 3min; 94 ℃, 15s, 55 ℃, 1min, 40 circulations.
4. regular-PCR sensitivity analysis
Use fluorescent probe the primer Foc4-0422F2/Foc4-0422R2 to carry out regular-PCR, the concentration gradient of FOC4 arranges respectively and adopts the plasmid DNA (100ng/ μ L) of extracting to carry out 1:10 serial dilution, be respectively 1, eight points such as 1:10,1:100,1:1000,1:10000,1:1000000,1:10000000,1:100000000, be respectively three repetitions.Adopt TAKARAExTaqPCR amplification kit, the explanation of reaction system reference reagent box is carried out, and expection amplified fragments is 100bp.Reaction conditions is as follows: 94 ℃ of denaturation 3min; 94 ℃ of sex change 1min, 55 ℃ of renaturation 1min, 72 ℃ are extended 20s, 25 circulations; Last 72 ℃ are extended 10min.
5. single tube dual real-time fluorescence PCR sensitivity analysis
The same regular-PCR of dilution gradient design.Adopting the plasmid DNA (100ng/ μ L) of extracting to carry out 1:10 serial dilution, be respectively 1, eight points such as 1:10,1:100,1:1000,1:10000,1:1000000,1:10000000,1:100000000, is respectively three repetitions.Reaction system and parameter arrange the same.Every sample is established three parallel reactors, and negative control and blank are set.Utilize ABI7500 type quantitative real time PCR Instrument to carry software and carry out interpretation of result, amplification curve and sample Ct value are generated automatically by software.Threshold value take Ct value 35 as judged result yin and yang attribute.According to standard amplification, Ct is worth linear equations, then can calculate template starting point concentration by the measured Ct value of testing sample.
6. detection by quantitative bacterial content (copy number) calculation formula
The molecular weight of supposing double-stranded DNA is 660Da, and copy number calculation formula is as follows:
7. detect reproducibility
Variation within batch adopts and detects lower bound template concentrations 0.00001ng/ μ L, each reaction system contains template 1 μ L, carries out first order fluorescence PCR process on 96 orifice plates, the repetition in each each 8 holes, Ct value to gained is analyzed, and calculates the variation coefficient of FOC4; Batch variation adopts template concentrations 0.000001ng/ μ L, and each reaction system contains template 1 μ L, is divided into 21 days and carries out, and carries out every three days PCR process one time, and the repetition in each each 3 holes, analyzes the Ct value of gained, calculates the variation coefficient of FOC4.
The present invention has designed the quantitative primer of TaqMan real-time fluorescence PCR of No. 4 physiological strains of Fusarium oxysporum Cuba specialized form; Standard quantitative detection system is provided.Reach following effect:
1. fluorescent PCR specificity checking
Fluorescent PCR detected result shows (as shown in Figure 1): obvious amplified signal and curve smoothing appear in FOC4 sample, and amplified signal does not appear in all the other allied specieses and the outer sample of genus.Therefore single tube double fluorescent PCR system can be in the Fusariums such as banana fusarium moniliforme, wax gourd wilt, bitter gourd wilt bacterium and Muskmelon Fusarium wilt bacterium, Hylocereus undatus wilt when Penicillium detects jointly outside allied species and Fusarium, FOC4 detected specifically, the amplification curve of finding FOC4 primer and probe is level and smooth, and fluorescent signal do not detected as health Brazil any of several broadleaf plants root tissue, bulb tissue, false stem tissue and the leaf texture of negative control.
The sensitivity of 2.FOC4 probe primer
The PCR product electrophoresis result of plasmid DNA shows (as shown in Figure 2 a): cloned plasmids DNA10
-2-10
-5four Cigarette dilution detections have arrived 100bp object band, and 10
-6-10
-8three extent of dilution have no object band, reach 10 therefore regular-PCR detects the lower bound of plasmid DNA sensitivity
-5extent of dilution, i.e. 0.001ng.
The fluorescent PCR result of plasmid DNA shows (as shown in Figure 2 b): plasmid DNA 10
-4-10
-7four dilution average Ct values are all less than 35(in table 1), be judged to the positive, and plasmid DNA 10
-8-10
-10three dilution average Ct values are all greater than 35(in table 1), be judged to and do not detect.Therefore the lower bound of detection sensitivity reaches 10
-7extent of dilution, i.e. 0.0001ng, 91258 copy numbers are high 100 times compared with regular-PCR primer.
For the starting point concentration of quantitative analysis template to be checked, corresponding relation in the Ct value (in table 1) of measuring according to each reaction tubes and pipe between contained template concentrations, application EXCEL software statistics is analyzed, be depicted as typical curve, obtain the linear equation of the typical curve (as schemed: as shown in the of 3) of FOC4 plasmid DNA fluoroscopic examination: Y=-3.584logX+18.051(Y is Ct value, X is template copy number), coefficient R 2=0.999, amplification efficiency Eff%=87.18.The linear relationship of typical curve is good as can be seen from the results, can calculate accurately template concentrations.
3. reproducibility
Detected result shows: in each processing (in table 2) of variation within batch (Intra-assay variation), the variation coefficient is 1.03%; Batch variation (Inter-assay variation) is each to be processed, 1-21 days, the variation coefficient (in table 3) within the 1st day, be up to 4.89%, the 21 day minimum be 0.78%.No matter be variation within batch, or batch variation, variation coefficient CV value is all less than 5%, illustrates that this system variability is little, good stability.Also verified that the detected result of lower concentration processing (detection lower bound) has higher repeatability, experimental result is all positive simultaneously, and false-positive situation does not appear in ultralow density processing (detecting 10 times of lower bound dilutions).
The sensitivity of the dual probe of table 1 single tube detects Ct value
Table 2 variation within batch is analyzed
Table 3FOC4 batch variation is analyzed
Accompanying drawing explanation
Fig. 1: fluorescent PCR specific detection figure;
Fig. 2: FOC4 primer regular-PCR and fluorescent PCR sensitivity detect figure (plasmid DNA): the PCR product electrophoresis result that a is plasmid DNA shows; B is that the fluorescent PCR result of plasmid DNA shows; Wherein M:DL2000Marker; 1:10
-2; 2:10
-3; 3:10
-4; 4:10
-5; 5:10
-6; 6:10
-7; 7:10
-8; 8:10
-9; 9:10
-10; 10:N;
Fig. 3: probe in detecting plasmid DNA typical curve;
Fig. 4: dual real-time fluorescence PCR detects application, wherein 1: dwarf banana; 2: Brazilian any of several broadleaf plants; 3: Brazilian any of several broadleaf plants; 4: dwarf banana; 5-8: Brazilian any of several broadleaf plants.
Embodiment
1, with FOC1 and FOC4 conidium 5 × 10
6spore liquid is hindered respectively root and is inoculated each 20 strains of dwarf banana of wide powder, inoculates and observes the symptoms respectively after 14 days, and two groups of inoculation plant see vascular bundle blackening, blade flavescence.From symptom, the yellowing degree inoculation FOC1 of blade will overweight inoculation FOC4's.The bulb tissue of getting disease symptom carries out TaqMan probe method single tube dual real-time fluorescence PCR and detects, the relatively content of FOC1 germ in bulb and the copy number difference of FOC4 germ.Result shows: the false stem of FOC1 inoculation detects that the average Ct value of FOC1 is 28.74, and copy number reaches 1130934; The false stem of FOC4 inoculation detects that the average Ct value of FOC4 is 34.22, and copy number reaches 31653.Therefore, inoculation coke breeze after 14 days, the content of FOC1 germ in dwarf banana bulb will be higher than FOC4 germ, and the former content is about 36 times of the latter.
2, gather the field 8 classes representative Banana Root tissue sample of totally 80 strains, and use the fluorescence PCR method set up, total DNA of gathered root tissue sample extraction is carried out to single tube multiple fluorescence PCR qualitative detection, and every sample is established three parallel reactors, and negative control and blank are set.
Field sample observation of symptoms result: No. 1 sample picks up from dwarf banana of the wide powder of morbidity, and disease is seen vascular bundle blackening, and blade flavescence is wilting; No. 2 samples pick up from the severe Brazilian any of several broadleaf plants that falls ill, and disease is seen vascular bundle blackening, blade flavescence; No. 3 sample picks up from their early stage Brazil any of several broadleaf plants, and disease is seen vascular bundle blackening, blade flavescence; No. 4 sample picks up from the dwarf banana of wide powder that do not fall ill, without disease; 5-8 sample picks up from any of several broadleaf plants without disease Brazil.
Large Tanaka is adopted to representative banana sample and carry out the detection of TaqMan probe method single tube dual real-time fluorescence PCR, result shows (as shown in Figure 4): No. 1 sample detection is FOC1 and FOC4 Combined Infection, No. 2 sample detection are that FOC4 infects, No. 3 sample detection are that FOC4 infects, No. 4 sample detection are that FOC1 infects, and 5-8 sample does not detect.Therefore, adopt single tube double fluorescent PCR method, can in a reaction system, detect two microspecies simultaneously, also can detect respectively two microspecies; Meanwhile, the method can also detect the pathogenic bacteria in the early stage Banana Root tissue of falling ill without disease, has the sensitivity higher than regular-PCR.
The fluorescent mark of the probe in this programme can substitute with cohort labelling thing, as Cy5 can use 5'-Biotin, 5'-Cy3,, the 5' fluorescent marker such as 5'-Digoxin, 5'-FAM, 5'-HEX, 5'-JOE, 5'-ROX, 5'-TAMRA, 5'-Amino, 5'-Phosphorylation, 5'-ROX, 5'-SHC6,5'-SIMA (HEX), 5'-TET is alternative; BHQ1 can use the 3' fluorescent markers such as 3'-JOE, 3'-BHQ2,3'-ROX, 3'-BHQ3,3'-TAMRA, 3'-Biotin, 3'-BiotinTEG, 3'Dabcyl, 3'Dabsyl, 3'-TET, 3'-Digoxin, 3'-Amino, 3'-diThiol, 3'-Eclipse, 3'-FAM, 3'-HEX, 3'-Phosphorylation, 3'-SHC6,3'-SHC3 to substitute.After substituting, can reach same detection effect.
Innovation point of the present invention is:
(1) by real time fluorescent PCR method success qualitative detection banana blight bacteria (No. 1 physiological strain of sharp fusarium Cuba specialized form);
(2) by real time fluorescent PCR method success detection by quantitative banana blight bacteria (No. 1 physiological strain of sharp fusarium Cuba specialized form).
(3) this cover primer and probe can coordinate other physiological strain TaqMan fluoroscopic examination primer to carry out bacterial content (copy number) dual or multiple fluorescence PCR detection by quantitative point fusarium Cuba specialized form.
Claims (7)
- One kind detect Fusarium oxysporum Cuba specialized form No. 4 physiological strains ( fusarium oxysporumf. sp. cubenserace 4) TaqMan primer and probe, wherein, described primer sequence is: upstream primer Foc4-0422F2 5 ' GGCTTCCAGACCGACAAGATAT3 ', downstream primer Foc4-0422R2 5 ' TGCTTGGCCTTGATTCTGACT3 ', probe sequence is 5 ' fluorescent mark, the DNA sequence dna of 3 ' quencher mark, described sequence is: Foc4-0422P2 5 ' Dye-ATAATCGAACAGTTTGCG-Quen 3 '.
- 2. probe according to claim 1, wherein said fluorescent agent is selected from Biotin, Cy3, Cy5, Digoxin, FAM, HEX, JOE, ROX, TAMRA, Amino, Phosphorylation, ROX, SHC6, SIMA (HEX), TET.
- 3. probe according to claim 1, wherein said quencher is selected from JOE, BHQ1, BHQ2, ROX, BHQ3, TAMRA, Biotin, BiotinTEG, Dabcyl, Dabsyl, TET, Digoxin, Amino, diThiol, Eclipse, FAM, HEX, Phosphorylation, SHC6, SHC3.
- 4. primer as claimed in claim 1 and probe, wherein said probe be Foc4-0422P2 5 ' FAM-ATAATCGAACAGTTTGCG-BHQ1 3 '.
- One kind detect Fusarium oxysporum Cuba specialized form No. 4 physiological strains ( fusarium oxysporumf. sp. cubenserace 4) test kit, it is characterized in that comprising primer and probe, described primer and the sequence of probe are: upstream primer Foc4-0422F2 5 ' GGCTTCCAGACCGACAAGATAT3 ', downstream primer Foc4-0422R2 5 ' TGCTTGGCCTTGATTCTGACT3 ', probe sequence is: Foc4-0422P2 5 ' FAM-ATAATCGAACAGTTTGCG-BHQ1 3 '.
- 6. the test kit of No. 4 physiological strains of detection Fusarium oxysporum Cuba specialized form as claimed in claim 5, characterized by further comprising and implement needed other reagent of quantitative fluorescent PCR, be specially TaqMan Universal PCR Master Mix, the plasmid template that comprises No. 4 physiological strain DNA fragmentations of Fusarium oxysporum Cuba specialized form, TE damping fluid.
- 7. a method that detects No. 4 physiological strains of Fusarium oxysporum Cuba specialized form, is characterized in that, utilizes probe and primer described in claim 1 to carry out the detection of TaqMan fluorescent quantitation, and concrete grammar comprises:1) sample DNA extracts and adopts CTAB two-step approach, before extracting, fully grinds with liquid nitrogen, after extracting, measures nucleic acid concentration with NanoDrop1000 type nucleic acid microdetermination instrument.Concrete extraction step is as follows:Take 1g sample (DNA content per sample can be adjusted sample size), join in the centrifuge tube of 50mL; Add 5mLCTAB extracting solution and 20 μ L Proteinase K solution, fully mix.Hatch 30min for 65 ℃, frequently vibration; 65 ℃ spend the night after, the centrifugal 10min of 8000r/min room temperature, gets 1ml supernatant liquor and enters in 2ml centrifuge tube; After adding 700 μ L chloroforms, use forced oscillation, the centrifugal 10min of 13000 × g, shifts supernatant liquor 600 μ L in new 2ml centrifuge tube; Add the CTAB precipitation solution of 2 times of volumes to put upside down the standing 60min of for several times rear room temperature, the centrifugal 10min of 13000 × g, supernatant discarded, add 350 μ LNaCl solution that precipitation is suspended, add 350 μ L chloroforms, vortex vibration mixes, the centrifugal 10min of 13000 × g again, shift and add after supernatant the Virahol of 0.8 times of volume to be used for precipitate nucleic acids, room temperature is placed 20min, the centrifugal 10min of 13000 × g, supernatant discarded, add 500 μ L70% ethanolic soln washing precipitations, be dissolved in 50 μ LTE solution;2) real-time fluorescence PCR detection method specificity checkingUse fluorescent probe Foc4-0422P2 and primers F oc4-0422F2/Foc4-0422R2 to carry out real-time fluorescence PCR, jointly detect allied species and the outer Penicillium of Fusarium in the Fusariums such as banana fusarium moniliforme, wax gourd wilt, bitter gourd wilt bacterium and Muskmelon Fusarium wilt bacterium, Hylocereus undatus wilt.Amplification kit adopts ABI test kit 2 × TaqMan Universal PCR Master Mix II,Real-time fluorescence PCR reaction system is as follows:
Reagent name Volume TE damping fluid 8.25μL Primer (upstream) 0.75μL Primer (downstream) 0.75μL Probe 0.25μL TaqMan?Universal?PCR?Master?Mix 12.5μL DNA profiling (10-100ng/ μ L) 2.5μL Cumulative volume 25μL Wherein, instrument adopts ABI7500 fluorescent PCR instrument, and the reaction parameter of real-time fluorescence quantitative PCR is: 50 ℃, and 5min; 94 ℃ of denaturations, 3min; 94 ℃, 15s, 55 ℃, 1min, 40 circulations.
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Cited By (4)
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| CN105441543A (en) * | 2015-12-04 | 2016-03-30 | 江苏省农业科学院 | Primers for identifying fusarium oxysporum watermelon forma specialis physiological races and application thereof |
| CN108841984A (en) * | 2018-06-29 | 2018-11-20 | 苏州百源基因技术有限公司 | It is a kind of for detecting nucleotide sequence group, kit and the method for Fusarium oxysporum |
| CN114480700A (en) * | 2021-12-27 | 2022-05-13 | 仲恺农业工程学院 | PCR primer, method and application for detecting and identifying physiological race 1 and 4 of banana fusarium oxysporum |
| CN116669541A (en) * | 2020-12-24 | 2023-08-29 | 贝霍种子有限公司 | Fusarium resistance in celery |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105441543A (en) * | 2015-12-04 | 2016-03-30 | 江苏省农业科学院 | Primers for identifying fusarium oxysporum watermelon forma specialis physiological races and application thereof |
| CN105441543B (en) * | 2015-12-04 | 2018-11-13 | 江苏省农业科学院 | It is a kind of identification Fusarium oxysporum f. sp. niveum biological strain primer and its application |
| CN108841984A (en) * | 2018-06-29 | 2018-11-20 | 苏州百源基因技术有限公司 | It is a kind of for detecting nucleotide sequence group, kit and the method for Fusarium oxysporum |
| CN116669541A (en) * | 2020-12-24 | 2023-08-29 | 贝霍种子有限公司 | Fusarium resistance in celery |
| CN114480700A (en) * | 2021-12-27 | 2022-05-13 | 仲恺农业工程学院 | PCR primer, method and application for detecting and identifying physiological race 1 and 4 of banana fusarium oxysporum |
| CN114480700B (en) * | 2021-12-27 | 2024-03-19 | 仲恺农业工程学院 | PCR primer, method and application for detecting and identifying banana fusarium wilt bacteria No.1 and No. 4 physiological race |
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