CN103789417B - 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 SYBR? The GREEN fluorescent dye method does not have the limitation of being able to perform two-fold or multiple assays. 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 the TaqMan probe real-time fluorescence primer of a kind of Rapid identification and detection by quantitative Fusarium oxysporum Cuba specialized form No. 4 physiological strains and detection method thereof.
Background technology
By Fusarium oxysporum No. 1 (Fusariumoxysporumf.sp.cubenserace1, FOC1) and No. 4 physiological strain (F.oxysporumf.sp.cubenserace4, FOC4) research of fusarium wilt disesase of banana caused, 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.Banana Industry oneself become the mainstay industry of China's torrid areas agricultural.But along with banana planting area increases year by year, the harm of banana disease is also day by day serious, and especially banana blight brings great threat to banana production.1876, JosephBancroft is banana blight in Australian reported first.Within 1904, also find that this is sick, 1910 at Hawaii, America, Panama is because of the large generation of banana blight, and cause heavy loss, thus banana blight is also called banana Panama disease.In the same year, from banana disease tissue, be separated to the pathogenic bacteria of this disease in Cuba by ErwinF.Smith, and by its called after Fusariumoxysporumf.cubenseE.F.Smith.Within 1913, Ashby has done detailed description first to this disease and pathogenic bacteria thereof, Brandes confirmed the pathogenic of this germ first in 1919, within 1940, advised this germ definite designation being F.oxysporumSchlecht.f.sp.cubense (E.F.Smith) SnyderetHansen by Snyder and Hansen.{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 1940-1950 in Central and South American countries outburst, and the large honey of international trade banana variety in the Shi Fengmi world is breathed out (GrosMichel) and stepped down from the stage of history.There is banana blight in Taiwan Province of China in 1967, popular after 20 century 70s, make the banana area in Taiwan from 50000 hectares of 5000 hectares of being down in recent years in climax period, and half wherein is still subject to the impact of blight.Also in succession locally there is blight in the banana of the countries and regions such as Philippines, Australia, Malaysia, Africa.
In China in 19th-century five sixties in Guangdong, Guangxi, Hainan finds that this disease infects dwarf banana of causing harm, there is blight in Brazil and No. 2, the Guangdong banana in Qing Sha town, Fanyu ten thousand, Guangdong in 1996, now diffuse to middle mountain, Zhuhai, Dongguan, Zhaoqing, Xinyi, the ground such as Gaozhou county and Zhanjiang (Lezhou Peninsula) 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 gardens 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, constitute a serious threat to China's banana planting industry.
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, the banana strains such as (AAA) and Apple (AAB), Silk (AAB), TaiwanLatundan (AAB) and IC2 (AAAA) breathed out by the large honey of Cultivar of infection dwarf banana, banana; No. 2 microspecies, in Central America Honduras, El Salvador, Puerto Rico, Dominica and Virgin Islands distribution, only infect Triploid induction Bluggoe (ABB) and Closely related variety thereof 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), the fragrant tooth any of several broadleaf plants (AAA) that large honey is breathed out all bananas such as (AAA), TaiwanLatundan (AAB), PisangLilin (AA), wild any of several broadleaf plants (BB) and plantain kind and other microspecies had to resistance can be infected, therefore dangerous and destructiveness is maximum, cause showing great attention to of each banana planting country of the world.Current China with No. 4, microspecies and No. 1 harm the most serious.
The prophylactico-therapeutic measures that this disease is not good at present, and the region of harm is in continuous expansion, and dwarf banana is more existed to the phenomenon of Combined Infection.Dwarf banana economic worth is high, and along with the increase of cultivated area, disease occurs also to be 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.Be the prerequisite and the basis that solve banana blight problem to the Testing and appraisal of banana blight and cause of disease thereof and monitoring epidemic preventing working, the foundation of detection method and application spread for prevention disease and have great importance.
Domestic Testing and appraisal research, as kingdom's sweet smell etc. forms primer pair A (FusF1/R1) and B (FusF1/R2) according to three primer: FusF1 (5 ' AACCCCTGTGAACATACCACTTG3 '), the FusR1 (5 ' GAGGAACGCGAATTAACGCGAC3 ') of banana blight bacteria ITS sequence design and synthesis and FusR2 (5 ' GACGATTACCAGTAGCGAGGGT3 '), effective PCR specific amplified test is carried out to Fusarium oxysporum Cuba specialized form germ.
Mainly concentrate on No. 4 physiological strains for physiological strain Testing and appraisal aspect, the regular-PCR established for FOC4 specificity 404bp sequence as Taiwan LinYinghong etc. detects primer, and applies SYBRGREEN fluorescence dye and carried out real-time PCR detection.The LAMP method established for FOC4 specificity 404bp sequence such as Fujian Li detect No. 4 physiological strains.
In recent years, studies have reported that the regular-PCR qualification problem solving two physiological strains.As physiological strain is divided in the trial RAPD technical areas such as Liu Jingmei, filter out 4 RAPD mark through random primer and be successfully converted into SCAR mark, wherein Race1-SCAR marks 1, Race4-SCAR marks 2, can identify the SCAR mark 1 of 2 microspecies simultaneously.Apply these 4 SCAR mark to detect 9 the germ isolates picking up from field simultaneously, Fusarium oxysporum Cuba specialized form Racel and Race4 of Guangdong Jiao Qu can be identified exactly.Li Minhui in 2012 etc. have established the regular-PCR Molecular Detection system can distinguishing two physiological strains.But field sample germ content is low, interfering substance is many, in endogenetic fungus, bacterium and soil, actinomycetes equal distribution is extensive, great interference is all constituted to the sensitivity detected and specificity, cause existing detection method often because of sensitivity and specific restriction to present the phenomenon of detected result instability, a huge obstacle is brought to the early detection of disease.
Real-time fluorescence PCR technology is the method using real-time fluorescence amplification device Real-Time Monitoring pcr amplification product carrying out to analyze.The method is more reliably sensitive compared with regular-PCR method, and comparatively loop-mediated isothermal amplification method is more stable, not easily pollutes, and can realize Multiple detection, is sensitive and reliable detection method of passing through now, is widely used.Especially probe method real-time fluorescence PCR technology because its specificity is high, can Multiple detection, therefore be usually used in SNP and resolve, virus, detection of pathogens.But not yet there is the report of probe method real-time PCR detection Fusarium oxysporum at present, more lack specially for the real-time fluorescence PCR detection method of Fusarium oxysporum Cuba specialized form No. 4 physiological strains.
Present method establishes can distinguish the TaqMan probe real-time fluorescence PCR detection method of Fusarium oxysporum Cuba specialized form No. 4 physiological strains by precise and high efficiency.Wish by the foundation of the method, the difficult problem that content is low, interference is many to be solved; Meanwhile, establish a kind of can the method for detection by quantitative Fusarium oxysporum Cuba specialized form No. 4 physiological strains to be applied to the comparative study of bacterial content.
Present method can be used for real-time fluorescence PCR method simultaneously and detect Fusarium oxysporum Cuba specialized form No. 4 physiological strains, and more stable, and sensitivity is higher, meets the detection general requirement of " fast, accurate and stable, province ".The method sensitiveer relative to other is as more stable in LAMP method, not easily pollutes, relative traditional PCR method and inoculated identification physiological strain method more sensitive and quick, can detect at Seedling Stage, for significant in banana production.Because banana belongs to triploid crop, do not breed by traditional cross-breeding, production adopt tissue cultured seedling do field planting, so for the control of field banana blight mainly in the monitoring of seedling, and the bacteria containing amount in seedling is very low, need the detection method that sensitivity is higher, and the method that this research is set up can address this problem preferably, application future is extensive.And because FOC4 endangers day by day serious within Chinese territory, field investigation finds that the harm of FOC4 has the trend expanded further.Present method establishes TaqMan probe real-time fluorescence detection method for FOC4 first, can be used for coordinating other physiological strain TaqMan fluorescent detector primers to carry out bacterial content (copy number) that is dual or multiple fluorescence PCR detection by quantitative point fusarium Cuba specialized form simultaneously.For monitoring and preventing spreading further of this physiological strain to be of great importance.
Present method establishes a kind of real-time fluorescence PCR qualitative checking method based on the special conservative primer of Fusarium oxysporum Cuba specialized form No. 4 physiological strains of this cause of disease and TaqMan probe, the quantitative and qualitative analysis detection of precise and high efficiency can be carried out respectively to Fusarium oxysporum Cuba specialized form No. 1 physiological strain in 1.5 hours simultaneously, there is high specific and highly sensitive, regular-PCR detect delay comparatively improves detection sensitivity and reaches 1-2 the order of magnitude, and can detection by quantitative and Multiple detection be carried out, breach SYBRGREEN fluorescent dye determination and can not carry out restriction that is double or Multiple detection.The capable precise and high efficiency of this real-time fluorescence PCR qualitative checking method, can be used in strengthening this disease prevention and control detection and quarantine.
Summary of the invention
Primary and foremost purpose of the present invention is the shortcoming and the weak point that overcome prior art, provides TaqMan probe primer and the detection method thereof of a kind of quick and precisely sensitive detection Fusarium oxysporum Cuba specialized form No. 4 physiological strains.
Another object of the present invention is to the application that described detection method is provided.
The present invention adopts following scheme to realize: the TaqMan probe detection method of a kind of rapid detection Fusarium oxysporum Cuba specialized form No. 4 physiological strains, comprises following steps:
1. primer and probe
Primer adopts PrimerExpress3.0 software design, is synthesized by Dalian TAKARA company.
FOC4 the primer and probe 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, fully grinds before extracting with liquid nitrogen, measures nucleic acid concentration after extracting with NanoDrop1000 type nucleic acid microdetermination instrument.Concrete extraction step is as follows:
Take 1g sample (DNA content per sample can adjust sample size), join in the centrifuge tube of 50mL; Add 5mLCTAB extracting solution and 20 μ L Proteinase K Solution, fully mix.Hatch 30min, frequently vibrate for 65 DEG C; 65 DEG C spend the night after, the centrifugal 10min of 8000r/min room temperature, gets 1ml supernatant liquor and enters in 2ml centrifuge tube; Use forced oscillation, the centrifugal 10min of 13000 × g after adding 700 μ L chloroforms, transfer supernatant liquor 600 μ L is in new 2ml centrifuge tube; The CTAB precipitation solution adding 2 times of volumes is put upside down room temperature for several times and is left standstill 60min, the centrifugal 10min of 13000 × g, supernatant discarded, add 350 μ LNaCl solution precipitation is suspended, add 350 μ L chloroforms again, vortex oscillation mixes, the centrifugal 10min of 13000 × g, the Virahol adding 0.8 times of volume after transfer supernatant is used for precipitate nucleic acids, room temperature places the centrifugal 10min of 20min, 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 verification
Fluorescent probe Foc4-0422P2 and primers F oc4-0422F2/Foc4-0422R2 is used to carry out real-time fluorescence PCR, allied species and the outer Penicillium of Fusarium in common detection banana fusarium moniliforme, wax gourd wilt, bitter gourd wilt bacterium and the Fusarium such as Melon fusarium Wilt, Hylocereus undatus wilt.Amplification kit adopts ABI test kit 2 × TaqManUniversalPCRMasterMixII.
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 DEG C, 5min; Denaturation 94 DEG C, 3min; 94 DEG C, 15s, 55 DEG C, 1min, 40 circulations.
4. regular-PCR sensitivity analysis
Fluorescent probe the primer Foc4-0422F2/Foc4-0422R2 is used to carry out regular-PCR, the concentration gradient of FOC4 arranges respectively and adopts the plasmid DNA (100ng/ μ L) extracted 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, reaction system reference reagent box illustrates and carries out, and expection amplified fragments is 100bp.Reaction conditions is as follows: 94 DEG C of denaturation 3min; 94 DEG C of sex change 1min, 55 DEG C of renaturation 1min, 72 DEG C extend 20s, 25 circulations; Last 72 DEG C extend 10min.
5. single tube dual real-time fluorescence PCR sensitivity analysis
The same regular-PCR of dilution gradient design.Adopt the plasmid DNA (100ng/ μ L) extracted 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.Reaction system and optimum configurations the same.Three parallel reactors established by every sample, and arrange negative control and blank.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.With the threshold value that Ct value 35 is judged result yin and yang attribute.Be worth linear equations according to standard amplification Ct, then the Ct value measured by testing sample can calculate template starting point concentration.
6. detection by quantitative bacterial content (copy number) calculation formula
Assuming that the molecular weight of double-stranded DNA is 660Da, copy number calculation formula is as follows:
7. detect reproducibility
Intra-assay variability adopts detect and track template concentrations 0.00001ng/ μ L, each reaction system contains template 1 μ L, and one piece of 96 orifice plate carries out first order fluorescence PCR process, at every turn the repetition in each 8 holes, the Ct value of gained is analyzed, 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 within 21 days, carry out, and carries out a PCR process every three days, at every turn the repetition in each 3 holes, analyzes the Ct value of gained, calculates the variation coefficient of FOC4.
The present invention devises the quantitative primer of TaqMan real-time fluorescence PCR of Fusarium oxysporum Cuba specialized form No. 4 physiological strains; Provide standard quantitative detection system.Reach following effect:
1. fluorescent PCR specificity verification
Fluorescent PCR detected result display (as shown in Figure 1): curve smoothing appears obvious amplified signal and in FOC4 sample, there is not amplified signal in all the other allied specieses and the outer sample of genus.Therefore single tube double fluorescent PCR system can when in banana fusarium moniliforme, wax gourd wilt, bitter gourd wilt bacterium and the Fusarium such as Melon fusarium Wilt, Hylocereus undatus wilt, the outer Penicillium of allied species and Fusarium detects jointly, 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 of negative control, bulb tissue, false stem tissue and leaf texture.
The sensitivity of 2.FOC4 probe primer
PCR primer electrophoresis result display (as shown in Figure 2 a) of plasmid DNA: 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, therefore the lower bound that regular-PCR detects plasmid DNA sensitivity reaches 10
-5extent of dilution, i.e. 0.001ng.
Fluorescent PCR result display (as shown in Figure 2 b) of plasmid DNA: 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, comparatively common PCR primers is high 100 times.
In order to the starting point concentration of quantitative analysis template to be checked, corresponding relation in the Ct value (see table 1) measured 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 shown in figure: 3) of FOC4 plasmid DNA fluoroscopic examination: Y=-3.584logX+18.051(Y is Ct value, X is template copy numbers), 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 template concentrations accurately.
3. reproducibility
Detected result shows: in each process (see table 2) of intra-assay variability (Intra-assayvariation), the variation coefficient is 1.03%; During batch variation (Inter-assayvariation) respectively processes, 1-21 days, the variation coefficient (see table 3) is up to 4.89% on the 1st day, and the 21st day minimum is 0.78%.No matter be intra-assay variability, or batch variation, variation coefficient CV value is all less than 5%, illustrates that this system variability is little, good stability.The detected result simultaneously also demonstrating lower concentration process (detect and track) has higher repeatability, and experimental result is the positive, and false-positive situation does not appear in ultralow density process (detect and track dilutes 10 times).
The sensitivity technique Ct value of table 1 single tube dual probe
The analysis of table 2 intra-assay variability
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 technique figure (plasmid DNA): a is the PCR primer electrophoresis result display of plasmid DNA; B is the fluorescent PCR result display of plasmid DNA; 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 root respectively and is inoculated each 20 strains of wide powder dwarf banana, inoculates and observes the symptoms respectively after 14 days, and two groups of inoculation plant see vascular bundle blackening, and blade turns yellow.From symptom, will the overweighting of yellowing degree inoculation FOC1 of blade inoculates FOC4's.The bulb tissue getting disease symptom carries out TaqMan probe method single tube dual real-time fluorescence PCR and detects, and compares the 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 values of FOC1 is 28.74, and copy number reaches 1130934; The false stem of FOC4 inoculation detects that the Average Ct values of FOC4 is 34.22, and copy number reaches 31653.Therefore, inoculation coke breeze is after 14 days, and the content of FOC1 germ in dwarf banana bulb is higher than FOC4 germ, and the former content is about 36 times of the latter.
2, the field 8 class representative Banana Root tissue sample of totally 80 strains is gathered, and use the fluorescence PCR method set up, carry out single tube multiple fluorescence PCR qualitative detection to the STb gene of gathered root tissue sample extraction, three parallel reactors established by every sample, and arrange negative control and blank.
Field sample observation of symptoms result: No. 1 sample picks up from morbidity wide powder dwarf banana, and disease sees vascular bundle blackening, and blade turns yellow wilting; No. 2 samples pick up from severe and to fall ill Brazilian any of several broadleaf plants, and disease sees vascular bundle blackening, and blade turns yellow; No. 3 samples pick up from their early stage Brazil any of several broadleaf plants, and disease sees vascular bundle blackening, and blade turns yellow; No. 4 samples pick up from the wide powder dwarf banana that do not fall ill, without disease; 5-8 sample picks up from without disease Brazil any of several broadleaf plants.
Representative Banana swatches is adopted to large Tanaka and carries out the detection of TaqMan probe method single tube dual real-time fluorescence PCR, result display (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, two microspecies can be detected in a reaction system simultaneously, also can detect two microspecies respectively; Meanwhile, the method can also detect without disease be in morbidity early stage Banana Root tissue in pathogenic bacteria, there is 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'-Cy3,5'-Digoxin, 5'-FAM, 5'-HEX, 5'-JOE, 5'-ROX, 5'-TAMRA, 5'-Amino, 5'-Phosphorylation, 5'-ROX, 5'-SHC6,5'-SIMA (HEX), the 5' fluorescent marker such as 5'-TET be alternative; BHQ1 can substitute with 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.Same Detection results can be reached after substituting.
Innovation point of the present invention is:
(1) with real time fluorescent PCR method success qualitative detection banana blight bacteria (No. 1 physiological strain of sharp fusarium Cuba specialized form);
(2) have detected banana blight bacteria (No. 1 physiological strain of sharp fusarium Cuba specialized form) with real time fluorescent PCR method successful quantitation.
(3) this cover primer and probe can coordinate other physiological strain TaqMan fluorescent detector primers to carry out bacterial content (copy number) that is dual or multiple fluorescence PCR detection by quantitative point fusarium Cuba specialized form.
Sequence table
<110> The People's Republic of China Zhongshan Entry-Exit Inspection & Quarantine Bure
<120> detects TaqMan probe real-time fluorescence primer and the application thereof of Fusarium oxysporum Cuba specialized form No. 4 physiological strains
<160>3
<170>PatentInversion3.5
<210>1
<211>22
<212>DNA
<213> synthetic
<400>1
ggcttccagaccgacaagatat22
<210>2
<211>21
<212>DNA
<213> synthetic
<400>2
tgcttggccttgattctgact21
<210>3
<211>18
<212>DNA
<213> synthetic
<400>3
ataatcgaacagtttgcg18
Claims (2)
1. one kind is detected TaqMan primer and the probe of Fusarium oxysporum Cuba specialized form No. 4 physiological strains (Fusariumoxysporumf.sp.cubenserace4), wherein, described primer sequence is: upstream primer Foc4 ?0422F25 ’ ?GGCTTCCAGACCGACAAGATAT ?3 ', downstream primer Foc4 ?0422R25 ’ ?TGCTTGGCCTTGATTCTGACT ?3 ', probe sequence is 5 ' fluorescent mark, the DNA sequence dna of 3 ' quencher labels, described sequence is: Foc4 ?0422P25 ’ ?Ying Guang Ji ?ATAATCGAACAGTTTGCG ?cancellation Ji ?3 '.
2. primer as claimed in claim 1 and probe, wherein said probe be Foc4 ?0422P25 ’ ?FAM ?ATAATCGAACAGTTTGCG ?BHQ1 ?3 '.
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| 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 |
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| 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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Non-Patent Citations (2)
| Title |
|---|
| A molecular diagnosis method using real-time PCR for quantification and detection of Fusarium oxysporum f. sp. cubense race 4;Ying-Hong Lin，et al;《Eur J Plant Pathol》;20120926;395-405页 * |
| Development of a Real-Time Fluorescence Loop-Mediated Isothermal Amplification Assay for Rapid and Quantitative Detection of Fusarium oxysporum f.sp.cubense Tropical Race 4 In Soil;Xin Zhang，et al;《PLOS ONE》;20131220;第8卷(第12期);1-10页 * |
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