CN107022624A - The LAMP methods and kit of quick detection bakanae disease of rice germ from seed rice - Google Patents
The LAMP methods and kit of quick detection bakanae disease of rice germ from seed rice Download PDFInfo
- Publication number
- CN107022624A CN107022624A CN201710316754.1A CN201710316754A CN107022624A CN 107022624 A CN107022624 A CN 107022624A CN 201710316754 A CN201710316754 A CN 201710316754A CN 107022624 A CN107022624 A CN 107022624A
- Authority
- CN
- China
- Prior art keywords
- rice
- primer
- bakanae disease
- lamp
- rice germ
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Botany (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of LAMP specific primer sets things for bakanae disease of rice germ quick determination method, LAMP specific primer sets thing is made up of upstream and downstream outer primer F3, B3 and upstream and downstream inner primer FIP, BIP this four primers.The present invention further simultaneously discloses loop-mediated isothermal amplification kit and corresponding loop-mediated isothermal amplification method：Then method one, the first dyestuff hydroxynaphthol blue that added before amplification carry out LAMP amplified reactions as reaction indicator；After reaction terminates, color changes, and is judged as the positive, otherwise is judged as feminine gender；Method two, amplified production is taken, detected with 2% agarose gel electrophoresis, the positive is judged as if there is trapezoid-shaped strips, otherwise be judged as feminine gender.
Description
Technical field
The invention belongs to plant epiphyte technical field of molecular biological detection, it is related to ring mediated isothermal amplification (LAMP) skill
Art detects the loop-mediated isothermal amplification (LAMP) primer group and its application method of fusarium moniliforme bacterium, belongs to plant disease detection, identification
And the early warning technical field of preventing and treating.
Background technology
Bakanae disease of rice is a kind of transmissibility fungal disease, has generation in each paddy rice main producing region, seedling stage to heading stage is all
Can occur, fall ill light field underproduction 5-50%.The disease is mainly invaded by rattan storehouse Fusariumsp (Fusraium.fujikuroi)
Dye causes, carry pathogen seed be bakanae disease of rice main source of infection, if by the seed carried disease germs and by health
Seed is soaked seed together, and disease-free seed can be contaminated.When seed grows sprouting, germ just can invade seedling by bud scale, with band
The growth of bacterium seedling, mycelia gradually spreads to complete stool, and a series of symptom, such as internode elongation occurs, and section portion exposes to the open air and leaf sheath
Outside, tillering ability weakens, and leaf color jaundice, root system development is abnormal.The light plant of occurring degree does sth. in advance heading, and fringe shape is small and unreal.
At heading stage, it is also possible to occur disease, grain ear browning when serious, it is impossible to solid.Paddy infection in 21 days after rice strain heading
The probability of germ is highest, and the seed carried disease germs in threshing turns into the primary source of infection in next season.Therefore bakanae disease of rice is set up
Bacterium rapid detection system, is quick and precisely detected to infected rice paddy seed in early days, and to prediction disease, a situation arises, in time
The propagation and prevalence, reduction economic loss for taking effectively preventing controlling measurement pathogen all have important theoretical and actual meaning
Justice.
Still continuing to use traditional tissue mostly, which is separately cultured and Morphological Identification method, is detected to fusarium moniliforme at present,
But, time-consuming for this authentication method, and the general one-time detection that completes wants more than one week, and workload is big, and Success rate of virus isolation is not
Height, there is significant limitation.If separated disease sample is stale, it is more difficult to make a definite diagnosis pathogen, it is difficult to meet to paddy rice
Bakanae disease diagnosis is actually needed, and this detection and prevention and control to pathogen is very unfavorable.With the related authentication method of nucleic acid not
Disconnected development, the method based on regular-PCR has been used successfully to the detection of pathogen, but PCR method specificity etc. and has detected time-consuming partially long,
6~8h is needed, while PCR method needs expensive instrument PCR instrument, detection process is more numerous and diverse.And most of PCR detection methods are all
Carried out with the culture of germ, early stage needs to carry out the separation of germ, culture and purified, it is impossible to carry out Site Detection, actual to answer
With having little significance.
Loop-mediated isothermal amplification technique (Loop-mediated isothermal amplification, LAMP) is Japan
A kind of new nucleic acid amplification technologies of Rong Yan Co., Ltd. invention, because its expansion is easy to operate, quick, specific high, cost is low
The advantages of, as PCR new nucleic acid amplification technologies can be substituted.It is 6 regions design, 4 pairs of specificity for target gene
Primer, causes in the presence of Bst Large fragment polymerases in self-loopa strand replacement reaction, 60-65 DEG C of scope 60min, can be a large amount of
Synthesize target dna.The detection of amplified production is typically using fluorescent dye visual observations, agarose gel electrophoresis and turbidity observation etc.
Method.Because LAMP amplification procedures rely on identification 6 isolated areas of target sequence, so atopic is very strong, and nucleic acid expands
Increasing process is carried out under constant temperature, and common water-bath or isothermal vacuum flask can just meet reaction and require, testing cost drop
Low, required time is short.Because reaction is simple, quick, efficient, economic dispatch feature, thus with extremely wide application prospect.
The content of the invention
The technical problem to be solved in the present invention is to provide a set of ring mediated isothermal amplification for being used to detect fusarium moniliforme
Primer composition and the kit containing the primer, and a kind of quick detection bakanae disease of rice germ from seed rice is provided
LAMP methods.
In order to solve the above-mentioned technical problem, the present invention provides a kind of following LAMP primer composition for detecting fusarium moniliforme
Thing, by upstream and downstream outer primer, four primer compositions of upstream and downstream inner primer, primer sequence is as follows：
Upstream outer primer (F3)：5’—CGGCCCTTATACCCCATTC—3’；
Downstream outer primer (B3)：5’—GCGCCACTATTGCTGTCT—3’；
Upstream inner primer (FIP)：5’—ACTTGCGCTGGAAAGACCAGAACATCCGCCACCTCACTGA—3’；
Downstream inner primer (BIP)：5’—GGTCACCAATCCTCGGCTACAGCCCTAGGAGATGGTCGCTTA—3’.
The present invention also provides one kind simultaneously to be used to detect bakanae disease of rice germ loop-mediated isothermal amplification kit, comprising
Above-mentioned LAMP specific primer sets thing.
It is used as the improvement of the kit of the present invention：It is just interior to primers F IP, 1.6 μM of reverse inner primer comprising 1.6 μM
BIP, 0.2 μM of positive outer primer F3,0.2 μM of reverse outer primer B3；Also include：10×ThermoPol Buffer、1mmol/L
dNTPs、4mmol/L MgCl2, 0.6mmol/L glycine betaines, 150mmol/L hydroxynaphthol blues (HNB), 8U/ μ L Bst DNA gather
Synthase, ddH2O。
That is, LAMP kit includes loop-mediated isothermal amplification (LAMP) primer mixed liquor and loop-mediated isothermal amplification premixed liquid structure
Into detection solution, primer combination mixed liquid concentration be：1.6 μM just interior to primers F IP, 1.6 μM of reverse inner primer BIP,
0.2 μM of positive outer primer F3,0.2 μM of reverse outer primer B3；Loop-mediated isothermal amplification premixed liquid：10×ThermoPol
Buffer、1mM dNTPs、4mM MgCl2, 0.6M glycine betaines, 150 μM of hydroxynaphthol blues (HNB), 8U/ μ L Bst DNA polymerizations
Enzyme, ddH2O。
The present invention also provides the application of above-mentioned primer combination or the kit in detection rice paddy seed Carriage.
The present invention also simultaneously there is provided the loop-mediated isothermal amplification method for being used to detect bakanae disease of rice germ, detects solution
Totally 24 μ L, add the μ L of DNA profiling 1 to be measured, constitute 25 μ L detection reaction systems；
The 25 μ L reaction systems contain the 8U/ μ L μ L (4U) of Bst archaeal dna polymerases 0.5,10 × ThermoPol
The μ L of Buffer 2.5,20 μM of FIP primer 2 .0 μ L, 20 μM of BIP primer 2 .0 μ L, 10 μM of the μ L of F3 primers 0.5,10 μM of B3
Primer 0.5 μ L, 25mM MgCl24.0 μ L, 10mM dNTPs 2.5 μ L, 5M glycine betaine 3.0 μ L, 3.75mM hydroxyl naphthalene
Phenol indigo plant (HNB) 1 μ L, the μ L of DNA profiling 1, distilled water complement to 25 μ L；
LAMP amplified reactions are carried out using above-mentioned 25 μ L detection reaction systems；LAMP amplified reaction programs are：63 DEG C,
60min；80 DEG C, 10min.
It is used as the improvement of the loop-mediated isothermal amplification method of the present invention：
LAMP amplified reactions are carried out using above-mentioned 25 μ L detection reaction systems, following either method is selected：
Method one, the first dyestuff hydroxynaphthol blue (HNB) that added before amplification are become as reaction indicator with HNB color
Change as result judgement standard, then carry out LAMP amplified reactions；After reaction terminates, color is changed, that is, the result that develops the color is seen
Observe sky blue and be judged as the positive, that is, be determined as paddy rice germ containing bakanae disease of rice to be measured；Color does not change, colour developing knot
Fruit is still judged as feminine gender for bluish violet, that is, is determined as that paddy rice to be measured is disliked without paddy rice contained by bakanae disease of rice germ or paddy rice to be measured
Seedling diseases germ content is not up to the minimal detectable concentration 0.01ng/ μ L of detection；
Method two, 5~10 μ L amplified productions are taken, detected with 2% agarose gel electrophoresis, judged if there is trapezoid-shaped strips
For the positive, that is, it is determined as paddy rice germ containing bakanae disease of rice to be measured；It is judged as feminine gender if without amplified band, that is, is judged to treating
Paddy rice is surveyed without bakanae disease of rice germ, or bakanae disease of rice germ contained by paddy rice to be measured does not reach minimal detectable concentration 10fg/ μ L.
The solution of the present invention includes：
(1) specific primer is designed according to the DNA sequence dna of bakanae disease of rice germ NPRS31 genes；
(2) LAMP amplifications and the detection of amplified production；
(3) specific detection of LAMP primer；
(4) LAMP amplifications and PCR amplifications sensitivity compare；
(5) rice paddy seed carries the detection of bakanae disease germ.
The solution of the present invention is specific as follows：
1st, the design of primer：
Inventor obtains the NPRS31 gene orders of fusarium moniliforme first, and primerexplorer V5 can be used
(http://primerexplorer.jp/lampv5e/index.html) the multigroup primer of design, then according to primer place sequence
The composite factors such as the conservative of column region, the hairpin structure of primer, dimer G/C content and Tm values further select primer.Most
Afterwards 4 primers, including 2 outer primers (F3 and B3) and 2 inner primers (FIP and BIP) are have selected for bakanae disease of rice germ.Institute
State primer information and be shown in Table 1.
Table 1, primer sequence information
2nd, the specific detection of primer：
It is specific for detection primer, using rattan storehouse sickle-like bacteria (Fusarium fujikuroi) as positive control, with Teng Cang
The sick sickle of Fusariumsp (F.fujikurio), Fusarium oxysporum (F.oxysporum), fusarium prolifertum (F.proliferatum), eggplant
Knife bacterium (F.solani), Fusarium graminearum (F.graminearum), Penicillium notatum (Penicilliu sp.), ustilaginoidea virens
(Ustilaginoidea virens), Pyricularia oryzae (Pyricularia grisea), alternaric bacteria (Alternaria
Alternate), this seven kinds of pathogens of sheath blight fungus (Rhizoctonia solani) make as negative control, and with distilled water
Tested for blank control, it is found that the primer has the specificity of detection rattan storehouse sickle-like bacteria (Fusarium fujikuroi)
(Fig. 1).
3rd, LAMP reaction systems are：
The μ L (4U) of Bst archaeal dna polymerases 0.5, the μ of 10 × ThermoPol Buffer 2.5 containing 8U/ μ L in 25 μ L systems
L, 20.0 μM of FIP primer 2 .0 μ L, 20.0 μM of BIP primer 2 .0 μ L, 10 μM of the μ L of F3 primers 0.5,10 μM of B3 primers 0.5
μ L, 25.0mM MgCl 4.0 μ L, 10.0mM dNTPs 2.5 μ L, 5.0M glycine betaine 3.0 μ L, 3.75mM hydroxyl naphthols
Indigo plant (HNB) 1 μ L, the μ L of DNA profiling 1, distilled water complement to 25 μ L；Amplification condition is 63 DEG C and incubates reaction at 60min, 80 DEG C
10min。
After reaction terminates, the specificity of primer is judged with agarose gel electrophoresis method and HNB developing dyes method respectively.Agar
Sugared gel electrophoresis：5 μ L LAMP amplified productions are taken, are detected with 2% agarose gel electrophoresis, EB dyeing, in gel imaging system
Observed in system, it is the positive characteristic scalariform band occur, it is feminine gender not occur band；HNB developing dye methods：Visual color
The color change of LAMP reaction mixtures, generation sky blue is positive reaction, and bluish violet is negative reaction.
4th, LAMP amplifications and standard PCR amplification sensitivity compare：
The fusarium moniliforme genomic DNA of extraction is determined after concentration with NanoDrop 1000, by 10 times of gradient dilutions
Into 7 concentration (10ng/ μ L~1ng/ μ L), the measure for LAMP sensitivity.LAMP reaction systems and amplification condition are ibid.
Meanwhile, using above-mentioned dilution DNA as template, using F3 and B3 as primer, standard PCR amplification is carried out, control is used as.Reaction
System is：The system of PCR reactions is 25.0 μ L, wherein the μ L of 2 × Taq MasterMix 12.5, each 1.0 μ of 10 μM of upstream and downstream primer
L, the μ L of DNA profiling 1.0, the μ L of distilled water 9.5；PCR response procedures are：95 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 56 DEG C of annealing
30s, 72 DEG C of extension 1min, carries out 35 circulations altogether；72 DEG C of extension 10min.
After amplification terminates, LAMP amplifications and PCR is taken to expand each μ L of concentration gradient product 5 electric on 2% Ago-Gel
Swimming analysis, after ethidium bromide staining, result is observed using gel imaging system.
5th, the optimization of response procedures：
For the purpose of the LAMP optimal reaction temperatures that fusarium moniliforme can be detected to set up.Combined using above-mentioned primer, together
Reaction premixed liquid constitutes detection liquid, adds the μ L of fusarium moniliforme DNA profiling 1, by reaction temperature be respectively set to 59 DEG C, 60
DEG C, 61 DEG C, 62 DEG C, 63 DEG C, 64 DEG C, 65 DEG C, 66 DEG C, after amplified reaction terminates, observing response pipe color change is further used
The detection of 2% agarose gel electrophoresis is used as checking.
Reaction result shows (Fig. 2), and when reaction temperature is 63 DEG C, reaction tube color change is most obvious, electrophoresis LAMP bars
Band is clear.
For the purpose of the LAMP optimum reacting times that fusarium moniliforme can be detected to set up.Closed using with above-mentioned primer sets,
Constitute detection liquid with reaction premixed liquid, add the μ L of fusarium moniliforme DNA profiling 1, will be set in the reaction time 30min,
40min, 50min, 60min, after amplified reaction terminates, observing response pipe color change.Further use 2% agarose gel electrophoresis
Detection is used as checking.
Reaction result shows (Fig. 3), when 60min is reached in the reaction time and be longer, and reaction tube color change is obvious, electrophoresis
LAMP bands are clear, are proliferation time to save detection time, i.e. 60min.
According to above-mentioned response procedures optimum results, response procedures are 63 DEG C, 60min in the present invention.
6th, rice paddy seed carries the detection of Fusarium moniliforme Sheld：
Seed of the rice paddy seed of inoculation Fusarium moniliforme Sheld with health without Fusarium moniliforme Sheld is taken to mix in varing proportions respectively, with
Machine takes seed sample after mixing, and then seed sample is cracked using 5% Chelex-10 solution, and extracts DNA, such as
Upper progress LAMP detections.
The present invention establishes the quick, easy of fusarium moniliforme, high specificity, sensitivity is high and can detect by an unaided eye
Monitoring technology system, the bacterial bearing rate before rice paddy seed presoaking and germinating can be detected in agricultural production, the foundation pair of this method
The prevention and control tool of bakanae disease of rice is of great significance.
The present invention compared with prior art, with following technical advantage：
(1) high specificity：Two kinds of specific detection implementation results are all shown, only carry Fusarium moniliforme Sheld on rice paddy seed
Just produce positive findings.
(2) sensitivity is high：Sensitivity technique, LAMP technology are carried out by doubling dilution seed Fusarium moniliforme Sheld genomic DNA
Can detect minimum Fusarium moniliforme Sheld DNA concentration is 64fg/ μ L, and 100 times are higher by than Standard PCR.
(3), can be with direct visual perception reaction result after quick, convenient reaction terminates, all processes only need 60min, greatly
Shorten detection time greatly.
(4) it is with low cost：Because LAMP technology does not need PCR instrument, gel electrophoresis and gel electrophoresis imaging system etc. to hold high
Your instrument, reaction can be completed using water-bath and common thermos cup, be adapted to common lab and field mass detection.
(5) Carriage of rice paddy seed can be directly detected using this technology, needs not move through separation, the culture of germ,
Without DNA extraction procedures such as cumbersome centrifugations, it is possible to detect the bacterial bearing rate of 0.06% and the above.
Brief description of the drawings
The embodiment to the present invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 is that primer specificity determines comparison diagram；
A：For LAMP colour developing variation diagrams；
B：For LAMP agarose gel electrophoresis figures；
M represents DL 2000DNA Marker, and detection sample is respectively with 1. rattan storehouse Fusariumsps (F.fujikurio), 2. points
Fusarium oxysporum (F.oxysporum), 3. fusarium prolifertums (F.proliferatum), 4. Fusarinm solanis (F.solani), 5.
Fusarium graminearum (F.graminearum), 6. Penicillium notatums (Penicilliu sp.), 7. ustilaginoidea virens (Ustilaginoidea
Virens), 8. Pyricularia oryzaes (Pyricularia grisea), 9. alternaric bacterias (Alternaria alternate), 10. lines
This 9 kinds of pathogens of rot bacterium (Rhizoctonia solani) are used as blank control as negative control, and using distilled water (11).
Fig. 2 is that reaction temperature optimizes figure；
A：For LAMP colour developing variation diagrams；
B：For LAMP agarose gel electrophoresis figures；
M represents DL 2000DNA Marker；
1st, 2,3,4,5,6,7,8 59 DEG C, 60 DEG C, 61 DEG C, 62 DEG C, 63 DEG C, 64 DEG C, 65 DEG C, 66 DEG C are represented respectively；
63 DEG C of colour developing changes are the most obvious, and the agarose gel electrophoresis band of its LAMP product becomes clear.
Fig. 3 is to optimize figure in the reaction time；
A：For LAMP colour developing variation diagrams；
B：For LAMP agarose gel electrophoresis figures；
M represents DL 2000DNA Marker；
1st, 2,3,4 60min, 50min, 40min, 30min are represented respectively；
Being shown in after 60min all has bright LAMP agarose gel electrophoresis figure bands.
Embodiment
Below by example with reference, the present invention is described further, and the protection model to the present invention is described below
The restriction not constituted in all senses is enclosed, is only illustrated.
Used in following examples main agents and instrument Bst archaeal dna polymerases (New England Biolabs,
MgCl2(Sigma), dNTPs, glycine betaine, DEPC water, molecular mass standard DNA Maker (TaKaRa bio-engineering corporations genes
Group), fungal genomic DNA Rapid extraction kit (Sheng Gong bioengineering limited company), eppendorf regular-PCRs expand
Increase instrument, the grand laboratory apparatus factory DK-8D types electrical heating thermostat water bath of upper Nereid.
Embodiment 1：
The sensitivity technique of primer
The DNA of bakanae disease of rice germ (Fusraium fujikuroi) genome is extracted, measurement DNA solution concentration is
200ng/μL.DNA solution is diluted, gradient is 0.0001ng/ μ L, 0.001ng/ μ L, 0.01ng/ μ L, 0.1ng/ μ L, 1ng/ μ
L、10ng/μL、100ng/μL.It is configured to the 25 μ L reaction systems under：The μ L (4U) of 8U/ μ L Bst archaeal dna polymerases 0.5,10 ×
ThermoPol Buffer 2.5μL、20μM FIP 2.0μL、20μM BIP 2.0μL、10μM F3 0.5μL、10μM B3
0.5μL、25mM MgCl24.0 μ L, the μ of 2.5 μ L, 5M glycine betaines of 10mM dNTPs, 3.0 μ L, 3.75mM hydroxynaphthol blue (HNB) 1
L, above-mentioned each concentration gradient the μ L of DNA profiling 1 (being used as blank control using the μ L of distilled water 1), finally respectively complement to 25 μ with distilled water
L。
Closed and tested with following primer sets：
Upstream outer primer (F3)：5’—CGGCCCTTATACCCCATTC—3’；
Downstream outer primer (B3)：5’—GCGCCACTATTGCTGTCT—3’；
Upstream inner primer (FIP)：5’—ACTTGCGCTGGAAAGACCAGAACATCCGCCACCTCACTGA—3’；
Downstream inner primer (FIP)：5’—GGTCACCAATCCTCGGCTACAGCCCTAGGAGATGGTCGCTTA—3’.
The LAMP amplified reaction programs used are：63 DEG C of amplification 60min；80 DEG C of inactivation 10min.
It is 0.01ng/ μ L, 0.1ng/ μ L, 1ng/ μ L, 10ng/ μ L, 100ng/ μ L in DNA profiling concentration after the completion of reaction
When reaction result be illustrated as the positive, the color of reaction tube is changed into sky blue from bluish violet before and after reaction, and is coagulated with 2% agarose
Gel electrophoresis detection is respectively provided with specific band.When concentration is less than 0.01ng/ μ L, the color of reaction tube does not occur change realization and is
Feminine gender, does not have specific band appearance with the detection of 2% agarose gel electrophoresis.
As a result show that to bakanae disease of rice germ (F.fujikuroi) DNA detection least concentration be 0.01ng/ μ L.
Embodiment 2：The specificity analysis of primer
Extract rattan storehouse Fusariumsp (F.fujikurio), Fusarium oxysporum (F.oxysporum), fusarium prolifertum
(F.proliferatum), Fusarinm solani (F.solani), Fusarium graminearum (F.graminearum), Penicillium notatum
(Penicilliu sp.), ustilaginoidea virens (Ustilaginoidea virens), Pyricularia oryzae (Pyricularia
Grisea), alternaric bacteria (Alternaria alternate), 9 kinds of pathogens of sheath blight fungus (Rhizoctonia solani)
Blank control is used as negative control, and using distilled water.
Using 25 μ L reaction systems, formed by following component proportioning：The μ L (4U) of 8U/ μ L Bst archaeal dna polymerases 0.5,10 ×
ThermoPol Buffer 2.5μL、20μM FIP 2.0μL、20μM BIP 2.0μL、10μM F3 0.5μL、10μM B30.5
μL、25mM MgCl24.0 μ L, the μ L of 2.5 μ L, 5M glycine betaines of 10mM dNTPs, 3.0 μ L, 3.75mM hydroxynaphthol blue (HNB) 1,
DNA solution adds 1 μ L as template, and distilled water complements to 25 μ L.
Closed and tested with following primer sets：
Upstream outer primer (F3)：5’—CGGCCCTTATACCCCATTC—3’；
Downstream outer primer (B3)：5’—GCGCCACTATTGCTGTCT—3’；
Upstream inner primer (FIP)：5’—ACTTGCGCTGGAAAGACCAGAACATCCGCCACCTCACTGA—3’；
Downstream inner primer (FIP)：5’—GGTCACCAATCCTCGGCTACAGCCCTAGGAGATGGTCGCTTA—3’.
The LAMP amplified reaction programs used are：63 DEG C of amplification 60min；80 DEG C of inactivation 10min.
Template of the result of the test in addition to bakanae disease of rice germ (F.fujikuroi) is all shown as feminine gender, reaction tube
Middle reaction solution color does not change, does not have specific band appearance by the detection of 2% agarose gel electrophoresis.Represent that this draws
The ring mediated isothermal amplification system that thing combination is constituted can detect bakanae disease of rice germ (F.fujikuroi), and with spy
The opposite sex.
Comparative example 1-1,
Closed and tested with following primer sets：
Upstream outer primer (F3-1)：5’—CGGCCCTTATACCCCATTT—3’；
Downstream outer primer (B3-1)：5’—GCGCCACTATTGCTGTCA—3’；
Upstream inner primer (FIP)：5’—ACTTGCGCTGGAAAGACCAGAACATCCGCCACCTCACTGA—3’；
Downstream inner primer (FIP)：5’—GGTCACCAATCCTCGGCTACAGCCCTAGGAGATGGTCGCTTA—3’.
Note：The 3 ' of two outer primers hold last base C and T to replace with T and A respectively, to verify the specificity of primer.
Using 25 μ L reaction systems, formed by following component proportioning：The μ L (4U) of 8U/ μ L Bst archaeal dna polymerases 0.5,10 ×
ThermoPol Buffer 2.5μL、20μM FIP 2.0μL、20μM BIP 2.0μL、10μM F3-1 0.5μL、10μM B3-
1 0.5μL、25mM MgCl24.0 μ L, 2.5 μ L, 5M glycine betaines of 10mM dNTPs 3.0 μ L, 3.75mM hydroxynaphthol blue (HNB)
1 μ L, using concentration be respectively 1ng/ μ L, 10ng/ μ L, 100ng/ μ L DNA solution as template plus 1 μ L, distilled water complements to 25 μ
L。
Result of the test is all shown as feminine gender, and reaction solution color does not change in reaction tube, by 2% Ago-Gel electricity
Swimming detection does not have specific band appearance.Represent that the ring mediated isothermal amplification system that primer combination is constituted is unable to effective detection
Go out bakanae disease of rice germ (F.fujikuroi), still can not be effective even if the concentration of DNA profiling is increased into 100ng/ μ L
Distinguish.
Comparative example 1-2,
Closed and tested with following primer sets：
Upstream outer primer (F3)：5’—CGGCCCTTATACCCCATTC—3’；
Downstream outer primer (B3)：5’—GCGCCACTATTGCTGTCT—3’；
Upstream inner primer (FIP-1)：5’—ACTTGCGCTGGAAAGACCAGAACATCCGCCACCTCACTGT—3’；
Downstream inner primer (FIP-1)：5’—GGTCACCAATCCTCGGCTACAGCCCTAGGAGATGGTCGCTTC—3’.
Note：The 3 ' of two outer primers hold last base A and A to replace with T and C respectively, to verify the special of primer
Property.
Using 25 μ L reaction systems, formed by following component proportioning：The μ L (4U) of 8U/ μ L Bst archaeal dna polymerases 0.5,10 ×
ThermoPol Buffer 2.5μL、20μM FIP 2.0μL、20μM BIP 2.0μL、10μM F3 0.5μL、10μM B30.5
μL、25mM MgCl24.0 μ L, the μ L of 2.5 μ L, 5M glycine betaines of 10mM dNTPs, 3.0 μ L, 3.75mM hydroxynaphthol blue (HNB) 1,
Using concentration be respectively 1ng/ μ L, 10ng/ μ L, 100ng/ μ L DNA solution as template plus 1 μ L, distilled water complements to 25 μ L.
Result of the test is all shown as feminine gender, and reaction solution color does not change in reaction tube, by 2% Ago-Gel electricity
Swimming detection does not have specific band appearance.Represent that the ring mediated isothermal amplification system that primer combination is constituted is unable to effective detection
Go out bakanae disease of rice germ (F.fujikuroi), still can not be effective even if the concentration of DNA profiling is increased into 100ng/ μ L
Distinguish.
Embodiment 3, rice paddy seed carry the detection of bakanae disease germ
(1) preparation of rice paddy seed sample
Beaten in culture 5d bakanae disease bacterial strain flat board and take 0.5cm2Bacteria cake, by bacteria cake and 10g health without bakanae disease
After the rice paddy seed mixing of bacterium, 25 DEG C of culture 5d.Then the rice paddy seed with Fusarium moniliforme Sheld 1 being obtained as described above respectively with 0,
200th, 400 after, 800,1600 and 3200 health are without the seed mixing of Fusarium moniliforme Sheld, 300rpm concussions 60min.
(2) rice paddy seed bakanae disease DNA rapid extraction
A seed is taken to be placed in 200 μ L 5% at random respectively in the seed sample obtained from the mixing of as above different proportion
In Chelex-100, shaken in whirlpool concussion instrument after 10s, boil 5min, then shake 10s, boiled after 5min, natural cooling,
Save backup.
(3) each sample gene group extracted is subjected to LAMP amplifications respectively, uses agarose gel electrophoresis method and HNB
Dye method observing response result, detection method be the same as Example 1.
(4) take a 16, seed mixed to be seeded on 9cm PDA plate respectively, seed bakanae disease band is counted after 2 days
Bacterium rate.
As a result show, LAMP reactions can't detect Fusarium moniliforme Sheld in the seed sample that health is not polluted by Fusarium moniliforme Sheld,
Biased sample after seed with Fusarium moniliforme Sheld and healthy seed are polluted by it detects Fusarium moniliforme Sheld, and inner reaction tube liquid is from purple
Discoloration is sky blue, and scalariform band occurs in 2% agarose gel electrophoresis, and Fusarium moniliforme Sheld carrying rate is 0.0625% kind increment
Product (that is, after 1 rice paddy seed with Fusarium moniliforme Sheld is mixed with 1600 health without the seed of Fusarium moniliforme Sheld) can still use the party
Method carries out quick diagnosis, i.e. detection is limited to 1/1600=0.0625%.
Finally, in addition it is also necessary to it is noted that listed above is only several specific embodiments of the invention.Obviously, this hair
It is bright to be not limited to above example, there can also be many deformations.One of ordinary skill in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
<110>Zhejiang A ＆ F University
<120>The LAMP methods and kit of quick detection bakanae disease of rice germ from seed rice
<160> 4
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>Upstream outer primer F3
<400> 1
cggcccttat accccattc 19
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>Downstream outer primer B3
<400> 2
gcgccactat tgctgtct 18
<210> 3
<211> 40
<212> DNA
<213>Artificial sequence
<220>
<223>Upstream inner primer FIP
<400> 3
acttgcgctg gaaagaccag aacatccgcc acctcactga 40
<210> 4
<211> 42
<212> DNA
<213>Artificial sequence
<220>
<223>Downstream inner primer BIP
<400> 4
ggtcaccaat cctcggctac agccctagga gatggtcgct ta 42
Claims (6)
1. the LAMP specific primer sets things for bakanae disease of rice germ quick determination method, it is characterised in that：It is described
LAMP specific primer sets thing is made up of upstream and downstream outer primer, upstream and downstream inner primer this four primers；
Upstream outer primer (F3)：5’—CGGCCCTTATACCCCATTC—3’；
Downstream outer primer (B3)：5’—GCGCCACTATTGCTGTCT—3’；
Upstream inner primer (FIP)：5’—ACTTGCGCTGGAAAGACCAGAACATCCGCCACCTCACTGA—3’；Draw in downstream
Thing (BIP)：5’—GGTCACCAATCCTCGGCTACAGCCCTAGGAGATGGTCGCTTA—3’.
2. the loop-mediated isothermal amplification kit for detecting bakanae disease of rice germ, it is characterised in that include such as claim 1
Described LAMP specific primer sets things.
3. loop-mediated isothermal amplification kit according to claim 2, it is characterised in that：Draw comprising 1.6 μM of positive introversion
Thing FIP, 1.6 μM of reverse inner primer BIP, 0.2 μM of positive outer primer F3,0.2 μM of reverse outer primer B3.
4. the loop-mediated isothermal amplification kit according to Claims 2 or 3, it is characterised in that the kit is also included：
10×ThermoPol Buffer、1mmol/L dNTPs、4mmol/L MgCl2, 0.6mmol/L glycine betaines, 150mmol/L hydroxyls
Base naphthol blue, 8U/ μ L Bst archaeal dna polymerases, ddH2O。
5. the loop-mediated isothermal amplification method for detecting bakanae disease of rice germ, it is characterised in that：
LAMP reaction systems are 25 μ L, are：The μ L of 8U/ μ L Bst archaeal dna polymerases 0.5, the μ of 10 × ThermoPol Buffer 2.5
L、20μM FIP 2.0μL、20μM BIP 2.0μL、10μM F3 0.5μL、10μM B3 0.5μL、25mM MgCl2 4.0μL、
The μ L of 2.5 μ L, 5M glycine betaines of 10mM dNTPs, 3.0 μ L, 3.75mM hydroxynaphthol blue 1, the μ L of bakanae disease of rice germ DNA profiling 1,
Distilled water complements to 25 μ L；
Amplification condition is incubates 60min under the conditions of 63 DEG C, 80 DEG C are reacted 10min.
6. loop-mediated isothermal amplification method according to claim 5, it is characterised in that：
LAMP amplified reactions are carried out using 25 μ L detection reaction systems, following either method is selected：
Method one, the first dyestuff hydroxynaphthol blue that added before amplification are as reaction indicator, with HNB color change as result
Criterion, then carries out LAMP amplified reactions；After reaction terminates, color changes, that is, the result that develops the color observes sky blue
It is judged as the positive, that is, is determined as paddy rice germ containing bakanae disease of rice to be measured；Color does not change, and colour developing result is still royal purple
Color is judged as feminine gender, that is, is determined as that paddy rice to be measured contains without bakanae disease of rice germ contained by bakanae disease of rice germ or paddy rice to be measured
Amount is not up to the minimal detectable concentration 0.01ng/ μ L of detection；
Method two, 5~10 μ L amplified productions are taken, detected with 2% agarose gel electrophoresis, sun is judged as if there is trapezoid-shaped strips
Property, that is, it is determined as paddy rice germ containing bakanae disease of rice to be measured；It is judged as feminine gender if without amplified band, that is, is determined as water to be measured
Rice is without bakanae disease of rice germ, or bakanae disease of rice germ contained by paddy rice to be measured does not reach minimal detectable concentration 10fg/ μ L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710316754.1A CN107022624B (en) | 2017-05-08 | 2017-05-08 | LAMP method and kit for rapidly detecting bakanae disease bacteria of rice from rice seeds |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710316754.1A CN107022624B (en) | 2017-05-08 | 2017-05-08 | LAMP method and kit for rapidly detecting bakanae disease bacteria of rice from rice seeds |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107022624A true CN107022624A (en) | 2017-08-08 |
| CN107022624B CN107022624B (en) | 2021-06-11 |
Family
ID=59529684
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710316754.1A Active CN107022624B (en) | 2017-05-08 | 2017-05-08 | LAMP method and kit for rapidly detecting bakanae disease bacteria of rice from rice seeds |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107022624B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109897889A (en) * | 2019-04-17 | 2019-06-18 | 北京天恩泽基因科技有限公司 | A kind of LAMP(ring mediated isothermal amplification) product visible detection method |
| CN110117592A (en) * | 2019-06-04 | 2019-08-13 | 中国农业大学 | A method of quickly detecting fusarium moniliforme rattan storehouse fusarium based on RPA |
| CN110527630A (en) * | 2019-05-24 | 2019-12-03 | 浙江工业大学 | One plant of Gibberella fujikuroi mutant strain and application using the breeding of ARTP induced-mutation technique |
| CN110982922A (en) * | 2019-12-20 | 2020-04-10 | 浙江大学 | Primer composition and method for rapidly detecting rice bakanae disease pathogenic bacteria fusarium granatum based on LAMP |
| CN111378776A (en) * | 2019-10-25 | 2020-07-07 | 安徽省农业科学院植物保护与农产品质量安全研究所 | RPA primer and detection method for detecting rice bakanae disease Gibberella fujikuei |
| CN111549163A (en) * | 2020-04-29 | 2020-08-18 | 浙江农林大学 | LAMP-based method for rapidly detecting prochloraz-resistant S312T genotype Fusarium celandi |
| CN114231653A (en) * | 2021-12-17 | 2022-03-25 | 海南热带海洋学院 | LAMP primer group, kit and method for detecting rice aroma gene badh2 allelic variation |
| CN114317688A (en) * | 2022-01-27 | 2022-04-12 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Visual mixed dye and application thereof in LAMP detection |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630369A (en) * | 2015-02-13 | 2015-05-20 | 湖南农业大学 | PCR detection method of fussrium moniliforme |
-
2017
- 2017-05-08 CN CN201710316754.1A patent/CN107022624B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630369A (en) * | 2015-02-13 | 2015-05-20 | 湖南农业大学 | PCR detection method of fussrium moniliforme |
Non-Patent Citations (4)
| Title |
|---|
| EVA-MARIA NIEHAUS等: "Apicidin F: Characterization and Genetic Manipulation of a New Secondary Metabolite Gene Cluster in the Rice Pathogen Fusarium fujikuroi", 《PLOS ONE》 * |
| SUNIL KUMAR SUNANI: "Understanding the seed borne infection of Fusarium fujikuroi inciting bakanae disease of rice", 《DIVISION OF PLANT PATHOLOGY》 * |
| 田擎等: "利用环介导等温扩增技术检测黑白轮枝菌", 《南京农业大学学报》 * |
| 郑永允: "《渔业技术与健康养殖》", 31 January 2014, 海洋出版社 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109897889A (en) * | 2019-04-17 | 2019-06-18 | 北京天恩泽基因科技有限公司 | A kind of LAMP(ring mediated isothermal amplification) product visible detection method |
| CN110527630A (en) * | 2019-05-24 | 2019-12-03 | 浙江工业大学 | One plant of Gibberella fujikuroi mutant strain and application using the breeding of ARTP induced-mutation technique |
| CN110527630B (en) * | 2019-05-24 | 2021-04-20 | 浙江工业大学 | Aleurites lutescens mutant strain bred by ARTP mutagenesis technology and application thereof |
| CN110117592A (en) * | 2019-06-04 | 2019-08-13 | 中国农业大学 | A method of quickly detecting fusarium moniliforme rattan storehouse fusarium based on RPA |
| CN111378776A (en) * | 2019-10-25 | 2020-07-07 | 安徽省农业科学院植物保护与农产品质量安全研究所 | RPA primer and detection method for detecting rice bakanae disease Gibberella fujikuei |
| CN110982922A (en) * | 2019-12-20 | 2020-04-10 | 浙江大学 | Primer composition and method for rapidly detecting rice bakanae disease pathogenic bacteria fusarium granatum based on LAMP |
| CN110982922B (en) * | 2019-12-20 | 2021-08-17 | 浙江大学 | Primer composition and method for rapidly detecting rice bakanae disease pathogenic bacteria fusarium granatum based on LAMP |
| CN111549163A (en) * | 2020-04-29 | 2020-08-18 | 浙江农林大学 | LAMP-based method for rapidly detecting prochloraz-resistant S312T genotype Fusarium celandi |
| CN111549163B (en) * | 2020-04-29 | 2023-05-05 | 浙江农林大学 | LAMP-based method for rapidly detecting prochloraz-resistant S312T genotype fusarium graminearum |
| CN114231653A (en) * | 2021-12-17 | 2022-03-25 | 海南热带海洋学院 | LAMP primer group, kit and method for detecting rice aroma gene badh2 allelic variation |
| CN114317688A (en) * | 2022-01-27 | 2022-04-12 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Visual mixed dye and application thereof in LAMP detection |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107022624B (en) | 2021-06-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107022624A (en) | The LAMP methods and kit of quick detection bakanae disease of rice germ from seed rice | |
| CN106350588B (en) | The method of the ash arrhizus bacteria of benzimidazoles-resisting bactericide is quickly detected based on LAMP | |
| CN104611428B (en) | A kind of loop-mediated isothermal amplification (LAMP) primer compositions detecting colletotrichum gloeosporioides Penz and application thereof | |
| CN106434993B (en) | For detecting LAMP primer composition object and its application of cucumber fusarium axysporum | |
| CN101880711A (en) | Nucleic acid screening method of staphylococcus aureus, salmonella, shigella and listeria monocytogenes | |
| CN106868142A (en) | Detect the loop-mediated isothermal amplification method of hickory nut dry rot germ | |
| CN101831493A (en) | Loop-mediated isothermal amplification (LAMP) primer pair of bacillus cereus and detection method | |
| CN108676910A (en) | A kind of LAMP detection primer of fusarium prolifertum and its application | |
| AU2020102244A4 (en) | Primer group, kit and detection method for rapidly detecting carbapenemases genes | |
| CN108085410A (en) | Seedling stage Strawberry anthracnose latent infection and its fast detection method of medication | |
| CN108359717A (en) | A kind of direct expansion RPA visualization of presence detection methods | |
| CN109280715B (en) | LAMP primer group for detecting peanut black rot, and rapid detection method and kit thereof | |
| CN104593502B (en) | Loop-mediated isothermal amplification primer composition capable of detecting colletotrichum truncatum and application thereof | |
| CN109680056A (en) | A kind of kit, detection method and its application detecting MCR gene | |
| Rong et al. | Rapid diagnosis of rice bakanae caused by Fusarium fujikuroi and F. proliferatum using loop‐mediated isothermal amplification assays | |
| CN102010910A (en) | Loop-mediated isothermal amplification technology-based plasmodium genus and species nucleic acid screening method | |
| CN106381340B (en) | Botrytis cinerea LAMP detection primer, detection kit and its application | |
| CN106755339B (en) | Cucumber anthracnose LAMP detection primer and its application | |
| CN104328171A (en) | Loop-mediated isothermal amplification primer, kit and method for detecting rat staphylococcus aureus | |
| CN108315473A (en) | It is a kind of to be used for the Primer composition and its application that rice green smut germ LAMP is quickly detected | |
| CN102121051A (en) | Multiplex fluorescent quantitative PCR detection method for main pathogenic bacteria in aquatic product | |
| CN107828905A (en) | Tobacco smoke pollution LAMP detection primer and detection method | |
| CN104293957B (en) | A kind of early stage rapid molecular detection method of Botrytis cinerea | |
| CN108411017A (en) | Detect the LAMP primer and method of pseudomonas syringae tomato pvs oryzae and oryzicola | |
| CN108330178A (en) | It is a kind of detection tomato early blight bacterium loop-mediated isothermal amplification (LAMP) primer and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |