CN106868142A - Detect the loop-mediated isothermal amplification method of hickory nut dry rot germ - Google Patents
Detect the loop-mediated isothermal amplification method of hickory nut dry rot germ Download PDFInfo
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Abstract
The invention discloses a kind of loop-mediated isothermal amplification method and primer for detecting hickory nut dry rot germ, LAMP primer composition thing is made up of 4 primers.The present invention further simultaneously discloses a kind of loop-mediated isothermal amplification kit for detecting hickory nut dry rot germ, in addition to comprising above-mentioned LAMP primer composition thing, also including 10 × ThermoPol Buffer, dNTPs, MgCl2, glycine betaine, hydroxynaphthol blue, 8U/ μ L Bst archaeal dna polymerases, ddH2O.The present invention designs LAMP primer with β tubulin genes conserved region fragment sequence, and carry out the optimization of rapid molecular detection technique system and reaction condition, LAMP detections can be carried out to hickory nut dry rot germ, course of reaction simplicity, short detection cycle, high specificity, sensitivity are high, and can detect by an unaided eye testing result.
Description
Technical field
The invention belongs to biological technical field, it is related to ring mediated isothermal amplification (LAMP) technology for detection hickory nut dry rot disease
The loop-mediated isothermal amplification (LAMP) primer group and its application method of bacterium, belong to the early warning skill of plant disease detection, identification and preventing and treating
Art field.
Background technology
Hickory nut dry rot generally occurred since 2002 in the hickory nut producing region such as Linan, Chunan, Tonglu, injured area
Progressively expand, current occurring area is up to 270,000 mu, and the serious whole strain of generation is withered, light influence growth causes shedding, according to measuring and calculating
Generating region average product reduces by 24% or so, the bottleneck as hickory nut industry development.There are different researchs to report or speculate not
Same factor is relevant with the generation of hickory nut dry rot.This laboratory research finds currant grape seat chamber bacterium
(Botryosphaeria dothidea), i.e. hickory nut dry rot germ (B dothidea) is the master for causing hickory nut dry rot
Cause of disease is wanted, and the generation order of severity of hickory nut is mainly relevant with its biology and popular characteristic.Continuous 5 years it has been observed that
The pathogen is survived the winter with mycelium in diseased tissues, and Second Year spring temperature rises germ and starts actively, to be formed in bark surface
Fructification simultaneously discharges spore, then infected.Spore is propagated by means of wind and rain, and performance is obvious after invading bark cell or tree body wound
" latent infection " characteristic, has different degrees of disease since late March to November.Mid-April is to temperature mid-June
It is the peak of the peak period that spores release infects at 25 DEG C -30 DEG C, the hot weather that more than 30 DEG C of 7~August temperature is unfavorable for disease
The development of bacterium, autumn air temperature drops to less than 30 DEG C and can produce the development (not reporting data) of germ.Because the germ is long-term
In " latent infection " state, once the commensalism for waiting its latent is converted to saprophytic pathogenic state, macroscopic disease is formed
Spot, then prophylactico-therapeutic measures is taken, it is late.Prevention effect can be very undesirable.So, efficiently to prevent and treat hickory nut dry rot
Disease, it is just extremely important in the early stage quick detection of its latent commensalism phase.
As the related authentication method of nucleic acid is continued to develop, the method based on regular-PCR has been used successfully to detect mountain core
Dried peach maize ear rot bacterium, but the detection such as PCR method specificity is time-consuming partially long, it is necessary to 6~8h, while PCR method needs expensive instrument PCR
Instrument, and detection sensitivity is relatively low, detection process is more numerous and diverse.
Loop-mediated isothermal amplification technique (Loop-mediated isothermal amplification, LAMP) is Japan
Rong Yan Co., Ltd. invention a kind of new nucleic acid amplification technologies because its expansions it is easy to operate, quick, it is specific height, low cost
The advantages of, as the new nucleic acid amplification technologies that can substitute PCR.Design 4 pairs of specificity in 6 regions that it is directed to target gene
Primer, causes self-loopa strand replacement reaction in the presence of Bst Large fragment polymerases, in 60-65 DEG C of scope 60min, can be a large amount of
Synthesis target dna.Because LAMP amplification procedures rely on identification 6 isolated areas of target sequence, so atopic is very strong, and
Amplification process is carried out under constant temperature, and common water-bath or isothermal vacuum flask can just meet reaction and require, detection
Cost reduction, required time is short.But, the design of primer is not readily available.
Additionally, common PCR reaction carries out gel electrophoresis to product easily causes product amplification, this is laboratory pollution
One main source；And ethidium bromide (EB) has huge poison, can accumulate carcinogenic, significant damage is produced to human body；Long-term observation is purple
Outer lamp can also cause a certain degree of injury to experimenter's health.And LAMP reactions need to only be carried out in thermostat water bath,
Add dyestuff HNB (hydroxynaphthol blue) as reaction indicator before amplification, be as result judgement standard with the color change of HNB
Can, reach the purpose of quick detection.
The selection of target gene is one of key factor of LAMP detections.The conventional target gene of regular-PCR has β micro-pipe eggs
White gene, after progressively grow up as molecular labeling, the advantage is that：With high copy number；Simultaneously comprising holding and variation
Sequence；The variant sites special primer of design that can be in conserved sequence carries out specific amplification comparing.Although the β of fungi-
Tublin is generally speaking relatively conservative, but has enough variant sites to can be used for distinctive therebetween to compare, and it is more
Copy number makes the amplification of distinctive sensitiveer.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of ring mediated isothermal amplification for detecting hickory nut dry rot germ
(LAMP) Primer composition；The present invention also provides the application of primer combination；The present invention also provides a kind of detection hickory nut dry rot
The LAMP kit of germ.
In order to solve the above-mentioned technical problem, the present invention provides a kind of ring mediation for detecting hickory nut dry rot germ etc.
Warm amplimer：LAMP primer composition thing is made up of upstream and downstream outer primer, four primers of upstream and downstream inner primer, and primer sequence is such as
Under：
Upstream outer primer F3：5’—CGCGTTCAGAAGATTGCCATA—3’；
Downstream outer primer B3：5’—TTGTTGCCAAAACACCCGC—3’；
Upstream inner primer FIP：5’—GCGTGGGAGAACATCAATGAC-GCTCGAGCTGAAGGTCCG—3’；
Downstream inner primer FIB：5’—CTTACACGCCAGAGCCGT-AACGCGAATCGACACCACAG—3’.
The present invention also provides a kind of loop-mediated isothermal amplification kit for detecting hickory nut dry rot germ simultaneously,
Comprising above-mentioned LAMP primer composition thing.
As the improvement of kit of the invention：LAMP primer composition thing is：1.6 μM just interior to primers F IP, 1.6 μM
Reverse inner primer BIP, 0.2 μM of positive outer primer F3,0.2 μM of reverse outer primer B3.
As the further improvement of kit of the invention：Kit is also comprising as follows：10×ThermoPol Buffer、
dNTPs(1mM)、MgCl2(4mM), glycine betaine (0.6M), hydroxynaphthol blue (150 μM, HNB), 8U/ μ L Bst archaeal dna polymerases,
ddH2O。
Remarks：Mentioned component constitutes loop-mediated isothermal amplification premixed liquid.
The present invention also provides a kind of loop-mediated isothermal amplification method for detecting hickory nut dry rot germ, 25 simultaneously
μ L reaction systems are consisted of the following composition：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 MgCl2
4.0 μ L, 2.5 μ L, 5M glycine betaines of 10mM dNTPs 3.0 μ L, 3.75mM hydroxynaphthol blue (HNB) 1 μ L, the μ L of DNA profiling 1,
ddH2O (distilled water) complements to 25 μ L.
As the improvement of loop-mediated isothermal amplification method of the invention：LAMP is carried out using 25 μ L detections reaction system
Amplified reaction, selects following either method：
Method one, elder generation add dyestuff hydroxynaphthol blue (HNB) as reaction indicator before amplification, with hydroxynaphthol blue
(HNB) then color change carries out LAMP amplified reactions as result judgement standard, and after reaction terminates, color changes,
I.e. colour developing result observes that sky blue is judged as the positive, judges that display detects hickory nut dry rot germ；Color does not occur
Change, colour developing result judges display sample without hickory nut dry rot germ, or contained hickory nut still for bluish violet is judged as feminine gender
Dry rot germ content is not up to the minimal detectable concentration of detection；
Method two, 5 μ L amplified productions are taken, detected with 2% agarose gel electrophoresis, sun is judged as if there is trapezoid-shaped strips
Property, i.e. display detects hickory nut dry rot germ；Then it is judged as feminine gender without amplified band, that is, shows without hickory nut dry rot disease
Bacterium, or contained hickory nut dry rot germ does not reach minimal detectable concentration.
As the further improvement of loop-mediated isothermal amplification method of the invention：LAMP amplified reaction programs are：65 DEG C of expansions
Increase 60min；80 DEG C of inactivation 10min.
As the further improvement of loop-mediated isothermal amplification method of the invention：It is minimum in methods described one, method two
Detectable concentration is 0.01ng/ μ L.
The invention provides LAMP primer composition thing detect hickory nut dry rot germ LAMP kit in application, with
And there is provided a kind of LAMP kit for detecting hickory nut dry rot germ, comprising Primer composition of the present invention.
In the present invention, solution totally 24 μ L are detected, add the μ L of DNA profiling 1 to be measured, constitute 25 μ L detection reaction systems.
The solution of the present invention is specific as follows：
1st, the design of primer：
It is described for detecting that the specific primer group of hickory nut dry rot germ is to obtain hickory nut dry rot by expanding
β-Tublin gene nucleic acid the sequences of a large amount of bacterial strains of bacterium, and with the β-Tublin gene nucleic acid sequences of other currant grapes seat chamber bacterium
Row are compared, and are designed for conserved region fragment sequence.Wherein by drawing in a pair of upstream and downstream outer primers and a pair of upstream and downstream
The final concentration of thing, totally four primers composition, inner primer and outer primer is with 8:1 ratio is prepared.
For the purpose of for detection primer specificity, by right as the positive using hickory nut dry rot germ (B dothidea) respectively
According to, with apple alternaric bacteria (Alternaria mali), Lasiodiplodia theobromae (Lasiodiplodia therbromae),
Intend disk stey (Pestalotiopsis theae), Rhizoctonia solani Kuhn (Rhizoctonia Solani), gibberella fujikuroi
(Gibberella fujikuroi) and Sclerotium rolfsii (Sderotium rolfsii), colletotrichum gloeosporioides Penz
(Colletotrichum gloeosporioides) seven kinds of pathogens as negative control, and using distilled water as blank
Tested, it is found that the primer has the specificity (Fig. 1) to the detection of hickory nut dry rot.
2nd, the optimization of response procedures：
For the purpose of the LAMP optimal reaction temperatures of hickory nut dry rot germ can be detected to set up.Using above-mentioned primer sets
Close, detection liquid is constituted with reaction premixed liquid, add the μ L of hickory nut dry rot germ DNA profiling 1, reaction temperature is 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, closely
One step is detected as checking with 2% agarose gel electrophoresis.
Reaction result shows (Fig. 2), and when reaction temperature is 65 DEG C, reaction tube color change is most obvious, electrophoresis LAMP bars
Band is most clear.
For the purpose of the LAMP optimum reacting times of hickory nut dry rot germ can be detected to set up.Using using above-mentioned primer sets
Close, detection liquid is constituted with reaction premixed liquid, add the μ L of hickory nut dry rot germ DNA profiling 1, will be set in the reaction time
30min, 45min, 60min, 75min, after amplified reaction terminates, observing response pipe color change is further solidifying with 2% agarose
Gel electrophoresis detection is used as checking.
Reaction result shows (Fig. 3), the reaction time reach 60min and it is longer when, reaction tube color change substantially, 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 65 DEG C, 60min in the present invention.
In the present invention, LAMP is carried out by the conserved region fragment sequence of hickory nut dry rot germ β-tubulin genes
Primer specificity is designed, and filters out that high specificity, sensitivity is high, primer suitable for the detection of LAMP rapid moleculars, and then is set up
The technology for being available for the bacterium rapid molecular to detect.The present invention designs LAMP primer simultaneously with β-tubulin genes conserved region fragment sequence
Rapid molecular detection technique system and reaction condition optimization are carried out, LAMP detections are carried out to hickory nut dry rot germ, reacted
Journey simplicity (65 DEG C of constant temperature), detection cycle short (only needing 60min), high specificity, sensitivity are high, and can detect by an unaided eye detection knot
Really.
The present invention compared with prior art, with following technical advantage：
1), practicality is good.The method of traditional detection of pathogens is mainly by being separately cultured pathogen, then carries out shape
State identifies that the method detection needs technology specialty requirement higher, and there is certain uncertainty.Common PCR reaction pair
Pathogen DNA is expanded, reclaimed and is sequenced, it is necessary to ethidium bromide (EB) is dyeed and observed under ultraviolet light can just differentiate knot
Really, and detection time is long, testing cost is high, it is impossible to meet the demand of economical and efficient detection.And LAMP reactions only need to be in constant temperature
Carried out under (65 DEG C), reaction terminate after can by naked eyes under ordinary light direct judged result, energy quick detection go out pathogen so that
Increased its application value in Fields detection.
2), constant-temperature amplification.Unlike PCR methods have to thermal cycle, the dependence to thermal cycler instrument is thus broken away from, as long as
The thermal source for having stabilization can be completed if thermostat water bath LAMP reacts, it is not necessary to expensive instrument and equipment, thus be easy to basic unit
Agricultural production unit popularization and application.Why LAMP can react because adding in LAMP reaction solutions under constant thermal source
Glycine betaine is added, double-stranded DNA is in the dynamic equilibrium unwind, amplification has been realized in the presence of Bst archaeal dna polymerases.
3), accuracy is high.Traditional pathogen identification, time-consuming for authentication method, easily receives the factors such as artificial and environment
Interference.LAMP reactions recognize 6 isolated areas on target sequence by 4 primer specificities, know relative to common PCR primers
For 2 isolated areas of other target sequence, specificity and sensitivity are all significantly improved.
Brief description of the drawings
Specific embodiment of the invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 is that primer specificity determines comparison diagram；
Upper figure：It is LAMP colour developing variation diagrams；
Figure below：It is LAMP agarose gel electrophoresis figures；
M represents DL 2000DNA Marker, and detection sample is respectively hickory nut dry rot germ (B dothidea), apple
Rod method (Alternaria mali), the spore of cocoa two (Lasiodiplodia therbromae), plan disk stey
(Pestalotiopsis theae), vertical withered pyrenomycetes (Rhizoctonia Solani), gibberella fujikuroi (Gibberella
Fujikuroi), Sclerotium rolfsii (Sderotium rolfsii), colletotrichum gloeosporioides Penz (Colletotrichum
) and blank distilled water gloeosporioides.
Fig. 2 is that reaction temperature optimizes figure；
Upper figure：It is LAMP colour developing variation diagrams；
Figure below：It is LAMP agarose gel electrophoresis figures；
M represents DL 2000DNA Marker, and 64 DEG C of display, 65 DEG C of LAMP agarose gel electrophoresis band more become clear.
Fig. 3 is to optimize figure in the reaction time；
Upper figure：It is LAMP colour developing variation diagrams；
Figure below：It is LAMP agarose gel electrophoresis figures；
M represents DL 2000DNA Marker, all has bright LAMP agarose gel electrophoresis figures after being displayed in 45min
Band.
Specific embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This.
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.
All germs used below, detection demonstrates its qualitatively correctness in advance.
It is embodiment 1, a kind of for detecting hickory nut dry rot germ loop-mediated isothermal amplification kit, including for detecting
The loop-mediated isothermal amplification (LAMP) primer of hickory nut dry rot germ,
The LAMP primer composition thing is made up of following 4 primers：
Upstream outer primer (F3)：5’—CGCGTTCAGAAGATTGCCATA—3’；
Downstream outer primer (B3)：5’—TTGTTGCCAAAACACCCGC—3’；
Upstream inner primer (FIP)：5’—GCGTGGGAGAACATCAATGAC-GCTCGAGCTGAAGGTCCG—3’；
Downstream inner primer (FIB)：5’—CTTACACGCCAGAGCCGT-AACGCGAATCGACACCACAG—3’.
Kit is included：10×ThermoPol Buffer、1mM dNTPs、4mM MgCl2, 0.6mM glycine betaines, 150 μM
Hydroxynaphthol blue (HNB), 8U/ μ L Bst archaeal dna polymerases, ddH2In O, 1.6 μM just interior to primers F IP, 1.6 μM reverse
Primer BIP, 0.2 μM of positive outer primer F3,0.2 μM of reverse outer primer B3.
The 25 μ L reaction systems that experiment is used are formulated by following component：The μ L of 8U/ μ L Bst archaeal dna polymerases 0.5
(4U)、10×ThermoPol Buffer 2.5μL、20μM FIP 2.0μL、20μM BIP 2.0μL、10μM F3 0.5μL、
10μM B30.5μL、25mM MgCl24.0 μ L, μ L, 3.75mM hydroxynaphthol blues of 2.5 μ L, 5M glycine betaines of 10mM dNTPs 3.0
(HNB) 1 μ L, the μ L of DNA profiling 1, distilled water complement to 25 μ L.
That is, the μ L of DNA profiling 1 to be measured, detect solution totally 24 μ L, so as to constitute above-mentioned 25 μ L detections reaction system.
Embodiment 2, a kind of loop-mediated isothermal amplification method for detecting hickory nut dry rot germ：
LAMP amplified reactions are carried out using 25 μ L detections reaction system, following either method is selected：
Method one, elder generation add dyestuff hydroxynaphthol blue (HNB) as reaction indicator before amplification, with hydroxynaphthol blue
(HNB) then color change carries out LAMP amplified reactions as result judgement standard, and after reaction terminates, color changes,
That is colour developing result observes that sky blue is judged as the positive, i.e. display detects hickory nut dry rot germ；Color does not become
Change, colour developing result shows sample without hickory nut dry rot germ, or contained hickory nut dry rot still for bluish violet is judged as feminine gender
Germ content does not reach test limit；
Method two, 5 μ L amplified productions are taken, detected with 2% agarose gel electrophoresis, sun is judged as if there is trapezoid-shaped strips
Property, i.e. display detects hickory nut dry rot germ, feminine gender is then judged as without amplified band, that is, shows without hickory nut dry rot disease
Bacterium, or contained hickory nut dry rot germ does not reach test limit.
LAMP amplified reaction programs are：65 DEG C of amplification 60min；80 DEG C of inactivation 10min.
Experiment one,
The DNA of hickory nut dry rot germ (Botryosphaeria dothidea) genome is extracted, DNA solution is measured to obtain
Concentration is 200ng/ μ L.DNA solution is diluted, gradient be 0.0001ng/ μ L, 0.001ng/ μ L, 0.01ng/ μ L, 0.1ng/ μ L,
1ng/μL、10ng/μL、100ng/μL.25 μ L reaction systems are configured to by lower method：The μ L of 8U/ μ L Bst archaeal dna polymerases 0.5
(4U)、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, μ L, 3.75mM the hydroxyl naphthols of 2.5 μ L, 5M glycine betaines of 10mM dNTPs 3.0
Indigo plant (HNB) 1 μ L, the μ L of DNA profiling 1 of above-mentioned each concentration gradient, it is finally each with distilled water using the μ L of distilled water 1 as blank
Complement to 25 μ L.The LAMP amplified reaction programs for using are：65 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, there is not change realization and is in the color of reaction tube
Feminine gender, detects do not have specific band to occur with 2% agarose gel electrophoresis.
Result shows and is to the detection least concentration of hickory nut dry rot germ (Botryosphaeria dothidea) DNA
0.01ng/μL。
Experiment two,
Extract hickory nut dry rot germ (B dothidea), the black disk spore bacterium in square garden (Melanconium oblongum), intend
Phoma (Phomopsis juglandina), the spore of cocoa two (Lasiodiplodia therbromae), Qilian gold rust
Bacterium (Chrysomyxa qilianensis), Pestalotiopsis guepinii bacterium (Pestalotiopsis guepini), camellia charcoal
7 kinds of tree disease DNA of subcutaneous ulcer bacterium (Colletotrichum camelliae) are used as template.
Using 25 μ L reaction systems, it is formulated by following component：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, 2.5 μ L, 5M glycine betaines of 10mM dNTPs 3.0 μ L, 3.75mM hydroxynaphthol blue (HNB) 1 μ L,
DNA solution adds 1 μ L, distilled water to complement to 25 μ L as template.
Template of the result of the test in addition to hickory nut dry rot germ (B dothidea) all shown as feminine gender, in reaction tube
Reaction solution color does not change, and does not have specific band to occur by the detection of 2% agarose gel electrophoresis.Represent the primer
Combining the ring mediated isothermal amplification system for constituting can detect hickory nut dry rot germ (Botryosphaeria
Dothidea), and with specificity.
Comparative example 1-1,
Tested with the combination of following primer：
Upstream outer primer (F3-1)：5’—CGCGTTCAGAAGATTGCCTAT—3’；
Downstream outer primer (B3-1)：5’—TTGTTGCCAAAACACCGCG—3’；
Upstream inner primer (FIP)：5’—GCGTGGGAGAACATCAATGAC-GCTCGAGCTGAAGGTCCG—3’；
Downstream inner primer (FIB)：5’—CTTACACGCCAGAGCCGT-AACGCGAATCGACACCACAG—3’.
Using 25 μ L reaction systems, it is formulated by following component：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, the DNA solution of 1ng/ μ L, 10ng/ μ L, 100ng/ μ L is respectively using concentration as template plus 1 μ L, distilled water complements to 25 μ
L。
All shown as feminine gender, reaction solution color does not change result of the test in reaction tube, by 2% Ago-Gel electricity
Swimming detection does not have specific band to occur.Represent that the ring mediated isothermal amplification system that primer combination is constituted is unable to effective detection
Come out of retirement and take up an official post Dry-rot Phenomena of Persian Walnut bacterium (Botryosphaeria dothidea), even if the concentration of DNA profiling is increased into 100ng/ μ L,
Still cannot effectively distinguish.
Comparative example 1-2,
Tested with the combination of following primer：
Upstream outer primer (F3-2)：5’—CGCGTTCAGAAGATTGCCATT—3’；
Downstream outer primer (B3-2)：5’—TTGTTGCCAAAACACCCGG—3’；
Upstream inner primer (FIP)：5’—GCGTGGGAGAACATCAATGAC-GCTCGAGCTGAAGGTCCG—3’；
Downstream inner primer (FIB)：5’—CTTACACGCCAGAGCCGT-AACGCGAATCGACACCACAG—3’.
Using 25 μ L reaction systems, it is formulated by following component：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 F3-2 0.5μL、10μM B3-
2 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, the DNA solution of 1ng/ μ L, 10ng/ μ L, 100ng/ μ L is respectively using concentration as template plus 1 μ L, distilled water complements to 25 μ
L。
All shown as feminine gender, reaction solution color does not change result of the test in reaction tube, by 2% Ago-Gel electricity
Swimming detection does not have specific band to occur.Represent that the ring mediated isothermal amplification system that primer combination is constituted is unable to effective detection
Come out of retirement and take up an official post Dry-rot Phenomena of Persian Walnut bacterium (Botryosphaeria dothidea), even if the concentration of DNA profiling is increased into 100ng/ μ L,
Still cannot effectively distinguish.
Comparative example 2-1,
Tested with the combination of following primer：
Upstream outer primer (F3-3)：5’—TGCCTGTTCGAGCGTCATTA—3’；
Downstream outer primer (B3-3)：5’—TTCAGAAGGTTCGTCCGGC—3’；
Upstream inner primer (FIP-3)：5’—CCGAGGTCTTTGAGGCGC-GGTATTGGGCACCGTCCTTT—3’；
Downstream inner primer (FIB-3)：5’—GCGTCTTGCCTCAAGCG-GCTCCGAAGCGAGATGTATGT—3’.
Using 25 μ L reaction systems, it is formulated by following component：The μ L (4U) of 8U/ μ L Bst archaeal dna polymerases 0.5,10 ×
ThermoPol Buffer 2.5μL、20μM FIP-3 2.0μL、20μM BIP-3 2.0μL、10μM F3-3 0.5μL、10μM
B3-3 0.5μL、25mM MgCl24.0 μ L, μ L, 3.75mM hydroxynaphthol blues of 2.5 μ L, 5M glycine betaines of 10mM dNTPs 3.0
(HNB) 1 μ L, respectively with hickory nut dry rot germ (B dothidea), apple rod method (Alternaria mali), cocoa
Two spores (Lasiodiplodia therbromae), plan disk stey (Pestalotiopsis theae), vertical withered pyrenomycetes
(Rhizoctonia Solani), rattan storehouse sickle-like bacteria (Fusarium fujikuroi), Sclerotium rolfsii (Sderotium
Rolfsii), colletotrichum gloeosporioides Penz (Colletotrichum gloeosporioides) and blank distilled water are used as DNA moulds
Plate；ddH2O (distilled water) complements to 25 μ L.
All shown as the positive, reaction solution color changes result of the test in reaction tube, by 2% agarose gel electrophoresis
Detection has specific band to occur.Represent that the ring mediated isothermal amplification system that above-mentioned primer combination is constituted is unable to effective detection area
Separate " currant grape seat chamber bacterium (Botryosphaeria dothidea).
Comparative example 2-2,
Tested with the combination of following primer：
Upstream outer primer (F3-4)：5’—TGCCTGTTCGAGCGTCAAAA—3’；
Downstream outer primer (B3-4)：5’—TTCAGAAGGTTCGTCCGGG—3’；
Upstream inner primer (FIP-4)：5’—CCGAGGTCTTTGAGGCGC-GGTATTGGGCACCGTCCAAA—3’；
Downstream inner primer (FIB-4)：5’—GCGTCTTGCCTCAAGCG-GCTCCGAAGCGAGATGTATTG—3’.
Using 25 μ L reaction systems, it is formulated by following component：The μ L (4U) of 8U/ μ L Bst archaeal dna polymerases 0.5,10 ×
ThermoPol Buffer 2.5μL、20μM FIP-4 2.0μL、20μM BIP-4 2.0μL、10μM F3-4 0.5μL、10μM
B3-4 0.5μL、25mM MgCl24.0 μ L, μ L, 3.75mM hydroxynaphthol blues of 2.5 μ L, 5M glycine betaines of 10mM dNTPs 3.0
(HNB) 1 μ L, respectively with hickory nut dry rot germ (B dothidea), apple rod method (Alternaria mali), cocoa
Two spores (Lasiodiplodia therbromae), plan disk stey (Pestalotiopsis theae), vertical withered pyrenomycetes
(Rhizoctonia Solani), rattan storehouse sickle-like bacteria (Fusarium fujikuroi), Sclerotium rolfsii (Sderotium
Rolfsii), colletotrichum gloeosporioides Penz (Colletotrichum gloeosporioides) and blank distilled water are used as DNA moulds
Plate；ddH2O (distilled water) complements to 25 μ L.
All shown as the positive, reaction solution color changes result of the test in reaction tube, by 2% agarose gel electrophoresis
Detection has specific band to occur.Represent that the ring mediated isothermal amplification system that primer combination is constituted is unable to effective detection differentiation
Come out of retirement and take up an official post Dry-rot Phenomena of Persian Walnut bacterium (Botryosphaeria dothidea).
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
The all deformations directly derived or associate, are considered as protection scope of the present invention.
<110>Zhejiang A ＆ F University
<120>Detect the loop-mediated isothermal amplification method of hickory nut dry rot germ
<160> 4
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>Upstream outer primer F3
<400> 1
cgcgttcaga agattgccat a 21
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>Downstream outer primer B3
<400> 2
ttgttgccaa aacacccgc 19
<210> 3
<211> 39
<212> DNA
<213>Artificial sequence
<220>
<223>Upstream inner primer FIP
<400> 3
gcgtgggaga acatcaatga cgctcgagct gaaggtccg 39
<210> 4
<211> 38
<212> DNA
<213>Artificial sequence
<220>
<223>Downstream inner primer FIB
<400> 4
cttacacgcc agagccgtaa cgcgaatcga caccacag 38
Claims (8)
1. it is used to detect the loop-mediated isothermal amplification (LAMP) primer of hickory nut dry rot germ, it is characterised in that：LAMP primer composition thing
It is made up of following 4 primers：
Upstream outer primer F3：5’—CGCGTTCAGAAGATTGCCATA—3’；
Downstream outer primer B3：5’—TTGTTGCCAAAACACCCGC—3’；
Upstream inner primer FIP：5’—GCGTGGGAGAACATCAATGAC-GCTCGAGCTGAAGGTCCG—3’；
Downstream inner primer FIB：5’—CTTACACGCCAGAGCCGT-AACGCGAATCGACACCACAG—3’.
2. it is used to detect the loop-mediated isothermal amplification kit of hickory nut dry rot germ, it is characterised in that：Will comprising such as right
Seek the LAMP primer composition thing described in 1.
3. kit according to claim 2, it is characterised in that：LAMP primer composition thing is：1.6 μM just interior to primer
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 kit according to Claims 2 or 3, it is characterised in that：Kit is also comprising as follows：10×ThermoPol
Buffer、dNTPs、MgCl2, glycine betaine, hydroxynaphthol blue, 8U/ μ L Bst archaeal dna polymerases, ddH2O。
5. it is used to detect the loop-mediated isothermal amplification method of hickory nut dry rot germ, it is characterised in that：
25 μ L reaction systems are consisted of the following composition：The μ L 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 L of 2.5 μ L, 5M glycine betaines of 10mM dNTPs, 3.0 μ L, 3.75mM hydroxynaphthol blue 1, DNA profiling 1 μ L, ddH2O is complemented to
25μL。
6. loop-mediated isothermal amplification method according to claim 5, it is characterised in that：Reactant is detected using the 25 μ L
System carries out LAMP amplified reactions, selects following either method：
Method one, elder generation add dyestuff hydroxynaphthol blue as reaction indicator before amplification, with the color change of hydroxynaphthol blue
As result judgement standard, LAMP amplified reactions are then carried out, after reaction terminates, color changes, that is, the result that develops the color observation
It is judged as the positive to sky blue, judges that display detects hickory nut dry rot germ；Color does not change, and colour developing result is still
For bluish violet is judged as feminine gender, judge display sample without hickory nut dry rot germ, or contained hickory nut dry rot germ content
Not up to the minimal detectable concentration of detection；
Method two, 5 μ L amplified productions are taken, are detected with 2% agarose gel electrophoresis, the positive is judged as if there is trapezoid-shaped strips,
That is display detects hickory nut dry rot germ；Then it is judged as feminine gender without amplified band, that is, shows without hickory nut dry rot germ,
Or contained hickory nut dry rot germ does not reach minimal detectable concentration.
7. loop-mediated isothermal amplification method according to claim 6, it is characterised in that LAMP amplified reaction programs are：65℃
Amplification 60min；80 DEG C of inactivation 10min.
8. loop-mediated isothermal amplification method according to claim 7, it is characterised in that：
Minimal detectable concentration in methods described one, method two is 0.01ng/ μ L.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108315391A (en) * | 2018-04-27 | 2018-07-24 | 安徽省农业科学院植物保护与农产品质量安全研究所 | It is a kind of to be used for the Primer composition and its application that pomegranate dry rot germ LAMP is quickly detected |
| CN108754004A (en) * | 2018-05-10 | 2018-11-06 | 山东省烟台市农业科学研究院 | A method of Botryosphaeria berengeriana f. sp is quickly detected based on LAMP |
| CN109897908A (en) * | 2019-03-18 | 2019-06-18 | 浙江农林大学 | The qLAMP method and the primer for infecting and propagating for Quantitative Monitoring hickory nut dry rot |
| CN110699475A (en) * | 2019-07-29 | 2020-01-17 | 江苏省中国科学院植物研究所 | Padlock probe of pecan alternaria alternata and detection method thereof |
| CN110760604A (en) * | 2019-09-18 | 2020-02-07 | 江苏省中国科学院植物研究所 | Padlock probe for pecan anthracnose pathogen and detection method thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103509856B (en) * | 2013-06-13 | 2015-01-28 | 四川农业大学 | Specific primer used for rapid detection of kiwi fruit soft rot pathogenic bacterium Botryosphaeria dothidea, and detection method of Botryosphaeria dothidea |
| CN104611428A (en) * | 2015-01-21 | 2015-05-13 | 南京农业大学 | LAMP (loop-mediated isothermal amplification) primer composition for detecting colletotrichum gloeosporioides and application of LAMP primer composition |
| CN106350588A (en) * | 2016-08-30 | 2017-01-25 | 浙江农林大学 | Device for rapidly detecting benzimidazole fungicide-resistant botrytis cinerea Pers. based on LAMP |
-
2017
- 2017-03-03 CN CN201710124909.1A patent/CN106868142B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103509856B (en) * | 2013-06-13 | 2015-01-28 | 四川农业大学 | Specific primer used for rapid detection of kiwi fruit soft rot pathogenic bacterium Botryosphaeria dothidea, and detection method of Botryosphaeria dothidea |
| CN104611428A (en) * | 2015-01-21 | 2015-05-13 | 南京农业大学 | LAMP (loop-mediated isothermal amplification) primer composition for detecting colletotrichum gloeosporioides and application of LAMP primer composition |
| CN106350588A (en) * | 2016-08-30 | 2017-01-25 | 浙江农林大学 | Device for rapidly detecting benzimidazole fungicide-resistant botrytis cinerea Pers. based on LAMP |
Non-Patent Citations (3)
| Title |
|---|
| QINGHE CHEN ET AL.,: ""Development and evaluation of specific PCR and LAMP assays for the rapid detection of Phytophthora melonis"", 《EUR J PLANT PATHOL》 * |
| 朱致翔等: ""实时荧光定量PCR定量检测山核桃干腐病病菌潜伏侵染量方法的建立"", 《浙江农林大学学报》 * |
| 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》 * |
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| CN108315391B (en) * | 2018-04-27 | 2021-10-22 | 安徽省农业科学院植物保护与农产品质量安全研究所 | Primer composition for rapid LAMP detection of pomegranate dry rot and application thereof |
| CN108315391A (en) * | 2018-04-27 | 2018-07-24 | 安徽省农业科学院植物保护与农产品质量安全研究所 | It is a kind of to be used for the Primer composition and its application that pomegranate dry rot germ LAMP is quickly detected |
| CN108754004A (en) * | 2018-05-10 | 2018-11-06 | 山东省烟台市农业科学研究院 | A method of Botryosphaeria berengeriana f. sp is quickly detected based on LAMP |
| CN109897908A (en) * | 2019-03-18 | 2019-06-18 | 浙江农林大学 | The qLAMP method and the primer for infecting and propagating for Quantitative Monitoring hickory nut dry rot |
| CN109897908B (en) * | 2019-03-18 | 2022-05-27 | 浙江农林大学 | qLAMP method for quantitatively monitoring infection and spread of hickory nut dry rot and primers used by method |
| CN110699475A (en) * | 2019-07-29 | 2020-01-17 | 江苏省中国科学院植物研究所 | Padlock probe of pecan alternaria alternata and detection method thereof |
| CN110699475B (en) * | 2019-07-29 | 2022-06-14 | 江苏省中国科学院植物研究所 | Padlock probe of pecan alternaria alternata and detection method thereof |
| CN110760604B (en) * | 2019-09-18 | 2022-06-14 | 江苏省中国科学院植物研究所 | Padlock probe for pecan anthracnose pathogen and detection method thereof |
| CN110760604A (en) * | 2019-09-18 | 2020-02-07 | 江苏省中国科学院植物研究所 | Padlock probe for pecan anthracnose pathogen and detection method thereof |
| CN111020055A (en) * | 2020-01-07 | 2020-04-17 | 北京林业大学 | LAMP primer and kit for detecting Lasiodipia gonubiensis |
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