CN109897908A - The qLAMP method and the primer for infecting and propagating for Quantitative Monitoring hickory nut dry rot - Google Patents
The qLAMP method and the primer for infecting and propagating for Quantitative Monitoring hickory nut dry rot Download PDFInfo
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Abstract
The invention discloses one kind dynamically to quantify loop-mediated isothermal amplification (LAMP) primer for monitoring hickory nut dry rot germ spore quantity, and q-LAMP Primer composition is made of following 4 primers: upstream outer primer (F3), downstream outer primer (B3), upstream inner primer (FIP), downstream inner primer (FIB).The present invention goes back while providing the quantitative loop-mediated isothermal amplification kit comprising above-mentioned q-LAMP Primer composition, and provide and dynamically quantify loop-mediated isothermal amplification method using the hickory nut dry rot germ spore quantity that the kit carries out, to obtain spore quantity, DNA concentration.Using the hickory nut dry rot germ spore count amount of method energy Quantitative Monitoring rainwater, tree body and soil of the invention etc..
Description
Technical field
The invention belongs to biotechnology and Plant Disease Epidemiology field, it is related to quantitative ring mediated isothermal amplification (qLAMP)
The quantitative ring mediated isothermal amplification that technology is used to monitor rainwater, tree body and soil etc. hickory nut dry rot germ spore quantity draws
Object group and its application method belong to Plant Disease Epidemiology, dynamic monitoring and the technical field of early warning.
Background technique
Currant grape seat chamber bacterium (Botryosphaeria dothidea) is host's dependent form thryptophytism pathogenic
Fungi, host range is very wide, is to endanger the fungal pathogen of xylophyta health in a kind of world wide, wherein infecting Chinese walnut
It is dry to cause hickory nut dry rot.Hickory nut dry rot generally occurs in the hickory nuts main producing region such as West Zhejiang Province, and injured area gradually expands,
It is the bottleneck of hickory nut industry development.Pathogen is overwintering in Chinese walnut body with mycelium, and spring in next year hides in tree body
Germ come into play, break through bark formed ulceration, surface formed fructification simultaneously discharge spore, cause new infect.Spore
Son borrows wind and rain to propagate, and shows apparent " latent infection " characteristic after invading bark cell or tree body wound, since late March to
There is different degrees of disease November.Mid-April to mid-June, temperature was that spore discharges the height infected at 25 DEG C -30 DEG C
The peak of peak phase, 30 DEG C of 7~August temperature or more of hot weather are unfavorable for the development of germ, autumn air temperature drop to 30 DEG C with
The lower development that can generate germ again.It is badly in need of a kind of quick, accurate measurements rainwater, tree body etc. hickory nut dry rot germ spore at present
The technology of subnumber amount, to achieve the purpose that the propagation to dry rot and make scientific and reasonable prediction.
As the relevant identification method of nucleic acid continues to develop, the method based on regular-PCR has been used successfully to detection mountain core
Dried peach maize ear rot bacterium, but the detection such as PCR method specificity is time-consuming partially long, needs 6~8h, and detection sensitivity is relatively low, detection process compared with
It is many and diverse, it is critical that be also easy to the influence by various PCR mortifiers.Loop-mediated isothermal amplification technique (Loop-mediated
Isothermal amplification, LAMP) it is the new nucleic acid amplification technologies of one kind of Japanese Rong Yan Co., Ltd. invention, because
The advantages that easy to operate, quick, specific high, at low cost for its expansion, become the new nucleic acid amplification technologies that can substitute PCR.
It is 6 regions design, 4 pairs of specific primers for target gene, causes self-loopa under the action of Bst Large fragment polymerase
Strand replacement reaction in 60-65 DEG C of range 60min, can largely synthesize target dna.Since LAMP amplification procedure relies on identification target sequence
6 isolated areas of column, so atopic is very strong, and amplification process is to carry out under constant temperature conditions, common water-bath
Pot or isothermal vacuum flask are just able to satisfy reaction and require, and testing cost reduces, and required time is short.
Summary of the invention
The technical problem to be solved in the present invention is to provide one kind for monitoring rainwater, tree body etc. hickory nut dry rot germ spore
Quantitative ring mediated isothermal amplification (q-LAMP) method, Primer composition and the kit of subnumber amount.
In order to solve the above technical problem, the present invention provides dynamic for monitoring hickory nut dry rot germ spore quantity
Quantitative loop-mediated isothermal amplification (LAMP) primer, q-LAMP Primer composition be made of following 4 primers (that is, by upstream and downstream outer primer, on
Inner primer this four primers in downstream form):
Upstream outer primer (F3): 5 '-TGTCCATAGGTTCACCTCCA -3 '；
Downstream outer primer (B3): 5 '-AGATGGTCTGCCTGTGGT -3 '；
Upstream inner primer (FIP): 5 '-CAAAGTCAGCGCGATGCAGCGACCGGCCAATGCGTAAG -3 '；
Downstream inner primer (FIB): 5 '-AGGGTAACCAAATCGGTGCTGCGTCGATTCGCGTTAGCGG -3 '.
The present invention goes back while providing for monitoring dynamic quantitative loop mediation of hickory nut dry rot germ spore quantity etc.
Warm amplification kit includes above-mentioned q-LAMP Primer composition.
Improvement as kit of the invention: it is positive/negative it is interior to the concentration of primers F IP/BIP be 1.6 μM, it is positive/negative outward
The concentration of primers F 3/B3 is 0.125 μM.
Further improvement as kit of the invention: kit also includes quantitative loop-mediated isothermal amplification premix
Liquid: 10 × ThermoPol Buffer, dNTPs (1mM), MgCl2(6.25mM), glycine betaine (0.6M), 10 × SYBR Green
I, Bst archaeal dna polymerase (0.4U μ L-1)、ddH2O。
The present invention goes back while providing dynamic using the hickory nut dry rot germ spore quantity of mentioned reagent box progress
Quantitative loop-mediated isothermal amplification method:
1), q-LAMP reacts
Q-LAMP reaction system (20 μ L):
The MgCl of 10 × ThermoPol Buffer, 2.0 μ L, 2.0 μ L of dNTPs of 10mM, 25mM2The sweet tea of 5.0 μ L, 5M
2.4 μ L of dish alkali, 10 × SYBR Green I, 1.2 μ L, 8U/ μ L Bst archaeal dna polymerase 1 μ L, 20 μM of 1.6 μ L of FIP primer,
20 μM of 1.6 μ L of BIP primer, 10 μM of 0.25 μ L of F3 primer, 10 μM of 0.25 μ L of B3 primer, DNA profiling 1 μ L, ddH2O is supplied
To 20 μ L；
Amplification condition is 65.9 DEG C of constant temperature, 1min (this time point obtains fluorescence data, this process is a circulation), into
Row 80 circulations；80 DEG C of reaction 10min terminate reaction；
Note: it can get CTValue；
That is, 20 μ L detect reaction system, by above-mentioned 19 μ L detection solution and 1 μ L DNA profiling to be measured, (concentration is in 0.001ng/ μ
L~100ng/ μ L) composition.
2) spore quantity, DNA concentration, are obtained.
Improvement as quantitative loop-mediated isothermal amplification method of the invention:
Spore quantity (a) calculation formula is
DNA concentration calculation formula is
Y is CTValue.
In the present invention,
Dilute sample CTThe standard curve 1:y=- being worth between (y) and the Log value (x) of every milliliter of sample spore quantity
6.863x+49.937, spore quantity (a) calculation formula are
Calculation formula: building standard curve, the Log value with every milliliter of sample spore quantity is x-axis, corresponding CTValue is y
Axis mapping, display is successively decreased linear equality [y=mx+b or y=m (every milliliter of sample spore quantity of log)+b], linear from what is successively decreased
Equation can derive following equation, to determine the amount of unknown sample:
Standard curve 2: defining lg (DNA concentration value/0.0001) is x, corresponding CTValue is that y-axis is mapped, it may be assumed that y=-
3.5823x+37.233 DNA concentration calculation formula is
In the present invention, the analysis of amplification and determination method include:
SYBR Green I is added before amplification as reaction indicator, with fluorescence signal as result judgement foundation, instead
Instrument shows quantitative result C automatically according to standard curve after answeringTValue.According to q-LAMP amplified reaction time and gradient dilution
Standard curve is made in hickory nut dry rot germ spore, further according to the reaction time of sample to be tested, can accurately calculate the spore of the sample
Sub- initial concentration.
The hickory nut dry rot germ spore count amount of method energy Quantitative Monitoring rainwater, tree body and soil of the invention etc., gram
The deficiency of previous real-time fluorescence quantitative PCR detection method is taken, has high sensitivity, high specific, low pollution, stable reaction etc. excellent
Point, this method is not vulnerable to PCR mortifier and other pollutant effects.
Invention process of the invention includes:
(1) according to from the β-between the β-tubulin sequence that hickory nut dry rot germ expands and its bacterium his strain
Design specific primer in tubulin sequence difference site；
(2) screening of primer；
(3) specific detection of primer；
(4) q-LAMP reaction temperature optimizes；
(5) detection sensitivity of q-LAMP.
It is specific as follows:
1, the design of primer:
It is primarily based on red mould β-tubulin sequence alignment and goes out 2 higher sequences of homology, devise 1 pair of grape seat chamber
Bacterium primer A1 (dry A β-F1816 and dry A β-R), downloads quasi- pestalotia bacteria (Pestalotiopsis from GenBank
Microspora, accession number KU377338.1), Trichoderma (Trichoderma viride, accession number Z15055.1), layer
Raw sickle-like bacteria (Fusarium proliferatum, accession number KJ544178.1), Tuber Melanosporum (Nigrospora
Chinensis, accession number KY019579.1), wheel layer charcoal bacterium (Daldinia sp., accession number KU684130.1), chain lattice
β-tubulin the sequence of spore bacterium (Alternaria oregonensis, accession number JQ671993.1), according to hickory nut dry rot
β-tubulin sequence difference site between the β-tubulin sequence that germ expands and its bacterium his strain, designs Si Taote
The primer (for example, online primer-design software Primer Explore V5 can be used) of anisotropic grape seat chamber bacterium LAMP detection.
The primer information is shown in Table 1.
1 primer sequence information of table
2, the screening of primer
LAMP uses 25 μ l reaction systems, specific as follows:
LAMP response parameter is set as 65.9 DEG C, 60min.
Note: the screening of primer uses LAMP detection technique, using ABI 2720PCR instrument, according to the color of HNB indicator
Variation and electrophoretogram screen in turn obtains suitable primer.
LAMP determines whether positive amplification after reaction, by color change and electrophorogram.If hickory nut is dry
Maize ear rot bacterium sample LAMP amplified production shows that sky blue, electrophorogram should be scalariform band, and sample to be tested is the positive.Hickory nut is dry
Maize ear rot bacterium sample LAMP amplified production shows that purple, electrophorogram are expanded without band, determines that sample to be tested is feminine gender.By screening
Show that the primer sets 1 in table 1 are detected suitable for the LAMP of hickory nut dry rot germ.
3, the specific detection of primer
For the specificity of detection primer, LAMP verifying, hickory nut dry rot are carried out to each bacterial strain using the primer sets 1 in table 1
Germ bacterial strain (dry rot BDDH-1 bacterial strain, dry rot BDDH-2 bacterial strain, dry rot BDDH-3 bacterial strain) can amplify product, color
It is shown as sky blue, agarose gel electrophoretogram is in scalariform band；Reference strain cannot amplify product, and color is shown as purple
Color, agarose gel electrophoretogram is without scalariform band.Reaction result shows such as Fig. 1, and primer sets 1 have to hickory nut dry rot germ
Specificity.
Reference strain, including Trichoderma (Trichoderma viride), the raw sickle-like bacteria (Fusarium of layer
Proliferatum), intend pestalotia bacteria (Pestalotiopsis microspora), Tuber Melanosporum (Nigrospora
Chinensis), alternaric bacteria (Alternaria oregonensis), wheel layer charcoal bacterium (Daldinia sp.).
4, the optimization of response procedures
For the purpose of the q-LAMP optimal reaction temperature for establishing energy Quantitative Monitoring hickory nut dry rot germ, above-mentioned primer is utilized
Combination 1 constitutes detection liquid with reaction premixed liquid, and 1 μ L of hickory nut dry rot germ DNA profiling (concentration is 0.1ng/ μ L) is added, will
Reaction temperature is respectively set 67.0 DEG C, 66.6 DEG C, 65.9 DEG C, 64.5 DEG C, 62.8 DEG C, 61.4 DEG C, 60.5 DEG C and 60.0 DEG C, and each 80
A circulation, each circulation 60s, 80 DEG C 10 minutes.Reaction result shows such as Fig. 2, and when reaction temperature is 65.9 DEG C, fluorescence is most
By force, amplified production is most, and the reaction time is most short, and in 38min, amplification reaches peak value.65.9 DEG C are the best temperature expanded
Degree, subsequent dry rot disease q-LAMP reaction are carried out at 65.9 DEG C.
5, the sensitivity verification result of detection architecture
It is examined with DNA solution of the dry rot germ q-LAMP detection architecture after optimization to the pure mycelia of dry rot germ of gradient dilution
Survey the result shows that, DNA concentration be 100ng/ μ L, 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L and 0.001ng/ μ L
When, it can effectively expand, same sample repeatability is reliable.The corresponding proliferation time of each concentration be respectively 16.32min,
18.68min, 22.43min, 26.63min, 30.84min and 33.26min.The result shows that the q-LAMP detection architecture established
Theoretically the detection line of dry rot germ DNA is 1pg DNA.
6, the foundation of q-LAMP amplification and standard curve
It takes the dry rot germ pycnidia of certain amount maturation in 2.0mL centrifuge tube, 1.5mL sterile water is added and carries out
The spore of dry rot germ is diluted to 1.0 × 10 then with 3 layers of filter paper filtering elution spore by broken wall treatment5A/mL, 1.0 ×
104A/mL, 1.0 × 103A/mL, 1.0 × 102A/mL, 1 × 1016 series of concentrations gradients such as a/mL, 1/mL.With excellent
Dry rot germ q-LAMP detection architecture after change detects the spore DNA of gradient dilution, is 1.0 × 10 in spore concentration5A/
mL、1.0×104A/mL, 1.0 × 103A/mL, 1.0 × 102A/mL and 1.0 × 101When a/mL, can effectively it expand.Respectively
The corresponding proliferation time of concentration is respectively 16.67min, 21.51min, 28.69min, 36.26min and 43.61min, is existed
Within 60min.The result shows that the q-LAMP detection architecture established can theoretically detect that concentration is 1.0 × 101A/mL's
Spore.
The amplification of the dry rot germ spore DNA profiling of 6 gradient dilutions establishes dilute sample CTIt is worth (y) and every milli
Rise the standard curve between the Log value (x) of sample spore quantity, y=-6.863x+49.937, coefficient R2=0.9922,
I.e. good linear relationship is presented in the two.
The method can be used for the quantitative determination of rainwater, air, tree body etc. dry rot germ spore.
The present invention is based on the calibration curve equation that quantitative standard sample is made, the logarithm of standard concentration with react
Coefficient R between time2> 0.95, the DNA for quantitative unknown sample provides theoretical foundation.
The present invention establishes quick, specific, high sensitivity the Quantitative Monitoring technical system of hickory nut dry rot germ, should
The foundation of method has a very important significance the prediction of dry rot and prevention and control.
Compared with prior art, the present invention having following technical advantage:
(1) high specificity, identification are quick: detection method is the results show that common fungi is the negative findings of detection, only
There is hickory nut dry rot germ positive findings just occur.Entire detection process only needs to overcome biography within 1 hour without uncapping
A large amount of professional knowledge is needed to judge in the aobvious pathogen spore microexamination and counting process of system, result is difficult to determine
Disadvantage.
(2) high sensitivity: accurate quantitative analysis carries out sensitivity technique, q- by hickory nut dry rot germ spore genomic DNA
It is 1.0 × 10 that minimum hickory nut dry rot germ spore concentration, which can be detected, in LAMP technology1A/mL, and 1.0 × 101~1.0 ×
105There is good linear relationship in a/mL concentration range.Not only overcoming the suppressed object of normal PCR influences the problems such as big,
The limitation of the presence or absence of germ can only be determined by solving common LAMP；
Practicability is good, has a wide range of application: can be with hickory nut dry rot in Quantitative Monitoring soil, air and rainwater and tree body
Bacterium provides foundation suitable for the dynamic monitoring of hickory nut dry rot germ spore count amount for its prediction.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 is primer specificity measurement；
1: Trichoderma (Trichoderma viride)；2: the raw sickle-like bacteria of layer (Fusarium proliferatum)；3: quasi-
Pestalotia bacteria (Pestalotiopsis microspora)；4: Tuber Melanosporum (Nigrospora chinensis)；5: rod method
Bacterium (Alternaria oregonensis)；6: wheel layer charcoal bacterium (Daldinia sp.) 7:CK；8: dry rot BDDH-1 bacterial strain；9:
Dry rot BDDH-2 bacterial strain；10: dry rot BDDH-3 bacterial strain；
Fig. 2 is influence diagram of the temperature to dry rot germ q-LAMP detection architecture；
Fig. 3 is the specificity figure of dry rot germ q-LAMP detection architecture；
Fig. 4 is the sensitivity map of q-LAMP detection grape seat chamber bacterium DNA；
The corresponding concentration of curve from left to right be followed successively by 100ng/ μ L, 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ L,
0.01ng/ μ L and 0.001ng/ μ L.
Specific embodiment
Below by example with reference, the present invention is described further, is described below to protection model of the invention
The restriction not constituted in all senses is enclosed, is only illustrated.
Main agents used in the following embodiment and instrument are as follows: Bst archaeal dna polymerase (New England Biolabs,
MgCl2(Sigma), dNTPs (Nanjing Vazyme Biotechnology Co., Ltd.), glycine betaine, ddH2O water, molecular mass standard DNA
Maker (TaKaRa bio-engineering corporation genome), fungal genomic DNA Rapid extraction kit (raw work bioengineering share
Co., Ltd), BIO-RAD CFX96 real-time fluorescence quantitative PCR instrument.
The specificity analysis of embodiment 1, detection architecture
Using hickory nut dry rot germ (Botryosphaeria dothidea) as positive control, with Trichoderma
(Trichoderma viride), the raw sickle-like bacteria of layer (Fusarium proliferatum), quasi- pestalotia bacteria
(Pestalotiopsis microspora), Tuber Melanosporum (Nigrospora chinensis), alternaric bacteria (Alternaria
Oregonensis), layer charcoal bacterium (Daldinia sp.) six kinds of fungies are taken turns as negative control, and using distilled water as blank pair
Shine into capable test.
Solution totally 19 μ L is detected, DNA profiling to be measured is added, and (concentration is in 0.001ng/ μ L~100ng/ μ L, positive control, yin
Property control when, concentration is 0.1ng/ μ L) 1 μ L, constitute 20 μ L detect reaction system.Bst containing 8U/ μ L in 20 μ L systems
1 μ L of archaeal dna polymerase, 10 × ThermoPol Buffer, 2.0 μ L, 20 μM of 1.6 μ L of FIP primer, 20 μM of 1.6 μ of BIP primer
L, the MgCl of 10 μM of 0.25 μ L of F3 primer, 10 μM of 0.25 μ L of B3 primer, 25mM25.0 μ L, 10mM 2.0 μ L of dNTPs,
2.4 μ L of glycine betaine, 10 × SYBR Green I, 1.2 μ L, the 1 μ L of DNA profiling of 5M, distilled water complement to 20 μ L.Q-LAMP amplification
Response procedures are as follows: 65.9 DEG C, 80 circulations, every circulation 60s；80 DEG C, 10min.
Reaction result is as shown in figure 3, there is fluorescence signal (corresponding expansion by the sample detection of template of dry rot germ DNA
Increasing the time is that 23.94), testing result is the positive, using other 6 kinds of fungal DNAs template and ddH2The sample of O control is not detected
Fluorescence signal, testing result are feminine gender.It is dry that testing result shows that the LAMP primer group 1 screened can be detected specifically
Maize ear rot bacterium.
The sensitivity technique of embodiment 2, primer
It is pure to the dry rot germ of gradient dilution with the dry rot germ q-LAMP detection architecture (as described in Example 1) after optimization
The DNA solution testing result of mycelia is as shown in figure 4, be 100ng/ μ L, 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ in DNA template concentration
L, it when 0.01ng/ μ L and 0.001ng/ μ L, can effectively expand, same sample repeatability is reliable.When the corresponding amplification of each concentration
Between be respectively 16.32min, 18.68min, 22.43min, 26.63min, 30.84min and 33.26min.The result shows that being built
Vertical q-LAMP detection architecture theoretically dry rot germ DNA detection line be 1pg DNA.
The acquisition of embodiment 3, standard curve:
Standard curve 1, spore concentration be 1.0 × 105A/mL, 1.0 × 104A/mL, 1.0 × 103A/mL, 1.0 ×
102A/mL and 1.0 × 101When a/mL, the corresponding proliferation time of each concentration be respectively 16.67min, 21.51min,
28.69min, 36.26min and 43.61min, according to dilute sample CTIt is worth the Log value (x) of (y) and every milliliter of sample spore quantity
Between standard curve: y=-6.863x+49.937, coefficient R2=0.9922, spore quantity (a) calculation formula is
Calculation formula: building standard curve, the Log value with every milliliter of sample spore quantity is x-axis, corresponding CTValue is y
Axis mapping, display is successively decreased linear equality [y=mx+b or y=m (every milliliter of sample spore quantity of log)+b], linear from what is successively decreased
Equation can derive following equation, to determine the amount of unknown sample:
Standard curve 2, mycelia DNA concentration be 100ng/ μ L, 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L and
When 0.001ng/ μ L, can effectively expand, the corresponding proliferation time of each concentration be respectively 16.32min, 18.68min,
22.43min, 26.63min, 30.84min and 33.26min.Standard curve is constructed, defining lg (DNA concentration value/0.0001) is
X, corresponding CTValue is that y-axis is mapped, it may be assumed that y=-3.5823x+37.233, coefficient R2=0.9904, DNA concentration calculation formula
For
Spore in embodiment 4, q-LAMP detection woodland water sample
Placed in the hickory nut woodland infected by dry rot, at random selection 5 open canister (30 × 20 ×
10cm), set-point should be away from disease plant about 50cm at.Standing time is selected as before the flush period of prediction, second day will be each
Rainwater-collecting in container.The water sample that 100mL is collected is measured, conidial genomic DNA in water sample is extracted.
Extracting mode are as follows: 100mL sample is centrifuged 5min with 6800rcf, 90% water is discharged, by being concentrated into twice
1mL, and spore being resuspended in remaining 1mL water sample by the concussion that is vortexed, then take 1mL spore suspension with
6800rpm is centrifuged 5min, the water of 800 μ L is discharged, and spore is resuspended in remaining 200 μ L water sample by being vortexed concussion
In, 200 μ L10%Chelex-100 solution, 200 μ L10%SDS solution and 0.4g pickling pearl is added, is placed in Fsat-prep instrument
In, after shaking 40s with the speed of 6m/s, 5min is heated in 100 DEG C of water-baths, on ice after shaking heating stepses in triplicate
Place 2min.Then with 10000rpm centrifugation 5 minutes, 400 μ L of supernatant liquid is taken, 500 μ L buffer GP2 (TIANGEN examination is added
Agent box), it mixes well；The liquid of mixing is transferred in adsorption column CB3,12000rpm is centrifuged 30s, discards waste liquid；To adsorption column
500 μ L buffer GD, 12000rpm centrifugation 30s are added in CB3, discards waste liquid, adsorption column CB3 is put into collecting pipe；To suction
600 μ L buffer PW, 12000rpm centrifugation 30s are added in attached column CB3, discards waste liquid, adsorption column CB3 is put into collecting pipe,
Then it is primary to repeat this step；Adsorption column CB3 is put back in collecting pipe, 12000rpm is centrifuged 2min, discards waste liquid.By adsorption column
CB3, which is placed in, to be placed at room temperature for several minutes, thoroughly to dry rinsing liquid remaining in adsorbent material；Adsorption column CB3 is transferred to one to do
In net centrifuge tube, 50-200 μ L elution buffer TE is vacantly added dropwise to the intermediate position of adsorbed film, is placed at room temperature for 2-5 minutes,
12000rpm is centrifuged 2min, and solution is collected into centrifuge tube；100mL water sample finally obtains the lysate of 50 μ L.
Take 1 μ L lysate directly as DNA profiling, according to after optimization reaction system and amplification condition (such as 1 institute of embodiment
State) q-LAMP detection is carried out on BIO-RAD CFX96 real-time fluorescence quantitative PCR instrument, record proliferation time.According to spore quantity
With the corresponding relationship of proliferation time, conversing spore quantity, (y=-6.863x+49.937, spore quantity (a) calculation formula are).The proliferation time of institute sample Container A-E is respectively 30.93,33.22,34.00,37.45 and
39.09, the corresponding spore amount of each sample is respectively 588.17,272.77,209.99,65.99 and 38.107, with practical phase
Symbol.
It is illustrated below: 30.93=-6.863x+49.937, therefore x=2.7695；102.7695=588.17.
Embodiment 5 detects the sample amounts of artificial addition spore
Respectively toward the spore for artificially adding dry rot germ on 3 prepregs (2cm × 2cm), the spore quantity of each film
Respectively 1.0 × 103It is a, 1.0 × 102It is a, 1 × 101It is a.The film for being adsorbed with spore of collection is carried out cutting into side length small
It is put into 2mL centrifuge tube after the fractionlet of 1mm, then extracts the genomic DNA of spore on film.
Extraction process are as follows: 200 μ L10%Chelex-100 solution, 200 μ L10%SDS solution and 0.4g is added in centrifuge tube
Pickling pearl is placed in Fsat-prep instrument, after shaking 40s with the speed of 6m/s, heats 5min in 100 DEG C of water-baths, shakes
Heating stepses in triplicate after in placing 2min on ice.Then with 10000rpm centrifugation 5 minutes, 400 μ L of supernatant liquid is taken, is added
500 μ L buffer GP2 (TIANGEN kit), mix well；The liquid of mixing is transferred in adsorption column CB3,12000rpm from
Heart 30s, discards waste liquid；500 μ L buffer GD, 12000rpm centrifugation 30s are added into adsorption column CB3, discards waste liquid, will adsorb
Column CB3 is put into collecting pipe；600 μ L buffer PW, 12000rpm centrifugation 30s are added into adsorption column CB3, discard waste liquid, it will
Adsorption column CB3 is put into collecting pipe, and it is primary then to repeat this step；Adsorption column CB3 is put back in collecting pipe, 12000rpm centrifugation
2min discards waste liquid.Adsorption column CB3 is placed in and is placed at room temperature for several minutes, thoroughly to dry rinsing liquid remaining in adsorbent material；
Adsorption column CB3 is transferred in a clean centrifuge tube, 50-200 μ L elution buffer is vacantly added dropwise to the intermediate position of adsorbed film
Liquid TE is placed at room temperature for 2-5 minutes, and 12000rpm is centrifuged 2min, and solution is collected into centrifuge tube；The final cracking for obtaining 50 μ L
Liquid.
Take 1 μ L lysate directly as DNA profiling, according to after optimization reaction system and amplification condition (such as 1 institute of embodiment
State) q-LAMP detection is carried out on BIO-RAD CFX96 real-time fluorescence quantitative PCR instrument, record proliferation time.According to spore quantity
(y=-6.863x+49.937, spore quantity (a) calculation formula are corresponding relationship between proliferation time), it changes
It calculates spore quantity and is compared with the spore amount artificially added.Proliferation time 29.42,35.94 and of institute's sample
43.48, the corresponding spore amount of each film is respectively 976.11,109.49 and 8.73.The q-LAMP testing result of spore quantity
It is consistent with the volume trends artificially added.
Embodiment 6, the hickory nut woodland on Linan Long Gangzhen, Qingliangfeng National Nature Reserve town, the town Chang Hua, the town Tuan Kou, sun town and other places, point
Not from the trunk bottom of morbidity to the position below 2m, every the phloem tissue of 50cm acquisition morbidity, labeled as 0,50,100,
150, sample point at 200cm etc. 5；Separately from the trunk scab position of morbidity, the phloem tissue of trunk is acquired at a certain distance,
Labeled as 0,5,10, sample point at 20cm etc. 4.
Acquisition sample bast 3g is taken, is respectively put into FW100 type high speed Universal pulverizer, after crushing 2min, takes 400mg
It organizes liquid feeding nitrogen to roll sample with pestle, is quickly firmly ground when remaining a small amount of liquid nitrogen, repeat grinding 3-4 times, then use
Plant genome DNA extracts kit (TIANGEN) extracts DNA, to obtain the DNA profiling of each acquisition sample.
Standard sample is the DNA solution of the pure mycelia of dry rot germ；It is handled through 10 times of gradient dilutions, to obtain standard sample
DNA profiling, concentration be 100ng/ μ L to 0.001ng/ μ L.
Acquisition sample is reacted on quantitative fluorescent PCR simultaneously with standard sample, record will acquisition sample and standard
Sample amplification cycle threshold (CT), the standard curve 2 obtained by dilute sample (y=-3.5823x+37.233, DNA concentration meter
Calculating formula is) come determine acquisition sample in dry rot germ DNA amount.As a result with collection point it is continuous raising with
And the increase with acquisition scab position distance, the amount of DNA of the dry rot germ detected are constantly reduced.
Above-mentioned each sample point resulting amplification cycles threshold value (CT) and the resulting spore of conversion on quantitative fluorescent PCR
Amount is described in table 2 below.
Table 2
Comparative example 1-1,
It is tested with the combination of following primer:
Upstream outer primer (F3-1): 5 '-TGTCCATAGGTTCACCTCCT -3 '；
Downstream outer primer (B3-1): 5 '-AGATGGTCTGCCTGTGGG -3 '；
Upstream inner primer (FIP-1): 5 '-CAAAGTCAGCGCGATGCAGCGACCGGCCAATGCGTAAG -3 '；
Downstream inner primer (FIB-1): 5 '-AGGGTAACCAAATCGGTGCTGCGTCGATTCGCGTTAGCGG -3 '；
Note: the 3 ' of two outer primers hold the last one base A and T to replace with T and G respectively, to verify the specificity of primer.
With the primer (concentration, dosage remain unchanged) of above-mentioned primer alternate embodiment 1,20 μ L reaction systems remaining at
Divide and q-LAMP amplified reaction program is equal to embodiment 1；To the positive control in embodiment 1,6 negative controls, 1 blank
Control is detected.
Fluorescence signal is not detected all shown as feminine gender in test result.The ring mediated isothermal for indicating that primer combination is constituted expands
Increasing system cannot effectively detect dry rot germ, still can not effective district even if the concentration of DNA profiling is increased to 200ng/ μ L
Point.
Comparative example 1-2,
It is tested with the combination of following primer:
Upstream outer primer (F3-2): 5 '-TGTCCATAGGTTCACCTCCA -3 '；
Downstream outer primer (B3-2): 5 '-AGATGGTCTGCCTGTGGT -3 '；
Upstream inner primer (FIP-2): 5 '-CAAAGTCAGCGCGATGCAGCGACCGGCCAATGCGTAAT -3 '；
Downstream inner primer (FIB-2): 5 '-AGGGTAACCAAATCGGTGCTGCGTCGATTCGCGTTAGCGC -3 '；
Note: the 3 ' of two inner primers hold the last one bases G and G to replace with T and T respectively, to verify the special of primer
Property.
With the primer (concentration, dosage remain unchanged) of above-mentioned primer alternate embodiment 1,20 μ L reaction systems remaining at
Divide and q-LAMP amplified reaction program is equal to embodiment 1；To the positive control in embodiment 1,6 negative controls, 1 blank
Control is detected.
Fluorescence signal is not detected all shown as feminine gender in test result.The ring mediated isothermal for indicating that primer combination is constituted expands
Increasing system cannot effectively detect hickory nut dry rot germ, even if the concentration of DNA profiling is increased to 200ng/ μ L, still can not
Effectively distinguish.
The above list is only a few specific embodiments of the present invention for finally, it should also be noted that.Obviously, this hair
Bright to be not limited to above embodiments, acceptable there are many deformations.Those skilled in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Sequence table
<110>Zhejiang A & F University
<120>the qLAMP method and the primer for infecting and propagating for Quantitative Monitoring hickory nut dry rot
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tgtccatagg ttcacctcca 20
<210> 2
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
agatggtctg cctgtggt 18
<210> 3
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
caaagtcagc gcgatgcagc gaccggccaa tgcgtaag 38
<210> 4
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
agggtaacca aatcggtgct gcgtcgattc gcgttagcgg 40
Claims (6)
1. dynamically quantifying loop-mediated isothermal amplification (LAMP) primer for monitoring hickory nut dry rot germ spore quantity, feature exists
In:
Q-LAMP Primer composition is made of following 4 primers:
Upstream outer primer (F3): 5 '-TGTCCATAGGTTCACCTCCA -3 '；
Downstream outer primer (B3): 5 '-AGATGGTCTGCCTGTGGT -3 '；
Upstream inner primer (FIP): 5 '-CAAAGTCAGCGCGATGCAGCGACCGGCCAATGCGTAAG -3 '；
Downstream inner primer (FIB): 5 '-AGGGTAACCAAATCGGTGCTGCGTCGATTCGCGTTAGCGG -3 '.
2. dynamically quantifying loop-mediated isothermal amplification kit for monitoring hickory nut dry rot germ spore quantity, feature exists
In: it include q-LAMP Primer composition as described in claim 1.
3. kit according to claim 2, it is characterised in that: it is positive/negative it is interior to the concentration of primers F IP/BIP be 1.6 μM,
The concentration of positive/negative outside primers F 3/B3 is 0.125 μM.
4. kit according to claim 2 or 3, it is characterised in that: kit also includes quantitative ring mediated isothermal amplification
Reaction premixed liquid, the reaction premixed liquid include 10 × ThermoPol Buffer, dNTPs, MgCl2, glycine betaine, 10 × SYBR
Green I, Bst archaeal dna polymerase, ddH2O。
5. dynamically fixed using the hickory nut dry rot germ spore quantity that any kit of claim 2~4 carries out
Measure loop-mediated isothermal amplification method, it is characterised in that:
1), q-LAMP reacts
Q-LAMP reaction system:
The MgCl of 10 × ThermoPol Buffer, 2.0 μ L, 2.0 μ L of dNTPs of 10mM, 25mM2The glycine betaine of 5.0 μ L, 5M
Bst archaeal dna polymerase 1 the μ L, 20 μM of 1.6 μ L of FIP primer, 20 μM of 2.4 μ L, 10 × SYBR Green I, 1.2 μ L, 8U/ μ L
1.6 μ L of BIP primer, 10 μM of 0.25 μ L of F3 primer, 10 μM of 0.25 μ L of B3 primer, DNA profiling 1 μ L, ddH2O complements to 20
μL；
Amplification condition is 65.9 DEG C of constant temperature, 1min, carries out 80 circulations；80 DEG C of reaction 10min terminate reaction；
2) spore quantity, DNA concentration, are obtained.
6. quantitative loop-mediated isothermal amplification method according to claim 5, it is characterised in that:
Spore quantity (a) calculation formula is
DNA concentration calculation formula is
Y is CTValue.
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