CN104404157B - LAMP detection primer composition for detecting phytophthora drechsler tucker, LAMP detection kit, and LAMP detection method - Google Patents
LAMP detection primer composition for detecting phytophthora drechsler tucker, LAMP detection kit, and LAMP detection method Download PDFInfo
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Abstract
The invention discloses an LAMP detection primer composition for detecting phytophthora drechsler tucker, an LAMP detection kit and an LAMP detection method. The LAMP detection primer composition comprises a forward inner primer FIP, a backward inner primer BIP, a forward outer primer F3, a backward outer primer B3 and a backward loop primer. The detection method disclosed by the invention is high in accuracy, high in specificity, convenient to operate and good in practicality, achieves isothermal amplification, provides a new technical platform for detecting phytophthora drechsler tucker, is used for high-sensitivity fast detection of phytophthora drechsler tucker and the identification of pathogens at the initial stage of disease infection, and can be used to detect phytophthora drechsler tucker in field soil. The LAMP detection primer composition for detecting phytophthora drechsler tucker, the LAMP detection kit and the LAMP detection method disclosed by the invention have significance of disease control caused by phytophthora drechsler tucker and reducing of blind use of pesticides, can reduce production cost, and has the great importance in reducing environmental pollution caused by the pesticides.
Description
Technical field
The invention belongs to biological technical field and in particular to a kind of lamp detection primer compositionss of Phytophthora drechsleri bacterium and its
Lamp detection kit and lamp detection method.
Background technology
Phytophthora drechsleri (phytophthora drechsleri) be early 1931 by drechsler from potato tuberss
Upper separation obtains.Phytophthora drechsleri is nineteen eighty-two report in China, can infect a novel species of Fructus Cucumidis sativi.Phytophthora drechsleri is invaded at present
Contaminating the vegetable plague causing is that a kind of universal soil passes destructive disease, and harm host extensively, often makees to diversified economy
The production of thing (as Fructus Cucumidis sativi, Radix Betae, Rhizoma Solani tuber osi, Cedrus deoclar (Roxb.) G. Don etc.) brings heavy losses.Phytophthora drechsleri Pseudomonas is in oomycota
(oomycota), Oomycete (oomycetes), Peronosporales (peronosporales), pythiaceae (pythiaceae), Phytophthora
(phytophthora).The microbial plant disease of Phytophthora drechsleri is widely distributed, so setting up the rapid molecular of Phytophthora drechsleri bacterium
It is significant that detection technique causes the early stage of epidemic disease to control for it.At present this disease is not also fast and effectively prevented and treated
Measure, the most effective approach controlling this disease is exactly to strengthen quarantining, and prevents the propagation of pathogen and hinders its diffusion way.
The taxonomic identification of traditional Phytophthora drechsleri bacterium is mainly based upon morphological feature, Pathogenicity, Physiology and biochemistry spy
Levy.Traditional method has played important function in the detection of Phytophthora drechsleri bacterium, but wastes time and energy and require operator to possess
The phytophthora separation of specialty, Morphological Identification knowledge and rich experience；Time-consuming, sensitivity for traditional classification authentication method simultaneously
Interference that is low, being easily subject to the factors such as artificial and environment, it is impossible to make diagnosis in disease incubation period and their early stage, is difficult to disease
Evil is timely monitored and effective control.With the development of molecular biology, pcr technology diagnoses for pathogenic and provides
Quick, sensitive, accurate advantage.The method of common pcr has been used successfully to detection Phytophthora drechsleri bacterium although pcr method is in spy
It is greatly improved in the opposite sex and sensitivity, but detection time is still long, general 4～5h, pcr method relies on precision simultaneously
Temperature cycling device, its detection sensitivity is higher, detection process is complicated it is impossible to meet the demand of quick detection.
Loop-mediated isothermal amplification technique (loop-mediated isothermal amplification, lamp) is a kind of
New nucleic acid amplification technologies, because it is simple to operate, quick, specificity is high, low cost and other advantages, becomes and can substitute the new of pcr
Nucleic acid amplification technologies.It is 6 regions design 4 species specific primers for target gene, in the work of bst Large fragment polymerase
With under cause self-loopa strand replacement reaction, in 60～65 DEG C of scopes 80min, while a large amount of synthesis target dna, be accompanied by by-product
The magnesium pyrophosphate precipitation of thing -- white produces.Because lamp amplification procedure relies on identification 6 isolated areas of target sequence, so reaction
Specificity is very strong, and amplification process is to carry out under constant temperature, common water-bath or just have the equipment of stable thermal source
Reaction can be met require, testing cost substantially reduces.Because lamp reaction is simple, quick, efficient, economic dispatch feature, thus tool
There is extremely wide application prospect.Since lamp detection technique sets up 14 years, this technology has been widely used for viral, thin
The detection research of the pathogen such as bacterium, parasite, funguses, but the detection in pathogenic oomycetes is reported seldom, Phytophthora drechsleri bacterium
Detection is not reported both at home and abroad.
Content of the invention
Goal of the invention: special for cycle length, detection method needed for Phytophthora drechsleri bacterium biological detection method in prior art
The problem that the opposite sex is poor, sensitivity is low, it is an object of the invention to provide a kind of lamp detection primer compositionss of Phytophthora drechsleri bacterium.This
That invents another object is that the lamp detection kit providing above-mentioned Phytophthora drechsleri bacterium.The present invention another object is that the above-mentioned pick of offer
The lamp detection method of family name's phytophthora.
Technical scheme: in order to realize foregoing invention purpose, the technical solution used in the present invention is:
A kind of lamp detection primer compositionss for detecting Phytophthora drechsleri bacterium: by positive inner primer fip, reverse inner primer
Bip, positive outer primer f3, reverse outer primer b3 and reverse ring primer lb composition；Each primer sequence is specific as follows:
Fip:5 '-cggattttctagaacgtggtaccaa-aatgaagagtcgactctagca-3 '；
Bip:5 '-actattgagctggacggcaa-tcgatagcagcccaagag-3 '；
F3:5 '-gtgatcctttcaccctgg-3 '；
B3:5 '-ttacaaatgtcagctggatg-3 '；
Lb:5 '-tgtacgtctacagaggatttggat-3 '.
Application in detection Phytophthora drechsleri bacterium for the described lamp detection primer compositionss.
A kind of lamp detection kit of detection Phytophthora drechsleri bacterium: comprise 1ml detection solution, described detection solution bag
Include: 32mm forward direction inner primer fip, 32mm reverse inner primer bip, 8mm forward direction outer primer f3,8mm reverse outer primer b3,8mm ring
Primer lb, 48mm dntps, 0.8m tris-hc, 0.4mm kcl, 0.4mm (nh4)2so4、0.24mm mgso4, 4%triton
X-100, bst dna polymerase 320 unit, 200mm hydroxynaphthol blue；Wherein, each primer sequence is specific as follows:
Fip:5 '-cggattttctagaacgtggtaccaa-aatgaagagtcgactctagca-3 '；
Bip:5 '-actattgagctggacggcaa-tcgatagcagcccaagag-3 '；
F3:5 '-gtgatcctttcaccctgg-3 '；
B3:5 '-ttacaaatgtcagctggatg-3 '；
Lb:5 '-tgtacgtctacagaggatttggat-3 '.
Application in detection Phytophthora drechsleri bacterium for the lamp test kit of described detection Phytophthora drechsleri bacterium.
A kind of lamp detection method of detection Phytophthora drechsleri bacterium: include extracting the dna of microorganism to be checked, with the dna extracting
For template, carry out lamp using lamp detection primer compositionss or lamp detection kit；Amplified production carries out agarose gel
Electrophoresis, testing result under ultraviolet light, if there is the stepped band of characteristic, then prove there is Jue Shi epidemic disease in institute's detection sample
Mycete；The stepped band of atypism, then no Phytophthora drechsleri bacterium in institute's detection sample；Or observe the change of lamp reaction solution color
Change, sky blue represents positive findingses, there is Phytophthora drechsleri bacterium；Purple represents that testing result is negative, there is not Phytophthora drechsleri bacterium.
The lamp detection method of described detection Phytophthora drechsleri bacterium: extract the dna of microorganism to be checked, take 1 μ l dna solution,
The detection solution in 23 μ llamp test kits and 1 μ l sterile deionized water is added to carry out lamp, lamp response procedures are: 60 DEG C～
65 DEG C, 60～80min.
The method of the detection Phytophthora drechsleri bacterium of the present invention, including the dna extracting microorganism to be checked, with the dna of extraction as mould
Plate, carries out lamp using described lamp Primer composition；Hydroxynaphthol blue (hydroxylnaphthol blue, hnb) belongs to
One kind of Metal ion indicator.Hnb is mg2+Titrant, its color with solution ph change and change, therefore can by prison
Survey mg in lamp reaction system2+The change of concentration and solution ph and play the effect of color indicator.Before reaction, hnb is added to
In reactant liquor, reaction system is in purple, mg in course of reaction2+The by-product p reacting with lamp2o7 4-Precipitate in a large number in conjunction with producing,
Mg in solution2+Concentration reduces, and ph changes, so that the color from purple of hnb is changed into sky blue.Therefore, after reaction terminates
By the color change of reaction system, to judge the presence or absence of Phytophthora drechsleri bacterium: sky blue represents test positive, to there is Jue Shi epidemic disease
Mycete；Purple represents that testing result is negative, there is not Phytophthora drechsleri bacterium.
One of guardian technique of the present invention is the primer sequence of efficient specific amplified and its amplification side of Phytophthora drechsleri bacterium
Method.In order to verify the specific primer sequence of Phytophthora drechsleri bacterium, the present invention is with 8 plants of Phytophthora drechsleri bacteria strains and 14 kinds of other oomycetes
And 17 kinds of pathogenic fungi are material to be tested (table 1), extract the dna of Phytophthora drechsleri bacterium in incidence tissue using ctab method.Specifically
Method is as follows: take a small amount of mycelium powder, plus 900 μ l 2%ctab extracting solution and 90 μ l 10%sds, whirlpool mixes, in 55 DEG C of water-baths
1h, middle every 10min turns upside down several times.12000rpm be centrifuged 10min, take reset and add equal-volume phenol/chloroform/isoamyl alcohol (25:
24:1), overturn and mix, 12000rpm is centrifuged 10min；Supernatant is transferred to new pipe, plus equal-volume chloroform, gently overturn and mix,
12000rpm is centrifuged 5min.Supernatant is transferred in new pipe, plus the dehydrated alcohol of 2 times of volumes and the 3m naac (ph of 1/10 volume
5.2), -20 DEG C of precipitation (> 1h).12000rpm is centrifuged 10min, and incline supernatant, and precipitation with 70% washing with alcohol twice, dry in the air by room temperature
Dry.Plus sterilize in right amount ultra-pure water or te (ph 8.0) dissolution precipitation (containing 20 μ g/ml rnase), after 37 DEG C process 1h, -20 DEG C of guarantors
Deposit standby.All of pedotheque adoptsSpin test kit (q-biogene ltd, usa) carries out the extraction of dna.
Soil dna extraction step is referring to kit specification.This commercial soil microorganism dna extracts kit can carry in 0.5h
Get the microorganism in soil.
When there is lamp amplified reaction, producing substantial amounts of magnesium pyrophosphate white precipitate and leading to the turbidity of reactant liquor to rise,
By shown in the chromogenic reaction result of hnb, all in sky blue in the reaction tube of Phytophthora drechsleri bacterium, it is positive findingses, and other
Phytophthora kind, funguses, rotten mould and negative control bacterium reaction tube, all in purple, are negative findings it was demonstrated that designed lamp specificity
Primer has the specificity planted.Meanwhile, product is through 2% agarose gel electrophoresiies, the product of imaging amplification, pick
Reactant liquor in the reaction tube of family name's phytophthora occurs in that typical stairstepping band, and other phytophthora kind, funguses, rotten mould and negative
Trapezoid-shaped strips in comparison bacterium reaction tube.This illustrates that this primer sets can be used in incidence tissue and soil in production practices
The Testing and appraisal of the fast and reliable of Phytophthora drechsleri bacterium.When there is Phytophthora drechsleri bacterium in for incidence tissue, quick using naoh
Cracking process extracts the dna of Phytophthora drechsleri bacterium, and detailed process is as follows: takes the plant tissue of one section of morbidity, every milligram of tissue adds 10 μ
L 0.5m naoh, is transferred to after being fully ground in the ep pipe of 1.5ml in mortar, and 12000rpm is centrifuged 5min, takes 5 μ l supernatants
Liquid adds 495 μ l 0.1mm tris (ph8.0), takes 1 μ l to be directly used in pcr reaction after mixing.Each reaction at least repeats three
Secondary, simultaneously for determining that in plant, no pcr mortifier exists.
Table 1 is used for detecting the specific funguses of Phytophthora drechsleri bacterium and oomycetes bacterial strain
Beneficial effect: compared with prior art, advantages of the present invention and good effect show:
1) accuracy is high: because traditional Phytophthora drechsleri bacterium detection technique simply determines detection object according to morphological characteristic,
The interference of anthropic factor cannot be excluded, be difficult to distinguish the close kind of form, detection accuracy only has 60-80%；And the present invention according to
The sequence of the ypt1 gene (genebank:id dq162989.1) of Phytophthora drechsleri bacterium, using bioedit software by Phytophthora drechsleri
The ypt1 gene order of bacterium and the sequence of other phytophthora kinds are compared, and choose the distinctive one section of sequential design spy of Phytophthora drechsleri bacterium
The lamp primer of the opposite sex.Lamp reaction is by 6 independences on 4 primer (fip, bip, f3, b3) specific recognition target sequences
Region, its specificity and sensitivity are all higher.Additionally, reverse ring primer lb can improve reaction rate, and other four are drawn
Thing together, in the case of guaranteeing to react accuracy, enables the invention to be rapidly performed by Phytophthora drechsleri detection.
2) easy to operate: the lamp method of the detection Phytophthora drechsleri bacterium that the present invention provides overcomes Jue Shi epidemic disease in prior art
Cycle length needed for the biological detection method of mycete, waste time and energy, loaded down with trivial details, poor specificity problem and pcr detection technique needs
Thermal cycler instrument is it is impossible to the problem of quick detection Phytophthora drechsleri bacterium.Detection method, can be fast under 64 DEG C of isothermys
Speed, convenient, efficiently, high special, Phytophthora drechsleri bacterium is detected it is not necessary to complex instrument with sensitivity, can preferably meet to Jue Shi
The Site Detection of phytophthora.
3) achieve constant-temperature amplification, unlike pcr method has to thermal cycle, thus broken away to thermal cycler instrument according to
Rely, as long as have stable thermal source lamp to react just can occur, greatly extend the scope of lamp use, lamp why can
React under constant thermal source and be because with the addition of glycine betaine in lamp reactant liquor, make double-strand dna be in unwind dynamic
In state balance, realize amplification in the presence of bst dna polymerase.
4) practicality is good.Common pcr reaction carries out gel electrophoresiss and easily causes product diffusion to product, and this is laboratory
One main source of pollution；And ethidium bromide (eb) has huge poison, can accumulate carcinogenic；Long-term observation uviol lamp also can be to experiment
Personnel cause a certain degree of injury.And lamp reaction only need to be carried out in thermostat water bath, reaction passes through hnb's after terminating
Color change just can direct judged result, thus increased the using value that it detects in the plant carrying disease germs and soil.
5) present invention provides new technology platform for the detection of Phytophthora drechsleri bacterium, can be used for the highly sensitive of Phytophthora drechsleri bacterium
Degree quick detection, identifies pathogen it is also possible to detect to the pathogen in field soil at the disease infestation initial stage simultaneously.
The present invention uses to reducing pesticide blindness, reduces production cost, and the environmental pollution reducing pesticide is also significant.
Brief description
Fig. 1 is the specific gelose gel electrophoresis figure that lamp detects Phytophthora drechsleri bacterium；Wherein, m is 100bp dna
marker；In figure shows that the 1st swimming lane and the 9th swimming lane have typical trapezoidal shape band, is positive；Remaining swimming lane is negative；
Wherein, 1,9: Phytophthora drechsleri bacterium (p.drechsleri)；2: soybean phytophthora (p.sojae)；3: phytophthora parasitica
(p.parasitica)；4: phytophthora infestans (p.infestans)；5: Radix Aucklandiae phytophthora (p.tentaculata)；6: Fructus Fragariae Ananssae phytophthora
(p.fragariae)；7: ramie mould (p.boehmeriae)；10: Pythium ultimum (pythium ultimum)；11: Herba Equiseti Hiemalises' sickle
Spore bacterium (fusarium equiseti)；12: tack anthrax (colletotrichum truncatum)；13: Pyricularia oryzae
(magnaporthe grisea)；14: Rhizoctonia solani Kuhn (rhizoctonia solani)；15: verticillium dahliae
(verticilium dahliae)；8,16: negative control；
Fig. 2 is that color judges that lamp detects the specific chromogenic figure of Phytophthora drechsleri；In figure shows that the 1st pipe and the 9th pipe are aobvious
Sky blue, is positive；The aobvious purple of remaining pipe, is negative；Wherein, 1,9: Phytophthora drechsleri bacterium (p.drechsleri)；2: Semen sojae atricolor epidemic disease
Mould (p.sojae)；3: phytophthora parasitica (p.parasitica)；4: phytophthora infestans (p.infestans)；5: Radix Aucklandiae phytophthora
(p.tentaculata)；6: Fructus Fragariae Ananssae phytophthora (p.fragariae)；7: ramie mould (p.boehmeriae)；10: Pythium ultimum
(pythium ultimum)；11: Herba Equiseti Hiemalises' Fusariumsp (fusarium equiseti)；12: tack anthrax
(colletotrichum truncatum)；13: Pyricularia oryzae (magnaporthe grisea)；14: Rhizoctonia solani Kuhn
(rhizoctonia solani)；15: verticillium dahliae (verticilium dahliae)；8,16: negative control；
Fig. 3 is to detect lamp sensitivity based on agarose gel electrophoresiies and hnb color change；In Fig. 3, upper figure is lamp inspection
Survey Phytophthora drechsleri specific agarose gel electrophoresis figure；Wherein, m is 100bp dna marker；In Fig. 3, figure below is face
Color judges that lamp detects the sensitivity colour developing figure of Phytophthora drechsleri；10ng, 1ng Phytophthora drechsleri is contained respectively in the reaction system of 25 μ l
The reaction tube of bacterium dna shows sky blue, is positive, contain respectively in the reaction system of 25 μ l 100pg, 10pg, 1pg,
The reaction tube of 100fg, 10fg Phytophthora drechsleri bacterium dna shows purple, negative；Colour developing result shows the sensitivity of lamp reaction
Reach 1ng.
Specific embodiment
With reference to specific embodiment, the present invention is described further, but the present invention is not limited by following examples
System.
Embodiment 1
A kind of lamp detection kit for detecting Phytophthora drechsleri bacterium, by 1.6 μm of positive inner primer fip, 1.6 μm reversely
Inner primer bip, 0.2 μm of positive outer primer f3,0.2 μm of reverse outer primer b3,0.2 μm of reverse ring primer lb, 1.8mm dntps,
Tris-hcl, 10mm kcl, 10mm (nh of 20mm ph 8.84)2so4、6mm mgso4, 0.1%triton x-100, bst
Dna polymerase 16 unit, 5mm hydroxynaphthol blue, add ultra-pure water to be prepared into 25ul detection solution.Each primer sequence tool
Body is as follows:
Fip:5 '-cggattttctagaacgtggtaccaa-aatgaagagtcgactctagca-3 '；
Bip:5 '-actattgagctggacggcaa-tcgatagcagcccaagag-3 '；
F3:5 '-gtgatcctttcaccctgg-3 '；
B3:5 '-ttacaaatgtcagctggatg-3 '；
Lb:5 '-tgtacgtctacagaggatttggat-3 '.
Wherein, positive inner primer fip, reverse inner primer bip, positive outer primer f3, reverse outer primer b3 and reverse ring draw
Thing lb can directly form the lamp detection primer compositionss for detecting Phytophthora drechsleri bacterium.
The specific test of embodiment 2 Phytophthora drechsleri bacterium lamp reaction
In order to verify the specificity of lamp method, with 8 plants of Phytophthora drechsleri bacteria strains and 14 kinds of other oomycetes and 17 kinds of diseases
Fungal pathogenses are material to be tested (table 1), and lamp testing result shows that 8 plants of Phytophthora drechsleri bacteria strains all can be observed the sapphire positive
The stair-stepping band of lamp in reaction or agarose gel electrophoresiies, and remaining 14 kinds of oomycetes and 17 kinds of pathogenic fungi colour developings are tied
Fruit is the negative reaction of purple or amplified band in agarose gel electrophoresiies.Select not of the same race with Phytophthora drechsleri bacterium
(soybean phytophthora；Phytophthora parasitica；Phytophthora infestans；Radix Aucklandiae phytophthora；Fructus Fragariae Ananssae phytophthora；Ramie mould etc.) and bacterium (the tack charcoal that do not belong to together
Cellulitis bacterium；Eggplant Fusarium spp.；Pyricularia oryzae；Rhizoctonia solani Kuhn；Verticillium dahliae；Pythium ultimum) dna as template, take 1 μ l dna
Solution, adds the detection solution of 23 μ l embodiment 1 preparation and 1 μ l sterile deionized water to carry out lamp reaction, response procedures are: 64
℃60min.Based on reaction system agarose gel electrophoresiies and color reaction as result judgement standard, result is expanded with lamp primer
During the dna template of increasing Phytophthora drechsleri bacterium, all amplify typically stepped band；And not of the same race with Phytophthora drechsleri bacterium, do not belong to together
Bacterium and negative control all do not have to amplify purpose band (Fig. 1).Show the dna template of amplification Phytophthora drechsleri bacterium simultaneously
When, assume sky blue；Amplification all assumes purple with the dna template of bacterium that Phytophthora drechsleri bacterium is not of the same race, do not belong to together and negative control
(Fig. 2).
The sensitivity test of embodiment 3 Phytophthora drechsleri bacterium lamp reaction
In order to determine the sensitivity of lamp detection method, by the dna spectrophotometric determination of the Phytophthora drechsleri bacterium of extraction
Carry out 10 doubling dilutions with depc water, -70 DEG C preserve as template after concentration (1 μ g/ μ l).Take each after 10 doubling dilutions respectively
Concentration dna diluent 1 μ l, as template, adds the detection solution of 23 μ l embodiment 1 preparation and 1 μ l sterile deionized water to carry out
Lamp reacts, and response procedures are: 64 DEG C of 80min.Take 2 μ l amplified production loadings, result display agarose gel electrophoresiies and hnb show
Colour response shows that the sensitivity of lamp reaction reaches the dna (Fig. 3) of the Phytophthora drechsleri bacterium of 1ng.
Embodiment 4 Phytophthora drechsleri bacterium lamp reaction primer specificity checking and sensitivity checking
For the lamp primer sets of Phytophthora drechsleri bacterium, devise 11 groups of qualified primers altogether, finally filter out 1 group
It is that Primer composition used in embodiment 1 (includes positive inner primer fip, reversely for special and the high primer of sensitivity
Inner primer bip, positive outer primer f3, reverse outer primer b3, positive ring primer lf and reverse ring primer lb).Using design its
Remaining primer (select from remaining 10 to primer at random 1 to), primer sequence is as follows: fip1:5 '-
gtggtaccaaaatgcacaagtca-cgacaaggtccaatgaaga-3′；Bip1:5 '-
tagaaaatccgtactattgagctgg-agatccaaatcctctgtagac-3′；F31:5 '-gtgatcctttcaccctgg-
3′；B31:5 '-atgtcgatagcagcccaa-3 '；Lb1:5 '-acggcaagaccatcaagcttcag-3 '；With in embodiment 2
The bacterial strain being adopted is material to be tested (8 plants of Phytophthora drechsleri bacteria strains and 14 kinds of other oomycetes and 17 kinds of pathogenic fungi), lamp
Testing result show selected by primer specificity not high, sensitivity is also poor.Illustrate have relatively for Primer composition of the present invention
High specificity and sensitivity.
Embodiment 5 detects Phytophthora drechsleri bacterium from soil sample of carrying disease germs
The method that the Phytophthora drechsleri bacterium detection kit of embodiment 1 is used for detecting Phytophthora drechsleri bacterium, comprising:
1) in soil oospore enrichment: take 40～80 grams of pedotheque to be checked, grind, successively gone using 200 eye mesh screens
Locate larger grogs, be then passed through 400,500,800 mesh sieve net filtrations, repeatedly rinsed with 3～10 liters of water, from 800 eye mesh screens simultaneously
Upper collection oospore, uses 1ml aqueous suspension.Because oospore can not pass through 800 eye mesh screens, so processing to reach makes oospore
The effect of enrichment.
2) extract dna from micro oospore: in the centrifuge tube that 1.5ml will be transferred to the oospore of aseptic aqueous suspension,
In 12000r.min-1It is centrifuged 5 minutes under rotating speed, pour out liquid；Add 50 μ l ctab buffer, grind, add 500 μ l
Ctab buffer, water-bath 30 minutes；Add equal-volume chloroform, in 12000r.min-1It is centrifuged 10 minutes under rotating speed, draw
Supernatant；Add the 3m naac of 1/10 volume, the no water-ice ethanol of 2 times of volumes, precipitation at room temperature 30 minutes, 12000r.min-1Rotating speed
Lower centrifugation 10 minutes, dry liquids；Plus 1ml 70% (v/v) washing with alcohol, 12000r.min-1Centrifugation 10 minutes under rotating speed,
Dry liquids, dry to alcohol-free taste；Plus 10 μ l aseptic double-distilled water dissolving, for lamp amplification template.
3) Phytophthora drechsleri bacterium lamp detection, comprising: the lamp detection of Phytophthora drechsleri bacterium: take 1 μ l dna solution, add 23 μ l
Detection solution and 1 μ l sterile deionized water, cumulative volume be 25 μ l；Response procedures are: 64 DEG C of 60min；With hnb (hydroxyl naphthols
Blue) as reaction indicator, the color that amplification terminates rear lamp reaction system is assumed sky blue, is judged from soil sample of carrying disease germs with this
Positive reaction can be produced and contain Phytophthora drechsleri bacterium.
The lamp detection of Phytophthora drechsleri bacterium in embodiment 6 biological tissue
Inoculate the dna of the morbidity cucumber plant of Phytophthora drechsleri bacterium using naoh alkaline lysis method of extracting, use as template
In lamp amplification.Take 1ul dna solution, as described in Example 4, carry out lamp reaction.Result display inoculation Phytophthora drechsleri bacterium
Morbidity cucumber plant in carry out lamp, its color reaction also assumes positive sky blue；And health cucumber plant and negative control
Assume purple.
sequence listing
<110>Nanjing Forestry University
<120>a kind of lamp detection primer compositionss of Phytophthora drechsleri bacterium and its lamp detection kit and lamp detection
Method
<130> 100
<160> 10
<170> patentin version 3.3
<210> 1
<211> 46
<212> dna
<213> artificial
<220>
<223>fip primer sequence
<400> 1
cggattttct agaacgtggt accaaaatga agagtcgact ctagca 46
<210> 2
<211> 38
<212> dna
<213> artificial
<220>
<223>bip primer sequence
<400> 2
actattgagc tggacggcaa tcgatagcag cccaagag 38
<210> 3
<211> 18
<212> dna
<213> artificial
<220>
<223>f3 primer sequence
<400> 3
gtgatccttt caccctgg 18
<210> 4
<211> 20
<212> dna
<213> artificial
<220>
<223>b3 primer sequence
<400> 4
ttacaaatgt cagctggatg 20
<210> 5
<211> 24
<212> dna
<213> artificial
<220>
<223>lb primer sequence
<400> 5
tgtacgtcta cagaggattt ggat 24
<210> 6
<211> 42
<212> dna
<213> artificial
<220>
<223>fip1 primer sequence
<400> 6
gtggtaccaa aatgcacaag tcacgacaag gtccaatgaa ga 42
<210> 7
<211> 46
<212> dna
<213> artificial
<220>
<223>bip1 primer sequence
<400> 7
tagaaaatcc gtactattga gctggagatc caaatcctct gtagac 46
<210> 8
<211> 18
<212> dna
<213> artificial
<220>
<223>f31 primer sequence
<400> 8
gtgatccttt caccctgg 18
<210> 9
<211> 18
<212> dna
<213> artificial
<220>
<223>b31 primer sequence
<400> 9
atgtcgatag cagcccaa 18
<210> 10
<211> 23
<212> dna
<213> artificial
<220>
<223>lb1 primer sequence
<400> 10
acggcaagac catcaagctt cag 23
Claims (4)
1. a kind of lamp Primer composition for detecting Phytophthora drechsleri bacterium it is characterised in that: by positive inner primer fip, reversely
Inner primer bip, positive outer primer f3, reverse outer primer b3 and reverse ring primer lb composition；Each primer sequence is specific as follows:
Fip:5 '-cggattttctagaacgtggtaccaa-aatgaagagtcgactctagca-3 '；
Bip:5 '-actattgagctggacggcaa-tcgatagcagcccaagag-3 '；
F3:5 '-gtgatcctttcaccctgg-3 '；
B3:5 '-ttacaaatgtcagctggatg-3 '；
Lb:5 '-tgtacgtctacagaggatttggat-3 '.
2. application in detection Phytophthora drechsleri bacterium for the lamp Primer composition described in claim 1.
3. a kind of lamp test kit of detection Phytophthora drechsleri bacterium it is characterised in that: comprise 1ml detection solution, described detection is molten
Liquid includes: 32mm forward direction inner primer fip, 32mm reverse inner primer bip, 8mm forward direction outer primer f3,8mm reverse outer primer b3,
8mm ring primer lb, 48mm dntps, 0.8m tris-hc, 0.4mm kcl, 0.4mm (nh4)2so4、0.24mm mgso4, 4%
Triton x-100, bst dna polymerase 320 unit, 200mm hydroxynaphthol blue；Wherein, each primer sequence is concrete such as
Under:
Fip:5 '-cggattttctagaacgtggtaccaa-aatgaagagtcgactctagca-3 '；
Bip:5 '-actattgagctggacggcaa-tcgatagcagcccaagag-3 '；
F3:5 '-gtgatcctttcaccctgg-3 '；
B3:5 '-ttacaaatgtcagctggatg-3 '；
Lb:5 '-tgtacgtctacagaggatttggat-3 '.
4. application in detection Phytophthora drechsleri bacterium for the lamp test kit of the detection Phytophthora drechsleri bacterium described in claim 3.
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Non-Patent Citations (3)
| Title |
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| SchenaAssessing the potential of regions of the nuclear and mitochondrial;Schena L和Cooke D E L;《Journal of Microbiological Methods》;20060417;第67卷;70-85 * |
| 以Ypt1基因序列为靶标的辣椒疫病菌快速分子检测;李毛毛等;《植物病理学报》;20141015;第44卷(第5期);全文 * |
| 基于环介导等温扩增技术检测橡树疫霉菌;戴婷婷等;《南京农业大学学报》;20131231;第36卷(第3期);23-28 * |
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Application publication date: 20150311 Assignee: Beijing Huamei Wanxiang Technology Co., Ltd. Assignor: Nanjing Forestry University Contract record no.: 2018320000235 Denomination of invention: LAMP detection primer composition for detecting phytophthora drechsler tucker, LAMP detection kit, and LAMP detection method Granted publication date: 20170118 License type: Common License Record date: 20181024 Application publication date: 20150311 Assignee: Nanjing city Pukou District moon farm Assignor: Nanjing Forestry University Contract record no.: 2018320000234 Denomination of invention: LAMP detection primer composition for detecting phytophthora drechsler tucker, LAMP detection kit, and LAMP detection method Granted publication date: 20170118 License type: Common License Record date: 20181024 |
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