CN108588265A - The LAMP detection primer composition and its LAMP detection kit and LAMP detection method of a kind of cloves phytophthora - Google Patents
The LAMP detection primer composition and its LAMP detection kit and LAMP detection method of a kind of cloves phytophthora Download PDFInfo
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Abstract
The invention discloses a kind of LAMP detection primer composition of cloves phytophthora and its LAMP detection kits and LAMP detection method.The ring mediated isothermal amplification detection primer composition is made of positive inner primer FIP, reversed inner primer BIP, positive outer primer F3, reversed outer primer B3.The detection method accuracy height of the present invention, high specificity, easy to operate, practicability is good, realizes constant-temperature amplification, simultaneously also new technology platform is provided for the detection of quarantine phytophthora, it can be used for the high sensitivity quickly detection of cloves phytophthora, pathogen is identified at disease infestation initial stage simultaneously, the cloves phytophthora in entry and exit fruit sample can be detected.The present invention is blindly used to epidemic disease caused by cloves phytophthora and to reducing pesticide, reduces production cost, the environmental pollution for reducing pesticide is also of great significance.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of LAMP detection primer composition of cloves phytophthora and its
LAMP detection kit and LAMP detection method.
Background technology
Cloves phytophthora (Phytophthora ramorum) belong to oomycota (Oomycota), Oomycete (Oomycetes),
Peronosporales (Peronosporales), pythiaceae (Pythiaceae), Phytophthora (Phytophthora).The bacterium host distribution model
It encloses extensively, is distributed mainly on the states such as the U.S., Japan, Canada, Britain, France, Germany, Italy, Australia, does not have in China
It reports.The germ is mainly by being infected children's adult seedling, soil and the fruit long-distance communications of germ.Germ is mainly infected
The plants of 14 sections such as citrus fruit, Prunus class fruit, fiery spine, cloves, European Chinese chestnut, Oak Tree, 29 categories, cause to post root,
The a variety of lesions of stem, leaf, fruit are a kind of great important phytopathogens of harmfulness, China were put into 2007 and is entered the territory
Plant quarantine harmful organism list.2011, Tianjin inspection and quarantine bureau intercepted and captured cloves phytophthora on U.S.'s navel orange for the first time.Quilt
The citrus fruits that germ is infected can gradual brown rot, limb gummosis, rootstock foot is rotten and root-rot, plant-active reduce, occur withered
It withers, blade chlorisis is wilted, and can finally be led to the death of the whole strain of plant, be seriously affected fruit yield and quality, bring great economy
Loss, seriously threatens world's Orange Producing.China's fruits output occupies the first in the world, is the big country for producing fruit, cultivated area
Nearly 10,000,000 hm2, the income that yield every year on average is brought is more than 280,000,000,000 yuan.Application has become the cause of agricultural production industry
Rich important channel, the development of rural economy and social economy to China play the role of very important.The phytophthora is once
Incoming China simultaneously colonizes and will cause destructive strike perfume phytophthora that can infect cinnamon to the fruit-growing industry in China to cause root
Portion rots and the diseases such as limb ulcer, can cause crushing blow to cinnamon.Cloves phytophthora host is extensive, can infect
Thousands of kinds of plants.It is reported that cloves phytophthora has resulted in Australian southwest forest recurring structure and plant population variation,
Local natural ecosystems and bio-diversity are seriously threatened.To the disease, there are no effectively preventings to arrange at present
It applies, reinforces quarantine, it is the most effective measure for controlling the disease to prevent the Spreading and diffusion of pathogen.It should reinforce epidemic monitoring thus, build
Vertical rapid detection method, for disease risk and study decision foundation be provided, to advantageously reduce camphor tree epidemic disease caused by damage
It loses.
The taxonomic identification of traditional cloves phytophthora is mainly based upon morphological feature, Pathogenicity, physiological and biochemical property
Deng.The method of traditional separation phytophthora detaches phytophthora using mass trapping from soil, irrigation water and vegetable material.In order to carry
High trap effect, can be added a small amount of antibiotic in the soil liquid and fungicide inhibits varied bacteria growing.Suitable for doing the plant of bait
Object material is because different phytophthoras are also different, and pine needle is suitable for trapping cloves phytophthora.Conventional method is detected in cloves phytophthora
In played important function, but it is time-consuming and laborious and require that operator has the phytophthora separation of profession, Morphological Identification is known
Knowledge and abundant experience；Time-consuming, sensitivity is low, easily by factors such as artificial and environment for traditional classification identification method simultaneously
Interference, cannot make diagnosis in disease incubation period and early stage, it is difficult to occur to carry out timely monitoring and effectively control to disease
System.With the development of molecular biology, round pcr provides quick, sensitive, accurate advantage for pathogenic diagnosis.Commonly
The method of PCR has been used successfully to detection cloves phytophthora, although PCR methods are greatly improved in specificity and sensibility,
It is that detection time is still long, general 4~5h, while PCR methods rely on accurate temperature cycling device, detection sensitivity ratio
It is higher, detection process is complicated, cannot meet the needs of quickly detecting.The triple PCR of cloves phytophthora synchronizes molecular detecting method, with
Heat shock protein HSP90 sequences are detection target, are verified to primer specificity with 15 bacterial strains of 14 kinds of phytophthora germs, as a result
The detection sensitivity of Standard PCR and multiplex PCR is respectively 200pg and 2ng, and it is 12pg that Standard PCR, which detects lower bound, and the two difference is close
10 times.PCR fluoroscopic examinations and conventional detection have carried out quick detection on some inward Prunus, citrus fruit, together
When also carried out the test of the upper cloves phytophthora of other hosts, fluoroscopic examination and conventional detection are convenient in port laboratory applications and are pushed away
Extensively, specificity is preferable, high sensitivity.Triple PCR, which synchronizes molecular detecting method, can effectively facilitate speeding passage through customs for inward fruit, can
It to reliably detect the quarantine pathogen at port and field, but has a disadvantage in that primer is multipair, is easy to cause primer dimerization
Body or mispairing make its sensitivity decline.
Loop-mediated isothermal amplification technique (Loop-mediated isothermal amplification, ring mediated isothermal
Amplification) be a kind of new nucleic acid amplification technologies, it is easy to operate, quick, specific high, at low cost because of its advantages that, becoming can be with
Substitute the new nucleic acid amplification technologies of PCR.It is 6 regions design, the 4 species specific primers for target gene, in Bst sheets
Cause self-loopa strand replacement reaction under the action of section polymerase, in 64 DEG C of range 80min, a large amount of companions while synthesize target dna
With there is by-product -- white magnesium pyrophosphate, which precipitates, to be generated.Since process of the loop-mediated isothermal amplification relies on identification target sequence 6 solely
Vertical region, so atopic is very strong, and amplification process is carried out under constant temperature, common water-bath or is had steady
Reaction requirement can be met by determining the equipment of heat source, and testing cost substantially reduces.Since loop-mediated isothermal amplification is simple, fast
Speed, efficient, economic dispatch feature, thus there is extremely wide application prospect.From ring mediated isothermal amplification detection technique establish with
Come, which has been widely used for the research of the detection to pathogens such as virus, bacterium, parasite, fungies, the inspection of cloves phytophthora
Survey is not reported both at home and abroad.
Invention content
Goal of the invention：It is special for period length, detection method needed for cloves phytophthora biological detection method in the prior art
The problem that property is poor, sensitivity is low, the object of the present invention is to provide a kind of ring mediated isothermal amplification detection primer groups of cloves phytophthora
Close object.It is a further object of the present invention to provide the loop-mediated isothermal amplification detection kits of above-mentioned cloves phytophthora.The present invention is another
Purpose is to provide the loop-mediated isothermal amplification detection method of above-mentioned cloves phytophthora.
Technical solution：In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is：
A kind of loop-mediated isothermal amplification (LAMP) primer composition for detecting cloves phytophthora, it is characterised in that：By just inwardly drawing
Object FIP, reversed inner primer BIP, positive outer primer F3, reversed outer primer B3 compositions；Each primer sequence is specific as follows：
FIP：5 ＇-GCCGCGGTAGTAGCTGCTAGT-TTGTAGTGGGACACGGCT-3 '；
BIP：5 ＇-CGTGGTGTACGACGTGACGG-CGACGCACCTATCAATCTCG-3 ＇；
F3：5 ＇-CTGTAGAGCATGCTGCTGAT-3 ＇；
B3：5 '-CCCGCAGAGAAACCTATGG-3 ＇.
Application of the loop-mediated isothermal amplification (LAMP) primer composition in detecting cloves phytophthora.
A kind of loop-mediated isothermal amplification kit of detection cloves phytophthora, including volume is molten for detection more than ampoule
Liquid, wherein the detection solution of 1mL includes：48mM dNTPs、0.8M Tris-HC、0.4mM KCl、0.4mM(NH4)2SO4、
0.24mM MgSO4, 4%Triton X-100,320 units of Bst DNA polymerase, 200mM hydroxynaphthol blues, 32mM
The positive reversed reversed outer primer B3 of inner primer BIP, 8mM forward direction outer primer F3,8mM of inner primer FIP, 32mM；Wherein, each primer sequence
It arranges specific as follows：
FIP：5 ＇-GCCGCGGTAGTAGCTGCTAGT-TTGTAGTGGGACACGGCT-3 ＇；
BIP：5 ＇-CGTGGTGTACGACGTGACGG-CGACGCACCTATCAATCTCG-3 ＇：
F3：5 '-CTGTAGAGCATGCTGCTGAT-3 ＇；
B3：5 '-CCCGCAGAGAAACCTATGG-3 ＇.
Application of the loop-mediated isothermal amplification kit of the detection cloves phytophthora in detecting cloves phytophthora.
A method of detection cloves phytophthora extracts the DNA of microorganism to be checked, using the DNA of extraction as template, utilizes right
It is required that the loop-mediated isothermal amplification kit described in loop-mediated isothermal amplification (LAMP) primer composition or claim 3 described in 1 carries out
Ring mediated isothermal amplification；The variation of loop-mediated isothermal amplification solution colour is observed, sky blue indicates test positive, and there are fourths
Fragrant phytophthora；Purple indicates that testing result is feminine gender, and cloves phytophthora is not present.
The method of the detection cloves phytophthora：The DNA for extracting microorganism to be checked takes 2 μ LDNA solution, and 23 μ L rings are added
Detection solution in mediated isothermal amplification kit carries out ring mediated isothermal amplification, and loop-mediated isothermal amplification program is：64
DEG C, 60min, amplified production carries out the observation of color change.
The method of the detection cloves phytophthora of the present invention, includes the DNA of extraction microorganism to be checked, using the DNA of extraction as template,
Ring mediated isothermal amplification is carried out using the loop-mediated isothermal amplification (LAMP) primer composition；Hydroxynaphthol blue
(hydroxylnaphthol blue, HNB) belongs to one kind of Metal ion indicator.HNB is Mg2+Titrant, color with
Solution PH changes and changes, therefore can be by monitoring Mg in loop-mediated isothermal amplification system2+The variation of concentration and solution
PH and play the role of color indicator.HNB is added in reaction solution before reaction, reaction system is in purple, in reaction process
Mg2+With the by-product P of loop-mediated isothermal amplification2O7 4-It is largely precipitated in conjunction with generating, Mg in solution2+Concentration reduces, and pH occurs
Variation, to make the color of HNB become sky blue from purple.Therefore, come after reaction by the color change of reaction system
Judge the presence or absence of cloves phytophthora：Sky blue indicates test positive, and there are cloves phytophthoras；Purple indicates that testing result is feminine gender,
There is no cloves phytophthoras.
Advantageous effect：Compared with prior art, the advantages and positive effects of the present invention are shown：
1) accuracy is high：Since traditional cloves phytophthora detection technique only determines detection object, nothing according to morphological feature
Method excludes the interference of human factor, it is difficult to distinguish close kind of form, detection accuracy only has 60-80%；And the present invention chooses fourth
The loop-mediated isothermal amplification (LAMP) primer of the fragrant distinctive gene order design specificity of phytophthora, loop-mediated isothermal amplification pass through 4
6 isolated areas on primer (FIP, BIP, F3, B3) specific recognition target sequence, specificity and sensitivity are relatively high.
2) easy to operate：The loop-mediated isothermal amplification method of detection cloves phytophthora provided by the invention overcomes the prior art
The problem of period needed for the biological detection method of middle cloves phytophthora is long, time-consuming and laborious, cumbersome, poor specificity and PCR detect skill
Art needs thermal cycler instrument, the problem of can not quickly detecting cloves phytophthora.Detection method is under 64 DEG C of isothermys, energy
Quickly, conveniently, efficiently, it is high special, detect cloves phytophthora with sensitivity, do not need complex instrument, can preferably meet to cloves
The Site Detection of phytophthora.
3) realize constant-temperature amplification, unlike PCR methods have to thermal cycle, thus broken away to thermal cycler instrument according to
Rely, as long as there is stable heat source loop-mediated isothermal amplification, greatly extending ring mediated isothermal amplification makes
Range, it is because anti-in ring mediated isothermal amplification why ring mediated isothermal amplification can react under constant heat source
It answers and is added to glycine betaine in liquid, double-stranded DNA is made to be in the dynamic equilibrium of unwinding, realized under the action of Bst archaeal dna polymerases
Amplification.
4) practicability is good.Common PCR reaction carries out gel electrophoresis to product and easily causes product diffusion, this is laboratory
One main source of pollution；And ethidium bromide (EB) has huge poison, can accumulate carcinogenic；Long-term observation ultraviolet lamp also can be to experiment
Personnel cause a degree of injury.And loop-mediated isothermal amplification need to only carry out in thermostat water bath, reaction end
Afterwards by the color change of HNB can direct judging result, answering to increase that it detects in the plant and soil to carry disease germs
With value.
5) present invention provides new technology platform for the detection of cloves phytophthora, and the high sensitivity that can be used for cloves phytophthora is fast
Speed detection, while quickly preparation identifies pathogen in sample of entering and leaving the border.The present invention is blindly used reducing pesticide, reduces life
Cost is produced, the environmental pollution for reducing pesticide is also of great significance.
Description of the drawings
Fig. 1 is the specific chromogenic figure of color judgement ring mediated isothermal amplification detection cloves phytophthora；In figure show the 1st pipe with
And the 9th pipe show sky blue, be positive；Remaining pipe shows purple, is negative；Wherein, 1,9：Cloves phytophthora (Phytophthora
syringae)；2：Soybean phytophthora (P.sojae)；3：Phytophthora parasitica (P.parasitica)；4：Phytophthora infestans
(P.infestans)；5：Root of Aucklandia lappa Decne phytophthora (P.tentaculata)；6：Strawberry phytophthora (P.fragariae)；7：Ramie mould
(P.boehmeriae)；10：Pythium ultimum (Pythium ultimum)；11：Scouring rush's Fusariumsp (Fusarium equiseti)；
12：Tack anthrax-bacilus (Colletotrichum truncatum)；13：Verticillium dahliae (Verticilium dahliae)；
14：Rhizoctonia solani Kuhn (Rhizoctonia solani)；15：Pyricularia oryzae (Magnaporthegrisea)；8,16：It is negative right
According to；
Fig. 2 is to detect ring mediated isothermal amplification sensitivity based on HNB color changes；Fig. 2 is that color judges ring mediated isothermal
The sensitivity colour developing figure of augmentation detection cloves phytophthora；Contain 10ng, 1ng, 100pg cloves phytophthora in the reaction system of 25 μ L respectively
The reaction tube of DNA shows sky blue, is positive, and contains 10pg, 1pg, 100fg, 10fg fourth in the reaction system of 25 μ L respectively
The reaction tube of fragrant phytophthora DNA shows purple, negative.Colour developing is the result shows that the sensitivity of loop-mediated isothermal amplification reaches
100pg。
Fig. 3 is ring mediated isothermal amplification testing result figure；
Fig. 4 is artificial infection incidence tissue verification LAMP results.HNB chromogenic reactions show the shaddock extraction of artificial infection
The reaction tube of DNA shows sky blue, is positive, and the reaction solution in negative control bacterium reaction tube and healthy shaddock tissue DNA
Trapezoid-shaped strips do not occur, (2% agarose gel electrophoresis detects：1. incidence tissue's sample；2. health tissues sample；3. negative right
According to).
Specific implementation mode
With reference to specific embodiment, the present invention is described further, but the present invention is not limited by following embodiment
System.
Embodiment 1
It is a kind of for detecting the loop-mediated isothermal amplification detection kit of cloves phytophthora, by 1.6 μM of positive inner primer FIP,
1.6 μM of reversed inner primer BIP, 0.2 μM of positive outer primer F3,0.2 μM of reversed outer primer B3,1.8mM dNTPs, 20mM pH
8.8 Tris-HCl, 10mM KCl, 10mM (NH4)2SO4、6mM MgSO4, 0.1%Triton X-100, Bst
16 units of DNApolymerase, 5mM hydroxynaphthol blues are added ultra-pure water and are prepared into 25uL detection solution.Each primer sequence tool
Body is as follows：
FIP：5 ＇-GCCGCGGTAGTAGCTGCTAGT-TTGTAGTGGGACACGGCT-3 '；
BIP：5 ＇-CGTGGTGTACGACGTGACGG-CGACGCACCTATCAATCTCG-3 '；
F3：5 ＇-CTGTAGAGCATGCTGCTGAT-3 '；
B3：5 '-CCCGCAGAGAAACCTATGG-3 ＇.
Wherein, positive inner primer FIP, reversed inner primer BIP, positive outer primer F3, reversed outer primer B3 can be formed directly
Ring mediated isothermal amplification detection primer composition for detecting cloves phytophthora.
The specific test of 2 cloves phytophthora loop-mediated isothermal amplification of embodiment
In order to verify the specific primer sequence of cloves phytophthora, the present embodiment is other with 1 plant of cloves Mtr mutant and 14 kinds
Oomycetes and 17 kinds of disease fungus are material to be tested (table 1), and the DNA of cloves phytophthora in incidence tissue is extracted using CTAB methods, takes 1
μ LDNA solution, detection solution and the 1 μ L sterile deionized waters progress ring mediated isothermal amplification that the preparation of 23 μ L embodiments 1 is added are anti-
It answers, response procedures are：64℃ 60min.
Extracting the DNA of each material to be tested, the specific method is as follows：Take a small amount of hypha powder, add 900 μ L2%CTAB extracting solutions and
90 μ L10%SDS, whirlpool mixing are intermediate to turn upside down several times per 10min in 55 DEG C of water-bath 1h.12000rpm centrifuges 10min,
It takes and resets and add isometric phenol/chloroform/isoamyl alcohol (25：24：1) mixing, is overturned, 12000rpm centrifuges 10min；Supernatant is transferred to
New pipe, adds isometric chloroform, gently overturns mixing, and 12000rpm centrifuges 5min.Supernatant is transferred in new pipe, adds 2 times of volumes
The 3M NaAc (pH 5.2) of absolute ethyl alcohol and 1/10 volume, -20 DEG C of precipitations (＞ 1h).12000rpm centrifuge 10 min, incline on
Clearly, precipitation is washed twice with 70% ethyl alcohol, and room temperature is dried.Appropriate sterilizing ultra-pure water or TE (pH 8.0) dissolving precipitations is added (to contain 20 μ
G/mL RNase), after 37 DEG C handle 1h, -20 DEG C save backup.
Table 1 is used to detect fungi and the oomycetes bacterial strain of cloves phytophthora specificity
Ring mediated isothermal amplification testing result is as shown in Figure 1, sapphire sun can be observed in 1 plant of cloves Mtr mutant of display
Property reaction or agarose gel electrophoresis there is the stair-stepping band of ring mediated isothermal amplification, remaining 14 kinds of oomycetes and 17 kinds of diseases
Fungal pathogens colour developing result is that the negative reaction of purple or agarose gel electrophoresis amplified band do not occur.Designed by proving
Ring mediated isothermal amplification specific primer has the specificity of kind.This illustrates that the primer sets can be used for morbidity group in production practices
Knit and soil in cloves phytophthora fast and reliable Testing and appraisal.
The sensitivity test of 3 cloves phytophthora loop-mediated isothermal amplification of embodiment
In order to determine the sensitivity of loop-mediated isothermal amplification detection method, by the DNA of the cloves phytophthora of extraction light splitting light
Degree meter measured concentration (1 μ g/ μ L) carries out 10 doubling dilutions with DEPC water afterwards, and -70 DEG C preserve as template.Take 10 multiple proportions dilute respectively
Detection solution and 1 μ L sterilizing deionizations prepared by 23 μ L embodiments 1 is added as template in each 1 μ L of concentration DNA dilutions after releasing
Water carries out loop-mediated isothermal amplification, and response procedures are：64℃ 60min.2 μ L amplified production loadings are taken, as a result such as Fig. 2 institutes
Show, shows that agarose gel electrophoresis and HNB chromogenic reactions show that the sensitivity of loop-mediated isothermal amplification reaches the fourth of 100pg
The DNA of fragrant phytophthora.
4 cloves phytophthora loop-mediated isothermal amplification primer specificity verification of embodiment and sensitivity verification
For the loop-mediated isothermal amplification (LAMP) primer group of cloves phytophthora, 8 groups of qualified primers are devised altogether, are finally screened
Go out that 1 group the most special and Primer composition (including the positive inner primer used in the high primer of sensitivity i.e. embodiment 1
FIP, reversed inner primer BIP, positive outer primer F3, reversed outer primer B3).Using remaining primer of design (at random from 8 groups remaining
1 group is selected in primer), primer sequence is as follows：FIP-1：5 ,-GCCGCGGTAGTAGCTGCTAGT-TTGTAGTGGGACACGGCT-
3′；BIP-1：5 ＇-CGTGGTGTACGACGTGACGG-CGACGCACCTATCAATCTCG-3 ＇：F3-1：5 ＇-
CTGTAGAGCATGCTGCTGAT-3′；B3-1：5 '-CCCGCAGAGAAACCTATGG-3 ＇.With the bacterium employed in embodiment 2
Strain is material to be tested (1 plant of cloves Mtr mutant and 14 kinds of other oomycetes and 17 kinds of disease fungus), ring mediated isothermal amplification inspection
The results are shown in Figure 3 for survey, and the specificity of primer selected by display residue is not high, and sensitivity is also poor, illustrates embodiment 1 for this hair
Bright Primer composition has higher specificity and sensitivity.
Embodiment 5 detects cloves phytophthora from soil sample of carrying disease germs
Cloves phytophthora in soil is carried out using the cloves phytophthora detection kit or Primer composition of embodiment 1 to detect, mistake
Journey is as follows：
1) in soil egg spore enrichment：40~80 grams of pedotheque to be checked is taken, grinds, is successively gone using 200 mesh screens
Locate larger grogs, then passes through 400,500,800 mesh screens and filter, while being rinsed repeatedly with 3~10 liters of water, from 800 mesh screens
Upper collection egg spore, with 1mL aqueous suspensions.Since egg spore cannot penetrate 800 mesh screens, processing, which can reach, in this way makes egg spore
The effect of enrichment.
2) DNA is extracted from micro egg spore：It will be transferred in the centrifuge tube of 1.5mL with the egg spore of sterile aqueous suspension,
In 12000r.min-1It is centrifuged 5 minutes under rotating speed, pours out liquid；50 μ L CTAB buffer are added, grinds, adds 500 μ L
CTAB buffer, water-bath 30 minutes；Isometric chloroform is added, in 12000r.min-1It centrifuges 10 minutes, draws under rotating speed
Supernatant；Be added the 3M NaAc of 1/10 volume, 2 times of volumes without water-ice ethyl alcohol, precipitation at room temperature 30 minutes, 12000r.min-1Rotating speed
Lower centrifugation 10 minutes, dry liquids；1mL 70% (V/V) ethyl alcohol is added to wash, 12000r.min-1Centrifuge 10 minutes under rotating speed,
Dry liquids are dried to alcohol-free taste；Add 10 μ L aseptic double-distilled waters to dissolve, is used for the template of ring mediated isothermal amplification.
3) cloves phytophthora ring mediated isothermal amplification detects, including：The ring mediated isothermal amplification of cloves phytophthora detects：Take 1 μ
LDNA solution, is added the detection solution and 1 μ L sterile deionized waters of 23 μ L, and total volume is 25 μ L；Response procedures are：64℃
60min；Using HNB (hydroxynaphthol blue) as reaction indicator, the color of loop-mediated isothermal amplification system after amplification
Sky blue is presented, judges that can generate positive reaction from soil sample of carrying disease germs contains cloves phytophthora with this.
Embodiment 6 is inoculated with the ring mediated isothermal amplification detection of cloves phytophthora in shaddock sample tissue
There are the DNA of cloves phytophthora, tool when cloves phytophthora, are extracted using NaOH rapid cleavage methods in for incidence tissue
Body process is：The tissue sample of one section of morbidity is taken, 10 μ L 0.5M NaOH are added in every milligram of tissue, after being fully ground in mortar
It being transferred in the EP pipes of 1.5mL, 12000rpm centrifuges 5min, takes 5 μ L supernatants that 495 μ L 0.1mM Tris (pH 8.0) are added,
1 μ L are taken to be directly used in PCR reactions after mixing.Each reaction at least in triplicate, while being deposited to determine in plant without PCR mortifiers
.
The DNA that the morbidity shaddock of inoculation cloves phytophthora is extracted using the above method, mediated for ring as template etc.
Temperature amplification.1uL DNA solutions are taken, as described in Example 4, carry out loop-mediated isothermal amplification.The results are shown in Figure 4, shows
Show and carry out ring mediated isothermal amplification in the disease plant of inoculation cloves phytophthora, positive sky blue (sample is also presented in color reaction
1)；And purple (sample 2,3) is presented in healthy plant and negative control.
Claims (6)
1. a kind of loop-mediated isothermal amplification (LAMP) primer composition for detecting cloves phytophthora, it is characterised in that：By positive inner primer
FIP, reversed inner primer BIP, positive outer primer F3, reversed outer primer B3 compositions；Each primer sequence is specific as follows：
FIP：5 ＇-GCCGCGGTAGTAGCTGCTAGT-TTGTAGTGGGACACGGCT-3 '；
BIP：5 ＇-CGTGGTGTACGACGTGACGG-CGACGCACCTATCAATCTCG-3 ＇；
F3：5 ＇-CTGTAGAGCATGCTGCTGAT-3 ＇；
B3：5 ＇-CCCGCAGAGAAACCTATGG-3 ＇.
2. application of the loop-mediated isothermal amplification (LAMP) primer composition described in claim 1 in detecting cloves phytophthora.
3. a kind of loop-mediated isothermal amplification kit of detection cloves phytophthora, it is characterised in that：Including volume be ampoule with
On detection solution, wherein the detection solution of 1mL includes：48mM dNTPs、0.8M Tris-HC、0.4mM KCl、0.4mM
(NH4)2SO4、0.24mM MgSO4, 4%Triton X-100,320 units of Bst DNA polymerase, 200mM hydroxyl naphthalenes
Phenol indigo plant, the reversed reversed outer primer B3 of inner primer BIP, 8mM forward direction outer primer F3,8mM of 32mM forward direction inner primers FIP, 32mM；Its
In, each primer sequence is specific as follows：
FIP：5 '-GCCGCGGTAGTAGCTGCTAGT-TTGTAGTGGGACACGGCT-3 ＇；
BIP：5 ＇-CGTGGTGTACGACGTGACGG-CGACGCACCTATCAATCTCG-3 ＇；
F3：5 ＇-CTGTAGAGCATGCTGCTGAT-3 ＇；
B3：5 '-CCCGCAGAGAAACCTATGG-3 ＇.
4. loop-mediated isothermal amplification kit the answering in detecting cloves phytophthora of the detection cloves phytophthora described in claim 3
With.
5. a kind of method of detection cloves phytophthora, it is characterised in that：The DNA for extracting microorganism to be checked, using the DNA of extraction as mould
Plate utilizes the ring mediated isothermal amplification described in loop-mediated isothermal amplification (LAMP) primer composition described in claim 1 or claim 3
Kit carries out ring mediated isothermal amplification；The variation of loop-mediated isothermal amplification solution colour is observed, sky blue expression is detected as
The positive, there are cloves phytophthoras；Purple indicates that testing result is feminine gender, and cloves phytophthora is not present.
6. the method for detection cloves phytophthora according to claim 5, it is characterised in that：The DNA for extracting microorganism to be checked, takes
2 μ L DNA solutions, the detection solution being added in 23 μ L loop-mediated isothermal amplification kits carry out ring mediated isothermal amplification, and ring mediates
Isothermal amplification program is：64 DEG C, 60min, amplified production carries out the observation of color change.
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