CN106755342A - Based on the ring mediated isothermal amplification that color judges（LAMP）Technology for detection chestnut phytophthora Heisui River germ - Google Patents
Based on the ring mediated isothermal amplification that color judges（LAMP）Technology for detection chestnut phytophthora Heisui River germ Download PDFInfo
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Abstract
The invention discloses a kind of ring mediated isothermal amplification judged based on color（LAMP）The molecular detecting method and its primer of technology for detection chestnut phytophthora Heisui River germ, primer sequence is respectively as shown in the ID NO. 6 of SEQ ID NO. 1 to SEQ.Detection architecture of the invention is under 62 °C of isothermys, can be quick, it is convenient, efficiently, it is high special, chestnut phytophthora Heisui River germ is detected with sensitivity, and do not need complex instrument, for the detection of chestnut phytophthora Heisui River germ provides new technology platform, the Site Detection to chestnut phytophthora Heisui River germ can preferably be met, suitable for the inspection and quarantine and the investigation of disease of entry and exit plant and plant product, quick diagnosis and monitoring etc., to preventing chestnut phytophthora Heisui River incoming China of germ significant, simultaneously, the foundation of system of the present invention is also for the detection of other pathogens provides technological guidance and theoretical foundation.
Description
Technical field
The present invention relates to a kind of detection chestnut phytophthora Heisui River germ (Phytophthora lateralis Tucker et
Mibrath LAMP primer), and using the molecular detecting method of primer detection chestnut phytophthora Heisui River germ, belong to biological
Technical field.
Background technology
Chestnut phytophthora Heisui River germ is one to be used for a kind important crushing phytopathogen, can cause serious chestnut black canker
And root, the stem rot of many planting fruit-trees[1].Because the disease causes harm weight, propagation soon, and prevent and treat and eradicate extremely difficult, the cause of disease
Bacterium has caused the great attention of many countries such as European Union, takes measures to prevent that its is incoming one after another.In recent years, due to international nursery stock
And its allocation and transportation of plant packaging material are increased, the most probably incoming China of the pathogen, once it is incoming, will be to China's agricultural
Industry, ecological environment etc. are caused a significant threat.Because there is not a report that chestnut phytophthora Heisui River germ occurs in China, State General Administration for Quality Supervision in
List the germ in new revision within 2007《Enter the territory plant quarantine harmful organism register》In.In order to prevent the pathogen incoming
China, it is necessary to quickly and accurately detected to it, therefore for chestnut phytophthora Heisui River germ detection We conducted a series of
Research.
Chestnut phytophthora Heisui River germ [Phytophthora cambivora (Petri) Buisman] is subordinate to phycomycete circle
(Chromista), oomycota (Oomycota), Peronosporales (Peronosporales), pythiaceae (Pythiaceae), Phytophthora
(Phytophthora), it is the important phytopathogen of a class, its geographical distribution and host range are very extensive.Germ mycelium
Coralliform, produces mycelia to expand body on butter bean culture medium.Sporangium width egg shape, it is sub- it is spherical, fall pyriform or ellipse, spore
Capsule is without mastoid process, base portion blunt circle.48~83 × 32~61 μm of sporangium size, average 59~64 × 40~45 μm；Length-width ratio 1.4~
1.8.Sporangium internal layer goes out, sporangiophore Dan Sheng, and branch or sympodium are not given birth to.Heterothallism.Oogonium is spherical, most of oogonium
Outer wall has excipuliform to swell, 30~58 μm of diameter, average 40.5 ± 5.5 μm, 2 μm of the thickness average out to of oogonium wall.Egg spore is expired
Device, 35~39 μm of diameter, average 33.8 ± 5.6 μm.Spermary side is given birth to, generally extension, unicellular or double cells, about 60% hero
Device is double cells.16.6~29.4 μm of spermary length, average 27.3 μm (double cellular types), 19.3 μm (unicellular type).Germ is most
2 DEG C of low growth temperature, 22~24 DEG C of optimum growth temperature, 32 DEG C of maximum growth temperature[1]。
The classification of traditional pathogen, identification are based primarily upon morphological characteristic, Pathogenicity etc., cumbersome, time-consuming,
Sensitivity is low, easily by artificially and the factors such as environment are disturbed[2], additionally, also having black using serological method detection chestnut phytophthora
The report of edema bacterium[2], but detection time is more long, and detection process is complicated, it is impossible to meet the demand of quick detection.
Loop-mediated isothermal amplification technique (Loop-mediated isothermalamplification, LAMP) is Japan
A kind of new nucleic acid amplification technologies that can invent of Rong Yan plants of formula[3], because its simple to operate, quick, specific high, low cost
The advantages of, as the new nucleic acid amplification technologies that can substitute regular-PCR.Design 4 kinds of spies in 6 regions that it is directed to target gene
Different primer, causes self-loopa strand replacement reaction, 60~65 DEG C of 60~70min of scope in the presence of Bst Large fragment polymerases
It is interior, it is accompanied by accessory substance while a large amount of synthesis target dnas --- the magnesium pyrophosphate precipitation of white is produced[4].Due to LAMP amplifications
Process relies on identification 6 isolated areas of target sequence, so atopic is very strong, and amplification process is in constant temperature
Under carry out, common water-bath or the equipment for having stable thermal source just can meet reacts requirement, and testing cost substantially reduces.
The selection of target gene is one of key factor of LAMP detections.At present, the conventional target gene of Molecular Detection has
Internal Transcribed Spacer (Internal transcribed space, ITS), but many scholars think that the target does not have
There are enough variant sites to distinguish all of phytophthora kind[5].The modulation GTP (GTP) that the present invention is selected combines egg
White gene (Ypt1) is a gene related to proto-oncogene Ras (Rat sarcoma), in yeast, the gene code one
The individual gtp binding protein related to Ras.Ypt1 genes include multiple intrones, and noncoding region has enough specificity
Site, and its sequence is conservative in kind of inner height, is highly suitable as the Molecular Detection target of phytophthora[6]。
Bibliography
1.Erwin,D.C.；Ribeiro,O.K.Phytophthora lateralis.In:Phytophthora
diseases worldwide.American Phytopathological Society,St.Paul(US),1996,pp
365-367.
2.Daniells J,Davis D,Peterson R,et al..Goldfinger:not as resistant to
Sigatoka/yellow sigatoka as first thought.Infomusa, 1995,4 (1):6.
3.Notomi,T.,Okayama,H.,Masubuchi,H.,Yonekawa,T.,Watanabe,K.,Amino,N.,
and Hase,T.Loop-mediated isothermal amplification of DNA.Nucleic Acids
Research,2000,28:e63-e63.
4.6.Yasuyoshi Mori,Kentaro Nagamine,Norihiro Tomita,and Tsugunori
Notomi.Detection of Loop-Mediated Isothermal Amplification Reaction by
Turbidity Derived from Magnesium Pyrophosphate Formation.Biochemical and
Biophysical Research Communications,2001,289:e150-e154.
5.White,T.J.,Bruns,T.,Lee,S.,and Taylor,J.Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics.1990,Pages 315-
322in:PCR Protocols:A Guide to Methods and Applications.M.A.Innis,
D.H.Gelfand,J.J.Sninsky,and T.J.White,eds.Academic Press,San Diego,CA.
6.Chen Y,Roxby R.Characterization of a Phytophthora infestans gene
involved in vesicle transport.Gene,1996,181(1-2):89-94.
The content of the invention
The technical problems to be solved by the invention are needed for solving the detection method of chestnut phytophthora Heisui River germ in the prior art
Cycle is long, waste time and energy, cumbersome, poor specificity and PCR detection techniques need thermal cycler instrument, high cost, it is impossible to quick detection
The problem of chestnut phytophthora Heisui River germ, so that there is provided the new molecular detecting method of chestnut phytophthora Heisui River germ, to chestnut phytophthora black canker
Bacterium carries out LAMP detections, and the method detection cycle short (only needing 80min), high specificity, sensitivity is high, can visually observe detection knot
Fruit and to be not required to expensive instrument cost low, easy to operation.
The selection of modulation gene and the screening of primer are the key factors of LAMP detections.At present, Molecular Detection is extensive
Application is the target based on ribosomes transcribed spacer (ITS), but by ITS sequence does not have enough variant sites to distinguish institute
Some phytophthora kinds, it is therefore desirable to excavate out new Molecular Detection target.First, search pertinent literature and have chosen tRNA (transhipments
RNA) the availability such as EF1 α (translation elongation factor), Ypt1 (GTP binding-protein gene), beta microtubule protein genes
Target, then Screening target gene successively, final choice Ypt1 as detection chestnut phytophthora Heisui River germ target gene.Again to
Many set primers of target gene design are tested and verified, select the LAMP primer of a set of detection chestnut phytophthora Heisui River germ.
The present invention provide technical scheme be：
The present invention chooses modulation gene, designs and screen the LAMP primer for detecting chestnut phytophthora Heisui River germ, its
Including four specific primers F3, B3, FIP, BIP and two rings primer LF and LB, primer sequence respectively as SEQ ID NO.1 extremely
Shown in SEQ ID NO.6, table 1 below is specifically shown in.
The chestnut phytophthora Heisui River germ LAMP primer sequence of table 1
Meanwhile, the present invention also puies forward a kind of method using above-mentioned primer detection chestnut phytophthora Heisui River germ altogether, and its process is：Carry
The DNA of testing sample is taken as template, LAMP amplified reactions are carried out using described primer, after reaction, visually seen under ordinary light
Examine, result judgement is carried out with the color change of hydroxynaphthol blue (hydroxynaphthol blue, HNB)；Under ordinary light, sky blue
Testing result is represented to detect chestnut phytophthora Heisui River germ in positive (+), i.e. sample, purple represents that testing result is negative (-),
Chestnut phytophthora Heisui River germ (Fig. 1) is not detected by sample.
The method of above-mentioned detection chestnut phytophthora Heisui River germ, its LAMP reaction system is：2.5μL 10×ThermoPol
Buffer (0.1%Trion-X, 20mM Tris-HCl, 10mM KCl, 10mM (NH4) SO4, pH 8.8), 4 μ L MgSO4
(50mM), 4 μ L glycine betaines (5M), 3.5 μ L dNTPs (10mM), inner primer FIP and BIP (20 μM) each 2 μ L, outer primer F3 and B3
(10 μM) each 0.5 μ L, ring primer LF and LB (10 μM) the μ L Bst archaeal dna polymerases of each 1 μ L, 2 μ L HNB (2.4mM), 1 (8U μ
L-1), the μ L of template DNA 2.
The method of above-mentioned detection chestnut phytophthora Heisui River germ, its LAMP response procedures is：62 DEG C, 80min.
Beneficial effect
The present invention compared with prior art, has the advantages that：
(1) practicality is good.Common PCR reaction carries out gel electrophoresis to product and easily causes product diffusion, and this is experiment
One main source of chamber contamination；And ethidium bromide (EB) has severe toxicity, can accumulate carcinogenic；Long-term observation uviol lamp also can be to reality
The personnel of testing cause a certain degree of injury.And LAMP reactions need to only be carried out in thermostat water bath, reaction passes through HNB after terminating
Color change just can direct judged result, whole operation process is simple and easy to apply, it is adaptable to the port of chestnut phytophthora Heisui River germ
Quarantine and the quick diagnosis of disease.
(2) constant-temperature amplification.Unlike PCR methods have to thermal cycle, the dependence to thermal cycler instrument is thus broken away from, as long as
The thermal source LAMP reactions for having stabilization can just occur, and greatly extend the scope that LAMP is used, and why LAMP can be constant
Thermal source under react because with the addition of glycine betaine in LAMP reaction solutions, double-stranded DNA is in the dynamic equilibrium unwind
In, amplification is realized in the presence of Bst archaeal dna polymerases.
(3) accuracy is high.Because traditional chestnut phytophthora Heisui River germ detection technique is to be identified according to morphological feature,
Time-consuming, sensitivity is low, easily by artificially and the factors such as environment are disturbed；And the present invention is according to chestnut phytophthora Heisui River germ
Ypt1 sequences, guard very much in genome of the sequence in the germ of chestnut phytophthora Heisui River, using Bioedit softwares that chestnut phytophthora is black
The Ypt1 sequences of edema bacterium and the Ypt1 sequences of other phytophthoras are compared, the specific LAMP of design chestnut phytophthora Heisui River germ
Primer.LAMP reactions recognize 6 isolated areas on target sequence by 4 primer specificities, are recognized relative to common PCR primers
For 2 isolated areas of target sequence, specificity and sensitivity are all higher.
Under 62 DEG C of isothermys, energy is quick, convenient, efficient, height specifically, is with sensitivity detected for detection architecture of the invention
To chestnut phytophthora Heisui River germ, and complex instrument is not needed, for the detection of chestnut phytophthora Heisui River germ provides new technology platform, energy
Preferably meet to the Site Detection of chestnut phytophthora Heisui River germ, it is adaptable to chestnut epidemic disease in the inspection and quarantine of enter and leave the border plant and plant product
High sensitivity, the quick detection of mould Heisui River germ, while can be used for the investigation of chestnut phytophthora black canker, quick diagnosis and monitoring etc.,
To preventing chestnut phytophthora Heisui River incoming China of germ significant, meanwhile, the foundation of system of the present invention is also other pathogens
Detection provide technological guidance and theoretical foundation.
Brief description of the drawings
Fig. 1 LAMP reactions add the experiment of HNB chromogenic reactions, wherein ,-:Negative reaction is presented purple；+:Positive reaction is in
Existing sky blue.
Fig. 2 chestnut phytophthoras Heisui River germ Ypt1 gene nucleic acids sequence and primer location.
The versatility checking of Fig. 3 primers；
Wherein 1:P.cambivora(ATCC 38811)；2:P.cambivora(CBS 111329)；3:P.cambivora
(CBS 114093)；4:Negative control.
The inter-species specificity verification of Fig. 4 primers；
Wherein 1:Chestnut phytophthora Heisui River germ P.cambivora, 2-4:Deodar Phytophthora Root Rot P.lateralis, 5,6:
Robur sudden death pathogen P.ramorum, 7:Soybean phytophthora P.sojae, 8:Phytophthora infestans P.infestans, 9:Big hero phytophthora
P.megasperma,10:Melon phytophthora P.melonis, 11:Phytophthora capsici P.capsici, 12:Clover phytophthora
P.medicaginis,13:Phytophthora drechsleri P.drechsleri, 14:Palm mould P.palmivora, 15:Peronophythora Litchii
Peronophthora litchi,16:Phytophthora cactorum P.cactorum, 17:Ramie mould P.boehmeriae, 18:Hidden ground phytophthora
P.cryptogea,19:Phytophthora nicotianae P.nicotianae, 20:Negative control.
The specificity among genus checking of Fig. 5 primers；
Wherein 1:Chestnut phytophthora Heisui River germ P.cambivora；2:Fusarium graminearum Fusarium graminearum；3:Point
Fusariumsp F.oxysporum；4:Layer goes out Fusariumsp F.proliferatum；5:Scouring rush's Fusariumsp F.equiseti；6:Eggplant corruption sickle
Spore bacterium F.solani；7:Oat Fusariumsp F.avenaceum；8:Yellow Fusariumsp F.culmorum；9:Tack anthrax-bacilus
Colletotrichum truncatum；10:Colletotrichum gloeosporioides Penz C.gloeosporioides；11:More main rod spore bacterium
Corynespora cassiicola；12:Soybean charcoal rot bacterium Macrophomina phaseolina；13:Cercospora kikuchii
Cercospora kikuchii；14:Rhizoctonia solani Kuhn Rhizoctonia solani；15:Chinese ilex Caionectria bacterium
Calonectria ilicicola；16:Fusarium oxysporum Bipolaris maydis；17:Aspergillus oryzae
Aspergillus oryzae；18:Pyricularia oryzae Magnaporthe grisea；19:Southern stem canker of soybean
Diaporthe phaseolorum var.meridionalis；20:The northern stem canker Diaporthe of soybean
phaseolorum var.caulivora；21:Brown stem rot bacterium Phialophora gregata；22:Soybean intends stem point
Seed rot disease bacterium Phomopsis longicolla；23:Soybean rest fungus Phakopsora pachyrhiz；24:Negative control.
The sensitivity technique of Fig. 6 chestnut phytophthoras Heisui River germ LAMP detection method；
Wherein 1-8:100ng·μL-1、10ng·μL-1、1ng·μL-1、100pg·μL-1、10pg·μL-1、1pg·μL-1、100fg·μL-1、10fg·μL-1The chestnut Heisui River phytophthora DNA of concentration gradient is used as reaction template；10:Negative control.
Specific embodiment
Embodiment 1：Modulation gene is chosen, the LAMP primer for detecting chestnut phytophthora Heisui River germ is designed and screen,
And set up LAMP detection architectures
(1) selection of modulation gene and the design of primer and screening
The selection of modulation gene and the screening of primer are the key factors of LAMP detections.At present, Molecular Detection is extensive
Application is the target based on ribosomes transcribed spacer (ITS), but by ITS sequence does not have enough variant sites to distinguish institute
Some phytophthora kinds, it is therefore desirable to excavate out new Molecular Detection target.First, search pertinent literature and have chosen tRNA (transhipments
RNA) the availability such as EF1 α (translation elongation factor), Ypt1 (GTP binding-protein gene), beta microtubule protein genes
Target.By taking target Ypt1 as an example, the Ypt1 gene orders and other phytophthora kinds of chestnut phytophthora Heisui River germ are downloaded from GeneBank
Ypt1 gene orders, the Ypt1 gene orders of all downloads are analyzed using Bioedit softwares then, and use
PrimerExplore V4 software Photographing On-line primers, choose suitable according to primer length, primer free energy and G/C content etc.
Primer is tested, and therefrom versatility is good in screening kind, and inter-species high specificity, specificity among genus are strong, sensitivity primer high.According to
Secondary other targets of screening, final choice Ypt1 is used as the target gene (Fig. 2) for detecting chestnut phytophthora Heisui River germ.Again to the target
Many set primers of base design carry out test of many times and checking, select that a set of versatility is good, and high specificity, sensitivity is high, can be quick,
The accurate primer for detecting chestnut phytophthora Heisui River germ, sequence is as follows：
Detection primer is to ((F3, B3, FIP, BIP, LB))
F3 (positive outer primer)：CCTAGGTCCACCATGGCTA(SEQ ID NO.1)；
B3 (reverse outer primer)：CGTGTCCCACTACAATTCCG(SEQ ID NO.2)；
FIP (positive inner primer) (F1C+F2)：
TCCAGCTCGATCGTGCGAATTT-TTGACCTCCAGGCTGACG(SEQ ID NO.3)；
BIP (reverse inner primer) (B1C+B2)：
CGGCAAGACCATCAAGCTCCA-CCAGTTAGCTCCATGAAGCA(SEQ ID NO.4)；
LF (positive ring primer)：GCACAACAACGAGCACGATAA(SEQ ID NO.5)；
LB (reverse ring primer)：GATTGTGCGTGCATTCCCG(SEQ ID NO.6).
(2) chestnut phytophthora Heisui River germ LAMP detection architectures are set up
Chestnut phytophthora Heisui River germ LAMP reaction systems：2.5 μ 10 × ThermoPol of L Buffer (0.1%Trion-X,
20mM Tris-HCl, 10mM KCl, 10mM (NH4) SO4, pH 8.8), 4 μ L MgSO4 (50mM), 4 μ L glycine betaines (5M), 3.5
μ L dNTPs (10mM), inner primer FIP and BIP (20 μM) each 2 μ L, outer primer F3 and B3 (10 μM) each 0.5 μ L, ring primer LF and
LB (10 μM) the μ L Bst archaeal dna polymerases of each 1 μ L, 2 μ L HNB (2.4mM), 1 (8U μ L-1), 2 μ L template DNAs.By above-mentioned mixing
Thing is placed in the PCR reaction tubes of 0.2mL and is slightly vortexed on vortice, is then centrifuged for ensuring not having drop on tube wall.Will
Reaction tube is placed in 62 DEG C of isothermal reaction 80min in water-bath.
Embodiment 2：Prepare DNA profiling
The template that the DNA of sample reacts as LAMP is extracted, detailed process is as follows：
(1) prepared by strain culturing and hypha powder
LBA (butter bean culture medium (Zheng little Bo phytophthoras and its agriculture of investigative technique China will be gone to for examination phytophthora bacteria strain
Industry publishing house .1997)) on flat board, other bacterial strains go to PDA (potato glucose solid medium (Erwin, D.C.；
Ribeiro,O.K.Phytophthora lateralis.In:Phytophthora diseases
Worldwide.American Phytopathological Society, St.Paul (US), 1996, pp 365-367)) it is flat
On plate, 10 pieces of 2 × 2mm mycelia blocks are cut from colony edge after 25 DEG C of dark culturing 3d, the bacterial strain of phytophthora goes to the training of V8 liquid
Base (Zheng little Bo phytophthoras and its investigative technique Chinese agriculture publishing house .1997) is supported, other bacterial strains go to PDB (potato Portugals
Grape sugar liquors culture medium) in, 25 DEG C of 5~7d of shaken cultivation are collected by filtration mycelia, and chilled draining grinds to form hypha powder, -20 DEG C
Save backup.
(2) extraction of genomic DNA
A small amount of hypha powder, plus 900 μ L 2%CTAB extract solutions and 90 μ L 10%SDS are taken, whirlpool is mixed, in 60 DEG C of water-baths
1h, it is middle to be turned upside down several times per 10min.12000rpm·min-1Centrifugation 10min, take supernatant add isometric phenol/chloroform/
Isoamyl alcohol (25：24：1), overturn and mix, 12000rpmmin-1Centrifugation 10min；Supernatant is transferred in new pipe, add etc.
The chloroform of volume, gently overturns and mixes, 12000rpmmin-1Centrifugation 5min.Supernatant is taken to be transferred in new pipe, plus 2 times of volumes
Absolute ethyl alcohol and 1/10 volume 3molL-1NaAc (pH 5.2), -20 DEG C staticly settle (>1h).12000rpm·min-1
Centrifugation 10min, incline supernatant, and precipitation is washed 2 times with 70% ethanol, and room temperature is dried.Plus appropriate sterilizing ultra-pure water or TE (pH
8.0) dissolution precipitation (contains 20 μ gml-1RNase), after 37 DEG C for the treatment of 1h, -20 DEG C save backup.
Embodiment 3：The specificity of detection LAMP primer and sensitivity
(1) specific detection
Bacterial strain uses therefor of the present invention and relevant information are shown in Table 2.Using all for examination bacterium in the primer pair table 2 designed by the present invention
The genomic DNA of strain carries out LAMP amplified reactions, and the chestnut phytophthora Heisui River germ of the chromogenic reaction result display separate sources of HNB is anti-
Guan Juncheng sky blues are answered, is positive findings, negative control reaction tube does not change colour, be still purple (Fig. 3).Chestnut phytophthora Heisui River germ
Reaction tube be in sky blue, be positive findings, the reaction tube of other phytophthoras, fungi and negative control is in purple, is cloudy
Property result (Fig. 4,5), as a result show that the primer has specificity well, can by chestnut phytophthora Heisui River germ and its allied species and
Other correlation kinds are distinguished.
The strains tested of table 2 and LAMP testing results
Note：NJAU represents Agricultural University Of Nanjing；CAIQ represents Chinese Jian Ke institutes；* to indicate and (only provide bacterial strain without bacterial strain
DNA)+expression there occurs that LAMP is expanded；- indicate without amplification.
(2) sensitivity technique
The sensitivity of above-mentioned set up detection architecture is determined in the reaction system of 26 μ L, by 10 times of gradient dilutions
The genomic DNA of chestnut phytophthora Heisui River germ is (from 100ng μ L-1To 10fg μ L-1) LAMP amplifications are carried out for template, in 62 DEG C
80min is reacted under isothermy.Fig. 6 shows the HNB visualization colour developing figures of sensitivity technique result, when there is LAMP amplifications,
Positive reaction is changed into sky blue, and what LAMP amplifications did not occurred is still purple.Testing result shows that lowest detection sensitivity is
100pg·μL-1。
<110>P. R. of China Kunshan Entry-exit Inspection and Quarantine Bureau
<120>Based on the ring mediated isothermal amplification that color judges（LAMP）Technology for detection chestnut phytophthora Heisui River germ
<160> 6
<210> 1
<211> 19
<212> DNA
<400> 1
CCTAGGTCCACCATGGCTA 19
<210> 2
<211> 20
<212> DNA
<400> 2
CGTGTCCCACTACAATTCCG 20
<210> 3
<211> 41
<212> DNA
<400> 3
TCCAGCTCGATCGTGCGAATTT-TTGACCTCCAGGCTGACG 40
<210> 4
<211> 42
<212> DNA
<400> 4
CGGCAAGACCATCAAGCTCCA-CCAGTTAGCTCCATGAAGCA 41
<210> 5
<211> 21
<212> DNA
<400> 5
GCACAACAACGAGCACGATAA 21
<210> 6
<211> 19
<212> DNA
<400> 6
GATTGTGCGTGCATTCCCG 19
Claims (4)
1. a kind of LAMP primer for detecting chestnut phytophthora Heisui River germ, it is characterised in that：It include four specific primer F3,
B3, FIP, BIP and two rings primer LF and LB, primer sequence is respectively as shown in the ID NO. 6 of SEQ ID NO. 1 to SEQ.
2. it is a kind of based on color judge chestnut phytophthora Heisui River germ LAMP detection method, it is characterised in that：Extract testing sample
DNA as template, carry out LAMP amplified reactions using the primer described in claim 1, after reaction, visually seen under ordinary light
Examine, result judgement is carried out with the color change of hydroxynaphthol blue；Under ordinary light, sky blue represents that testing result is the positive, i.e. sample
In detect chestnut phytophthora Heisui River germ, purple represents that testing result is feminine gender, i.e., chestnut phytophthora Heisui River germ is not detected by sample.
3. LAMP detection method according to claim 2, it is characterised in that：Its LAMP amplification reaction system is：2.5 μL
10×ThermoPol Buffer（0.1% Trion-X, 20 mM Tris-HCl, 10 mM KCl, 10 mM（NH4）SO4,
pH 8.8）, 4 μ L MgSO4（50 mM）, 4 μ L glycine betaines（5 M）, 3.5 μ L dNTPs（10 mM）, inner primer FIP and BIP
（20 μM）Each 2 μ L, outer primer F3 and B3（10 μM）Each 0.5 μ L, ring primer LF and LB（10 μM）Each 1 μ L, 2 μ L HNB
（2.4 mM）, 1 μ LBstDNA polymerases（8 U·μL-1）, the μ L of template DNA 2.
4. LAMP detection method according to claim 3, it is characterised in that：Its LAMP amplified reaction program is：62 DEG C,
80min。
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