CN110079631A - A kind of molecular detecting method of 8 kinds of phytophthoras of high-throughput detection - Google Patents
A kind of molecular detecting method of 8 kinds of phytophthoras of high-throughput detection Download PDFInfo
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Abstract
The invention discloses a kind of molecular detecting methods of 8 kinds of phytophthoras of high-throughput detection.In the present invention, LAMP technology is established to a kind of high-throughput detection technique that can detect 8 kinds of phytophthoras simultaneously in conjunction with 96 orifice plates.For 8 kinds of phytophthoras (phytophthora parasitica Phytophthora parasitica, Phytophthora tentaculata, phytophthora infestans Phytophthora infestans, strawberry phytophthora Phytophthora fragariae, Phytophthora capsici Phytophthora capsici, Phytophthora drechsleri Phytophthora drechsleri, Phytophthora cactorum Phytophthora cactorum, big hero phytophthora Phytophthora megasperma) the same target gene Ypt1 establish Ypt1-LAMP detection primer, kit and method.Detection method accuracy height of the invention, high specificity, easy to operate, practicability is good, provides high-flux quick detection technique for the detection of phytophthora, has important and far reaching significance to phytophthora epidemic prevention and control and monitoring.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of phytophthora high throughput molecular detecting method.
Background technique
Phytophthora (Phytophthora) belongs to oomycota (Oomycota), Oomycete (Oomycetes), Peronosporales
(Peronosporales), pythiaceae (Pythiaceae), be it is a kind of host range is wide, severe disease and it is more difficult to control, to plant
The cause of disease oomycetes that yield brings about great losses.Phytophthora is one big category, at least 103 kinds recognized extensively at present.By
Stronger in destructiveness of the phytophthora (Phytophthora spp.) to host plant, harmfulness is big, often leads to agricultural production
Heavy losses, thus the plant disease caused by phytophthora is commonly referred to as " epidemic disease ".The host range of Phytophthora is very wide, mainly invades
The up to ten thousand kinds of plants such as fruit tree, forest, vegetables, flowers, cotton, fiber crops, oil plant are contaminated, but for different phytophthoras, host range
It differs greatly.The host ranges such as camphor tree phytophthora, Phytophthora nicotianae, Phytophthora cactorum are very wide, can infect thousands of kinds of plants；It is ramie mould, peppery
The host ranges such as green pepper phytophthora are relatively narrow；Soybean phytophthora, cowpea phytophthora etc. only infect 1-3 kind host plant.However phytophthora two can
When affinity fungus strain cooperates and is formed with sexual organ, it may occur however that genetic recombination, result will make pathogen have stronger existence
Ability, pathogenicity and wider host range.In the loss of China epidemic disease due to caused by phytophthora and phytophthora population
Complexity is all increasing year by year, seriously threatens the normal production and living of the mankind.Therefore, a kind of a variety of epidemic diseases of high-throughput Testing and appraisal are studied
The method of mould quickly understands and grasps local phytophthora population and ecology, to reinforce epidemic monitoring, for disease risk and grind
Study carefully decision to provide according to great and far reaching significance.
The method of traditional detection identification phytophthora is mainly based upon morphological feature, Pathogenicity, physiological and biochemical property
Etc. characters, separate the method for phytophthora using mass trapping and separate phytophthora from soil, irrigation water and vegetable material.In order to improve
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
Material has many materials to can be used for leaf butterfly mass trapping because different phytophthoras are also different, including Citrus leaf traps tobacco
Phytophthora, cucumber fruits trapping Phytophthora drechsleri, pine needle trapping camphor tree phytophthora, soybean leaves trapping soybean phytophthora of punching etc..In order at
The separation phytophthora of function, the method that tradition is learned need to obtain the pure culture of pathogen using selective medium, and complete procedure takes
When it is laborious and operator is required to have pathogenicbacteria separation, Morphological Identification knowledge and the experience abundant of profession.
With the development of molecular biology technology, the technologies such as regular-PCR and real-time fluorescence PCR be successfully applied to it is various
In the detection of phytophthora.Although there is specificity and sensitivity all using the detection method of round pcr and real-time fluorescent PCR technology
Higher advantage, but detection time is still long, it is time-consuming and laborious that electrophoresis later runs glue, and needs to rely on accurate and be worth
Higher PCR instrument is not able to satisfy the demand quickly detected.And many detection technique researchs at present are both for a kind of phytophthora
Bacterium, but in a practical situation, the demand to high-throughput Testing and appraisal phytophthora is more more and more intense.Katarzyna Sikora etc.
The high-throughput detection of 23 kinds of phytophthoras has been carried out using Padlock probe combination microarray chip technology.But these are diagnosed
Method or program is cumbersome, the period is long or at high cost, while being also required to using expensive instrument and reagent etc..
Ring mediated isothermal amplification (Loop-mediated isothermal amplification, LAMP) technology is a kind of
Efficient nucleic acid amplification method.It is a kind of novel molecular Biological Detection technology, and this method is 6 areas according to target gene
4 species-specific primers are designed in domain, and using the strand replacement reaction of Bst archaeal dna polymerase, the short time is (logical under 60-65 DEG C of constant temperature
Often in one hour) in carry out nucleic acid amplification, target gene can be expanded to 109Again, thus conclude whether amplified production is deposited
In target gene, and the method for available agarose gel electrophoresis and SYBR Green I fluorescent quantitation is sensitive to its product, amplification
Degree is analyzed.In order to keep the detection of LAMP final product simpler, intuitive, time saving, domestic and international scientist largely grind
Study carefully, successively proposes that the LAMP final product detection simple and easy to operate such as turbidometry, HNB indicator, wax-wrapped pill dyestuff and result are sentenced
Disconnected method, what application was more at present is that agarose gel electrophoresis, turbidometry, HNB indicator method and SYBR Green I refer to
Show agent method.The technology does not need stringent experimental situation and accurate expensive instrument and equipment, and operating procedure is easy to learn, has height
The features such as specificity, high sensitivity.The technology was primarily used for the clinical detection of medicine since 2003, achieves and induces one to infuse
Purpose effect.Many scholars have been widely used in the detection of the pathogens such as virus, bacterium, helminth, fungus.
LAMP technology has been successfully applied in the detection of individual phytophthoras, still lacks a complete high-throughput phytophthora
The LAMP of bacterium identifies detection architecture, for this purpose, the present invention establishes the height of the loop-mediated isothermal amplification technique based on same target Ypt1
Flux detect phytophthora, on this basis applied metal ion indicator hydroxynaphthol blue (hydroxynaphthol blue,
HNB) determining reaction result, and the specificity and sensitivity of this method are analyzed, entire detection reaction only needs 80min,
And it with the naked eye can directly determine positive findings.This method is to combine 96 hole plate techniques using using LAMP technology for the first time, simultaneously
To the research that a variety of phytophthoras are detected, application prospect is very wide.
Summary of the invention
Goal of the invention: identify for convenient, fast and efficiently high-throughput phytophthora a LAMP is lacked in the prior art
Detection architecture can not quickly understand and grasp local phytophthora population and ecology, be unfavorable for reinforcing phytophthora epidemic monitoring, be disease
The problem of harmful risk and research decision provides foundation, the application establishes the ring mediated isothermal amplification based on same target Ypt1
Technology high throughput detects the technical system of phytophthora, and an object of the present invention is to provide the LAMP inspection of 8 kinds of phytophthora high throughputs
Primer sets are surveyed, detect the phytophthora to specificity；It is a further object of the present invention to provide high-throughput 8 kinds of phytophthoras of detection
Kit once identifies a variety of phytophthoras；LAMP technology and 96 orifice plates are combined into high throughput the present invention also provides a kind of
The detection method of 8 kinds of phytophthoras is detected, conveniently and efficiently identifies a variety of phytophthoras.
Technical solution: to solve the above-mentioned problems, the technical scheme adopted by the invention is as follows:
A kind of molecular detecting method of 8 kinds of phytophthoras of high-throughput detection, the DNA including extracting microorganism to be checked, to extract
DNA be template, combined using 8 kinds of phytophthora LAMP detection primers with 96 orifice plates and high throughput carried out simultaneously to microorganism to be checked
Detection；LAMP reaction solution color change is observed, sky blue indicates that testing result is the positive, and there are target phytophthoras；Purple table
Show that for feminine gender, target phytophthora is not present in testing result；8 kinds of phytophthora LAMP detection primers include Phytophthora
parasitica、Phytophthora tentaculata、Phytophthora infestans、Phytophthora
fragariae、Phytophthora capsici、Phytophthora drechsleri、Phytophthora cactorum
With the LAMP detection primer of Phytophthora megasperma, the LAMP detection primer of every kind of phytophthora is by positive inner primer
FIP, reversed inner primer BIP, positive outer primer F3, reversed outer primer B3 and positive ring primer LF, reversed ring primer LB composition, respectively
Primer sequence is specific as follows:
The primer of Phytophthoraparasitica includes:
F3:5 '-CTTTGTAAGTGCCACCATAC-3 '；
B3:5 '-ACAGACACACACGTGATT-3 '；
FIP:
5′-TCGATCGTACGGATTTTCTGCAGAAAGATCCAGGTTTTCATCAGG-3′；
BIP:
5′-AGACCATCAAGCTCCAGATTGTACGTGGTACATCGGTTAGTTGAA-3′′；
LB:5 '-GACTTGTATCACTGCAGTTT-3 '；
The primer of Phytophthora tentaculata includes:
F3:5 '-TGCCTTATAGGAATAGCGC-3 '；
B3:5 '-AGCATAAGTGAATTGACCCA-3 '；
FIP:
5′-ATCGTACGGATTTTCTGAGCAAAGTAGATCCCGATTTCCATCAG-3′；
BIP:
5′-TGGACGGCAAGACCATCAAGTCCGTTAGTTAAATAAATACCTCGA-3′；
LB:5 '-CTCCAGATTGTACGTCCTTCGT-3 '；
The primer of Phytophthora infestans includes:
F3:5 '-TGTGAGTGTCTAACATATTTTACG-3 '；
B3:5 '-GTTAGTTAAATAGGAAATCACGC-3 '；
FIP:5 '-AATCGCGAAAGCCATGTGAGCCAAACGACCTTTTGTAAGG-3 '；
BIP:
5′-TTGCTTAGAAAATCCGTACGATCGGACAAATGTTTTTTTAGCGGC-3′；
LB:5 '-GGCAAGACCATCAAGCTCCAA-3 '；
The primer of Phytophthora fragariae includes:
F3:5 '-TGAGTGCTAGTAACTAGCCT-3 '；
B3:5 '-ACCAGTTAGCTCCATGAAGC-3 '；
FIP:
5′-CTGTGCACAACAACGAGCACGAATTCCTAGGTCCAAAAAGGC-3′；
BIP:5 '-AATTCGCACGATCGAGCTGGATTAGCCGGCGAAATGTTCC 1 '；
LF:5 '-CAGCAATCGGAGAGCAAATCTTA-3 '；
LB:5 '-CGGCAAGACTATCAAGCTCCAGA-3 '；
The primer of Phytophthora capsici includes:
F3:5 '-CTCTGTTGTATAGCAGAGGT-3 '；
B3:5 '-GCACAAGACAATTAGCACAAT-3 '；
FIP:
5′-TTCTGGGCGCGTACACAAACTTAGTGAGGGACAATTTATATCAGG-3′；
BIP:5 '-GATCGAGTTGGACGGCAAGATCCAGTGCTCTAACTAAAACG-3 ':
LB:5 '-CCATCAAGCTCCAGATTGTAAGC-3 ':
The primer of Phytophthora drechsleri includes:
F3:5 '-GTGATCCTTTCACCCTGG-3 '；
B3:5 '-TTACAAATGTCAGCTGGATG-3 ':
FIP:
5′-CGGATTTTCTAGAACGTGGTACCAAAATGAAGAGTCGACTCTAGCA-3′；
BIP:5 '-ACTATTGAGCTGGACGGCAATCGATAGCAGCCCAAGAG-3 '；
LB:5 '-TGTACGTCTACAGAGGATTTGGAT-3 '；
The primer of Phytophthora cactorum includes:
F3:5 '-TTCTGCGCTAGGCGACC-3 '；
B3:5 '-CACACAAGTGGACCGTTAG-3 '；
FIP:5 '-TCTGGGCACAACCGCAAAAATTTGCGAGCTCCAGATTTCC-3 '；
BIP:5 '-AATCCGTACGATCGAGCTGGACACACGCCACGTCTGCT-3 '；
LB:5 '-GGAAAGACCATCAAGCTCCAGAT-3 '；
The primer of Phytophthora megasperma includes:
F3:5 '-GCTCTGCTCTTCCGACTTG-3 '；
B3:5 '-GTTAGTTTCGTCCACGGCA-3 '；
FIP:5 '-CGGGCAAGAGCAACGTCAGTGTCCCATTGTGGTCCAGTAC-3 '；
BIP:
5′-TCCGTACGATCGAGCTGGACGAGAAGAAAGGAATGGAGGCC-3′；
LB:5 '-GCAAGACCATCAAGCTCCAGATTG-3 '.
Application of the 8 kinds of phytophthora LAMP detection primers in detection phytophthora.
A kind of LAMP detection kit of phytophthora, including at least primer solution and LAMP reaction more than ampoule
Liquid；Wherein, the primer in primer solution is 8 kinds of phytophthora LAMP detection primers described in claim 1.
Preferably, the LAMP reaction solution contains dNTPs, Tris-HCl, KCl, (NH4)2SO4、MgSO4、Triton
X-100, Bst DNA polymerase, hydroxynaphthol blue.
Application of the LAMP detection kit of the phytophthora in detection phytophthora.
A kind of molecular detecting method of 5 kinds of phytophthoras of high-throughput detection, the DNA including extracting microorganism to be checked, to extract
DNA be template, combined using 5 kinds of phytophthora LAMP detection primers with 96 orifice plates and high throughput carried out simultaneously to microorganism to be checked
Detection；LAMP reaction solution color change is observed, sky blue indicates that testing result is the positive, and there are target phytophthoras；Purple table
Show that for feminine gender, target phytophthora is not present in testing result；8 kinds of phytophthora LAMP detection primers include
Phytophthoraparasitica、Phytophthora tentaculata、Phytophthora fragariae、
The LAMP detection primer of Phytophthora capsici and Phytophthora megasperma, the LAMP of every kind of phytophthora
Detection primer is by positive inner primer FIP, reversed inner primer BIP, positive outer primer F3, reversed outer primer B3 and positive ring primer
LF, reversed ring primer LB composition, each primer sequence are specific as follows:
The primer of Phytophthoraparasitica includes:
F3:5 '-CTTTGTAAGTGCCACCATAC-3 '；
B3:5 '-ACAGACACACACGTGATT-3 '；
FIP:
5′-TCGATCGTACGGATTTTCTGCAGAAAGATCCAGGTTTTCATCAGG-3′；
BIP:
5′-AGACCATCAAGCTCCAGATTGTACGTGGTACATCGGTTAGTTGAA-3′′；
LB:5 '-GACTTGTATCACTGCAGTTT-3 '；
The primer of Phytophthora tentaculata includes:
F3:5 '-TGCCTTATAGGAATAGCGC-3 '；
B3:5 '-AGCATAAGTGAATTGACCCA-3 '；
FIP:
5′-ATCGTACGGATTTTCTGAGCAAAGTAGATCCCGATTTCCATCAG-3′；
BIP:
5′-TGGACGGCAAGACCATCAAGTCCGTTAGTTAAATAAATACCTCGA-3′；
LB:5 '-CTCCAGATTGTACGTCCTTCGT-3 '；
The primer of Phytophthora fragariae includes:
F3:5 '-TGAGTGCTAGTAACTAGCCT-3 '；
B3:5 '-ACCAGTTAGCTCCATGAAGC-3 '；
FIP:
5′-CTGTGCACAACAACGAGCACGAATTCCTAGGTCCAAAAAGGC-3′；
BIP:5 '-AATTCGCACGATCGAGCTGGATTAGCCGGCGAAATGTTCC-3 '；
LF:5 '-CAGCAATCGGAGAGCAAATCTTA-3 '；
LB:5 '-CGGCAAGACTATCAAGCTCCAGA-3 '；
The primer of Phytophthora capsici includes:
F3:5 '-CTCTGTTGTATAGCAGAGGT-3 '；
B3:5 '-GCACAAGACAATTAGCACAAT-3 '；
FIP:
5′-TTCTGGGCGCGTACACAAACTTAGTGAGGGACAATTTATATCAGG-3′；
BIP:5 '-GATCGAGTTGGACGGCAAGATCCAGTGCTCTAACTAAAACG-3 '；
LB:5 '-CCATCAAGCTCCAGATTGTAAGC-3 '；
The primer of Phytophthora megasperma includes:
F3:5 '-GCTCTGCTCTTCCGACTTG-3 '；
B3:5 '-GTTAGTTTCGTCCACGGCA-3 '；
FIP:5 '-CGGGCAAGAGCAACGTCAGTGTCCCATTGTGGTCCAGTAC-3 '；
BIP:
5′-TCCGTACGATCGAGCTGGACGAGAAGAAAGGAATGGAGGCC-3′；
LB:5 '-GCAAGACCATCAAGCTCCAGATTG-3 '.
Application of the 5 kinds of phytophthora LAMP detection primers in detection phytophthora.
A kind of LAMP detection kit of phytophthora, including at least primer solution and LAMP reaction more than ampoule
Liquid；Wherein, the primer in primer solution is 5 kinds of phytophthora LAMP detection primers as claimed in claim 6.
Preferably, the LAMP reaction solution contains dNTPs, Tris-HCl, KCl, (NH4)2SO4、MgSO4、Triton
X-100, Bst DNA polymerase, hydroxynaphthol blue.
Application of the LAMP detection kit of the phytophthora in detection phytophthora.
The utility model has the advantages that compared with prior art, the advantages and positive effects of the present invention are shown:
1) accuracy is high:, can not since traditional phytophthora detection technique only determines test object according to morphological feature
The interference for excluding human factor is difficult to distinguish close kind of form, and detection accuracy only has 60-80%；And the present invention is according to phytophthora
The sequence of the Ypt1 gene of bacterium, genome evolution area and conserved region of the sequence in phytophthora are spaced in turn, soft using Bioedit
The sequence of the Ypt1 gene order of 8 kinds of phytophthoras to be measured and other phytophthora kinds is compared by part, chooses the phytophthora distinctive one
The LAMP primer of section sequence design specificity.
2) high specificity: the present invention has attempted several genes target in design primer, transcribes and is spaced such as ribosomal gene
Area, transcriptional elongation factor, actin gene etc., but all do not screen suitably.Ypt1 gene is structurally characterized in that coding
Area and noncoding region are alternately present, so that the noncoding region of Ypt1 gene, which has, detects what target more changed relative to other
Site is as the molecular labeling of most of phytophthoras, and in the same phytophthora kind, Ypt1 gene is very conservative.Cause
This has preferably specificity using the phytophthora species-specific primers of Ypt1 gene design relative to other targets.Finally, than
When to Ypt1 gene, the special sequence of comparison is had found, multiple alternative primers are obtained by software as target.This is directed to again
A little alternative primers carry out preliminary experiment, and the specific primer group used in us has finally been determined.
3) easy to operate: the LAMP method of detection phytophthora infestans provided by the invention overcomes epidemic disease of causing a disease in the prior art
The problem of period needed for the biological detection method of mould is long, time-consuming and laborious, cumbersome, poor specificity and PCR detection technique need
Thermal cycler instrument, the problem of can not quickly detecting phytophthora infestans.Detection method under 64 DEG C of isothermys, can quickly,
Facilitate, is efficient, Gao Teyi, detecting phytophthora infestans with sensitivity, not needing complex instrument, can preferably meet to phytophthora infestans
On-site test.
4) practicability is good: common PCR reaction carries out gel electrophoresis to product and product is easily caused to spread, 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 LAMP reaction need to only carry out in thermostat water bath, reaction passes through HNB's after terminating
Color change can direct judging result, to increase its application value detected in the plant and soil to carry disease germs.
In conclusion being based on LAMP technology and 96 orifice plates, the present invention establishes the LAMP based on same target Ypt1 for the first time
The technology of high throughput detection phytophthora, and the LAMP detection primer and method of 8 kinds of phytophthora high throughputs are provided, compare conventional identification
Method and Standard PCR technology detecting method have stronger specificity, sensitivity and portability, the phytophthora strain disposably detected
More, it can be used for quickly understanding and grasping local phytophthora population, have to phytophthora epidemic prevention and control and monitoring important and far-reaching
Meaning.
Detailed description of the invention
Fig. 1 is that LAMP detection method combines progress 8 kinds of phytophthora result figures of high-throughput detection, positive reaction with 96 orifice plates
Reaction solution present sky blue, negative reaction is still purple；A1-A9: the LAMP system of phytophthora parasitic；B1-B9:
The LAMP system of Phytophthora tentaculata；C1-C9: the LAMP system of phytophthora infestans；D1-D9: strawberry phytophthora
The LAMP system of bacterium；E1-E9: the LAMP system of phytophthora blight of pepper；F1-F9: the LAMP system of Phytophthora drechsleri bacterium；G1-G9: epidemic disease is disliked
The LAMP system of mould；The LAMP system of H1-H9: great Xiong phytophthora.The target DNA that 1A-1H:LAMP system is added is parasitic epidemic disease
Mould；The target DNA that 2A-2H:LAMP system is added is Phytophthora tentaculata；3A-3H:LAMP system is added
Target DNA be phytophthora infestans；The target DNA that 4A-4H:LAMP system is added is strawberry phytophthora；5A-5H:LAMP system
The target DNA of addition is phytophthora blight of pepper；The target DNA that 6A-6H:LAMP system is added is Phytophthora drechsleri phytophthora；7A-7H:
The target DNA that LAMP system is added is Phytophthora cactorum bacterium；The target DNA that 8A-8H:LAMP system is added is great Xiong phytophthora；9A-
The addition of 9H:LAMP system is water as negative control.
Specific embodiment
The present invention is further described below combined with specific embodiments below.Do not make point illustrated in following embodiment
Sub- biological experimental method, it is specific referring to listed by one book of " Molecular Cloning:A Laboratory guide " (third edition) J. Pehanorm Brooker
Method carries out, or carries out according to kit and product description.
The design of the LAMP detection primer of embodiment 1:8 kind phytophthora
According to phytophthora parasitic Ypt1 gene GeneBank accession number: DQ162981.1；Phytophthora
Tentaculata Ypt1 gene GeneBank accession number: HQ850014.1；Phytophthora infestans Ypt1 gene GeneBank is logged in
Number: DQ162981.1；Strawberry phytophthora Ypt1 gene GeneBank accession number: DQ270305.1；Phytophthora blight of pepper Ypt1 gene
GeneBank accession number: FJ535572.1；Phytophthora drechsleri bacterium Ypt1 gene GeneBank accession number: DQ162989.1；Phytophthora cactorum
Bacterium Ypt1 gene GeneBank accession number: HQ850000.1；Great Xiong phytophthora Ypt1 gene GeneBank accession number:
DQ162986.1；Different LAMP primers is designed, specific primer is shown in Table 1.
1 LAMP primer sequence of table
Embodiment 2: bacterium DNA profiling preparation to be measured
In soil sample of carrying disease germs prepared by phytophthora DNA profiling, and the specific method is as follows:
All pedotheques useSPIN kit (Q-Biogene Ltd, USA) carries out mentioning for DNA
It takes.Soil DNA extraction step is referring to kit specification.This commercial soil microbial DNA extracts kit can be in 0.5h
Inside extract the microorganism in soil.
The enrichment of egg spore in soil: taking 20~100 grams of pedotheque to be checked, grinds, and successively uses 200 mesh screen places to go
Then larger grogs passes through 400,500,800 mesh net filtrations, while with 3~10 liters of water repeated flushing, from 800 mesh screens
Egg spore is collected, with 1mL aqueous suspension.Since egg spore cannot penetrate 800 mesh screens, processing, which can achieve, in this way keeps egg spore rich
The effect of collection.
DNA is extracted from micro egg spore: by the centrifuge tube for being transferred to 1.5mL with the egg spore of sterile aqueous suspension,
12000r.min-1It is centrifuged 5 minutes under revolving 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 is centrifuged 10 minutes, draws under revolving 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-1Revolving speed
Lower centrifugation 10 minutes, dry liquids；Add 1mL 70% (V/V) ethanol washing, 12000r.min-1It is centrifuged 10 minutes under revolving speed,
Dry liquids are dried to alcohol-free taste；10 μ L aseptic double-distilled waters are added to dissolve, the template for LAMP amplification.
The DNA of phytophthora infestans is extracted in incidence tissue, and using CTAB method, the specific method is as follows:
A small amount of hypha powder is taken, adds 900 μ L 2%CTAB extracting solutions and 90 μ L 10%SDS, whirlpool mixes, in 55 DEG C of water-baths
1h, intermediate every 10min turn upside down several times.12000rpm be centrifuged 10min, take reset and add isometric phenol/chloroform/isoamyl alcohol (25:
24: 1), being mixed by inversion, 12000rpm is centrifuged 10min；Supernatant is transferred to new pipe, adds isometric chloroform, is gently mixed by inversion,
12000rpm is centrifuged 5min.Supernatant is transferred in new pipe, adds the dehydrated alcohol of 2 times of volumes and the 3M NaAc (pH of 1/10 volume
5.2), -20 DEG C of precipitatings (> 1h).12000rpm is centrifuged 10min, and incline supernatant, and precipitating twice with 70% ethanol washing, dry in the air by room temperature
It is dry.Appropriate sterilizing ultrapure water or TE (pH 8.0) dissolution is added to precipitate (containing 20 μ g/mL RNase), after 37 DEG C of processing 1h, -20 DEG C of guarantors
It deposits spare.
When there are NaOH rapid cleavage method when phytophthora infestans, also can be used to extract phytophthora infestans in incidence tissue
DNA, detailed process is as follows: taking the plant tissue of one section of morbidity, every milligram of tissue is added 10 μ L 0.5M NaOH, fills in mortar
It is transferred in the EP pipe of 1.5mL after dividing grinding, 12000rpm is centrifuged 5min, takes 5 μ L supernatants that 495 μ L 0.1mM Tris are added
(pH 8.0) takes l μ L to be directly used in PCR reaction after mixing.Each reaction at least in triplicate, presses down to determine in plant without PCR
Object processed exists.
Embodiment 3: phytophthora LAMP detection kit preparation
1. a kind of for detecting the LAMP detection kit of phytophthora infestans, by 1.6 μM of positive inner primer FIP, 1.6 μM it is anti-
To inner primer BIP, 0.2 μM of positive outer primer F3,0.2 μM of reversed outer primer B3,0.2 μM of reversed ring primer LB, 1.4mM
DNTPs, the Tris-HCl of 20mM pH 8.8,10mM KCl, 10mM (NH4)2SO4、6mM MgSO4, 0.1%Triton X-
100,8U Bst DNA polymerase unit, 5mM hydroxynaphthol blue are added ultrapure water and are prepared into 25uL detection solution.Respectively draw
Object sequence is shown in Table l
2.LAMP detection primer specificity verification
The key technique of the application first is that LAMP primer composition object combined with 96 orifice plates simultaneously to 8 kinds of phytophthoras into
The high-throughput detection of row.In order to verify the specific primer sequence of phytophthora, the present invention is with provinces such as China Jiangsu, Shandong and Fujian
Mtr mutant and several other disease fungus are material to be tested (table 2), extract different samples using method mentioned in embodiment 2
In bacterium DNA to be checked.
When LAMP amplified reaction occurs, generating a large amount of magnesium pyrophosphate white precipitate causes the turbidity of reaction solution to rise,
Shown in chromogenic reaction result by HNB, the reaction tube of phytophthora infestans is in sky blue, is positive findings, and other epidemic diseases
Mould kind, fungi, rotten mould and negative control bacterium reaction tube it is purple, be negative findings, it was demonstrated that designed LAMP specificity is drawn
Object has the specificity of kind.Meanwhile reaction product, through 2% agarose gel electrophoresis, the reaction product of imaging amplification is caused a disease
There is typical stairstepping band in reaction solution in the reaction tube of phytophthora, and other phytophthora kinds, fungi, rotten mould and feminine gender are right
There are not trapezoid-shaped strips according to bacterium reaction tube.This illustrates that the primer sets can be used for causing in incidence tissue and soil in production practices
The fast and reliable detection box identification of sick phytophthora.
Table 2 is used to detect fungi and the oomycetes bacterial strain of phytophthora infestans specificity
The LAMP of the high-throughput phytophthora of embodiment 4 identifies detection architecture
Fig. 1 shows that LAMP detection method combines progress 8 kinds of phytophthoras of high-throughput detection with 96 orifice plates, as the result is shown such as table
3, show that LAMP detection method designed by the application mutually ties technology with 96 orifice plates and simultaneously a variety of phytophthoras are carried out with small-sized height
Flux testing result is the specificity that reliable, designed primer has kind, can be well by target phytophthora and other epidemic diseases
Mould distinguishes.
Table 3
Sequence table
<110>Nanjing Forestry University
<120>molecular detecting method of 8 kinds of phytophthoras of a kind of high-throughput detection
<130> 100
<160> 41
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>F3(Phytophthora parasitica) primer sequence (Artificial)
<400> 1
ctttgtaagt gccaccatac 20
<210> 2
<211> 18
<212> DNA
<213>B3(Phytophthora parasitica) primer sequence (Artificial)
<400> 2
acagacacac acgtgatt 18
<210> 3
<211> 45
<212> DNA
<213>FIP(Phytophthora parasitica) primer sequence (Artificial)
<400> 3
tcgatcgtac ggattttctg cagaaagatc caggttttca tcagg 45
<210> 4
<211> 45
<212> DNA
<213>BIP(Phytophthora parasitica) primer sequence (Artificial)
<400> 4
agaccatcaa gctccagatt gtacgtggta catcggttag ttgaa 45
<210> 5
<211> 20
<212> DNA
<213>LB(Phytophthora parasitica) primer sequence (Artificial)
<400> 5
gacttgtatc actgcagttt 20
<210> 6
<211> 19
<212> DNA
<213>F3 (Phytophthora tentaculata) primer sequence (Artificial)
<400> 6
tgccttatag gaatagcgc 19
<210> 7
<211> 20
<212> DNA
<213>B3 (Phytophthora tentaculata) primer sequence (Artificial)
<400> 7
agcataagtg aattgaccca 20
<210> 8
<211> 44
<212> DNA
<213>FIP (Phytophthora tentaculata) primer sequence (Artificial)
<400> 8
atcgtacgga ttttctgagc aaagtagatc ccgatttcca tcag 44
<210> 9
<211> 45
<212> DNA
<213>BIP (Phytophthora tentaculata) primer sequence (Artificial)
<400> 9
tggacggcaa gaccatcaag tccgttagtt aaataaatac ctcga 45
<210> 10
<211> 22
<212> DNA
<213>LB (Phytophthora tentaculata) primer sequence (Artificial)
<400> 10
ctccagattg tacgtccttc gt 22
<210> 11
<211> 24
<212> DNA
<213>F3 (Phytophthora infestans) primer sequence (Artificial)
<400> 11
tgtgagtgtc taacatattt tacg 24
<210> 12
<211> 23
<212> DNA
<213>B3 (Phytophthora infestans) primer sequence (Artificial)
<400> 12
gttagttaaa taggaaatca cgc 23
<210> 13
<211> 40
<212> DNA
<213>FIP (Phytophthora infestans) primer sequence (Artificial)
<400> 13
aatcgcgaaa gccatgtgag ccaaacgacc ttttgtaagg 40
<210> 14
<211> 45
<212> DNA
<213>BIP (Phytophthora infestans) primer sequence (Artificial)
<400> 14
ttgcttagaa aatccgtacg atcggacaaa tgttttttta gcggc 45
<210> 15
<211> 21
<212> DNA
<213>LB (Phytophthora infestans) primer sequence (Artificial)
<400> 15
ggcaagacca tcaagctcca a 21
<210> 16
<211> 20
<212> DNA
<213>F3 (Phytophthora fragariae) primer sequence (Artificial)
<400> 16
tgagtgctag taactagcct 20
<210> 17
<211> 20
<212> DNA
<213>B3 (Phytophthora fragariae) primer sequence (Artificial)
<400> 17
accagttagc tccatgaagc 20
<210> 18
<211> 42
<212> DNA
<213>FIP (Phytophthora fragariae) primer sequence (Artificial)
<400> 18
ctgtgcacaa caacgagcac gaattcctag gtccaaaaag gc 42
<210> 19
<211> 40
<212> DNA
<213>BIP (Phytophthora fragariae) primer sequence (Artificial)
<400> 19
aattcgcacg atcgagctgg attagccggc gaaatgttcc 40
<210> 20
<211> 23
<212> DNA
<213>LB (Phytophthora fragariae) primer sequence (Artificial)
<400> 20
cggcaagact atcaagctcc aga 23
<210> 21
<211> 23
<212> DNA
<213>LF (Phytophthora fragariae) primer sequence (Artificial)
<400> 21
cagcaatcgg agagcaaatc tta 23
<210> 22
<211> 20
<212> DNA
<213>F3 (Phytophthora capsici) primer sequence (Artificial)
<400> 22
ctctgttgta tagcagaggt 20
<210> 23
<211> 21
<212> DNA
<213>B3 (Phytophthora capsici) primer sequence (Artificial)
<400> 23
gcacaagaca attagcacaa t 21
<210> 24
<211> 45
<212> DNA
<213>FIP (Phytophthora capsici) primer sequence (Artificial)
<400> 24
ttctgggcgc gtacacaaac ttagtgaggg acaatttata tcagg 45
<210> 25
<211> 41
<212> DNA
<213>BIP (Phytophthora capsici) primer sequence (Artificial)
<400> 25
gatcgagttg gacggcaaga tccagtgctc taactaaaac g 41
<210> 26
<211> 23
<212> DNA
<213>LB (Phytophthora capsici) primer sequence (Artificial)
<400> 26
ccatcaagct ccagattgta agc 23
<210> 27
<211> 18
<212> DNA
<213>F3 (Phytophthora drechsleri) primer sequence (Artificial)
<400> 27
gtgatccttt caccctgg 18
<210> 28
<211> 20
<212> DNA
<213>B3 (Phytophthora drechsleri) primer sequence (Artificial)
<400> 28
ttacaaatgt cagctggatg 20
<210> 29
<211> 46
<212> DNA
<213>FIP (Phytophthora drechsleri) primer sequence (Artificial)
<400> 29
cggattttct agaacgtggt accaaaatga agagtcgact ctagca 46
<210> 30
<211> 38
<212> DNA
<213>BIP (Phytophthora drechsleri) primer sequence (Artificial)
<400> 30
actattgagc tggacggcaa tcgatagcag cccaagag 38
<210> 31
<211> 24
<212> DNA
<213>LB (Phytophthora drechsleri) primer sequence (Artificial)
<400> 31
tgtacgtcta cagaggattt ggat 24
<210> 32
<211> 17
<212> DNA
<213>F3 (Phytophthora cactorum) primer sequence (Artificial)
<400> 32
ttctgcgcta ggcgacc 17
<210> 33
<211> 19
<212> DNA
<213>B3 (Phytophthora cactorum) primer sequence (Artificial)
<400> 33
cacacaagtg gaccgttag 19
<210> 34
<211> 40
<212> DNA
<213>FIP (Phytophthora cactorum) primer sequence (Artificial)
<400> 34
tctgggcaca accgcaaaaa tttgcgagct ccagatttcc 40
<210> 35
<211> 38
<212> DNA
<213>BIP (Phytophthora cactorum) primer sequence (Artificial)
<400> 35
aatccgtacg atcgagctgg acacacgcca cgtctgct 38
<210> 36
<211> 23
<212> DNA
<213>LB (Phytophthora cactorum) primer sequence (Artificial)
<400> 36
ggaaagacca tcaagctcca gat 23
<210> 37
<211> 19
<212> DNA
<213>F3 (Phytophthora megasperma) primer sequence (Artificial)
<400> 37
gctctgctct tccgacttg 19
<210> 38
<211> 19
<212> DNA
<213>B3 (Phytophthora megasperma) primer sequence (Artificial)
<400> 38
gttagtttcg tccacggca 19
<210> 39
<211> 40
<212> DNA
<213>FIP (Phytophthora megasperma) primer sequence (Artificial)
<400> 39
cgggcaagag caacgtcagt gtcccattgt ggtccagtac 40
<210> 40
<211> 41
<212> DNA
<213>BIP (Phytophthora megasperma) primer sequence (Artificial)
<400> 40
tccgtacgat cgagctggac gagaagaaag gaatggaggc c 41
<210> 41
<211> 24
<212> DNA
<213>LB (Phytophthora megasperma) primer sequence (Artificial)
<400> 41
gcaagaccat caagctccag attg 24
Claims (10)
1. a kind of molecular detecting method of 8 kinds of phytophthoras of high-throughput detection, it is characterised in that: including extracting microorganism to be checked
DNA is combined to microorganism to be checked simultaneously using 8 kinds of phytophthora LAMP detection primers with 96 orifice plates using the DNA of extraction as template
Carry out high-throughput detection；LAMP reaction solution color change is observed, sky blue indicates that testing result is the positive, and there are target phytophthoras
Bacterium；Purple indicates that testing result is feminine gender, and target phytophthora is not present；8 kinds of phytophthora LAMP detection primers include
Phytophthora parasitica、Phytophthora tentaculata、Phytophthora infestans、
Phytophthora fragariae、Phytophthora capsici、Phytophthora drechsleri、
The LAMP detection primer of Phytophthora cactorum and Phytophthoramegasperma, the LAMP of every kind of phytophthora
Detection primer is by positive inner primer FIP, reversed inner primer BIP, positive outer primer F3, reversed outer primer B3 and positive ring primer
LF, reversed ring primer LB composition, each primer sequence are specific as follows:
The primer of Phytophthora parasitica includes:
F3:5 '-CTTTGTAAGTGCCACCATAC-3 '；
B3:5 '-ACAGACACACACGTGATT-3 '；
FIP:
5′-TCGATCGTACGGATTTTCTGCAGAAAGATCCAGGTTTTCATCAGG-3′；
BIP:
5′-AGACCATCAAGCTCCAGATTGTACGTGGTACATCGGTTAGTTGAA-3″；
LB:5 '-GACTTGTATCACTGCAGTTT-3 '；
The primer of Phytophthora tentaculata includes:
F3:5 '-TGCCTTATAGGAATAGCGC-3 '；
B3:5 '-AGCATAAGTGAATTGACCCA-3 '；
FIP:
5′-ATCGTACGGATTTTCTGAGCAAAGTAGATCCCGATTTCCATCAG-3′；
BIP:
5′-TGGACGGCAAGACCATCAAGTCCGTTAGTTAAATAAATACCTCGA-3′；
LB:5 '-CTCCAGATTGTACGTCCTTCGT-3 '；
The primer of Phytophthora infestans includes:
F3:5 '-TGTGAGTGTCTAACATATTTTACG-3 '；
B3:5 '-GTTAGTTAAATAGGAAATCACGC-3 '；
FIP:5 '-AATCGCGAAAGCCATGTGAGCCAAACGACCTTTTGTAAGG-3 '；
BIP:
5′-TTGCTTAGAAAATCCGTACGATCGGACAAATGTTTTTTTAGCGGC-3′；
LB:5 '-GGCAAGACCATCAAGCTCCAA-3 '；
The primer of Phytophthora fragariae includes:
F3:5 '-TGAGTGCTAGTAACTAGCCT-3 '；
B3:5 '-ACCAGTTAGCTCCATGAAGC-3 '；
FIP:
5′-CTGTGCACAACAACGAGCACGAATTCCTAGGTCCAAAAAGGC-3′；
BIP:5 '-AATTCGCACGATCGAGCTGGATTAGCCGGCGAAATGTTCC-3 '；
LF:5 '-CAGCAATCGGAGAGCAAATCTTA-3 '；
LB:5 '-CGGCAAGACTATCAAGCTCCAGA-3 '；
The primer of Phytophthora capsici includes:
F3:5 '-CTCTGTTGTATAGCAGAGGT-3 '；
B3:5 '-GCACAAGACAATTAGCACAAT-3 '；
FIP:
5′-TTCTGGGCGCGTACACAAACTTAGTGAGGGACAATTTATATCAGG-3′；
BIP:5 '-GATCGAGTTGGACGGCAAGATCCAGTGCTCTAACTAAAACG-3 '；
LB:5 '-CCATCAAGCTCCAGATTGTAAGC-3 '；
The primer of Phytophthora drechsleri includes:
F3:5 '-GTGATCCTTTCACCCTGG-3 '；
B3:5 '-TTACAAATGTCAGCTGGATG-3 '；
FIP:
5′-CGGATTTTCTAGAACGTGGTACCAAAATGAAGAGTCGACTCTAGCA-3′；
BIP:5 '-ACTATTGAGCTGGACGGCAATCGATAGCAGCCCAAGAG-3 '；
LB:5 '-TGTACGTCTACAGAGGATTTGGAT-3 '；
The primer of Phytophthora cactorum includes:
F3:5 '-TTCTGCGCTAGGCGACC-3 '；
B3:5 '-CACACAAGTGGACCGTTAG-3 '；
FIP:5 '-TCTGGGCACAACCGCAAAAATTTGCGAGCTCCAGATTTCC-3 '；
BIP:5 '-AATCCGTACGATCGAGCTGGACACACGCCACGTCTGCT-3 '；
LB:5 '-GGAAAGACCATCAAGCTCCAGAT-3 '；
The primer of Phytophthora megasperma includes:
F3:5 '-GCTCTGCTCTTCCGACTTG-3 '；
B3:5 '-GTTAGTTTCGTCCACGGCA-3 '；
FIP:5 '-CGGGCAAGAGCAACGTCAGTGTCCCATTGTGGTCCAGTAC-3 '；
BIP:
5′-TCCGTACGATCGAGCTGGACGAGAAGAAAGGAATGGAGGCC-3′；
LB:5 '-GCAAGACCATCAAGCTCCAGATTG-3 '.
2. application of the 8 kinds of phytophthora LAMP detection primers described in claim l in detection phytophthora.
3. a kind of LAMP detection kit of phytophthora, it is characterised in that: including at least it is more than ampoule primer solution and
LAMP reaction solution；Wherein, the primer in primer solution is 8 kinds of phytophthora LAMP detection primers described in claim 1.
4. the LAMP detection kit of phytophthora as claimed in claim 3, it is characterised in that: the LAMP reaction solution contains
dNTPs、Tris-HCl、KCl、(NH4)2SO4、MgSO4, Triton X-100, Bst DNA polymerase, hydroxynaphthol blue.
5. application of the LAMP detection kit of phytophthora as claimed in claim 3 in detection phytophthora.
6. a kind of molecular detecting method of 5 kinds of phytophthoras of high-throughput detection, it is characterised in that: including extracting microorganism to be checked
DNA is combined to microorganism to be checked simultaneously using 5 kinds of phytophthora LAMP detection primers with 96 orifice plates using the DNA of extraction as template
Carry out high-throughput detection；LAMP reaction solution color change is observed, sky blue indicates that testing result is the positive, and there are target phytophthoras
Bacterium；Purple indicates that testing result is feminine gender, and target phytophthora is not present；8 kinds of phytophthora LAMP detection primers include
Phytophthora parasitica、Phytophthora tentaculata、Phytophthora fragariae、
The LAMP detection primer of Phytophthora capsici and Phytophthora megasperma, the LAMP of every kind of phytophthora
Detection primer is by positive inner primer FIP, reversed inner primer BIP, positive outer primer F3, reversed outer primer B3 and positive ring primer
LF, reversed ring primer LB composition, each primer sequence are specific as follows:
The primer of Phytophthora parasitica includes:
F3:5 '-CTTTGTAAGTGCCACCATAC-3 '；
B3:5 '-ACAGACACACACGTGATT-3 '；
FIP:
5′-TCGATCGTACGGATTTTCTGCAGAAAGATCCAGGTTTTCATCAGG-3′；
BIP:
5′-AGACCATCAAGCTCCAGATTGTACGTGGTACATCGGTTAGTTGAA-3″；
LB:5 '-GACTTGTATCACTGCAGTTT-3 '；
The primer of Phytophthora tentaculata includes:
F3:5 '-TGCCTTATAGGAATAGCGC-3 '；
B3:5 '-AGCATAAGTGAATTGACCCA-3 '；
FIP:
5′-ATCGTACGGATTTTCTGAGCAAAGTAGATCCCGATTTCCATCAG-3′；
BIP:
5′-TGGACGGCAAGACCATCAAGTCCGTTAGTTAAATAAATACCTCGA-3′；
LB:5 '-CTCCAGATTGTACGTCCTTCGT-3 '；
The primer of Phytophthora fragariae includes:
F3:5 '-TGAGTGCTAGTAACTAGCCT-3 '；
B3:5 '-ACCAGTTAGCTCCATGAAGC-3 '；
FIP:
5′-CTGTGCACAACAACGAGCACGAATTCCTAGGTCCAAAAAGGC-3′；
BIP:5 '-AATTCGCACGATCGAGCTGGATTAGCCGGCGAAATGTTCC-3 '；
LF:5 '-CAGCAATCGGAGAGCAAATCTTA-3 '；
LB:5 '-CGGCAAGACTATCAAGCTCCAGA-3 '；
The primer of Phytophthora capsici includes:
F3:5 '-CTCTGTTGTATAGCAGAGGT-3 '；
B3:5 '-GCACAAGACAATTAGCACAAT-3 ':
FIP:
5′-TTCTGGGCGCGTACACAAACTTAGTGAGGGACAATTTATATCAGG-3′；
BIP:5 '-GATCGAGTTGGACGGCAAGATCCAGTGCTCTAACTAAAACG-3 '；
LB:5 '-CCATCAAGCTCCAGATTGTAAGC-3 '；
The primer of Phytophthora megasperma includes:
F3:5 '-GCTCTGCTCTTCCGACTTG-3 '；
B3:5 '-GTTAGTTTCGTCCACGGCA-3 '；
FIP:5 '-CGGGCAAGAGCAACGTCAGTGTCCCATTGTGGTCCAGTAC-3 '；
BIP:
5′-TCCGTACGATCGAGCTGGACGAGAAGAAAGGAATGGAGGCC-3′；
LB:5 '-GCAAGACCATCAAGCTCCAGATTG-3 '.
7. application of the 5 kinds of phytophthora LAMP detection primers as claimed in claim 6 in detection phytophthora.
8. a kind of LAMP detection kit of phytophthora, it is characterised in that: including at least it is more than ampoule primer solution and
LAMP reaction solution；Wherein, the primer in primer solution is 5 kinds of phytophthora LAMP detection primers as claimed in claim 6.
9. the LAMP detection kit of phytophthora according to any one of claims 8, it is characterised in that: the LAMP reaction solution contains
dNTPs、Tris-HCl、KCl、(NH4)2SO4、MgSO4, Triton X-100, Bst DNA polymerase, hydroxynaphthol blue.
10. application of the LAMP detection kit of phytophthora according to any one of claims 8 in detection phytophthora.
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