CN106755339A - Cucumber anthracnose LAMP detection primer and its application - Google Patents
Cucumber anthracnose LAMP detection primer and its application Download PDFInfo
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- CN106755339A CN106755339A CN201611081522.4A CN201611081522A CN106755339A CN 106755339 A CN106755339 A CN 106755339A CN 201611081522 A CN201611081522 A CN 201611081522A CN 106755339 A CN106755339 A CN 106755339A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses a kind of cucumber anthracnose LAMP detection primer and its application.A kind of Primer composition based on LAMP technology detection cucumber anthracnose is made up of 4 specific primers F3, B3, FIP, BIP.Present invention also offers a kind of cucumber anthracnose bacterium LAMP detection method, comprise the following steps：1）Extract testing sample DNA；2）The Primer composition provided using the present invention carries out ring mediated isothermal amplification to testing sample DNA；3）Result detects that colour developing result observes that the judgement that trapezoid-shaped strips occur in green fluorescence or electrophoresis is the positive, develops the color orange（Crocus）Or it is feminine gender the judgement of trapezoid-shaped strips do not occur.The present invention provides new molecular detecting method and Primer composition for cucumber anthracnose, can it is quick, convenient, efficiently, it is high specifically, detect cucumber anthracnose with sensitivity, provide reliable technology and theoretical foundation to prevent and treat cucumber anthracnose.
Description
Technical field
The invention belongs to corps diseases detection, identification and Prevention Technique field, and in particular to a kind of cucumber anthracnose
LAMP detection primer and its application.Can be used for quick, the sensitive and special Molecular Detection of cucumber anthracnose, while can be used for Huang
The early diagnosis and the monitoring and identification of germ of melon anthracnose.
Background technology
Cucumber（Cucumis sativusL.）Nutritious, smell delicate fragrance is eaten raw, prepared food, is deep by vast life
The economic worth vegetable crop high that product person and consumer like.Cucumber is because of its various variety type and its stronger growth adaptation
Property, and be widely used, thus cucumber is one of staple vegetable of global cultivation.With the tune of China's crop mix
The development of whole and industrialized agriculture, cucumber constantly expands in the cultivated area and multiple crop index of China, therewith the hair of Cucumber Pests And Diseases
Life is also on the rise, and pest and disease damage has turned into one of key factor of limitation cucumber industry development.
By Curcurbitaceae colletotrichum（Colletotrichum orbiculare）It is a kind of important to infect the anthracnose for causing
Cucumber disease, have generation throughout the country, main harm cucumber leaves, vines and fruit, the time of infertility can fall ill, especially
With the aggrieved most serious of Later growth.The infection process power is strong, breeding potential is high, with mycelium or plan sclerotium on seed or with invalid
Body is survived the winter in soil, and onset condition often results in plant middle and lower part a large amount of once suitably, will result in the pandemic of disease
Blade dries up, and fruit produces scab, reduces quality or loses commodity value completely, reduces cucumber yield, diseased plant rate when serious
Up to 100%, production loss more than 40%, or even total crop failure.The disease has the trend of continuous exacerbation in recent years, however, being directed to this
Disease there is no preferable disease-resistant variety and the specific pesticide can to utilize, and difficulty of prevention and cure is larger.Therefore, quickly and accurately in premorbid
Or the initial stage is monitored to the Dynamics of germ, take effectively preventing method to control disease in field and accumulating in time
Generation, reduce economic loss tool be of great significance.
The classification of traditional anthrax bacteria is based primarily upon morphological feature and Pathogenicity etc., this authentication method with identification
Take that more long, program is cumbersome, sensitivity is low and empirical strong, separated from disease plant and when pathogen identification needs a couple of days
Between, it is difficult to accomplish monitoring in time and the effectively propagation of control pathogen and plant disease epidemic to disease.With molecular biology
Continuing to develop for technology, the successful example that special and rapid molecular is detected is carried out to pathogen using technologies such as PCR, detection of plasma
Son is more and more, but these Protocols in Molecular Biologies there is also some shortcomings part to a certain extent.Such as sero-immunity
Learn detection technique to be taken time and effort in the preparation process of serum, be frequently subjected to the influence of antiserum quality, thereby increases and it is possible to there is intersection anti-
Should, specificity is poor, easily causes false positive；PCR Fast Detection Techniques mainly include Standard PCR, nest-type PRC（Nest-PCR）
With real-time fluorescence quantitative PCR etc., round pcr detection time is more long, it is necessary to by expensive instruments such as PCR instrument, gel imaging systems
Equipment, is unfavorable for the upper popularization and application of basic unit's production, and above-mentioned shortcoming limits the popularization and application of these advanced methods.
Ring mediated isothermal amplification（Loop-mediated Isothermal Amplification, LAMP）Technology is by day
A kind of easy, quick, the accurate and cheap nucleic acid efficient amplification technology of this Rong Yan companies exploitation, the technology can be 60 DEG C ~ 65
Under DEG C constant temperature, using high activity strand displacement archaeal dna polymerase (BstDNA polymerase) target DNA fragment is entered
Row specific amplification.In 1 hour, the genes of interest of a small amount of copy number can be expanded to 109~1010Individual copy number.LAMP is anti-
Answering product can not only be detected by transmissometer, real-time PCR instruments and gel-electrophoretic apparatus, but also can be passed through
After SYBR Green I, calcein, hydroxynaphthol blue dyeing, naked eyes are recognized.Because LAMP reactions are simple, quick, efficient, warp
Ji, and without special installation, testing result can be particularly well suited in the popularization and application of production division of basic unit by visually judging, thus tool
There is extremely wide application prospect.Current LAMP detections are mainly used in the inspection of people and animals' pathogen, food security and environmental sanitation
Survey, reported in phytopathogen detection less, LAMP on cucumber anthracnose detection is not reported both at home and abroad.
The present invention is based on ring mediated isothermal amplification（LAMP）Know-why, chooses anthrax-bacilusBeta-tubulinGene order
It is detection target,Beta-tubulin6 regions of gene order devise 4 cucumber anthracnose specific primers, lead to
The optimization to reaction system and reaction condition is crossed, the cucumber anthracnose for fluorescence developing indicator with SYBR Green I is set up
Visualization LAMP detection techniques.The technical operation is simple, sensitivity and special high, and without valuable instrument and equipment, is suitable for field
Between cucumber anthracnose early diagnosis and pathogen detection and identification, effectively preventing timely to cucumber anthracnose have weight
Want meaning.
The content of the invention
The purpose of the present invention to be directed to that be based primarily upon morphology to cucumber anthracnose detection and identification in the prior art special
Levy, time-consuming for method, program is cumbersome, the empirical strong, degree of accuracy is low, it is difficult to accomplish the timely monitoring and control disease occurred to disease
The propagation of opportunistic pathogen, popular problem, and existing PCR Molecular Detections are needed by expensive instruments such as amplification instruments, and detection time
More long the problems such as, there is provided a kind of cucumber anthracnose LAMP technology detection method and the Primer composition for using, present invention inspection
Survey method is easily operated, high specificity, sensitivity are high, result accurately and reliably.
To achieve the above object, the present invention is adopted the following technical scheme that：
1. cucumber anthracnose LAMP detection primer：
By determining cucumber anthracnose（Colletotrichum orbiculare）With other anthrax bacterias
（Colletotrichum spp）'sBeta-tubulinGene, to colletotrichum difference inter-speciesBeta-tubulinGene order
Compare, using online LAMP primer design software Primer software Explorer V4 (http://
primerexplorer.jp/elamp4.0.0/index.html;Eiken Chemical Co., Japan) design a set of Huang
Melon anthrax bacteria specificity LAMP primer, 4 primers such as including F3, B3, FIP and BIP, its sequence is：
F3:5 '-GGGGCTAACTGCTTGAACAG -3 ',
B3:5 '-GGTGCCGT TGTACCTGTT -3 ',
FIP:5 '-TGTGCGAAGCATCGTTGCCGGGTAACCAGATTGGTGCTGC -3 ',
BIP: 5’- AGGCAAAACATCTCTGGCGAGCCCATTCTTGACGTGGCCTTA - 3’。
2. cucumber anthracnose LAMP detection method, comprises the following steps：
（1）Extract testing sample genomic DNA.
For detecting during pathogen pure culture, carry out extraction genomic DNA using CTAB methods, specific method is as follows：Take
A small amount of hypha powder is in 1.5 mL centrifuge tubes（Hypha powder had just covered semicircular base and had been advisable）, add 900 μ L 2%CTAB（16
Alkyl trimethyl ammonium bromide）Extract solution（2% CTAB；100 mmol/L Tris-HCl, pH 8.0；20 mmol/L EDTA,
pH8.0；1.4 mol/L NaCl）With 90 μ L SDS（Neopelex）【Note：CTAB, SDS need 60 DEG C of preheatings】, make
Vibrated with oscillator and mixed, 60 DEG C of water-bath 1h（DNA is discharged into buffer solution）, 12000 rmin-115 min are centrifuged；Take supernatant
The μ L of liquid 700, plus isometric phenol, chloroform, isoamyl alcohol（25:24:1）, gently vibration mixing, 12000 rmin-19 min are centrifuged；
The μ L of supernatant 500 are taken, adds isometric chloroform to extract again once, 12000 rmin-15 min are centrifuged；Take the μ of supernatant 350
L, adds the mol L of 1/10 volume 3-1NaAc and 2 times of volume absolute ethyl alcohol, -20 DEG C of precipitations 30 min, 12000 rmin-1
5 min are centrifuged；Abandoning supernatant, adds the ethanol of 700 μ L ice 70% to be washed（Slightly it is centrifuged；Incline and fall supernatant）, in ultra-clean work
Make to be dried on platform to alcohol-free taste, add 30 ~ 60 μ L TE（10 mmol/L Tris-HCl, 0.1 mmol/L EDTA, pH
8.0）Solution is dissolved, and obtains DNA solution, with UV spectrophotometer measuring DNA concentration and is diluted to 100 ng/ μ
L is stand-by.
When there is cucumber anthracnose in plant tissue for detecting, DNA, specific mistake are extracted using NaOH rapid cleavages method
Journey is as follows：To the mol/L NaOH of 10 μ L 0.5 are added in every milligram of plant tissue, will be organized in mortar after being fully milled to paste
It is transferred in 1.5mL centrifuge tubes, 12,000 rpm are centrifuged 6 min, takes the μ l of supernatant 5 and add the mol/L Tris- of 495 μ L 0.1
HCl（pH=8.0）It is well mixed, take 1.0 μ L and expanded as pcr template；
When there is cucumber anthracnose in pedotheque for detecting, using soil DNA extracts kit, DNA is extracted.
（2）The foundation of LAMP reaction systems：With step（1）The DNA of extraction is template, using primers F 3, B3, FIP and BIP
LAMP amplifications are carried out, LAMP detects reaction system for 25 μ L, including 5 μM of F3 and B3 each 1.0 μ L, 40 μM of FIP and BIP each
1.0 μ L, LAMP reaction mixture 12.5 μ L, 8 UBstThe μ L of polymerase 1.0, the μ L of DNA profiling 1.0, are mended with sterilizing ultra-pure water
Enough to 25 μ L；
（3）LAMP reaction conditions：63.5 DEG C of incubations 60 min, 85 DEG C of inactivation 5min；
（4）The measure of reaction result：It is measured using fluorescent dye visual observations method or agarose gel electrophoresis method.Using glimmering
Photoinitiator dye visual observations method, after LAMP reactions terminate, adds developer SYBR green I in the amplified production of LAMP reactions
1.0 μ L, colour developing result observes that the judgement of green fluorescence is the positive, orange（Crocus）It is judged as feminine gender；It is solidifying using agarose
Gel electrophoresis method, takes 2.0 μ L LAMP amplified productions and detects trapezoid-shaped strips such as occur and be judged as sun with 2% agarose gel electrophoresis
Property, there is not band and be then judged as feminine gender.
Remarkable advantage of the invention
Beneficial effects of the present invention：The cucumber anthracnose LAMP detection primer high specificity that the present invention is provided；And use it for
Cucumber anthracnose LAMP detections overcome that the cycle needed for biological detection method in the prior art is long, waste time and energy, cumbersome, spy
The problem and PCR detection techniques of opposite sex difference need the expensive equipments such as thermal cycler, it is impossible to the problems such as being applied to basic unit.The present invention
Detection method under 63.5 DEG C of isothermys, can it is quick, convenient, efficiently, it is high it is special, detect cucumber anthracnose with sensitivity
Bacterium, it is not necessary to complex instrument, can preferably meet the Site Detection to cucumber anthracnose, for the detection of cucumber anthracnose is provided
New technology platform, can be used for the early diagnosis of field cucumber anthracnose and the monitoring of germ.
The present invention compared with prior art, with following technical advantage and good effect：
1st, high specificity, reliable results：LAMP primer designed by the present invention is based on anthrax bacteria（Colletotrichum spp）'sBeta-tubulin6 different zones design 4 specific primers, any region in 6 regions in gene order
Being mismatched with primer can not carry out nucleic acid amplification, for 2 isolated areas of PCR primer identification target sequence, specifically
Property and sensitivity are all higher.The LAMP primer that the present invention goes out designed by establishes the LAMP detection sides of cucumber anthracnose
Method has carried out specific test to cucumber anthracnose and disease fungus, and only cucumber anthracnose can be detected, other diseases
Opportunistic pathogen does not detect that test of many times result is consistent, illustrates LAMP detection method high specificity of the present invention, reliable results.
2nd, sensitivity is high：The present invention can reach 10fg/ μ to the detection sensitivity of cucumber anthracnose on DNA level
L, with sensitivity very high.
3rd, practicality is good：LAMP detection method of the invention needs to want thermal cycler unlike PCR methods（PCR instrument）Etc. valuable instrument
Device equipment, has thus broken away from the dependence to expensive equipments such as thermal cyclers；As long as there is the thermal source of stabilization, LAMP reactions can
To occur, the detection range of cucumber anthracnose is have greatly expanded.Meanwhile, LAMP reactions of the invention only need to be in thermostat water bath
In carry out, reaction terminate the color change that passes through afterwards just can direct judged result, so as to increased its plant for carrying disease germs with
The application value detected in soil.
4th, it is easy to operate quick：Detection method, can quick, convenient, efficient, Gao Te under 63.5 DEG C of isothermys
It is different, detect cucumber anthracnose with sensitivity, it is not necessary to complex instrument, only need a thermostatic equipment, can preferably meet
To the Site Detection of cucumber anthracnose.
Brief description of the drawings
Fig. 1 is testing result of the LAMP technology of the present invention to cucumber anthracnose.The upper figures of Fig. 1 are the fine jade after LAMP amplifications
Sepharose electrophoresis detection result；Fig. 1 figure below is the fluorescent dye visualization colour developing result after LAMP amplifications, and pipe 1 is green in figure
Color, pipe 2-4 is orange；Wherein：M is the DNA marker of DL 2000, and 1 is cucumber anthracnose, and 2-3 is other pathogens, and 4 are
Negative control.
Fig. 2 is the sensitivity measurement result that LAMP technology of the present invention detects cucumber anthracnose.The upper figures of Fig. 2 are that LAMP expands
Agarose gel electrophoresis testing result after increasing；Fig. 2 figure below is the fluorescent dye colour developing result after LAMP amplifications, pipe 1-4 in figure
In green, 5-8 is in orange；Wherein：M is respectively 10 pg, 1 pg, 100 for 2000 DNA marker, 1-7 template DNA concentration
Fg, 10 fg, 1 fg, 100ag, 10 ag, 8 is negative control.
Fig. 3 is detection of the detection method to disease plant and with cucumber anthracnose in soil bacteria.The upper figures of Fig. 3
It is the agarose gel electrophoresis testing result after LAMP amplifications；Fig. 3 figure below is the fluorescent dye colour developing result after LAMP amplifications, figure
, in green, pipe 3,5,7 is in orange for middle pipe 1-2,4,6,8；Wherein：M is the DNA marker of DL 2000, and 1 is positive control, and 2 are
Incidence of leaf, 3 is healthy leaves, and 4 is incidence of leaf, and 5 is healthy leaves, and 6 is band soil bacteria, and 7 is negative control, and 8 is to carry disease germs
Soil.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is further elaborated, but is not limited to the scope of the present invention.Below
Embodiment is according to conventional laboratory conditions, or has delivered the operating technology code described in pertinent literature, or is built according to manufacturer
The experiment condition of view.
Embodiment 1：Cucumber anthracnose ring mediated isothermal amplification（LAMP）Detect specific primer sets thing design and
The specificity verification of primer pair cucumber anthracnose
1. the extraction of strains tested genomic DNA
Strains tested is extracted using CTAB methods（Table 1）Genomic DNA, specific method is as follows：A small amount of hypha powder is taken in 1.5 mL
In centrifuge tube（Hypha powder had just covered semicircular base and had been advisable）, add 900 μ L 2%CTAB（Cetyl trimethylammonium bromide）
Extract solution（2% CTAB；100 mmol/L Tris-HCl, pH 8.0；20 mmol/L EDTA, pH8.0；1.4 mol/L
NaCl）With 90 μ L SDS（Neopelex）【Note：CTAB, SDS need 60 DEG C of preheatings】, vibrated using oscillator and mixed,
60 DEG C of water-bath 1h（DNA is discharged into buffer solution）, 12000 rmin-115 min are centrifuged；The μ L of supernatant 700 are taken, plus in equal volume
Phenol, chloroform, isoamyl alcohol（25:24:1）, gently vibration mixing, 12000 rmin-19 min are centrifuged；The μ L of supernatant 500 are taken, plus
Enter isometric chloroform to extract again once, 12000 r min-15 min are centrifuged；The μ L of supernatant 350 are taken, 1/10 volume 3 is added
mol·L-1NaAc and 2 times of volume absolute ethyl alcohol, -20 DEG C of precipitations 30 min, 12000 rmin-15 min are centrifuged；Discard
Clear liquid, adds the ethanol of 700 μ L ice 70% to be washed（Slightly it is centrifuged；Incline and fall supernatant）, dried on superclean bench alcohol-free
Taste, adds 30 ~ 60 μ L TE（10 mmol/L Tris-HCl, 0.1 mmol/L EDTA, pH 8.0）Solution is dissolved, and is obtained
To DNA solution, with UV spectrophotometer measuring DNA concentration and to be diluted to 100 ng/ μ L stand-by.
The strains tested of table 1
2. cucumber anthracnose ring mediated isothermal amplification（LAMP）The design of special primer
By determining cucumber anthracnose（Colletotrichum orbiculare）With other anthrax bacterias
（Colletotrichum spp）'sBeta-tubulinGene, to colletotrichum difference inter-speciesBeta-tubulinGene order
Compare, using online LAMP primer design software Primer software Explorer V4 (http://
primerexplorer.jp/elamp4.0.0/index.html;Eiken Chemical Co., Japan) design a set of Huang
Melon anthrax bacteria specificity LAMP primer, including 4 primers of F3, B3, FIP and BIP, its sequence is：
F3:5 '-GGGGCTAACTGCTTGAACAG -3 ',
B3:5 '-GGTGCCGTTGTACCTGTT -3 ',
FIP:5 '-TGTGCGAAGCATCGTTGCCGGGTAACCAGATTGGTGCTGC -3 ',
BIP: 5’- AGGCAAAACATCTCTGGCGAGCCCATTCTTGACGTGGCCTTA - 3’。
3. the foundation of cucumber anthracnose LAMP detection method and primer specificity are verified
DNA with the strains tested of table 1 carries out LAMP amplifications, LAMP inspections as template using Primer composition F3, B3, FIP and BIP
Survey reaction system is 25 μ L, including 5 μM of F3 and B3 each 1.0 μ L, 40 μM of FIP and BIP each 1.0 μ L, LAMP reaction mixing
Liquid 12.5 μ L, 8 UBstThe μ L of polymerase 1.0, the μ L of DNA profiling 1.0,25 μ L are complemented to sterilizing ultra-pure water；LAMP reacts bar
Part：63.5 DEG C of incubations 60 min, 85 DEG C of inactivation 5min；The measure of reaction result：Using fluorescent dye visual observations method or agar
Sugared gel electrophoresis are measured.Using fluorescent dye visual observations method, after LAMP reactions terminate, in the amplification of LAMP reactions
The μ L of developer SYBR green I 1.0 are added in product, colour developing result observes that the judgement of green fluorescence is the positive, orange（Tangerine
Yellow）It is judged as feminine gender；Using agarose gel electrophoresis method, 2.0 μ L 2% agarose gel electrophoresis of LAMP amplified productions are taken
, such as there are trapezoid-shaped strips and is judged as the positive in detection, band does not occur and is then judged as feminine gender.
4. primer specificity the result
LAMP amplifications show that the cucumber anthracnose colour developing result for trying can be observed green fluorescence or Ago-Gel electricity
There is the trapezoid belt of LAMP features in swimming, and remaining pathogen develops the color result for amplification bar does not occur in orange or agarose gel electrophoresis
Band（Accompanying drawing 1）, illustrate that designed cucumber anthracnose specific primer sets thing F3, B3, FIP and BIP can be by cucumber charcoal
Subcutaneous ulcer germ makes a distinction with other pathogens, with plant specificity, can be used for the fast and reliable detection of cucumber anthracnose and
Identification.
Embodiment 2：Cucumber anthracnose ring mediated isothermal amplification（LAMP）Detection sensitivity is determined
1. the preparation of various concentrations genomic DNA
Cucumber anthracnose genomic DNA is diluted with aseptic ultra-pure water, the series concentration for being configured to 10 times of orders of magnitude is standby
With；
2. LAMP detection method sensitivity determination and result are observed
Cucumber anthracnose genomic DNA with various concentrations is carried out as template using Primer composition F3, B3, FIP and BIP
LAMP is expanded, and LAMP detects reaction system for 25 μ L, including 5 μM of F3 and B3 each 1.0 μ L, 40 μM of FIP and BIP each 1.0
μ L, LAMP reaction mixture 12.5 μ L, 8 UBstThe μ L of polymerase 1.0, the μ L of various concentrations DNA profiling 1.0, it is ultrapure with sterilizing
Water complements to 25 μ L；LAMP reaction conditions：63.5 DEG C of incubations 60 min, 85 DEG C of inactivation 5min；The measure of reaction result：Using glimmering
Photoinitiator dye visual observations method or agarose gel electrophoresis method are measured.Using fluorescent dye visual observations method, treat that LAMP reacts
After end, the μ L of developer SYBR green I 1.0 are added in the amplified production of LAMP reactions, colour developing result observes that green is glimmering
The judgement of light is the positive, orange（Crocus）It is judged as feminine gender；Using agarose gel electrophoresis method, 2.0 μ L LAMP amplifications are taken
Product detects trapezoid-shaped strips such as occur and be judged as the positive with 2% agarose gel electrophoresis, band does not occur and is then judged as feminine gender.
3. LAMP expands sensitivity technique result
LAMP expands sensitivity technique result and shows, 10 pg, 1 pg, 100 fg, the cucumber anthracnose base of 10 fg/ μ L concentration
Because a group DNA colour developing result can be observed green fluorescence or the trapezoid belt of LAMP features occurs in agarose gel electrophoresis, remaining concentration
And negative control develops the color result for amplified band does not occur in orange or agarose gel electrophoresis, illustrates designed cucumber anthracnose
Germ Primer composition F3, B3, FIP and BIP are expanded by LAMP, to the detection sensitivity of cucumber anthracnose up to 10 fg/
μL（Accompanying drawing 2）.
Embodiment 3：The LAMP detections of cucumber anthracnose in incidence of leaf
Sample collection：The typical blade of cucumber anthracnose disease symptom and healthy leaves are gathered from Fujian Foochow, ZhangZhou and Quanzhou
Take back laboratory standby；
The extraction of fruit DNA：DNA is extracted using NaOH rapid cleavages method, detailed process is as follows：Add in every milligram of plant tissue
Enter the mol/L NaOH of 10 μ L 0.5, be transferred in 1.5mL centrifuge tubes after tissue is fully milled into paste in mortar, 12,000
Rpm is centrifuged 6 min, takes the μ l of supernatant 5 and adds the mol/L Tris-HCl of 495 μ L 0.1（pH=8.0）It is well mixed, take 1.0 μ
L is expanded as pcr template.
Augmentation detection and observation：DNA with said extracted is carried out as template using Primer composition F3, B3, FIP and BIP
LAMP is expanded, and LAMP detects reaction system for 25 μ L, including 5 μM of F3 and B3 each 1.0 μ L, 40 μM of FIP and BIP each 1.0
μ L, LAMP reaction mixture 12.5 μ L, 8 UBstThe μ L of polymerase 1.0, the μ L of DNA profiling 1.0, are complemented to sterilizing ultra-pure water
25μL；LAMP reaction conditions：63.5 DEG C of incubations 60 min, 85 DEG C of inactivation 5min；The measure of reaction result：Using fluorescent dye
Visual observations method or agarose gel electrophoresis method are measured.Using fluorescent dye visual observations method, after LAMP reactions terminate,
The μ L of developer SYBR green I 1.0 are added in the amplified production of LAMP reactions, colour developing result observes sentencing for green fluorescence
It is the positive to break, orange（Crocus）It is judged as feminine gender；Using agarose gel electrophoresis method, 2.0 μ L LAMP amplified productions use is taken
2% agarose gel electrophoresis detects, trapezoid-shaped strips such as occurs and be judged as the positive, band do not occur and be then judged as feminine gender.
Testing result：Testing result（Accompanying drawing 3）Show, the typical leaves genomic DNA of cucumber anthracnose disease symptom leads to
LAMP amplifications are crossed, green fluorescence can be observed for colour developing result or the trapezoid belt of LAMP features occurs in agarose gel electrophoresis, exists
Cucumber anthracnose, and healthy leaves and negative control develop the color result for amplification bar does not occur in orange or agarose gel electrophoresis
Band, in the absence of cucumber anthracnose, illustrates that the set technology can be used for the rapid molecular detection of cucumber anthracnose in plant tissue.
Embodiment 4：LAMP detections with cucumber anthracnose in soil bacteria
Preparation with soil bacteria：To be incubated in PDA plate for the cucumber anthracnose of examination, 28 DEG C of dark culturing 7d treat mycelia
After covering with flat board, the conidium in flat board is washed down with appropriate sterilized water, be made conidial suspension, with appropriate soil
Earth mixes, and natural air drying, is made the pedotheque that carries disease germs indoors, standby；
Soil DNA is extracted：Using the soil DNA extracts kit of Sigma companies（Sigma,DNB100,Soil DNA
Isolation Kit）The STb gene in soil is extracted, 1.0 μ L is taken and is expanded as pcr template.
Augmentation detection and observation：DNA with said extracted is carried out as template using Primer composition F3, B3, FIP and BIP
LAMP is expanded, and LAMP detects reaction system for 25 μ L, including 5 μM of F3 and B3 each 1.0 μ L, 40 μM of FIP and BIP each 1.0
μ L, LAMP reaction mixture 12.5 μ L, 8 UBstThe μ L of polymerase 1.0, the μ L of DNA profiling 1.0, are complemented to sterilizing ultra-pure water
25μL；LAMP reaction conditions：63.5 DEG C of incubations 60 min, 85 DEG C of inactivation 5min；The measure of reaction result：Using fluorescent dye
Visual observations method or agarose gel electrophoresis method are measured.Using fluorescent dye visual observations method, after LAMP reactions terminate,
The μ L of developer SYBR green I 1.0 are added in the amplified production of LAMP reactions, colour developing result observes green fluorescence
It is judged as the positive, it is orange（Crocus）It is judged as feminine gender；Using agarose gel electrophoresis method, 2.0 μ L LAMP amplified productions are taken
Detect trapezoid-shaped strips such as occur and be judged as the positive with 2% agarose gel electrophoresis, band do not occur and be then judged as feminine gender.
Testing result：Testing result（Accompanying drawing 3）Show, the soil DNA for carrying cucumber anthracnose is expanded by LAMP, is shown
Color result can be observed green fluorescence or the trapezoid belt of LAMP features occurs in agarose gel electrophoresis, there is cucumber anthracnose,
And autoclaving soil and negative control develop the color result for amplified band does not occur in orange or agarose gel electrophoresis, do not exist
Cucumber anthracnose, illustrates that the set technology can be used for the rapid molecular detection of cucumber anthracnose in soil.
The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with
Modification, should all belong to covering scope of the invention.
SEQUENCE LISTING
<110>Inst. of Plant Protection, fujian Academy of Agricultural Science
<120>Cucumber anthracnose LAMP detection primer and its application
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> F3
<400> 1
ggggctaact gcttgaacag 20
<210> 2
<211> 18
<212> DNA
<213> B3
<400> 2
ggtgccgttg tacctgtt 18
<210> 3
<211> 40
<212> DNA
<213> FIP
<400> 3
tgtgcgaagc atcgttgccg ggtaaccaga ttggtgctgc 40
<210> 4
<211> 42
<212> DNA
<213> BIP
<400> 4
aggcaaaaca tctctggcga gcccattctt gacgtggcct ta 42
Claims (5)
1. cucumber anthracnose LAMP detection primer, it is characterised in that its nucleotides sequence is classified as：
F3:5 '-GGGGCTAACTGCTTGAACAG -3 ',
B3:5 '-GGTGCCGT TGTACCTGTT -3 ',
FIP:5 '-TGTGCGAAGCATCGTTGCCGGGTAACCAGATTGGTGCTGC -3 ',
BIP: 5’- AGGCAAAACATCTCTGGCGAGCCCATTCTTGACGTGGCCTTA - 3’。
2. a kind of method that cucumber anthracnose LAMP detections are carried out using primer described in claim 1, it is characterised in that including
Step in detail below：
（1）Extract testing sample genomic DNA；
（2）The foundation of LAMP reaction systems：LAMP detection reaction systems are 25 μ L, with step（1）The DNA of extraction is template,
Reaction system includes 5 μM of F3 and B3 each 1.0 μ L, each 1.0 μ L, LAMP reaction mixture of 40 μM of FIP and BIP 12.5 μ L, 8
U BstThe μ L of polymerase 1.0, the μ L of DNA profiling 1.0,25 μ L are complemented to sterilizing ultra-pure water；
（3）LAMP reaction conditions：63.5 DEG C of incubations 60 min, 85 DEG C of inactivation 5min；
（4）The measure of reaction result：It is measured using fluorescent dye visual observations method or agarose gel electrophoresis method：
Using fluorescent dye visual observations method, after LAMP reactions terminate, developer is added in the amplified production of LAMP reactions
The μ L of SYBR green I 1.0, colour developing result observes that the judgement of green fluorescence is the positive, and orange or crocus is judged as the moon
Property；
Using agarose gel electrophoresis method, take 2.0 μ L LAMP amplified productions and detected with 2% agarose gel electrophoresis, ladder such as occur
Shape band is judged as the positive, band does not occur and is then judged as feminine gender.
3. a kind of cucumber anthracnose LAMP detection method as claimed in claim 2, it is characterised in that described LAMP is anti-
Answer mixed liquor composed of the following components：40 mM Tris-HCl, 20 mM (NH4)2SO4, 20 mM KCl, 16 mM MgSO4,
1.6 mol/L glycine betaines, 2.0 mM dNTPs, 0.2% Trion X-100.
4. the early diagnosis and disease using the cucumber anthracnose LAMP detection primer described in claim 1 in cucumber anthracnose
Application in the monitoring of bacterium, identification.
5. the early diagnosis and disease using the cucumber anthracnose LAMP detection method described in claim 2 in cucumber anthracnose
Application in the monitoring of bacterium, identification.
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Cited By (4)
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| CN107815504A (en) * | 2017-11-30 | 2018-03-20 | 福建省农业科学院植物保护研究所 | A kind of anthracnose of peach collarium mediated isothermality amplification detection primer and its detection method |
| CN108754004A (en) * | 2018-05-10 | 2018-11-06 | 山东省烟台市农业科学研究院 | A method of Botryosphaeria berengeriana f. sp is quickly detected based on LAMP |
| CN108977508A (en) * | 2018-09-10 | 2018-12-11 | 福建省农业科学院植物保护研究所 | Primer combination and its application based on LAMP detection succulent Pathogen |
| CN117511935A (en) * | 2024-01-04 | 2024-02-06 | 韶关学院 | LAMP (loop-mediated isothermal amplification) detection primer and rapid detection method for pepper anthracnose based on TUB2 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107815504A (en) * | 2017-11-30 | 2018-03-20 | 福建省农业科学院植物保护研究所 | A kind of anthracnose of peach collarium mediated isothermality amplification detection primer and its detection method |
| CN108754004A (en) * | 2018-05-10 | 2018-11-06 | 山东省烟台市农业科学研究院 | A method of Botryosphaeria berengeriana f. sp is quickly detected based on LAMP |
| CN108977508A (en) * | 2018-09-10 | 2018-12-11 | 福建省农业科学院植物保护研究所 | Primer combination and its application based on LAMP detection succulent Pathogen |
| CN117511935A (en) * | 2024-01-04 | 2024-02-06 | 韶关学院 | LAMP (loop-mediated isothermal amplification) detection primer and rapid detection method for pepper anthracnose based on TUB2 |
| CN117511935B (en) * | 2024-01-04 | 2024-04-05 | 韶关学院 | LAMP (loop-mediated isothermal amplification) detection primer and rapid detection method for pepper anthracnose based on TUB2 |
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