CN102899416A - Cucumber phytophthora LAMP (Loop-mediated Isothermal Amplification) primer and rapid detection method thereof - Google Patents
Cucumber phytophthora LAMP (Loop-mediated Isothermal Amplification) primer and rapid detection method thereof Download PDFInfo
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- CN102899416A CN102899416A CN2012104096451A CN201210409645A CN102899416A CN 102899416 A CN102899416 A CN 102899416A CN 2012104096451 A CN2012104096451 A CN 2012104096451A CN 201210409645 A CN201210409645 A CN 201210409645A CN 102899416 A CN102899416 A CN 102899416A
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Abstract
The invention discloses cucumber phytophthora LAMP (Loop-mediated Isothermal Amplification) primer and a rapid detection method thereof, which are specifically used for the specific detection of cucumber phytophthora. The rapid detection method is characterized in that a cucumber phytophthora LAMP primer is mainly adopted and designed, detailed information can be seen in SEQ NO.1-SEQ NO.4. Green fluorescent light or an LAMP characterized ladder-shaped pattern can be observed through the LAMP and the color developing by adding an SYBR green I color agent for developing color or the agarose gel electrophoresis detection. The LAMP primer and the rapid detection method, which are disclosed by the invention, can be used for quickly, sensitively and accurately detecting the cucumber phytophthora in plants and soil which are affected by the cucumber phytophthora in production practice and can be simultaneously used for the early diagnosis of diseases in filed and the monitoring and the identification of germs, and reliable technical and theoretical basis is provided for preventing the disease caused by the cucumber phytophthora.
Description
Technical field
The present invention relates to a kind of cucumber phytophthora LAMP primer and method for quick thereof, being exclusively used in cucumber phytophthora highly sensitive rapid molecular detects, can be used for simultaneously the early diagnosis of field Cucumber Blight and monitoring and the evaluation of germ, belong to corps diseases detection, evaluation and Prevention Technique field.
Background technology
The cucumber epidemic disease mould (
Phytophthora melonisKatsura) obtained from the cucumber separation of falling ill first in nineteen sixty-eight by Katsura, and be accredited as novel species, the states such as China, Japan, Egypt, Turkey, Korea S, India also report this cause of disease in succession subsequently.Be that one of the most serious disease occurs in Chinese cucumber producing region by this microbial Cucumber Blight, stem, leaf and the fruit of the plant of mainly causing harm all can occur from seedling stage to the strain phase.This germ survives among the soil, short incubation period, and velocity of propagation is fast, and invasiveness is strong, and is destructive large to plant.Can cause disease to be very popular in short period of time at the utmost point in the situation that humiture is fit to, because epidemic disease causes harm the cucumber yield that loses up to 80%.The cucumber epidemic disease is mould to infect the crops such as cucumber, summer squash, hami melon, wax gourd.The morbidity plant is carried out fast and accurately disease screening, is that the control Plant diseases occurs, the popular and important foundation of causing disaster to the timely detection of pathogenic bacteria in anosis vegetable material and the plant growth environment and monitoring.Therefore set up cucumber phytophthora rapid detection system, in early days disease plant and soil are carried out phytophthora and quick and precisely detect, to the prediction disease a situation arises, in time take effectively preventing measure control pathogenic bacteria propagation and popular, reduce financial loss and all have important theoretical and practical significance.
At present traditional cultivation and authentication method are still continued to use in the detection of cucumber phytophthora mostly, traditional detection of pathogens technology is to obtain judging the kind of pathogen by morphological observation and Koch's Postulates on the basis of pathogen in separation.Whole process usually needs to expend a large amount of labor forces and time, generally needs just can finish in several days.And require the operator to possess pathogenicbacteria separation, Morphological Identification knowledge and the rich experience of specialty.Therefore, take the conventional disease screening technology of morphological specificity as the basis, because its length consuming time, efficient is low, is difficult to satisfy the actual needs to the Cucumber Blight diagnosis, because it is easy to miss the best period of disease control.Therefore, set up a cover fast, sensitive, accurately the cucumber phytophthora to detect diagnostic techniques not only very necessary, and very urgent.
Round pcr is for pathogenic diagnosis provides fast, sensitive, advantage accurately, yet PCR specific detection technology still needs expensive professional instrument and the molecular biology reagent such as PCR instrument, electrophoresis and gel imaging system at present, and need molecular biology Specialty Experiment personnel operation, limited applying of PCR detection method.Circulation constant temperature amplification technique (Ioop-mediated isothermal amplification is called for short LAMP) is a kind of New Cycle constant temperature nucleic acid amplification technology of being developed by people such as the Japanese Rong Yan Notomi of Co., Ltd. in 2000.LAMP reaction is designed 4 primers for 6 sites of target gene, utilize a kind of chain type substitute activity archaeal dna polymerase (
BstDNA polymerase), (65 ℃ of 60 –) insulation 30 – 90 minutes under constant temperature, can finish amplified reaction.Because high efficiency and the isothermal rapid amplifying of LAMP reaction can increase 10 in 90 minutes
9– 10
10Times product.The methods such as fluorescence dye visual observations, agarose gel electrophoresis and turbidity observation are generally adopted in the detection of LAMP amplified production.Because the LAMP reaction is simple, quick, efficient, the economic dispatch feature, thereby has very widely application prospect.LAMP detects and is mainly used in people and animals' pathogen, food safety and sanitary detection at present, and report is few in phytopathogen detects, and the detection of cucumber phytophthora is not reported both at home and abroad.
Summary of the invention
The objective of the invention is, detection method poor specificity long for the required cycle of the biological detection method of cucumber phytophthora in the prior art, the problem that sensitivity is low provides a kind of LAMP of cucumber phytophthora to detect the method for quick of primer and reliable results, easy handling, high specificity, highly sensitive cucumber phytophthora.
Realize that purpose of the present invention comprises the following steps:
1.LAMP the design of primer
We download the cucumber epidemic disease from GenBank mould
Ypt(accession number: EF649778) gene order, mould according to the cucumber epidemic disease
YptGene order adopts a kind of LAMP of PrimerExplorer V4 software design to detect primer, comprises 2 outer primers (F3 and B3) and 2 inner primers (FIP and BIP), and primer sequence is respectively:
F3：5’-AAATTCGCACGATCGAGCT-3’
B3：5’-CCGTGACGTCGTACACCA-3’
FIP：5’-GCGCTAAGTCGCGAATGTACCG-GACGGCAAGACCATCAAGC-3’
BIP：5’-CTATTGTAGTGGGACACGGCCG-CGATAATACCGTGGGCACC-3’
2. the foundation of cucumber phytophthora rapid detection system
LAMP detects: each 0.2-0.25uM of F3 and B3 in the 25ul reaction system, each 1.5-1.7uM of FIP and BIP, 20mM Tris-HCl, 10mM (NH
4)
2SO
4, 10mM KCl, 8mM MgSO
4, 0.1% Tween-20,0.8M Betaine, 1.4 mM dNTPs, 8U
BstDNA polysaccharase large fragment, the 10-50ng dna profiling, insufficient section is supplied with aseptic double-distilled water; The LAMP reaction conditions is at 60-65 ℃ of incubation 30-90 min, 80 ℃ of insulation 5-10min.
More preferably, each 0.2uM of F3 and B3 in the 25ul reaction system, each 1.6uM of FIP and BIP, 20mM Tris-HCl, 10mM (NH
4)
2SO
4, 10mM KCl, 8mM MgSO
4, 0.1% Tween-20,0.8M Betaine, 1.4 mM dNTPs, 8U
BstDNA polysaccharase large fragment, the 25ng dna profiling, insufficient section is supplied with aseptic double-distilled water; The LAMP reaction conditions is at 63 ℃ of incubation 60 min, 80 ℃ of insulation 10min.
Described detection method is fluorescence dye visual observations method and agarose gel electrophoresis method.Described fluorescence dye visual observations method: add the 1ul developer in the final amplified production of LAMP reaction, described developer is SYBR green I, and the colour developing result observes green fluorescence and is judged as the positive, the orange feminine gender that is judged as.Described agarose gel electrophoresis method: get the 2ul pcr amplification product and detect with 2% agarose gel electrophoresis, be judged as the positive if there is the distinctive trapezoid belt of LAMP, amplified band do not occur and be judged as feminine gender.
The highly sensitive rapid detection of the plant that the method can be used for carrying disease germs and soil.Set up that the cucumber phytophthora is quick, easy, high specificity, highly sensitive Monitoring techniques system, cause early monitoring before the aobvious disease of disease for the cucumber phytophthora, determine that disease control best period tool is of great significance.
Guardian technique of the present invention is primer sequence and the amplification method thereof of the efficient specific amplified of cucumber phytophthora.In order to verify the special primer sequence of cucumber phytophthora, the present invention adopts the CTAB method to extract the DNA of cucumber phytophthora in the tissue take 36 strain cucumber phytophthoras of the provinces such as China Fujian, Guangdong, Jiangsu and 22 kinds of different fungies and 11 kinds of phytophthoras and pythium oomycetes as for the examination material.Concrete grammar is as follows: get hypha powder after the 50mg lyophilize in the 1.5ml centrifuge tube, (prescription of extracting solution is: 2% CTAB to add 900ul 2% CTAB (cetyl trimethylammonium bromide) extracting solution; 100 m mol/L Tris-HCl(Tri(Hydroxymethyl) Amino Methane Hydrochlorides), PH 8.0; 20mmol/L EDTA(disodium ethylene diamine tetraacetate), pH8.0; 1.4 mol/L NaCl) and 90ul 10% SDS(Sodium dodecylbenzene sulfonate) after mixing, in 55～60 ℃ of water-bath 1.5 h, per 10 min vibration mixing once, behind the water-bath 1.5h centrifugal (12,000rpm) 15min, getting supernatant liquor adds and the isopyknic phenol/chloroform of supernatant liquor/primary isoamyl alcohol (phenol, the volume ratio of chloroform and primary isoamyl alcohol is 25:24:1), centrifugal (12,000rpm) 5 min, get supernatant liquor (water), add and the isopyknic chloroform extracting of supernatant liquor once (12,000rpm) centrifugal 5min, suct clearly (350ul), add the 3mol/L NaAC solution of 0.1 volume (35ul) and the ice dehydrated alcohol of 2 volumes (700ul), behind-20 ℃ of lower precipitation 30min 12, centrifugal 5 min of 000rpm, remove lightly supernatant liquor, adding 700ul ices 70% ethanol and washs (slightly centrifugal, incline and fall supernatant), after naturally drying alcohol-free flavor on the Bechtop, use 1 * TE(10mmol/L Tris-HCl, 0.1mmol/L EDTA, pH8.0) solution dissolves, obtain dna solution, with UV spectrophotometer measuring DNA concentration and to be diluted to 50ng/ul stand-by.
Through the specificity of strains tested and 36 strain cucumber phytophthoras being carried out the LAMP checking.
LAMP reaction system 25ul: comprise each 0.2uM of F3 and B3, each 1.6uM of FIP and BIP, 20mM Tris-HCl, 10mM (NH
4)
2SO
4, 10mM KCl, 8mM MgSO
4, 0.1% Tween-20,0.8M Betaine, 1.4 mM dNTPs, 8U
BstDNA polysaccharase large fragment, the 25ng dna profiling, insufficient section is supplied with aseptic double-distilled water.
Detect and occur the distinctive trapezoid belt of LAMP except can be observed green fluorescence or agarose gel electrophoresis from 36 strain cucumber phytophthoras of the provinces such as China Fujian, Guangdong, Jiangsu colour developing result, it is orange having detected 22 kinds of fungal bacterial strains and 11 kinds of other oomycetes colour developing results or amplified band does not appear in agarose gel electrophoresis.This illustrates that this primer can be used to fast and reliable detection and the evaluation of cucumber phytophthora in the incidence tissue and soil in the production practice.
1. when when having the cucumber phytophthora for the cucumber tissue, adopt the quick cracking process of NaOH to extract the DNA of cucumber phytophthora, detailed process is as follows: (1) is cleaned the sick leaf of cucumber or sick stem, dry, the clip site of pathological change; (2) press the sick leaf of 1mg and add 10ul(0.5mol/L NaOH, 0.5%PVP) metering, will organize and fully be milled to paste, centrifugal 5min in 12,000g whizzer; (3) get the Tris-HCl(pH8.0 of supernatant 20ul and isopyknic 0.1 mol/L) mix; (4) 10 times, 100 times, 1000 times liquid of dilution are got respectively 1ul stoste, 10 times, 100 times, 1000 times liquid increase as pcr template.
2. when when having the cucumber phytophthora for cucumber soil, adopt the soil DNA extraction method to extract the DNA of banana blight bacteria in the soil.Concrete grammar is as follows: add a small amount of quartz sand after getting the freezing 24-48 of the draining h of the soil that sieves, pour liquid nitrogen into and fully grind, the soil fine powder after grinding is divided be filled in the 1.5 ml centrifuge tubes, every pipe adds 500 ul, 0.4% skim-milk solution, vortex mixing.Centrifugal 15 min of 12000 rpm.Get supernatant and add equal-volume Proteinase K damping fluid, adding final concentration is 10 ug/ml Proteinase Ks, 55 ℃ of water-bath 1-3 h.After water-bath finishes, add 7.5 M NH of 1/2 volume
4AC solution, mixing turns upside down.Centrifugal 15 min of 12000 rpm.Suct and reset and add 2 times of volume dehydrated alcohols-20 ℃ precipitation (sedimentation time 1.5 h).After precipitation finishes, centrifugal 15 min of 12000 rpm.Go with the hypsokinesis of 70% washing with alcohol precipitation, room temperature is dried.DNA that every duplicate samples is carried is with 10 ul TE(or aseptic ultrapure water) dissolving, 20 ℃ save backup.
Detect with designed primer by following LAMP reaction system and reaction conditions:
1. LAMP reaction system 25ul: comprise each 0.2uM of F3 and B3, each 1.6uM of FIP and BIP, 20mM Tris-HCl, 10mM (NH
4)
2SO
4, 10mM KCl, 8mM MgSO
4, 0.1% Tween-20,0.8M Betaine, 1.4 mM dNTPs, 8U
BstDNA polysaccharase large fragment, the 25ng dna profiling, insufficient section is supplied with aseptic double-distilled water; The LAMP reaction conditions is at 63 ℃ of incubation 60 min, 80 ℃ of insulation 10min.
2. in the final amplified production of LAMP reaction, add the 1ul developer, described developer is SYBR green I, the colour developing result observes green fluorescence, or get the 2ul amplified production and detect with 2% agarose gel electrophoresis, the distinctive trapezoid belt of LAMP appears in the result, can judge in described cucumber tissue or the soil to have the cucumber phytophthora; Otherwise there is not the cucumber phytophthora in described cucumber tissue or the soil.
Beneficial effect of the present invention: the inventive method is applicable to fast and reliable detection and the evaluation of cucumber phytophthora in incidence tissue or the soil, and the disease control that causes for cucumber phytophthora in the agriculture production has important practical value.The present invention compared with prior art has following technical superiority and positively effect:
1, reliable results: the designed LAMP that goes out of the present invention detects primer, carried out testing authentication to the cucumber phytophthora that comes from the ground such as Fujian China, Guangdong, Jiangsu with soil sample, the plant tissue of cucumber phytophthora, so result reliability has sufficient assurance;
2, high specificity: LAMP primer of the present invention is for the cucumber phytophthora
Ypt6 different zones are designed 4 Auele Specific Primers in the gene order, and any zone and primer do not mate all and can not carry out nucleic acid amplification in 6 zones, therefore specificity is high.
3, highly sensitive: LAMP can reach 10fg to the detection sensitivity of cucumber phytophthora on dna level, detects high 1000 times than conventional PCR.
4, practicality is good: the designed LAMP primer that goes out of the present invention, can be used for the highly sensitive rapid detection with tissue and the soil of cucumber phytophthora, therefore present method is practical, can satisfy carry out the needs of fast and reliable detection and evaluation with the cucumber phytophthora that exists in hyphostroma and the soil;
5, easy and simple to handle quick: as to use the inventive method, tissue and soil with the cucumber phytophthora are detected and can finish in 1 hour, and the LAMP nucleic acid amplification is to carry out under isothermal condition, only need a water-bath to get final product, do not need complicated plant and instrument and expensive molecular agents, naked eyes directly as seen as a result.
Description of drawings
Fig. 1 is the special LAMP detected result figure of Causal Organism of Cucumber Blight of the present invention.A represents the agarose gel electrophoresis detected result among Fig. 1, and wherein: swimming lane M is DL 2000 DNA marker, swimming lane 1 negative contrast, and swimming lane 2 positive contrasts, swimming lane 3-8 is the cucumber phytophthora, swimming lane 9-16 is other oomycetes and fungal bacterial strain; B represents the result that develops the color among Fig. 1, wherein: the negative contrast of the 1st pipe, the positive contrast of the 2nd pipe, the 3rd – 8 pipes are the cucumber phytophthora, the 9th – 16 pipes are other oomycetes and fungal bacterial strain.
Fig. 2 is the LAMP susceptibility detected result figure of Causal Organism of Cucumber Blight of the present invention.A represents the agarose gel electrophoresis detected result among Fig. 2, and wherein: swimming lane M is DL 2000 DNA marker, and swimming lane 1 – 10 template concentrations are respectively 1000ng, 100ng, 10ng, 1ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg; B represents the result that develops the color among Fig. 2, and wherein: the 1st – 10 pipe die plate concentration are respectively 1000ng, 100ng, 10ng, 1ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg.
Fig. 3 is that the present invention is to the detected result figure of disease plant and potato piece.A represents the agarose gel electrophoresis detected result among Fig. 3, wherein: swimming lane 1 positive contrast, swimming lane 2 negative contrasts, swimming lane 3 is incidence tissue, and swimming lane 4 is morbidity soil, and swimming lane 5 is health tissues; Swimming lane M is DL 2000 DNA marker; B represents the result that develops the color among Fig. 3, wherein: the positive contrast of the 1st pipe, the negative contrast of the 2nd pipe, the 3rd pipe is incidence tissue, the 4th pipe is health tissues for morbidity soil, the 5th pipe.
Embodiment
Technology contents of the present invention comprises that the LAMP of cucumber phytophthora detects primer, and LAMP primer and sequence thereof are respectively:
F3：5’-AAATTCGCACGATCGAGCT-3’
B3：5’-CCGTGACGTCGTACACCA-3’
FIP：5’-GCGCTAAGTCGCGAATGTACCG-GACGGCAAGACCATCAAGC-3’
BIP：5’-CTATTGTAGTGGGACACGGCCG-CGATAATACCGTGGGCACC-3’
The result can be observed green fluorescence or the distinctive trapezoid belt of LAMP appears in agarose gel electrophoresis to utilize LAMP primer detection cucumber phytophthora to develop the color.
Main agents:
BstDNA polysaccharase large fragment is available from Britain NEB company; DNA marker is available from precious biotechnology Dalian company limited; All the other reagent are all given birth to worker Bioisystech Co., Ltd available from Shanghai.Primer is given birth to worker Bioisystech Co., Ltd by Shanghai and is synthesized.
The specific amplification of embodiment 1:LAMP primer pair cucumber phytophthora
1. the LAMP specific detection of cucumber phytophthora
1. LAMP reaction system 25 ul: comprise each 0.2uM of F3 and B3, each 1.6uM of FIP and BIP, 20mM Tris-HCl, 10mM (NH
4)
2SO
4, 10mM KCl, 8mM MgSO
4, 0.1% Tween-20,0.8M Betaine, 1.4 mM dNTPs, 8U
BstDNA polysaccharase large fragment, the 25ng dna profiling, insufficient section is supplied with aseptic double-distilled water; The LAMP reaction conditions is at 63 ℃ of incubation 60 min, 80 ℃ of insulation 10min.
2. add the 1ul developer in the final amplified production of LAMP reaction, described developer is SYBR green I, and the colour developing result observes green fluorescence and is judged as the positive, the orange feminine gender that is judged as.Or get the 2ul amplified production and detect with 2% agarose gel electrophoresis, be judged as the positive if there is the distinctive trapezoid belt of LAMP, amplified band do not occur and be judged as feminine gender.
2. detected result
The specificity that detects: the result can be observed green fluorescence or agarose gel electrophoresis occurs the distinctive trapezoid belt of LAMP except 36 strain cucumber phytophthoras from provinces such as China Fujian, Guangdong, Jiangsu develop the color, it is orange having detected 11 kinds of other oomycetes and 22 kinds of fungal bacterial strains colour developing result or amplified band (partial results is seen Fig. 1) does not appear in agarose gel electrophoresis, illustrates that this primer has very strong specificity.
The susceptibility of embodiment 2:LAMP primer pair cucumber phytophthora detects
1. the LAMP susceptibility of cucumber phytophthora detects
Adopt 10 times of concentration series dilution methods that the cucumber phytophthora DNA that extracts is diluted to 1000ng, 100ng, 10ng, 1ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg is totally 10 different concns gradients.
1. LAMP reaction system 25ul: comprise each 0.2uM of F3 and B3, each 1.6uM of FIP and BIP, 20mM Tris-HCl, 10mM (NH
4)
2SO
4, 10mM KCl, 8mM MgSO
4, 0.1% Tween-20,0.8M Betaine, 1.4 mM dNTPs, 8U
BstDNA polysaccharase large fragment, the 25ng dna profiling, insufficient section is supplied with aseptic double-distilled water; The LAMP reaction conditions is at 63 ℃ of incubation 60 min, 80 ℃ of insulation 10min.
2. add the 1ul developer in the final amplified production of LAMP reaction, described developer is SYBR green I, and the colour developing result observes green fluorescence and is judged as the positive, the orange feminine gender that is judged as.Or get the 2ul amplified production and detect with 2% agarose gel electrophoresis, be judged as the positive if there is the distinctive trapezoid belt of LAMP, amplified band do not occur and be judged as feminine gender.
2. detected result: cucumber phytophthora LAMP susceptibility detects, and the colour developing result can be observed green fluorescence or the distinctive trapezoid belt of LAMP appears in agarose gel electrophoresis, and detection sensitivity can reach 10fg(and see Fig. 2).
Embodiment 3: the detection of cucumber phytophthora in incidence tissue or the soil.
1. sample collecting: the plant tissue sample picks up from cucumber production base, Fuzhou City, Fujian; Pedotheque picks up from cucumber production base, Longhai City, Fujian Province.
2.DNA extract and detect
The morbidity plant tissue adopts the quick cracking process of NaOH to extract cucumber phytophthora DNA, and morbidity soil adopts the soil DNA extraction method to extract the DNA of cucumber phytophthora.
Carrying out as follows LAMP detects:
1. LAMP reaction system 25 ul: comprise each 0.2uM of F3 and B3, each 1.6uM of FIP and BIP, 20mM Tris-HCl, 10mM (NH
4)
2SO
4, 10mM KCl, 8mM MgSO
4, 0.1% Tween-20,0.8M Betaine, 1.4 mM dNTPs, 8U
BstDNA polysaccharase large fragment, the 25ng dna profiling, insufficient section is supplied with aseptic double-distilled water; The LAMP reaction conditions is at 63 ℃ of incubation 60 min, 80 ℃ of insulation 10min.
2. add the 1ul developer in the final amplified production of LAMP reaction, described developer is SYBR green I, and the colour developing result observes green fluorescence and is judged as the positive, the orange feminine gender that is judged as.Or get the 2ul amplified production and detect with 2% agarose gel electrophoresis, be judged as the positive if there is the distinctive trapezoid belt of LAMP, amplified band do not occur and be judged as feminine gender.
3. detected result
As shown in Figure 3, infect the cucumber phytophthora in incidence tissue or the soil, the colour developing result can be observed green fluorescence or the distinctive trapezoid belt of LAMP appears in agarose gel electrophoresis, and health tissues colour developing result then observes fluorescent orange or the distinctive trapezoid belt of LAMP does not appear in agarose gel electrophoresis.
＜110〉Inst. of Plant Protection, fujian Academy of Agricultural Science
＜120〉a kind of cucumber phytophthora LAMP primer and method for quick thereof
<160> 4
<170> PatentIn?version?3.5
<210> 1
<211> 19
<212> DNA
＜213〉artificial sequence
<400> 1
aaattcgcac?gatcgagct 19
<210> 2
<211> 18
<212> DNA
＜213〉artificial sequence
<400> 2
ccgtgacgtc?gtacacca?18
<210> 3
<211> 41
<212> DNA
＜213〉artificial sequence
<400> 3
gcgctaagtc?gcgaatgtac?cggacggcaa?gaccatcaag?c?41
<210> 4
<211> 41
<212> DNA
＜213〉artificial sequence
<400> 4
ctattgtagt?gggacacggc?cgcgataata?ccgtgggcac?c?41
Claims (3)
1. the LAMP primer of a cucumber phytophthora, it is characterized in that: described LAMP primer is as follows:
F3：5’-AAATTCGCACGATCGAGCT-3’?；
B3：5’-CCGTGACGTCGTACACCA-3’；
FIP：5’-GCGCTAAGTCGCGAATGTACCG-GACGGCAAGACCATCAAGC-3’；
BIP：5’-CTATTGTAGTGGGACACGGCCG-CGATAATACCGTGGGCACC-3’。
2. the LAMP detection method of a cucumber phytophthora, it is characterized in that: utilize LAMP primer claimed in claim 1 to carry out LAMP reaction, each 0.2-0.25uM of F3 and B3 in the 25ul reaction system, each 1.5-1.7uM of FIP and BIP, 20mM Tris-HCl, 10mM (NH
4)
2SO
4, 10mM KCl, 8mM MgSO
4, 0.1% Tween-20,0.8M Betaine, 1.4 mM dNTPs, 8U
BstDNA polysaccharase large fragment, the 10-50ng dna profiling, insufficient section is supplied with aseptic double-distilled water; The LAMP reaction conditions is at 60-65 ℃ of incubation 30-90 min, 80 ℃ of insulation 5-10min.
3. cucumber phytophthora LAMP detection method according to claim 2, it is characterized in that: in the amplified production of LAMP reaction, add the 1ul developer, described developer is SYBR green I, and the colour developing result observes green fluorescence and is judged as the positive, the orange feminine gender that is judged as; Or get the 2ul amplified production and detect with 2% agarose gel electrophoresis, as trapezoid-shaped strips occurs and be judged as the positive, feminine gender does not appear then not being judged as.
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| CN103525913A (en) * | 2013-09-22 | 2014-01-22 | 福建省农业科学院植物保护研究所 | Phytophthora capsici LAMP (Loop-mediated isothermal Amplification) primer and rapid detection method thereof |
| CN104962639A (en) * | 2015-07-09 | 2015-10-07 | 天津市植物保护研究所 | LAMP primer group and detection method for rapidly detecting corynespora cassiicola |
| CN105039331A (en) * | 2015-08-11 | 2015-11-11 | 华南农业大学 | Peronophythora litchi LAMP primer as well as rapid detection method and application thereof |
| CN106987653A (en) * | 2017-06-09 | 2017-07-28 | 福建省农业科学院植物保护研究所 | A kind of phytophthora infestans LAMP detection primer and its visible detection method |
| CN108103140A (en) * | 2017-11-09 | 2018-06-01 | 广东省农业科学院蔬菜研究所 | A kind of Rapid identification cucumber is to the method for epidemic disease resistance |
| CN109486985A (en) * | 2018-08-07 | 2019-03-19 | 天津出入境检验检疫局动植物与食品检测中心 | The LAMP primer pair and its amplification method and purposes of detection phytophthora hibernalis bacterium |
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| CN109486985A (en) * | 2018-08-07 | 2019-03-19 | 天津出入境检验检疫局动植物与食品检测中心 | The LAMP primer pair and its amplification method and purposes of detection phytophthora hibernalis bacterium |
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