CN106636378A - LAMP (Loop-mediated Isothermal Amplification) primer combination for detecting tomato phytophthora infestans and application - Google Patents
LAMP (Loop-mediated Isothermal Amplification) primer combination for detecting tomato phytophthora infestans and application Download PDFInfo
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Abstract
The invention discloses an LAMP (Loop-mediated Isothermal Amplification) primer combination for detecting tomato phytophthora infestans and application. The LAMP primer combination for detecting the tomato phytophthora infestans consists of four specific primers, and the primer combination disclosed by the invention is applied in the LAMP detection of the tomato phytophthora infestans. A detection system disclosed by the invention is incubated under the isothermal condition of 64 DEG C for 1 hour, SYBR green I developer is adopted for detection by development, and with whether green fluorescence is observed as a standard, a detection result is judged. The LAMP primer combination has the advantages of high accuracy, high specificity, convenience in operation and high practicability, does not need complex instruments, realizes isothermal amplification, provides a novel molecular detection technique for the tomato phytophthora infestans, can be used in rapid, sensitive and accurate detection in the early stage or infection incubation of the tomato phytophthora infestans in production practice, and provides a reliable technical and theoretical basis for the prevention and treatment of the tomato phytophthora infestans.
Description
Technical field
The invention belongs to corps diseases detection, identification and Prevention Technique field, and in particular to the ring of Phytophthora infestans germ
Mediated isothermality amplification（LAMP）Detection primer composition and its application, can be used for Phytophthora infestans germ quick, sensitive and special
Molecular Detection, while can be used for the early diagnosis of tomato late blight and the monitoring and identification of germ.
Background technology
Tomato (Lycopersicon esculentum Miller) tomato, tomato are called, on plant classification
Belong to Solanaceae (Solanaceae) tomato genus (Lycopersicon).Tomato contains abundant nutrient content, is both that vegetables are ripe
Food and eat raw, be again fruit, extremely broad masses like.Determine according to the study, the fresh tomatoes of 50～100 g eaten for each person every day,
Human body can be met to several vitamins and the needs of mineral matter.Also containing abundant antioxidant in tomato, can prevent certainly
By destruction of the base to skin, with obvious beauty anti-wrinkle effect.With continuous expansion of the tomato in China's cultivated area and kind
The not severed finger for planting index is high, and tomato pest species and the extent of injury are also presented the trend for increasing year by year and increasing, wherein, by evening
Epidemic disease bacterium (Phytophthora infestans) to infect the tomato late blight for causing be that most universal, harm occurs in tomato production
One of disease of most serious, in open country or protecting field heavy losses are all caused, and are a kind of very strong destructivenesses of popularity
Disease, general time underproduction 20%-30%, up to 40%-60% when serious, or even total crop failure.Often other are sick with tomato for tomato late blight
Evil（Such as gray mold, early blight）Mixing occurs, and early stage symptom has certain similitude, often to the correct diagnosis of disease
Great difficulty is caused, aborning often because disease screening is inaccurate, the correct preventing and treating for failing to reach enforcement " suiting the remedy to the case " is arranged
Apply and cause disease to cause the phenomenon of heavy loss to happen occasionally.Phytophthora infestans germ rapid detection system is hence set up, is being sent out
Whether late disease bacteria is carried to plant before sick initial stage or manifestation of symptoms and is fast and accurately detected that this is accurate for disease
Prediction, the in good time effectively preventing measure of formulation, the propagation of control disease and prevalence（Spread）And reduce what disease was caused
Economic loss all has important theoretical and practical significance.
The traditional detection method of phytopathogen is separated from soil, plant tissue or water body using selective medium
Bacterial strain, then the form to these bacterial strains etc. identifies to determine whether there is pathogen, or with the naked eye and by microscope
Technology is judged disease symptom.Traditional detection method is not only time-consuming, the degree of accuracy is low, and it is rich to require that testing staff will have
Rich experience, the more most important disease for being easily to omit incubation period or hidden disease, so that the preventing and treating of delay disease, causes the sudden and violent of disease
Send out, therefore traditional pathogeny detection method can not meet the needs of modern plants pathological research.
With the continuous development of Protocols in Molecular Biology, pathogen is carried out specifically using technologies such as PCR, detection of plasma
It is more and more with the successful examples of rapid molecular detection, but these Protocols in Molecular Biologies there is also to a certain extent one
A little weak points, such as immunoassay technology takes time and effort in the preparation process of serum, is frequently subjected to antiserum quality
Affect, thereby increases and it is possible to there is cross reaction, specificity is poor, easily cause false positive, PCR Fast Detection Techniques mainly include conventional
PCR, nest-type PRC（Nest-PCR）With real-time fluorescence quantitative PCR etc., round pcr detection time is longer, needs by PCR instrument, solidifying
The valuable instrument and equipments such as glue imaging system, are unfavorable for the upper popularization and application of basic unit's production, and above-mentioned shortcoming limits these advanced methods
Popularization and application.
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 highly active 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-
Answer product to be detected by transmissometer, real-time PCR instruments and gel-electrophoretic apparatus, but also can pass through
After SYBR Green I, calcein, hydroxynaphthol blue dyeing, naked eyes are recognized.Because LAMP reactions are simple, quick, efficient, Jing
Ji, and without the need for 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.LAMP detections at present are mainly used in people and animals' pathogen, food security and sanitary inspection
Survey, phytopathogen detection in report it is less, with regard to Phytophthora infestans germ LAMP detection there is not been reported.In LAMP detections
In, both at home and abroad majority researcher mainly designs special primer come to phytopathogen using rDNA/ITS sequences for target gene
It is used for quickly detecting, identifies.But the PCR molecular detection technology results of study of Phytophthora oomycetes show that rDNA/ITS is in phytophthora
Vary less between the sibling species of category, primer is designed from ITS sequence and is difficult to distinguish two similar high kinds.Therefore it is right
Phytophthora pathogen carries out high sensitivity and specific detection, primer should be designed from phytophthora other genes and be detected.Mesh
The front target gene for phytophthora PCR Molecular Detections mainly has elicitin genes, Ras families relatedYpt1Coding base
Cause, mitochondriaCox1WithCox2Encoding gene and may code storage albumenLpvGene etc..
It is contemplated that finding Molecular Detection target gene new in Phytophthora infestans germ.β-tubulinIt is eucaryote
It is special（House keeper）One of gene, in being widely present in eucaryote, guards very much on eucaryote is evolved, and there is no be directed at present
The target gene carries out the report of late disease bacteria Molecular Detection.
The present invention is based on ring mediated isothermal amplification（LAMP）Know-why, chooses phytophthoraβ-tubulinGene is detection
Target,β-tubulin6 regions of gene order devise 4 Phytophthora infestans germ specific primers, by reactant
System and the optimization of reaction condition, set up the Phytophthora infestans germ visualization LAMP with SYBR Green I as fluorescence developing indicator
Detection technique.The technical operation is simple, sensitivity and special high, and without the need for valuable instrument and equipment, is suitable for tomato field late blight
The early diagnosis of disease and the detection and identification of pathogen, effectively preventing timely to tomato late blight is significant.
The content of the invention
It is an object of the invention to provide LAMP primer composition thing and the application for detecting Phytophthora infestans germ, for existing
Morphological feature is based primarily upon to the detection of Phytophthora infestans germ and identification in technology, time-consuming for method, program is loaded down with trivial details, empirical
By force, the degree of accuracy is low, it is difficult to accomplish propagation, the popular problem of the timely monitoring and control pathogen to disease generation, Yi Jixian
Have PCR Molecular Detections need by the expensive instrument such as amplification instrument, and detection time it is longer the problems such as, there is provided Phytophthora infestans germ
New molecular detecting method, LAMP detections are carried out to Phytophthora infestans germ, and detection cycle is short, accuracy is high, sensitivity is high, naked eyes
Observation testing result.
Realize that the purpose of the present invention comprises the following steps（Technical scheme）：
1. Phytophthora infestans germ LAMP detects the design of specific primer sets thing：By determining Phytophthora infestans germ
（Phytophthora infestans）With other Fusariumsps（Phytophthora spp）'sβ-tubulinGene order is right
Phytophthora and other pathogen difference inter-speciesβ-tubulinGene order compares, and is designed using online LAMP primer
Software Primer software Explorer V4 (http://primerexplorer.jp/elamp4.0.0/
index.html;Eiken Chemical Co., Japan) a set of Phytophthora infestans germ specificity LAMP primer group is designed, by
1 pair of outside primers F 3/B3 and 1 pair of inner side primers F IP/BIP composition, F3/B3 and FIP/BIP primer sequences are as follows：F3: 5’-
GCCGATGAGGTCATGTGC-3 ', B3:5 '-GTTCACGGCCAGCTTACG-3 ', FIP: 5’-AGTGGGGGTGGTGAGCTT
CACTG-GACAATGAGGCCCTGTA-3 ', BIP: 5’-GGTGACCTGAACCACTTGGTGTGTTC- AGCTGACCGGGGAA
-3’。
2. the foundation of Phytophthora infestans germ 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, concrete grammar 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 L 2%CTAB（16
Alkyl trimethyl ammonium bromide）Extract（2% CTAB；100 mmol/L Tris-HCl, pH 8.0；20 mmol/L EDTA,
pH8.0；1.4 mol/L NaCl）With 90 L SDS（Neopelex）【Note：CTAB, SDS need 60 DEG C of preheatings】, make
Mixed with oscillator vibration, 60 DEG C of water-bath 1h（DNA is discharged into buffer solution）, 12000 rmin-115 min are centrifuged；Take supernatant
The L of liquid 700, plus equal-volume phenol, chloroform, isoamyl alcohol mixed liquor（Volume ratio 25:24:1）, gently vibration mixing, 12000 r
min-19 min are centrifuged；The L of supernatant 500 is taken, adds equal-volume chloroform to extract again once, 12000 rmin-1Centrifugation 5
min；The L of supernatant 350 is taken, the molL of 1/10 volume 3 is added-1NaAc and 2 times of volume absolute ethyl alcohol, -20 DEG C of precipitations 30
Min, 12000 rmin-15 min are centrifuged；Abandoning supernatant, adds the ethanol of 700 L ice 70% to be washed（Slightly it is centrifuged；Incline and
Supernatant）, alcohol-free taste is dried on superclean bench, add 30 ~ 60 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 it is stand-by to be diluted to 100 ng/ L.
When there is Phytophthora infestans germ in for detecting plant tissue, DNA, concrete mistake are extracted using NaOH rapid cleavages method
Journey is as follows：The mol/L NaOH of 10 L 0.5 are added in every milligram of plant tissue, tissue is fully milled to after paste in mortar
In proceeding to 1.5mL centrifuge tubes, 12,000 rpm are centrifuged 6 min, take the L of supernatant 5 and add the mol/L Tris- of 495 L 0.1
HCl（pH=8.0）It is well mixed, takes 1.0 L and expanded as pcr template；
When there is Phytophthora infestans germ in for detecting pedotheque, 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 Outside primer F3/B3 and interior
Side primers F IP/BIP carries out LAMP amplifications, and LAMP detects reaction system for 25 μ L, including 5 μM of Outside primer F3 and B3 each 1.0
The each 1.0 μ L of μ L, 40 μM of inner primers FIP and BIP, LAMP reaction mixtures【40 mM Tris-HCl, 20 mM (NH4)2SO4, 20 mM KCl, 16 mM MgSO4, 1.6 mol/L glycine betaines (Betaine), 2.0 mM dNTPs, 0.2% Trion X-
100】12.5 μ L, 8 UBstThe μ L of polymerase 1.0, the μ L of DNA profiling 1.0, with sterilizing ultra-pure water 25 μ L are complemented to；
（3）LAMP reaction conditions：64 DEG C of 60 min of incubation；
（4）The measure of reaction result：Using fluorescent dye visual observations method, after LAMP reactions terminate, in the expansion of LAMP reactions
Add the μ L of developer SYBR green I 1.0, the result that develops the color to observe that the judgement of green fluorescence is the positive in volume increase thing, exist kind
Eggplant blight bacterium, it is orange（Crocus）It is judged as feminine gender, there is no Phytophthora infestans germ.
Beneficial effects of the present invention：The present invention establishes the quick, easy of Phytophthora infestans germ, high specificity, sensitivity
High LAMP detection technique systems, the detection of Phytophthora infestans germ in can be used to carrying disease germs plant tissue and soil, or for tomato
The morbidity early stage of late blight, initial stage detection, for the best period tool for determining disease control is of great significance.
The present invention compared with prior art, with following technical advantage and good effect：
1st, high specificity, reliable results：The selection of target gene is one of key factor of LAMP detections.What regular-PCR was commonly used
Target gene has an Internal Transcribed Spacer (Internal transcribed space, ITS), but Phytophthora
The molecular detection technology result of study of oomycetes shows that rDNA-ITS is varied less between the sibling species of Phytophthora, from ITS sequence
Upper design primer is difficult to distinguish two similar high kinds.Therefore high sensitivity and specificity are carried out to Phytophthora pathogen
Detection, primer should be designed from phytophthora other genes and be detected.β-tubulinIt is that eucaryote is looked after the house（House keeper）Gene it
One, in being widely present in eucaryote, guard very much on eucaryote is evolved, there is abundant change in inter-species, be to compare
The more preferable Molecular Detection targets of rDNA-ITS.The present invention analyzes Phytophthora infestans germβ-tubulinGene and other pathogens
Difference in sequence, choose 6 specific regions, devise the LAMP primer of 4 specificity, in 6 regions any region with
Primer is mismatched and can not carry out nucleic acid amplification, with very strong specificity.The present invention using it is designed go out LAMP primer base
Phytophthora infestans germ LAMP detection method is established on plinth, only Phytophthora infestans germ can detect, and other pathogens are not
Detect, test of many times result is consistent, illustrate 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 Phytophthora infestans germ on DNA level
L, with very high sensitivity.
3rd, practicality is good：The LAMP detection method of the present invention needs to want thermal cycler unlike PCR detection methods（PCR instrument）Etc. expensive
Weight instrument and equipment, has thus broken away from the dependence to expensive equipments such as thermal cyclers, as long as there is stable thermal source, LAMP reactions
Can just occur, have greatly expanded the scope that LAMP is used.The LAMP reactions of the present invention simultaneously only need to enter in thermostat water bath
OK, reaction terminate the color change that passes through afterwards just can direct judged result, so as to increased it in the plant and soil for carrying disease germs
The using value of middle detection.
4th, it is easy to operate quick：The LAMP method of the detection Phytophthora infestans germ that the present invention is provided is overcome in prior art
Cycle length needed for the biological detection method of Phytophthora infestans germ, waste time and energy, loaded down with trivial details, poor specificity and PCR detection techniques are needed
Want the expensive equipments such as thermal cycler, it is impossible to the problem of quick detection Phytophthora infestans germ.Detection method is in 64 DEG C of isothermals
Under the conditions of, energy is quick, convenient, efficient, height specifically, with sensitivity detects Phytophthora infestans germ, it is not necessary to complex instrument, only needs
One thermostatic equipment, can preferably meet the Site Detection to Phytophthora infestans germ.
Description of the drawings
Fig. 1 is the LAMP specific detections of Phytophthora infestans germ of the present invention.1,2 is Phytophthora infestans germ in figure, and 3-7 divides
Not Wei phytophthora blight of pepper, soyabean phytophthora, Phytophthora nicotianae Breda, Phytophthora cactorum bacterium, palm mould bacterium, 8 is negative control, and 1-2 shows
Green fluorescence.
Fig. 2 is Phytophthora infestans germ LAMP detection sensitivities of the present invention.In figure 1-9 template DNAs concentration be respectively 1ng,
100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg, 100ag, 10 ag, 10 is negative control, and 1-6 shows green fluorescence.
Fig. 3 is detection method to disease plant and the detection with Phytophthora infestans germ in soil bacteria.1 is in figure
Positive control, 2-3 is tomato late blight incidence of leaf, and 4 is tomato late blight morbidity field soil, and 5-6 is healthy tomato leaf,
7 is autoclaving soil, and 8 is negative control, and 1-4 shows green fluorescence.
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：Tomato late blight collarium mediated isothermality amplification（LAMP）Detection specific primer sets thing design and
Primer specificity is verified
1. the extraction of strains tested genomic DNA
Strains tested is extracted using CTAB methods（Table 1）Genomic DNA, concrete grammar 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 L 2%CTAB（Cetyl trimethylammonium bromide）
Extract（2% CTAB；100 mmol/L Tris-HCl, pH 8.0；20 mmol/L EDTA, pH8.0；1.4 mol/L
NaCl）With 90 L SDS（Neopelex）【Note：CTAB, SDS need 60 DEG C of preheatings】, mixed using oscillator vibration,
60 DEG C of water-bath 1h（DNA is discharged into buffer solution）, 12000 rmin-115 min are centrifuged；Take the L of supernatant 700, plus equal-volume
Phenol, chloroform, isoamyl alcohol mixed liquor（Volume ratio 25:24:1）, gently vibration mixing, 12000 rmin-19 min are centrifuged；Take
The L of clear liquid 500, adds equal-volume chloroform to extract again once, 12000 rmin-15 min are centrifuged；The L of supernatant 350 is taken, plus
Enter the molL of 1/10 volume 3-1NaAc and 2 times of volume absolute ethyl alcohol, -20 DEG C of precipitations 30 min, 12000 rmin-1Centrifugation 5
min；Abandoning supernatant, adds the ethanol of 700 L ice 70% to be washed（Slightly it is centrifuged；Incline and fall supernatant）, on superclean bench
Alcohol-free taste is dried, 30 ~ 60 L TE are added（10 mmol/L Tris-HCl, 0.1 mmol/L EDTA, pH 8.0）Solution
Dissolved, obtained DNA solution, with UV spectrophotometer measuring DNA concentration and to be diluted to 100 ng/ L stand-by.
The strains tested of table 1
2. tomato late blight collarium mediated isothermality amplification（LAMP）The design of Specific primer pair thing
By determining Phytophthora infestans germ（Phytophthora infestans）With other Fusariumsps（Phytophthora spp）'sβ-tubulinGene order, to Phytophthora and other pathogen difference inter-speciesβ-tubulinGene order is compared
Analysis, 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
Cucurbit wilt bacterium specificity LAMP primer group, is made up of 1 pair of outside primers F 3/B3 and 1 pair of inner side primers F IP/BIP, F3/B3 and
FIP/BIP primer sequences are given：F3:5 '-GCCGATGAGGTCATGTGC-3 ', B3: 5’-GTTCACGGCCAGCTTACG-
3 ', FIP:5 '-AGTGGGGGTGGTGAGCTTCACTG-GACAATGAGGCCCTGTA-3 ', BIP:5’-GGTGACCTGAACCAC
TTGGTGTGTTC- AGCTGACCGGGGAA- 3’。
3. the foundation of Phytophthora infestans germ LAMP detection method and primer specificity are verified
DNA with the strains tested of table 1 carries out LAMP amplifications as template using Outside primer F3/B3 and inner primer FIP/BIP,
LAMP detection reaction systems are 25 μ L, including 5 μM of Outside primers F3 and B3 each 1.0 μ L, 40 μM of inner primers FIP and BIP
Each 1.0 μ L, LAMP reaction mixtures【40 mM Tris-HCl, 20 mM (NH4)2SO4, 20 mM KCl, 16 mM MgSO4,
1.6 mol/L glycine betaines (Betaine), 2.0 mM dNTPs, 0.2wt.% Trion X-100】12.5 μ L, 8 UBstPolymerization
The μ L of enzyme 1.0, the μ L of DNA profiling 1.0, with sterilizing ultra-pure water 25 μ L are complemented to；LAMP reaction conditions：64 DEG C of 60 min of incubation；Instead
Answer the measure of result：It is 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, the result that develops the color is added to observe that the judgement of green fluorescence is the positive, there is tomato in product
Late disease bacteria, it is orange（Crocus）It is judged as feminine gender, there is no Phytophthora infestans germ.
4. primer specificity the result
LAMP amplifications show, for only having Phytophthora infestans germ colour developing result that green fluorescence can be observed in the bacterial strain of examination, its
Remaining phytophthora and fungi colour developing result are orange（Accompanying drawing 1）, illustrate designed Phytophthora infestans germ Outside primer F3/B3 and interior
Side primers F IP/BIP can make a distinction Phytophthora infestans germ with other pathogens, with the specificity planted, can be used for tomato
The fast and reliable detection of late disease bacteria and identification.
Embodiment 2：Tomato late blight collarium mediated isothermality amplification（LAMP）Detection sensitivity is determined
1. the preparation of variable concentrations genomic DNA
Phytophthora infestans germ 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
Phytophthora infestans germ genomic DNA with variable concentrations as template, using Outside primer F3/B3 and inner primer FIP/
BIP carries out LAMP amplifications, and LAMP detection reaction systems are 25 μ L, including each 1.0 μ L of 5 μM of Outside primers F3 and B3,40 μM
The each 1.0 μ L of inner primer FIP and BIP, LAMP reaction mixtures【40 mM Tris-HCl, 20 mM (NH4)2SO4, 20 mM
KCl, 16 mM MgSO4, 1.6 mol/L glycine betaines (Betaine), 2.0 mM dNTPs, 0.2% Trion X-100】12.5μ
L, 8 UBstThe μ L of polymerase 1.0, the μ L of variable concentrations DNA profiling 1.0, with sterilizing ultra-pure water 25 μ L are complemented to；LAMP reacts bar
Part：64 DEG C of 60 min of incubation；The measure of reaction result：It is measured using fluorescent dye visual observations method, treats LAMP reaction knots
Shu Hou, adds the μ L of developer SYBR green I 1.0, colour developing result to observe green fluorescence in the amplified production of LAMP reactions
Judgement for the positive, it is orange（Crocus）It is judged as feminine gender.
3. LAMP expands sensitivity technique result
LAMP expands sensitivity technique result and shows, 1ng, 100pg, 10 pg, 1 pg, 100 fg, the tomato of 10 fg/ μ L concentration
Late disease bacteria genomic DNA colour developing result can be observed green fluorescence, and remaining concentration and negative control colour developing result are orange, are said
Bright designed Phytophthora infestans germ Outside primer F3/B3 and inner primer FIP/BIP is expanded by LAMP, to tomato late blight
The detection sensitivity of bacterium is up to 10 fg/ μ L（Accompanying drawing 2）.
Embodiment 3：The LAMP detections of Phytophthora infestans germ in incidence tissue
Sample collection：From Fujian Zhouning County, Sanming City, the typical blade of Liancheng collection tomato late blight disease symptom and healthy leaves band
Go back to laboratory standby；
The extraction of plant tissue DNA：DNA is extracted using NaOH rapid cleavages method, detailed process is as follows：To every milligram of plant tissue
The middle addition mol/L NaOH of 10 L 0.5, will organize in mortar and are fully milled to be proceeded in 1.5mL centrifuge tubes after paste, and 12,
000 rpm is centrifuged 6 min, takes the L of supernatant 5 and adds the mol/L Tris-HCl of 495 L 0.1（pH=8.0）It is well mixed, takes
1.0 L are expanded as pcr template.
LAMP augmentation detections and observation：DNA with said extracted as template, using Outside primer F3/B3 and inner primer
FIP/BIP carries out LAMP amplifications, and LAMP detection reaction systems are 25 μ L, including each 1.0 μ L of 5 μM of Outside primers F3 and B3, and 40
μM each 1.0 μ L of inner primer FIP and BIP, LAMP reaction mixtures【40 mM Tris-HCl, 20 mM (NH4)2SO4, 20 mM
KCl, 16 mM MgSO4, 1.6 mol/L glycine betaines (Betaine), 2.0 mM dNTPs, 0.2wt.% Trion X-100】
12.5 μ L, 8 UBstThe μ L of polymerase 1.0, the μ L of DNA profiling 1.0, with sterilizing ultra-pure water 25 μ L are complemented to；LAMP reaction conditions：
64 DEG C of 60 min of incubation；The measure of reaction result：It is measured using fluorescent dye visual observations method, treats that LAMP reactions terminate
Afterwards, the μ L of developer SYBR green I 1.0, colour developing result is added to observe green fluorescence in the amplified production of LAMP reactions
It is judged as the positive, it is orange（Crocus）It is judged as feminine gender.
Testing result：Testing result（Accompanying drawing 3）Show, the blade of tomato late blight morbidity is expanded by LAMP, colour developing knot
Fruit can be observed green fluorescence, illustrate there is Phytophthora infestans germ, and healthy leaves and negative control colour developing result are orange, are said
Bright to there is no Phytophthora infestans germ, the set technology can be used for the rapid molecular detection of Phytophthora infestans germ in plant tissue.
Embodiment 4：LAMP detections with Phytophthora infestans germ in soil bacteria
Sample collection：From the field collection plant root soil band that Fujian Zhouning County, Sanming City and Liancheng tomato late blight are fallen ill serious
Laboratory is gone back to standby, with autoclaved soil as control；
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 L is taken and is expanded as pcr template.
LAMP augmentation detections and observation：DNA with said extracted as template, using Outside primer F3/B3 and inner primer
FIP/BIP carries out LAMP amplifications, and LAMP detection reaction systems are 25 μ L, including each 1.0 μ L of 5 μM of Outside primers F3 and B3, and 40
μM each 1.0 μ L of inner primer FIP and BIP, LAMP reaction mixtures【40 mM Tris-HCl, 20 mM (NH4)2SO4, 20 mM
KCl, 16 mM MgSO4, 1.6 mol/L glycine betaines (Betaine), 2.0 mM dNTPs, 0.2% Trion X-100】 12.5
μ L, 8 UBstThe μ L of polymerase 1.0, the μ L of DNA profiling 1.0, with sterilizing ultra-pure water 25 μ L are complemented to；LAMP reaction conditions：64
DEG C incubate 60 min；The measure of reaction result：It is measured using fluorescent dye visual observations method, after LAMP reactions terminate,
The μ L of developer SYBR green I 1.0, colour developing result is added to observe sentencing for green fluorescence in the amplified production of LAMP reactions
Break as the positive, it is orange（Crocus）It is judged as feminine gender.
Testing result：Testing result（Accompanying drawing 3）Show, tomato late blight falls ill serious field soil DNA by LAMP expansions
Increase, colour developing result can be observed green fluorescence, illustrate there is Phytophthora infestans germ, and autoclaving soil and negative control colour developing
As a result it is orange, illustrates there is no Phytophthora infestans germ, the set technology can be used for the rapid molecular of Phytophthora infestans germ in soil
Detection.
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 the covering scope of the present invention.
SEQUENCE LISTING
<110>Inst. of Plant Protection, fujian Academy of Agricultural Science
<120>For detecting LAMP primer composition thing and the application of Phytophthora infestans germ
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence
<400> 1
gccgatgagg tcatgtgc 18
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> 2
gttcacggcc agcttacg 18
<210> 3
<211> 40
<212> DNA
<213>Artificial sequence
<400> 3
agtgggggtg gtgagcttca ctggacaatg aggccctgta 40
<210> 4
<211> 40
<212> DNA
<213>Artificial sequence
<400> 4
ggtgacctga accacttggt gtgttcagct gaccggggaa 40
Claims (4)
1. it is used to detect the LAMP primer composition thing of Phytophthora infestans germ, it is characterised in that the Primer composition is by 4 spies
Specific primer is constituted, including 1 pair of outside primers F 3/B3 and 1 pair of inner side primers F IP/BIP, F3/B3 and FIP/BIP primer sequences
It is as follows：F3:5 '-GCCGATGAGGTCATGTGC-3 ', B3:5 '-GTTCACGGCCAGCTTACG-3 ', FIP: 5’-AGTGGG
GGTGGTGAGCTTCACTG-GACAATGAGGCCCTGTA-3 ', BIP:5’-GGTGACCTGAACCACTTGGTGTGTTC-AGCT
GACCGGGGAA- 3’。
2. a kind of Phytophthora infestans germ LAMP detection method, it is characterised in that comprise the following steps：
（1）Extract testing sample genomic DNA；
（2）The foundation of LAMP reaction systems：With step（1）The DNA of extraction is template, is drawn using the outside described in claim 1
Thing F3/B3 and inner primer FIP/BIP carry out LAMP amplifications, and LAMP detects reaction system for 25 μ L, including 5 μM of Outside primers
The each 1.0 μ L of F3 and B3,40 μM of inner primers FIP and BIP each 1.0 μ L, LAMP reaction mixtures 12.5 μ L, 8 UBstPolymerization
The μ L of enzyme 1.0, the μ L of DNA profiling 1.0, with sterilizing ultra-pure water 25 μ L are complemented to；
（3）LAMP reaction conditions：64 DEG C of 60 min of incubation；
（4）The measure of reaction result：It is measured using fluorescent dye visual observations method, after LAMP reactions terminate, in LAMP
The μ L of developer SYBR green I 1.0, the result that develops the color is added to observe that the judgement of green fluorescence is sun in the amplified production of reaction
Property, there is Phytophthora infestans germ, orange or crocus is judged as feminine gender, there is no Phytophthora infestans germ.
3. a kind of Phytophthora infestans germ LAMP detection method according to claim 2, it is characterised in that step（2）It is described
LAMP reaction mixtures it is 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, 0.2wt.% Trion X-100.
4. Primer composition as claimed in claim 1 is in the early diagnosis and the monitoring of germ, identification of tomato late blight
Using.
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| CN110878373A (en) * | 2019-11-14 | 2020-03-13 | 南京农业大学 | Recombinase polymerase amplification detection kit for phytophthora infestans and application thereof |
| CN111057788A (en) * | 2020-02-21 | 2020-04-24 | 福建省农业科学院植物保护研究所 | LAMP primer group for detecting panax notoginseng rust rot and detection method |
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| CN104313175A (en) * | 2014-11-13 | 2015-01-28 | 南京林业大学 | LAMP detection primer composition for phytophthora infestans and LAMP detection kit and LAMP detection method of LAMP detection primer composition |
| CN105063040A (en) * | 2015-09-08 | 2015-11-18 | 福建省农业科学院植物保护研究所 | Tomato late blight PCR detection primer, kit and tomato late blight PCR detection method |
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| CN108330178A (en) * | 2017-10-26 | 2018-07-27 | 福建省农业科学院植物保护研究所 | It is a kind of detection tomato early blight bacterium loop-mediated isothermal amplification (LAMP) primer and application |
| CN110878373A (en) * | 2019-11-14 | 2020-03-13 | 南京农业大学 | Recombinase polymerase amplification detection kit for phytophthora infestans and application thereof |
| CN111057788A (en) * | 2020-02-21 | 2020-04-24 | 福建省农业科学院植物保护研究所 | LAMP primer group for detecting panax notoginseng rust rot and detection method |
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