CN106520977B - The primer and method of ring mediated isothermal amplification method detection causing root rot disease of Medicago sativa bacterium - Google Patents
The primer and method of ring mediated isothermal amplification method detection causing root rot disease of Medicago sativa bacterium Download PDFInfo
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Abstract
The invention discloses the primer and method of a kind of ring mediated isothermal amplification method detection causing root rot disease of Medicago sativa bacterium, the primer includes one group of outer primer F3/B3 and one group of inner primer FIP/BIP, and the base sequence of the primer is as shown in SEQ ID NO.1-4.The described method includes: extracting sample to be tested DNA；Using DNA as template, LAMP isothermal duplication is carried out using the primer；Fluorescent dye is added in LAMP amplified production to develop the color, observes the color of LAMP amplified production, product is analyzed, causing root rot disease of Medicago sativa bacterium is judged whether there is.The primer and its quick LAMP detection method can carry out quick, sensitive, accurate detection to causing root rot disease of Medicago sativa bacterium in production practice, it can be used for the early diagnosis of field diseases and the monitoring and identification of germ simultaneously, and it is easy to operation, reliable technology and theoretical foundation are provided for the prevention and treatment of causing root rot disease of Medicago sativa.
Description
Technical field
The invention belongs to corps diseases detection, identification and Prevention Technique fields, and in particular to the ring of causing root rot disease of Medicago sativa bacterium
It is quick, sensitive and special to can be used for causing root rot disease of Medicago sativa bacterium for mediated isothermality amplification (LAMP) detection primer and its rapid detection method
Molecular Detection, while can be used for causing root rot disease of Medicago sativa early diagnosis and germ monitoring and identification.
Background technique
Clover (Medicago sativa) it is pulse family clover category herbaceos perennial, it is that the world and China are most important
Legume Forage Species are known as " King of Pasture ".Alfalfa output is high, and protein content is high, and palatability is good, and well developed root system,
With biological nitrogen fixation ability, be conducive to the growth and water and soil conservation of other plant, in production of developing animal husbandry, improve the ecological environment
Etc. play irreplaceable role.30% or more of the China clover cultivated area Yi Zhan perennial grass at present, accounts for whole
The 25% of herbage, with the continuous introduction of New alfalfa cultivars, the continuous improvement of cultivation technique, liquid manure level, the hair of causing root rot disease of Medicago sativa
Life is also on the rise, and causing root rot disease of Medicago sativa is a kind of destructive fungi soil-borne disease in ALFALFA PRODUCTION, and it is big to often result in alfalfa output
Width decline, quality reduce, and Field diseases are typically up to 5%~7%, grave illness up to 15%~21%, more serious disease incidence up to 92%,
The extensive use for seriously affecting clover constitutes certain threat to ALFALFA PRODUCTION, becomes the exploitation of limitation alfalfa industry and ecological construction
One of principal element.
Domestic and foreign scholars have carried out a large amount of research for the cause of disease of causing root rot disease of Medicago sativa, so far, cause the disease of the disease
Opportunistic pathogen substantially has following several: clover phytophthora (Phytophthora megasperma), fusarium fungus (Fusarium spp.), Rhizoctonia solani Kuhn (Rhizoctonia solani) and pythium oomycetes (Pythoum spp.) etc..Causing root rot disease of Medicago sativa disease
Opportunistic pathogen is complex, but mostly related with sickle-like bacteria, wherein eggplant sickle-like bacteria (Fusarium solani) it is one of Dominantpathogen.
This research is using causing root rot disease of Medicago sativa bacterium --- and eggplant sickle-like bacteria establishes quick detection, the identification technology of the pathogen, is as research object
The Accurate Diagnosis of causing root rot disease of Medicago sativa and in time prevention and treatment provide technical support and theoretical foundation.
The Accurate Diagnosis of disease is the basis of disease prevention and control, for a long time, the classical way of classification of fungi and identification be according to
It is carried out according to the morphology and anatomy phenotypic characteristic and habitat characteristics of fungi.It is identified with morphology, method is simply easy
Row does not need expensive instrument, but needs symptom or obtain the pure culture of fungi, more to there is identification experience abundant and enough
Documents and materials.Categorizing system based on morphosis is to a certain extent by the interference of human factor, and fungi
That structure is complicated is changeable for form and dissection, obtains that its is sexual or need the long period without sexual organ, some fungal species are not easy even not
Sexual propagation structure can be formed, similar kind of form and mutation are difficult to differentiate between；The variation of some fungal morphologies is big, classification, identification
According to less stable；Some fungal morphology features are difficult to be quantitatively described, and are easy to cause error, these factors are to taxonomic identification
Bring inconvenience so that the traditional morphology and anatomy classification of pathogen, identification method be not able to satisfy disease quickly, it is quasi-
It really identifies, the demand of diagnosis.
With the development of biochemistry, science of heredity and molecular biology, technology is provided for the taxonomic identification of pathogen
Condition.Such as serology immunological technique, Standard PCR, nest-type PRC and real-time fluorescence quantitative PCR technology are the classification of pathogen
Identification provides high sensitivity, quick detection technique.But there is also one to a certain extent for these Protocols in Molecular Biologies
A little shortcomings, the preparation process such as immunoassay technology in serum take time and effort, and are frequently subjected to antiserum quality
It influences, thereby increases and it is possible to which there are cross reactions, and specificity is poor, be easy to cause false positive, PCR Fast Detection Technique mainly includes routine
PCR, nest-type PRC (Nest-PCR) and real-time fluorescence quantitative PCR etc., round pcr detection time is longer, needs to rely on valuable, smart
The valuable instrument and equipments such as close PCR instrument, gel imaging system and corresponding new and high technology personnel, thus testing cost is improved,
And limit its scope of application, it is unfavorable for promoting and applying in base's production, 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, accurate and cheap nucleic acid efficient amplification technology of this Rong Yan company exploitation, which can be 60 DEG C ~ 65
Under DEG C constant temperature, using high activity strand displacement archaeal dna polymerase (BstDNA polymerase) to target DNA fragment into
Row specific amplification.In 1 hour, the target gene of a small amount of copy number can be expanded to 109~1010A copy number.Due to
LAMP amplification procedure relies on identification 6 isolated areas of target sequence, so atopic is very strong, and amplification process be
It is carried out under constant temperature, common water-bath or there is the equipment of stable heat source to be just able to satisfy reaction to require, testing cost drops significantly
Low, LAMP reaction product can not only be detected by transmissometer, real-time PCR instrument and gel-electrophoretic apparatus, but also
After can be by SYBR Green I, calcein, hydroxynaphthol blue dyeing, naked eyes be identified, greatly reduce to experimenter
Injury, and increase the application value in field.At present LAMP detection be mainly used in people and animals' pathogen, food safety and
The detection of environmental sanitation, phytopathogen detection in report it is less, about causing root rot disease of Medicago sativa bacterium LAMP detection both at home and abroad it is equal
It has not been reported.
Summary of the invention
The object of the present invention is to provide the primers and method of ring mediated isothermal amplification method detection causing root rot disease of Medicago sativa bacterium, for existing
Have in technology and morphological feature is based primarily upon to causing root rot disease of Medicago sativa bacterium detection and identification, time-consuming for method, program is cumbersome, empirical
By force, accuracy is low, it is difficult to accomplish the propagation for the timely monitoring and control pathogen that disease occurs, the problem of prevalence, Yi Jixian
There is PCR Molecular Detection to need by the expensive instruments such as amplification instrument, and the problems such as detection time is longer, provides causing root rot disease of Medicago sativa bacterium
New molecular detecting method carries out LAMP detection to causing root rot disease of Medicago sativa bacterium, and detection cycle is short, accuracy is high, sensitivity is high, naked eyes
Observe testing result.
It achieves the object of the present invention and includes the following steps (technical solution):
1. the design of causing root rot disease of Medicago sativa bacterium LAMP detection specific primer sets object: by measuring causing root rot disease of Medicago sativa bacterium
(Fusarium solani) and other Fusariumsps (Fusarium spp) translation elongation factor 1-
alpha gene(EF-1alpha) gene order, to reaping hook category and other pathogen difference inter-speciesEF-1alphaGene order into
Row compare analysis, using online LAMP primer design software Primer software Explorer V4 (http: //
primerexplorer.jp/elamp4.0.0/index.html;Eiken Chemical Co., Japan) a set of lucerne of design
Mu pine root fungus specificity LAMP primer group is made of, primer sequence primers F IP/BIP in 1 external primers F 3/B3 and 1 pair
It is as follows: F3:5 '-CACCAAACCCTCTTGGCG-3 ', B3:5 '-GGCGACATACCAATGACGG-3 ', FIP:5 '-
TTACCGAGCTCAGCGGCTTC-CGC ATCACGTGGTTCACA-3 ', BIP:5 '-TGGGTCCTTGACAAGCTCAAGG-
GGGAG TCTCGAACTTCCAGA- 3’。
2. the foundation of causing root rot disease of Medicago sativa bacterium LAMP detection method, comprising the following steps:
(1) sample to be tested genomic DNA is extracted.
When for detecting pathogen pure culture, genomic DNA is extracted using CTAB method, the specific method is as follows: taking
A small amount of hypha powder (hypha powder, which had just covered semicircular base, to be advisable) in 1.5 mL centrifuge tubes, is added 900 μ L 2%CTAB(16
Alkyl trimethyl ammonium bromide) extracting solution (2% CTAB；100 mmol/L Tris-HCl, pH 8.0；20 mmol/L EDTA,
pH8.0；1.4 mol/L NaCl) and 90 μ L SDS(neopelexes) [note: CTAB, SDS need 60 DEG C of preheatings], make
Vibrated and mixed with oscillator, 60 DEG C of water-bath 1h(DNA are discharged into buffer), 12000 rmin-1It is centrifuged 15 min；Take supernatant
700 μ L of liquid adds isometric phenol, chloroform, isoamyl alcohol (25:24:1), and gently oscillation mixes, 12000 rmin-1It is centrifuged 9 min；
500 μ L of supernatant is taken, isometric chloroform is added and extracts again once, 12000 rmin-1It is centrifuged 5 min；Take 350 μ of supernatant
1/10 volume, 3 molL is added in L-1NaAc and 2 times of volume dehydrated alcohol, -20 DEG C of precipitatings 30 min, 12000 rmin-1
It is centrifuged 5 min；Liquid is discarded supernatant, addition 700 μ L ice, 70% ethyl alcohol, which is washed, (to be slightly centrifuged；Incline and fall supernatant), in ultra-clean work
Make to dry alcohol-free taste on platform, 30 ~ 60 μ L TE(10 mmol/L Tris-HCl, 0.1 mmol/L EDTA, pH 8.0 are added)
Solution is dissolved, and DNA solution is obtained, and with UV spectrophotometer measuring DNA concentration and is diluted to 100 ng/ μ L and is waited for
With.
For detecting in plant tissue there are when causing root rot disease of Medicago sativa bacterium, DNA, specific mistake are extracted using NaOH rapid cleavage method
Journey is as follows: 10 μ L, 0.5 mol/L NaOH being added into every milligram of plant tissue, after tissue is sufficiently milled to paste in mortar
It is transferred in 1.5mL centrifuge tube, 12,000 rpm are centrifuged 6 min, take 5 μ l of supernatant that 495 μ L, 0.1 mol/L Tris- is added
HCl(pH=8.0) it is uniformly mixed, take 1.0 μ L to be expanded as pcr template；
For detecting in pedotheque there are when causing root rot disease of Medicago sativa bacterium, using soil DNA extracts kit, DNA is extracted.
(2) foundation of LAMP reaction system: the DNA extracted using step (1) using outer primer F3/B3 and interior draws as template
Object FIP/BIP carries out LAMP amplification, and LAMP detects reaction system as 25 μ L, including 5 μM of each 1.0 μ L of Outside primer F3 and B3,
Each 1.0 μ L, LAMP reaction mixture of 40 μM of inner primers FIP and BIP [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 UBst1.0 μ L of polymerase, 1.0 μ L of DNA profiling complement to 25 μ L with sterilizing ultrapure water；
(3) LAMP reaction condition: 64 DEG C of 60 min of incubation；
(4) measurement of reaction result: using fluorescent dye visual observations method, after reaction to LAMP, reacts in LAMP
Amplified production in I 1.0 μ L of color developing agent SYBR green is added, colour developing result observes that the judgement of green fluorescence for the positive, is deposited
In causing root rot disease of Medicago sativa bacterium, orange (crocus) is judged as negative, and causing root rot disease of Medicago sativa bacterium is not present.
Beneficial effects of the present invention: the present invention establishes the quick, easy of causing root rot disease of Medicago sativa bacterium, high specificity, sensitivity
High LAMP detection technique system, the detection of causing root rot disease of Medicago sativa bacterium in can be used for carrying disease germs plant tissue and soil, or it is used for clover
Morbidity early period of root rot, initial stage detection, for determining that the best period of disease control has a very important significance.
Compared with prior art, the present invention having technical advantage below and good effect:
1, high specificity, result are reliable: one of an important factor for selection of target gene is LAMP detection.Regular-PCR is normal
Target gene has Internal Transcribed Spacer (Internal transcribed space, ITS), however is permitted
More scholars think that the target can not explicitly distinguish Fusarium fungi and Fusarium oxysporum difference specialized form very much.Inventor is with general
It is found in the research of logical round pcr detection causing root rot disease of Medicago sativa bacteriumEF-1alphaGene order existsFusariumThe interior and sharp spore sickle of kind
Between spore bacterium difference specialized form different strains have certain conservative, inter-species there are variation abundant, be compared to rDNA-ITS,β‐ tubulinThe better Molecular Detection target of sequence.The present invention analyzes causing root rot disease of Medicago sativa bacteriumEF-1alphaGene and other fusariums
Difference of the bacterium in sequence chooses 6 specific regions, devises 4 specific LAMP primers, any region in 6 regions
It not can be carried out nucleic acid amplification with primer mismatch, there is very strong specificity.The present invention utilizes designed LAMP primer out
On the basis of establish causing root rot disease of Medicago sativa bacterium LAMP detection method, causing root rot disease of Medicago sativa bacterium can detected, and other pathogens are not examined
It measures, test of many times result is consistent, illustrates LAMP detection method high specificity of the present invention, as a result reliably.
2, high sensitivity: the present invention can reach 10fg/ μ L to the detection sensitivity of causing root rot disease of Medicago sativa bacterium on DNA level,
With very high sensitivity；
3, quickly: detection method is applied, testing result can be obtained in 1~1.5h, and previous PCR or nido
PCR detection needs 4~6h that testing result just can be obtained, and the method for the invention substantially reduces operating time, fast and easy；
4, application is good: LMAP amplified reaction of the present invention expands at one temperature, does not need expensive PCR amplification instrument,
Convenient for base's popularization and use；
5, testing result is intuitive, visually can determine whether: amplified production of the present invention can be by adding color developing agent to be dyed, and green is
The positive, i.e. bacterium containing causing root rot disease of Medicago sativa in sample to be tested, orange is feminine gender, is illustrated in sample to be tested without causing root rot disease of Medicago sativa bacterium, meat
Eye can determine whether testing result, be not necessarily to electrophoresis detection and gel imaging.
Detailed description of the invention
Fig. 1 is the LAMP specific detection of causing root rot disease of Medicago sativa bacterium of the present invention.1-3 is causing root rot disease of Medicago sativa bacterium, and 4-5 is respectively
Cucumber fusarium axysporum, wheat scab, fusarium avenaceum, 6 be negative control, No. 1-3 display green fluorescence.
Fig. 2 is causing root rot disease of Medicago sativa bacterium LAMP detection sensitivity of the present invention.1-9 template DNA concentration is respectively 1ng, 100
Pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg, 100ag, 10 ag, 10 be negative control, No. 1-6 display green fluorescence.
Fig. 3 is detection of the detection method to disease plant and with causing root rot disease of Medicago sativa bacterium in soil bacteria.1 is the positive
Control, 2 be negative control, and 3 be morbidity root tissue, and 4 be healthy root tissue, and 5 be morbidity stem tissue, and 6 be healthy stem
Tissue, 7 be morbidity root soil, and 8 be high pressure sterilization soil, 1,3,5, No. 7 display green fluorescence.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is further elaborated, but is not intended to limit the scope of the invention.Below
Embodiment is according to conventional laboratory conditions, or has delivered operating technology regulation described in pertinent literature, or built according to manufacturer
The experiment condition of view.
Embodiment 1: causing root rot disease of Medicago sativa collarium mediated isothermality amplification (LAMP) detects design and the primer spy of specific primer
Opposite sex verifying
1. the extraction of strains tested genomic DNA
Strains tested (table 1) genomic DNA is extracted using CTAB method, the specific method is as follows: taking a small amount of hypha powder in 1.5
In mL centrifuge tube (hypha powder, which had just covered semicircular base, to be advisable), 900 μ L 2%CTAB(cetyl trimethyl brominations are added
Ammonium) extracting solution (2% CTAB；100 mmol/L Tris-HCl, pH 8.0；20 mmol/L EDTA, pH8.0；1.4 mol/L
NaCl) and 90 μ L SDS(neopelexes) [note: CTAB, SDS need 60 DEG C preheating], using oscillator vibrate mix,
60 DEG C of water-bath 1h(DNA are discharged into buffer), 12000 rmin-1It is centrifuged 15 min；700 μ L of supernatant is taken, is added isometric
Phenol, chloroform, isoamyl alcohol (25:24:1), gently oscillation mixes, 12000 rmin-1It is centrifuged 9 min；500 μ L of supernatant is taken, is added
Enter isometric chloroform to extract again once, 12000 rmin-1It is centrifuged 5 min；350 μ L of supernatant is taken, 1/10 volume 3 is added
mol·L-1NaAc and 2 times of volume dehydrated alcohol, -20 DEG C of precipitatings 30 min, 12000 rmin-1It is centrifuged 5 min；It discards
Clear liquid, addition 700 μ L ice, 70% ethyl alcohol, which is washed, (to be slightly centrifuged；Incline and fall supernatant), it is dried on superclean bench alcohol-free
30 ~ 60 μ L TE(10 mmol/L Tris-HCl, 0.1 mmol/L EDTA, pH 8.0 are added in taste) solution dissolved, obtained
To DNA solution, with UV spectrophotometer measuring DNA concentration and to be diluted to 100 ng/ μ L stand-by.
1 strains tested of table
The design of causing root rot disease of Medicago sativa collarium mediated isothermality amplification 2. (LAMP) special primer
By measurement causing root rot disease of Medicago sativa bacterium (Fusarium solani) and other Fusariumsps (Fusarium spp)
translation elongation factor 1-alpha gene(EF-1alpha) gene order, to reaping hook category and other
Pathogen difference inter-speciesEF-1alphaGene order is compared, and utilizes online LAMP primer design software Primer
software Explorer V4 (http://primerexplorer.jp/elamp4.0.0/index.html; Eiken
Chemical Co., Japan) a set of causing root rot disease of Medicago sativa bacterium specificity LAMP primer of design, by 1 external primers F 3/B3 and 1 pair
Inner primer FIP/BIP composition, primer sequence are as follows: F3:5 '-CACCAAACCCTCTTGGCG-3 ', B3:5 '-
GGCGACATACCAATGACGG-3 ', FIP:5 '-TTACCGAGCTCAGCGGCTTC-CGC ATCACGTGGTTCACA-3 ',
BIP: 5’- TGGGTCCTTGACAAGCTCAAGG-GGGAG TCTCGAACTTCCAGA- 3’。
3. the foundation of causing root rot disease of Medicago sativa bacterium LAMP detection method and primer specificity verifying
Using the DNA of 1 strains tested of table as template, LAMP amplification is carried out using outer primer F3/B3 and inner primer FIP/BIP,
It is 25 μ L, including 5 μM of outer primer F3 and B3 each 1.0 μ L, 40 μM of inner primer FIP and BIP each 1.0 that LAMP, which detects reaction system,
μ L, LAMP reaction mixture [40 mM Tris-HCl, 20 mM (NH4)2SO4, 20 mM KCl, 16 mM MgSO4, 1.6
Mol/L glycine betaine (Betaine), 2.0 mM dNTPs, 0.2% Trion X-100] 12.5 μ L, 8 UBst1.0 μ of polymerase
L, 1.0 μ L of DNA profiling complement to 25 μ L with sterilizing ultrapure water；LAMP reaction condition: 64 DEG C of 60 min of incubation；Reaction result
Measurement: use fluorescent dye visual observations method, after reaction to LAMP, LAMP reaction amplified production in be added colour developing
I 1.0 μ L of agent SYBR green, colour developing result observes that the judgement of green fluorescence is the positive, orange there are causing root rot disease of Medicago sativa bacterium
(crocus) is judged as negative, and causing root rot disease of Medicago sativa bacterium is not present.
4. primer specificity verification result
LAMP amplification shows that green fluorescence can be observed in the causing root rot disease of Medicago sativa bacterium colour developing result for examination, remaining reaping hook
Bacterium and fungi colour developing result are orange (attached drawing 1), illustrate designed causing root rot disease of Medicago sativa bacterium outer primer F3/B3 and inner primer FIP/
BIP can distinguish causing root rot disease of Medicago sativa bacterium and other pathogens, have the specificity of kind, it is fast to can be used for causing root rot disease of Medicago sativa bacterium
The reliable detection and identification of speed.
Embodiment 2: causing root rot disease of Medicago sativa collarium mediated isothermality amplification (LAMP) detection sensitivity measurement
1. the preparation of various concentration genomic DNA
Causing root rot disease of Medicago sativa bacterium genomic DNA is diluted with sterile ultrapure water, the series for being configured to 10 times of orders of magnitude is dense
It spends spare.
2. LAMP detection method sensitivity determination and result observation
Using the causing root rot disease of Medicago sativa bacterium genomic DNA of various concentration as template, outer primer F3/B3 and inner primer FIP/ are utilized
BIP carries out LAMP amplification, and it is 25 μ L, including 5 μM of outer primer F3 and B3 each 1.0 μ L that LAMP, which detects reaction system, in 40 μM
Each 1.0 μ L, LAMP reaction mixture of primers F IP and BIP [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] and 12.5 μ L, 8 UBst1.0 μ L of polymerase, 1.0 μ L of various concentration DNA profiling complement to 25 μ L with sterilizing ultrapure water；LAMP reaction condition: 64
DEG C incubate 60 min；The measurement of reaction result: using fluorescent dye visual observations method, after reaction to LAMP, anti-in LAMP
I 1.0 μ L of color developing agent SYBR green is added in the amplified production answered, colour developing result observes that the judgement of green fluorescence is the positive,
Orange (crocus) is judged as negative.
3. LAMP expands sensitivity technique result
LAMP expand sensitivity technique the result shows that, 1ng, 100pg, 10 pg, 1 pg, 100 fg, 10 fg/ μ L concentration
Green fluorescence can be observed in causing root rot disease of Medicago sativa bacterium genomic DNA colour developing result, remaining concentration and negative control colour developing result are orange
Color illustrates that designed causing root rot disease of Medicago sativa bacterium outer primer F3/B3 and inner primer FIP/BIP is expanded by LAMP, to clover root-rot
The detection sensitivity of germ is up to 10 fg/ μ L(attached drawings 2).
Embodiment 3: the LAMP detection of causing root rot disease of Medicago sativa bacterium in incidence tissue
Sample acquisition: from Shaanxi, Gansu, the typical root of Ningxia acquisition causing root rot disease of Medicago sativa disease symptom, stem (from earth's surface
At 4-6cm) and healthy root and stem to take back laboratory spare；
The extraction of plant tissue DNA: DNA is extracted using NaOH rapid cleavage method, detailed process is as follows: to every milligram of plant
10 μ L, 0.5 mol/L NaOH is added in tissue, is transferred in 1.5mL centrifuge tube after tissue is sufficiently milled to paste in mortar,
12,000 rpm are centrifuged 6 min, take 5 μ l of supernatant that 0.1 Tris-HCl(pH=8.0 mol/L 495 μ L are added) it is uniformly mixed,
1.0 μ L are taken to be expanded as pcr template.
Augmentation detection and observation: using the DNA of said extracted as template, using outer primer F3/B3 and inner primer FIP/BIP into
Row LAMP amplification, it is 25 μ L, including 5 μM of each 1.0 μ L of outer primer F3 and B3,40 μM of inner primers that LAMP, which detects reaction system,
Each 1.0 μ L, LAMP reaction mixture of FIP and BIP [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] and 12.5 μ L, 8 UBst
1.0 μ L of polymerase, 1.0 μ L of DNA profiling complement to 25 μ L with sterilizing ultrapure water；LAMP reaction condition: 64 DEG C incubate 60
min；The measurement of reaction result: using fluorescent dye visual observations method, after reaction to LAMP, produces in the amplification of LAMP reaction
I 1.0 μ L of color developing agent SYBR green is added in object, colour developing result observes that the judgement of green fluorescence is the positive, orange (orange
Color) it is judged as negative.
Testing result: testing result (attached drawing 3) shows that the cucumber root of clover root-rot morbidity, stem are expanded by LAMP, shows
Green fluorescence can be observed in color result, illustrates there are causing root rot disease of Medicago sativa bacterium, and healthy root, stem and negative control colour developing result are orange
Color illustrates that there is no causing root rot disease of Medicago sativa bacterium, the set technology can be used for the rapid molecular detection of causing root rot disease of Medicago sativa bacterium in plant tissue.
Embodiment 4: the LAMP detection with causing root rot disease of Medicago sativa bacterium in soil bacteria
Sample acquisition: it is taken back from Shaanxi, Gansu, the serious field acquisition plant root soil of Ningxia causing root rot disease of Medicago sativa morbidity
Laboratory is spare, is control with autoclaved soil；
Soil DNA extracts: using soil DNA extracts kit (Sigma, DNB100, the Soil DNA of Sigma company
Isolation Kit) extract soil in total DNA, take 1.0 μ L to be expanded as pcr template.
Augmentation detection and observation: using the DNA of said extracted as template, using outer primer F3/B3 and inner primer FIP/BIP into
Row LAMP amplification, it is 25 μ L, including 5 μM of each 1.0 μ L of outer primer F3 and B3,40 μM of inner primers that LAMP, which detects reaction system,
Each 1.0 μ L, LAMP reaction mixture of FIP and BIP [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] and 12.5 μ L, 8 UBst
1.0 μ L of polymerase, 1.0 μ L of DNA profiling complement to 25 μ L with sterilizing ultrapure water；LAMP reaction condition: 64 DEG C incubate 60
min；The measurement of reaction result: using fluorescent dye visual observations method, after reaction to LAMP, produces in the amplification of LAMP reaction
I 1.0 μ L of color developing agent SYBR green is added in object, colour developing result observes that the judgement of green fluorescence is the positive, orange (orange
Color) it is judged as negative.
Testing result: testing result (attached drawing 3) shows that the serious field soil DNA of causing root rot disease of Medicago sativa morbidity passes through LAMP and expands
Increasing, green fluorescence can be observed in colour developing result, illustrate there are causing root rot disease of Medicago sativa bacterium, and high pressure sterilization soil and negative control colour developing
As a result to be orange, illustrate that causing root rot disease of Medicago sativa bacterium is not present, which can be used for the rapid molecular of causing root rot disease of Medicago sativa bacterium in soil
Detection.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
SEQUENCE LISTING
<110>Inst. of Plant Protection, fujian Academy of Agricultural Science
<120>primer and method of ring mediated isothermal amplification method detection causing root rot disease of Medicago sativa bacterium
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213>artificial sequence
<400> 1
caccaaaccc tcttggcg 18
<210> 2
<211> 19
<212> DNA
<213>artificial sequence
<400> 2
ggcgacatac caatgacgg 19
<210> 3
<211> 38
<212> DNA
<213>artificial sequence
<400> 3
ttaccgagct cagcggcttc cgcatcacgt ggttcaca 38
<210> 4
<211> 42
<212> DNA
<213>artificial sequence
<400> 4
tgggtccttg acaagctcaa gggggagtct cgaacttcca ga 42
Claims (4)
1. a kind of primer of loop-mediated isothermal amplification method detection causing root rot disease of Medicago sativa bacterium, which is characterized in that including one group of outer primer
F3/B3 and one group of inner primer FIP/BIP, the primer sequence are as follows: F3:5 '-CACCAAA CCCTCTTGGCG-3 ', B3:
5 '-GGCGACATACCAATGACGG-3 ', FIP:5 '-TTACCG AGCTCAGCGGCTTC-CGCATCACGTGGTTCACA-
3 ', BIP:5 '-TGGGTCCTTG ACAAGCTCAAGG-GGGAGTCTCGAACTTCCAGA- 3 '.
2. a kind of method of ring mediated isothermal amplification method detection causing root rot disease of Medicago sativa bacterium characterized by comprising
(1) sample to be tested genomic DNA is extracted；
(2) foundation of LAMP reaction system: the DNA extracted using step (1) utilizes outer primer described in claim 1 as template
F3/B3 and inner primer FIP/BIP carries out LAMP amplification, and it is 25 μ L, including 5 μM of outer primers F3 and B3 that LAMP, which detects reaction system,
Each 1.0 μ L, 40 μM of inner primers FIP and BIP each 1.0 μ L, LAMP reaction mixture 12.5 μ L, 8 UBst1.0 μ of polymerase
L, 1.0 μ L of DNA profiling complement to 25 μ L with sterilizing ultrapure water；
(3) LAMP reaction condition: 64 DEG C of 60 min of incubation；
(4) measurement of reaction result: using fluorescent dye visual observations method, after reaction to LAMP, in the expansion of LAMP reaction
Increase production and I 1.0 μ L of color developing agent SYBR green is added in object, colour developing result observes the judgement of green fluorescence as the positive, and there are lucernes
Mu pine root fungus, orange or crocus are judged as negative, causing root rot disease of Medicago sativa bacterium are not present.
3. a kind of method of ring mediated isothermal amplification method detection causing root rot disease of Medicago sativa bacterium according to claim 2, feature
It is, LAMP reaction mixture described in step (2) 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. application of the primer as described in claim 1 in the monitoring early diagnosed with germ, identification of causing root rot disease of Medicago sativa.
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| CN113005218A (en) * | 2021-04-02 | 2021-06-22 | 山西农业大学 | LAMP (loop-mediated isothermal amplification) detection primer, kit and detection method for fusarium solani |
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| CN114164293B (en) * | 2021-12-03 | 2022-11-04 | 陕西师范大学 | LAMP (loop-mediated isothermal amplification) combined detection primer, detection kit and detection method for monkshood |
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-
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Non-Patent Citations (3)
| Title |
|---|
| A guide to LAMP primer designing;EIKEN CHEMICAL Co.,Ltd,;《百度文库》;20160428;全文 * |
| 玉米顶腐镰孢菌rDNA-ITS 和EF-1a 基因序列及UP-PCR 遗传多样性分析;陈璐;《沈阳农业大学学报》;20111230;第42卷(第1期);摘要 * |
| 苜蓿根腐病菌(Fusarium solani )生物学特性研究;李万苍;《草业学报》;20051231;第14卷(第4期);摘要 * |
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