CN108977508A - Primer combination and its application based on LAMP detection succulent Pathogen - Google Patents
Primer combination and its application based on LAMP detection succulent Pathogen Download PDFInfo
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- CN108977508A CN108977508A CN201811047812.6A CN201811047812A CN108977508A CN 108977508 A CN108977508 A CN 108977508A CN 201811047812 A CN201811047812 A CN 201811047812A CN 108977508 A CN108977508 A CN 108977508A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
Abstract
The present invention provides primer combinations and its application based on LAMP detection succulent Pathogen.LAMP primer composition for detecting succulent Pathogen is made of 1 couple of outside primers F 3/B3 and 1 couple of inside primers F IP/BIP, F3/B3 and FIP/BIP primer sequence is as shown in SEQ ID NO.1-4.Detection architecture of the invention is under 65 DEG C of isothermys, it is incubated by 1 h, can quickly, conveniently, efficiently, it is high special, detect succulent Pathogen with sensitivity, the instrument of complex and expensive is not needed, the on-site test to succulent Pathogen can be better meet, it can be widely applied to the early diagnosis of grass-roots unit's field diseases and the monitoring of germ, identification, provide reliable technology and theoretical foundation for prevention and treatment succulent stem rot.
Description
Technical field
The invention belongs to garden crop Defect inspection, identification and Prevention Technique fields, and in particular to succulent stem rot
The primer combination of ring mediated isothermal amplification (Loop-mediated Isothermal Amplification, the LAMP) detection of bacterium
And its application, it can be used for the Molecular Detection that succulent Pathogen is quick, sensitive and special, while can be used for succulent stem
The early diagnosis and monitoring identification of maize ear rot.
Background technique
Succulent refers to the higher plant of vegetable nutritorium's hypertrophy, usually have root, stem, three kinds of nutrition organs of leaf and
Three kinds of flower, fruit, seed organs of multiplication.On gardening, also known as succulent plant or fleshiness flowers, but with this title of succulent
It is the most commonly used.Succulent is very popular all over the world as potting, and type common at present belongs to Crassulaceae and celestial being mostly
Ren Zhang section.
The cause of disease of stem rot be Fusarium oxysporum (Fusarium oxysporum), be endanger succulent main property it is true
Fungus diseases belong to Deuteromycotina fungi, and a kind of universal fungi, and distributed pole is wide, is prevalent in soil and moves
On plant organism, or even it is present in the arctic of severe cold and the desert of extreme arid torrid climate, belongs to simultaneous parasitic or saprogen.It is not only
Can overwintering in the soil and more summer, different specialized forms can be also shown as on different ornamental plants；It draws in specific manner
Ornamental plant vascular bundle diseases are played, the conducting tissue of plant is destroyed, and generate toxin in plant growth and development metabolic process
Damage to crops causes plant wilt dead, influences yield and quality, the disease be prevented and treated in current ornamental flower production it is relatively difficult
One of important disease.Succulent one meets this disease and can largely damping off, and atrophy is dead, and base of the plant can start brown disease occur
Spot, then internal to rot, the death until complete stool is dampinged off.It is main in prevention and treatment since succulent stem rot is a kind of soil-borne disease
It aims at prevention, whether prevention and treatment is timely, directly affects the control efficiency to soil-borne disease.Therefore there is an urgent need to establish in production
A method of succulent Pathogen fast and accurately being detected, the early diagnosis and prevention and treatment, monitoring, prediction for being disease are in advance
Report, which provides, to be supported and services, and to the production of development succulent, is increased flower-grower's income, reduces environmental pollution and is remained agriculture sustainable
Development all has great production meaning.
Currently, the detection method of pathogen is more, and traditional fungi strain identification method is mainly using morphology as foundation, by bacterium
Kind identifies guiding principle, mesh, section, genus and species.The morphological feature of fungi is complicated, and some mushroom morphological features and physiological and biochemical property with
The variation of environment and it is unstable, therefore, the inconsistent of categorizing system is often caused in traditional classification of fungi.Tradition point simultaneously
Time-consuming for class identification method, sensitivity is low, vulnerable to the interference of the factors such as artificial and environment, cannot be intuitively latent in disease
Phase and initial phase make diagnosis, are difficult that disease occurs to carry out timely monitoring and effectively control.
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.
Loop-mediated isothermal amplification technique (LAMP) is by a kind of easy, quick, accurate and honest and clean of Japanese Rong Yan company exploitation
The nucleic acid efficient amplification technology of valence, the technology can be polymerize under 60 DEG C ~ 65 DEG C constant temperatures using the strand displacement DNA of high activity
Enzyme (BstDNA polymerase) specific amplification is carried out to target DNA fragment.In 1 hour, it will can copy on a small quantity
Several target gene are expanded to 109~1010A copy number.Since LAMP amplification procedure relies on identification 6 isolated areas of target sequence,
So atopic is very strong, and amplification process is to carry out under constant temperature conditions, common water-bath or have stablize heat
The equipment in source is just able to satisfy reaction and requires, and testing cost substantially reduces, LAMP reaction product not only can by transmissometer,
Real-time PCR instrument and gel-electrophoretic apparatus are detected, but also can pass through SYBR Green I, calcein, hydroxyl naphthalene
After the dyeing of phenol indigo plant, naked eyes are identified, greatly reduce the injury to experimenter, and increase the application value in field.Mesh
Application of the preceding LAMP in phytopathogen detection increases year by year, and domestic and international about the LAMP of succulent Pathogen detection
It is not reported.
Summary of the invention
The purpose of the present invention is be directed to be based primarily upon form to succulent Pathogen detection and identification in the prior art
Learn feature, time-consuming for method, program is cumbersome, it is empirical it is strong, accuracy is low, it is difficult to accomplish the timely monitoring that disease occurs and control
Pathogen processed propagates, popular problem, and PCR Molecular Detection is needed by expensive instruments such as amplification instruments, and detection time is longer etc.
Problem provides the primer combination based on loop-mediated isothermal amplification technique (LAMP) detection succulent Pathogen and its answers
With to the progress LAMP detection of succulent Pathogen, with detection cycle is short, accuracy is high, sensitivity is high, visually observes inspection
The advantages that surveying result.
To achieve the above object, using following technical scheme:
1. the design of succulent Pathogen LAMP detection specific primer sets: by measuring succulent Pathogen
(Fusarium oxysporum) and other sickle-like bacteria (Fusarium Spp translation elongation factor)
1-alpha gene(EF-1alpha) gene order, to Fusarium and other pathogen difference inter-speciesEF-1alphaGene sequence
Column are compared, using online LAMP primer design software Primer software Explorer V4 (http: //
Primerexplorer.jp/elamp4.0.0/ index.html, Eiken Chemical Co., Japan) design it is a set of
Succulent Pathogen specificity LAMP primer composition, by 1 couple of outside primers F 3/B3 and 1 pair of inside primers F IP/BIP group
At F3/B3 and FIP/BIP primer sequence is as follows:
F3:5'- CAGTATTCTGGCGGGCATG-3',
B3: 5'- ACCTGATCCGAGGTCAACAT-3';
FIP:5'-TGGGGAACGCGAATTAACGCGGAGCGTCATTTCAACCCTCA-3',
BIP:5'-GTCACGTCGAGCTTCCATAGCGAGAAGTTGGGGTTTAACGGC-3'。
2. the foundation of succulent Pathogen 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(ten
Six alkyl trimethyl ammonium bromides) and extracting solution (it includes: 2% CTAB；100 mmol·L-1Tris-HCl, pH 8.0；20
mmol·L-1EDTA, pH8.0；1.4 mol·L-1NaCl) and 90 μ L SDS(neopelexes) [note: CTAB,
SDS needs 60 DEG C of preheatings], vibrated and mixed using oscillator, 60 DEG C of water-bath 1h(DNA are discharged into buffer), 12000 rmin-1
It is centrifuged 15 min；700 μ L of supernatant is taken, isometric phenol, chloroform, isoamyl alcohol mixed liquor (each component volume ratio 25:24:1) are added,
Gently oscillation mixes, 12000 rmin-1It is centrifuged 9 min；500 μ L of supernatant is taken, 1/10 volume, 3 molL is added-1
NaAc and 2 times of volume dehydrated alcohol, -20 DEG C of precipitatings 30 min, 12000 rmin-1It is centrifuged 5 min；Liquid is discarded supernatant, is added
700 μ L ice, 70% ethyl alcohol is washed and (is slightly centrifuged, incline and fall supernatant), is dried on superclean bench to alcohol-free taste, is added
60 μ L aseptic double-distilled waters (DDW) are dissolved, and DNA solution is obtained, with UV spectrophotometer measuring DNA concentration and dilute
It releases to 100 ng μ L-1For use.
When for detecting succulent Pathogen in plant tissue, DNA, specific mistake are extracted using NaOH rapid cleavage method
Journey is as follows: 10 μ L, 0.5 molL being added into every milligram of plant tissue-1Tissue is sufficiently milled to by NaOH in mortar
It is transferred to after paste in 1.5 mL centrifuge tubes, 12,000 rpm are centrifuged 6 min, take 5 μ l of supernatant that 495 μ L, 0.1 molL is added-1
Tris-HCl(pH=8.0) it is uniformly mixed, take 1.0 μ L to be expanded as pcr template.
(2) foundation of LAMP reaction system: the DNA extracted using step (1) utilizes Outside primer F3/B3 and interior as template
Side primers F IP/BIP carries out LAMP amplification, and it is 25 μ L, including 20 mM Tris-HCl, 10 mM that LAMP, which detects reaction system,
(NH4)2SO4、8.0 mM MgSO4, 50 mM KCl, 0.8 mM glycine betaine (Betaine), 1.75 mmolL-1 dNTPs、8
U Bst archaeal dna polymerase, 1.6 μm of olL-1FIP and BIP, 0.2 μm of olL-1F3 and B3,1 μ L template DNA (50-
100 ng), aseptic double-distilled water is supplemented to 25 μ L, and 1000 × SYBR Green I that 2 μ L are added is covered in PCR pipe.
(3) LAMP reaction condition: 65 DEG C of incubations 60 min, 85 DEG C of 10 min of inactivation terminate reaction on ice.
(4) it the measurement of reaction result: is measured using fluorescent dye visual observations method or agarose gel electrophoresis method.It adopts
With fluorescent dye visual observations method, after reaction to LAMP, the bottom of PCR pipe is arrived in the centrifugation of SYBR Green I by brief centrifugation
Color change is observed in portion, and colour developing result observes the judgement of green fluorescence as the positive, and there are succulent Pathogens；It is orange
Or crocus is judged as negative, and succulent Pathogen is not present.Using agarose gel electrophoresis method, 2.0 μ L LAMP are taken
Amplified production is detected with 2% agarose gel electrophoresis, trapezoid-shaped strips is occurred and is judged as positive, there are succulent Pathogens；Not yet
It occurs band and is then judged as negative, succulent Pathogen is not present.
Remarkable advantage of the invention
Beneficial effects of the present invention: the present invention establishes the quick, easy of succulent Pathogen, high specificity, sensitivity
High LAMP detection technique method, the detection of succulent Pathogen in can be used for carrying disease germs plant tissue and soil, or be used for
Morbidity early period of succulent stem rot, initial stage detection, for determining that the best period of disease control has highly important meaning
Justice.
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 common
Target gene has Internal Transcribed Spacer (Internal transcribed space, ITS), however much
Person thinks that the target can not explicitly distinguish reaping hook category fungi and Fusarium oxysporum difference specialized form very much.Inventor's discoveryEF- 1alphaGene order existsFusariumThere is certain conservative in kind and between Fusarium oxysporum difference specialized form different strains,
Inter-species there are variation abundant, be compared to rDNA-ITS,β‐tubulinThe better Molecular Detection target of sequence.Present invention analysis
Succulent PathogenEF-1alphaGene and difference of other Fusariumsps in sequence, choose 6 specific regions, if
4 specific LAMP primers are counted, any region and primer mismatch not can be carried out nucleic acid amplification in 6 regions, have
Very strong specificity.The present invention establishes the detection side succulent Pathogen LAMP on the basis of designed LAMP primer
Method, only succulent Pathogen can detected, and other pathogens do not detect, and test of many times result is consistent, say
Bright LAMP detection method high specificity of the present invention, as a result reliably.
2, high sensitivity: the present invention can reach 10 to the detection sensitivity of succulent Pathogen on DNA level
fg·μL-1, there is very high sensitivity.
3, practicability is good: on the basis of keeping PCR technological merit, LAMP method does not need heat circulating equipment, saves
It is time used in heating and cooling link, more time saving, amplified reaction can be completed in 60 min.Simultaneously as LAMP
Technology uses 4 ~ 6 primers, has equal or higher sensitivity compared with PCR technology, further enhances reaction
Specificity.It joined calcein (Calcein) or SYBR Green I during constant-temperature amplification simultaneously, after the completion of reaction
Without opening the direct observed result of lid energy, keep detection process more efficient and convenient, therefore this method is particularly suitable in base
It is applied in unit or flowers company.
4, easy to operate quick: the LAMP method of detection succulent Pathogen provided by the invention overcomes existing skill
The period needed for the biological detection method of succulent Pathogen is long in art, time-consuming and laborious, cumbersome, poor specificity and PCR inspection
The problem of survey technology needs the expensive equipments such as thermal cycler, can not quickly detect succulent Pathogen.Present invention detection side
Method under 65 DEG C of isothermys, can quickly, conveniently, efficiently, it is high special, detect succulent Pathogen with sensitivity, no
Need complex instrument, only need a thermostatic equipment, can preferably meet the on-site test to succulent Pathogen, have compared with
High application value has good application prospect in base and on-site test.
Detailed description of the invention
Fig. 1 is the LAMP specific detection result of succulent Pathogen of the present invention.Fig. 1-a is after LAMP is expanded
Agarose gel electrophoresis testing result, Fig. 1-b are the fluorescent dye visualization colour developing result after LAMP amplification, in which: M DL
2000 DNA marker, 1 is succulent Pathogen, shows green fluorescence, 2 be negative control.
Fig. 2 is the LAMP specificity verification of succulent Pathogen of the present invention.Fig. 2-a is the agar after LAMP amplification
Sugared detected through gel electrophoresis is as a result, Fig. 2-b is the fluorescent dye visualization colour developing result after LAMP amplification, in which: M is DL 2000
DNA marker, 1-8 swimming lane is respectively as follows: succulent Pathogen, fleshiness anthrax bacteria, fleshiness alternaria, tomato leaf mold
Bacterium, orchid alternaria, cowpea phytophthora, Phytophthora cactorum bacterium and aseptic double-distilled water (DDW), wherein 1 display green fluorescence.
Fig. 3 is succulent Pathogen LAMP detection sensitivity of the present invention.Fig. 3-a is the agarose after LAMP amplification
Detected through gel electrophoresis is as a result, Fig. 3-b is the fluorescent dye colour developing result after LAMP amplification, in which: M is 2000 DNA
Marker, 1-8 template DNA concentration are respectively 10 ng μ L-1、1 ng·μL-1、100 pg·μL-1、10 pg·μL-1、1
pg·μL-1、100 fg·μL-1、10 fg·μL-1、1 fg·μL-1, 1-7 display green fluorescence.
Fig. 4 is the LAMP detection of succulent Pathogen in incidence tissue.Fig. 4-a is the agarose after LAMP amplification
Detected through gel electrophoresis result, in which: M be 2000 DNA marker, 1-5 swimming lanes be respectively as follows: succulent root incidence tissue,
Incidence tissue of succulent stem, succulent root health tissues, succulent stem health tissues, aseptic double-distilled water
(DDW)；Fig. 4-b is that the fluorescent dye after LAMP amplification develops the color as a result, 1-5 is respectively as follows: succulent root incidence tissue, fleshiness
Plant root health tissues, aseptic double-distilled water (DDW), succulent stem health tissues, incidence tissue of succulent stem,
In 1,5 display green fluorescences.
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: the design of succulent Pathogen ring mediated isothermal amplification (LAMP) detection specific primer sets
And primer specificity verifying
1. the extraction of strains tested genomic DNA
When for detecting pathogen pure culture, genomic DNA being extracted using CTAB method, the specific method is as follows: taken a small amount of
900 μ L, 2% CTAB(hexadecane is added in hypha powder (hypha powder, which had just covered semicircular base, to be advisable) in 1.5 mL centrifuge tubes
Base trimethylammonium bromide) (it contains 2% CTAB to extracting solution；100 mmol·L-1Tris-HCl, pH 8.0；20 mmol·L-1EDTA, pH 8.0；1.4 mol·L-1NaCl) and 90 μ L SDS(neopelexes) [note: CTAB, SDS need 60
DEG C preheating], vibrated and mixed using oscillator, 60 DEG C of water-bath 1h(DNA discharge into buffer), 12000 rmin-1Centrifugation 15
min；700 μ L of supernatant is taken, adds isometric phenol, chloroform, isoamyl alcohol mixed liquor (each component volume ratio is 25:24:1), gently shakes
Swing mixing, 12000 rmin-1It is centrifuged 9 min；500 μ L of supernatant is taken, 1/10 volume, 3 molL is added-1NaAc and 2 times
Volume dehydrated alcohol, -20 DEG C of precipitatings 30 min, 12000 rmin-1It is centrifuged 5 min；Liquid is discarded supernatant, 700 μ L ice are added
70% ethyl alcohol, which is washed, (to be slightly centrifuged；Incline and fall supernatant), it is dried on superclean bench to alcohol-free taste, it is sterile that 60 μ L is added
Water (DDW) is dissolved, and DNA solution is obtained, and with UV spectrophotometer measuring DNA concentration and is diluted to 100 ng μ
L-1For use.
2. the design of succulent Pathogen ring mediated isothermal amplification (LAMP) Specific primer pair is logical
Cross measurement succulent Pathogen (Fusarium oxysporum) and other sickle-like bacteria (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 succulent Pathogen specificity LAMP primer group of design, by 1 pair of outside primers F 3/
B3 and 1 pair of inside primers F IP/BIP composition, F3/B3 and FIP/BIP primer sequence are as follows:
F3:5'- CAGTATTCTGGCGGGCATG-3',
B3: 5'- ACCTGATCCGAGGTCAACAT-3'；
FIP:5'-TGGGGAACGCGAATTAACGCGGAGCGTCATTTCAACCCTC A-3',
BIP:5'-GTCACGTCGAGCTTCCATAGCGAGAAGTTGGGGTTTAACGGC-3'。
3. the foundation of succulent Pathogen LAMP detection method and primer specificity verifying
With succulent Pathogen, fleshiness anthrax bacteria, fleshiness alternaria, Cladosporium fulvum, orchid alternaria, cowpea
The DNA of the strains testeds such as phytophthora and Phytophthora cactorum bacterium is template, and Negative control Sterile distilled water (DDW) utilizes Outside primer F3/
B3 and inner primer FIP/BIP carries out LAMP amplification, and it is 25 μ L, 20 mM Tris-HCl, 10 mM that LAMP, which detects reaction system,
(NH4)2SO4、8.0 mM MgSO4, 50 mM KCl, 0.8 mM glycine betaine (Betaine), 1.75 mmolL-1 dNTPs、8U
Bst archaeal dna polymerase, 1.6 μm of olL-1FIP and BIP, 0.2 μm of olL-1F3 and B3,1 μ L template DNA (50-100
Ng), aseptic double-distilled water is supplemented to 25 μ L, and 1000 × SYBR Green I that 2 μ L are added is covered in PCR pipe；LAMP reacts item
Part: 65 DEG C of incubations 60 min, 85 DEG C of 10 min of inactivation terminate reaction on ice.The measurement of reaction result: fluorescent dye mesh is used
It surveys observation or agarose gel electrophoresis method is measured.Using fluorescent dye visual observations method, after reaction to LAMP, wink
When be centrifuged, by SYBR Green I centrifugation arrive PCR pipe bottom, observe color change.Colour developing result observes sentencing for green fluorescence
Break as the positive, there are succulent Pathogens；Orange or crocus is judged as negative, and succulent Pathogen is not present.
Using agarose gel electrophoresis method, takes 2.0 μ L LAMP amplified productions to be detected with 2% agarose gel electrophoresis, trapezoidal item such as occur
Band is judged as positive, and there are succulent Pathogens；There is not band and be then judged as negative, succulent stem rot is not present
Germ.
4. primer specificity verification result
LAMP amplification shows that green fluorescence can be observed in the succulent Pathogen colour developing result for examination or agarose is solidifying
There is the trapezoid belt of LAMP feature in gel electrophoresis, remaining sickle-like bacteria and fungi colour developing result are that orange or agarose gel electrophoresis does not have
Occur amplified band (attached drawing 1 and Fig. 2), illustrates designed succulent Pathogen Outside primer F3/B3 and inner primer
FIP/BIP can distinguish succulent Pathogen and other pathogens, have the specificity of kind, can be used for fleshiness plant
The fast and reliable detection and identification of object Pathogen.
Embodiment 2: succulent Pathogen ring mediated isothermal amplification (LAMP) detection sensitivity measurement
1. the preparation of various concentration genomic DNA
Succulent Pathogen genomic DNA is diluted with sterile ultrapure water (DDW), be configured to 10 times of orders of magnitude is
Column concentration is spare.
Detection method sensitivity determination and result observation
Using the succulent Pathogen genomic DNA of various concentration as template, Outside primer F3/B3 and inner primer are utilized
FIP/BIP carries out LAMP amplification, and it is 25 μ L, including 20 mM Tris-HCl, 10 mM (NH that LAMP, which detects reaction system,4)2SO4、8.0 mM MgSO4, 50 mM KCl, 0.8 mM glycine betaine (Betaine), 1.75 mmolL-1 dNTPs、8 U Bst
Archaeal dna polymerase, 1.6 μm of olL-1FIP and BIP, 0.2 μm of olL-1F3 and B3,1 μ L template DNA (difference dilution
10 ng μ L of concentration template-1、1 ng·μL-1、100 pg·μL-1、10 pg·μL-1、1 pg·μL-1、100 fg·μL-1、
10 fg·μL-1 、1 fg·μL-1), aseptic double-distilled water (DDW) is supplemented to 25 μ L, PCR pipe cover be added 2 μ L 1000 ×
SYBR GreenⅠ.65 DEG C of incubations 60 min, 85 DEG C of inactivation 10min terminate reaction on ice.The measurement of reaction result: fluorescence is used
Dyestuff visual observations method or agarose gel electrophoresis method are measured.Using fluorescent dye visual observations method, reacts and tie to LAMP
The centrifugation of SYBR Green I is arrived the bottom of PCR pipe, observes color change by Shu Hou, brief centrifugation.Colour developing result observes green
The judgement of fluorescence is the positive, and there are succulent Pathogen, orange or crocus is judged as negative, and succulent stem is not present
Maize ear rot bacterium；Using agarose gel electrophoresis method, 2.0 μ L LAMP amplified productions is taken to be detected with 2% agarose gel electrophoresis, such as gone out
Existing trapezoid-shaped strips are judged as positive, and there are succulent Pathogens, band do not occur and are then judged as negative, fleshiness is not present
Plant Pathogen.
3. LAMP expands sensitivity technique result
LAMP expand sensitivity technique the result shows that, 10 ng μ L-1、1 ng·μL-1、100 pg·μL-1、10 pg·μL-1、
1 pg·μL-1、100 fg·μL-1、10 fg·μL-1The succulent Pathogen genomic DNA colour developing result of concentration is considerable
It observes green fluorescence or the trapezoid belt of LAMP feature, 1 fg μ L occurs in agarose gel electrophoresis-1Concentration colour developing result is orange
Or there is not amplified band in agarose gel electrophoresis, illustrate designed succulent Pathogen Outside primer F3/B3 and
Inner primer FIP/BIP is expanded by LAMP, to the detection sensitivity of succulent Pathogen up to 10 fg μ L-1(attached drawing
3).
Embodiment 3: the LAMP detection of succulent Pathogen in incidence tissue
Sample acquisition: from the typical root of the ground such as Zhangzhou, Fujian Longhai City town Jiu Hu acquisition succulent stem rot disease symptom, stem
It is spare that portion (at earth's surface 2-6 cm) and healthy plant root and stem take back laboratory.
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 molL is added in tissue-1NaOH is transferred to 1.5 mL centrifuge tubes after tissue is sufficiently milled to paste in mortar
In, 12,000 rpm are centrifuged 6 min, take 5 μ l of supernatant that 495 μ L, 0.1 molL is added-1Tris-HCl(pH=8.0) it is mixed
It closes uniformly, 1.0 μ L is taken to be expanded as pcr template.
Augmentation detection and observation: using the DNA of said extracted as template, Outside primer F3/B3 and inner primer FIP/ are utilized
BIP carries out LAMP amplification, and it is 25 μ L, including 20 mM Tris-HCl, 10 mM (NH that LAMP, which detects reaction system,4)2SO4、8.0
mM MgSO4, 50 mM KCl, 0.8 mM glycine betaine (Betaine), 1.75 mmolL-1DNTPs, 8U Bst DNA polymerization
Enzyme, 1.6 μm of olL-1FIP and BIP, 0.2 μm of olL-1F3 and B3,1 μ L template DNA (50-100 ng) is sterile
Distilled water (DDW) is supplemented to 25 μ L, and 1000 × SYBR Green I that 2 μ L are added is covered in PCR pipe.65 DEG C of 60 min of incubation,
85 DEG C of 10 min of inactivation terminate reaction on ice.The measurement of reaction result: fluorescent dye visual observations method or Ago-Gel are used
Electrophoresis is measured.Using fluorescent dye visual observations method, after reaction to LAMP, brief centrifugation, by SYBR Green I
It is centrifuged the bottom of PCR pipe, observes color change.Colour developing result observes the judgement of green fluorescence as the positive, and there are fleshiness plants
Object Pathogen, orange or crocus are judged as negative, succulent Pathogen are not present；Using agarose gel electrophoresis
Method takes 2.0 μ L LAMP amplified productions to be detected with 2% agarose gel electrophoresis, trapezoid-shaped strips such as occurs and is judged as positive, exists
There is not band and is then judged as negative, succulent Pathogen is not present in succulent Pathogen.
Testing result: testing result (attached drawing 4) shows that the root of succulent stem rot morbidity, stem are expanded by LAMP,
Green fluorescence can be observed for colour developing result or the trapezoid belt of LAMP feature occurs in agarose gel electrophoresis, illustrates that there are succulents
Pathogen, and healthy root, stem and negative control colour developing result are that orange or agarose gel electrophoresis amplification item does not occur
Band illustrates there is no succulent Pathogen, which can be used for the quick of succulent Pathogen in plant tissue
Molecular 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 combination and its application based on LAMP detection succulent Pathogen
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213>artificial sequence
<400> 1
cagtattctg gcgggcatg 19
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
acctgatccg aggtcaacat 20
<210> 3
<211> 41
<212> DNA
<213>artificial sequence
<400> 3
tggggaacgc gaattaacgc ggagcgtcat ttcaaccctc a 41
<210> 4
<211> 42
<212> DNA
<213>artificial sequence
<400> 4
gtcacgtcga gcttccatag cgagaagttg gggtttaacg gc 42
Claims (4)
1. the primer combination based on LAMP detection succulent Pathogen, it is characterised in that: the primer combination is by 1 pair of outside
Primers F 3/B3 and 1 pair of inside primers F IP/BIP composition, F3/B3 and FIP/BIP primer sequence are as follows:
F3:5'- CAGTATTCTGGCGGGCATG-3',
B3: 5'- ACCTGATCCGAGGTCAACAT-3'；
FIP:5'-TGGGGAACGCGAATTAACGCGGAGCGTCATTTCAACCCTCA-3',
BIP:5'-GTCACGTCGAGCTTCCATAGCGAGAAGTTGGGGTTTAACGGC-3'。
2. application of the primer combination as described in claim 1 in succulent Pathogen LAMP detection.
3. application according to claim 2, which is characterized in that detection method includes the following steps for it:
(1) sample to be tested genomic DNA is extracted；
(2) foundation of LAMP reaction system: the DNA extracted using step (1) is drawn as template using outside described in claim 1
Object F3/B3 and inner primer FIP/BIP carries out LAMP amplification, and it is 25 μ L that LAMP, which detects reaction system, includes in reaction system:
20 mM Tris-HCl、10 mM (NH4)2SO4、8.0 mM MgSO4, 50 mM KCl, 0.8 mM glycine betaine, 1.75
mmol·L-1DNTPs, 8 U Bst archaeal dna polymerases, 1.6 μm of olL-1FIP and BIP, 0.2 μm of olL-1F3 and
B3,1 μ L template DNA 50-100 ng, aseptic double-distilled water are supplemented to 25 μ L, and 1000 × SYBR that 2 μ L are added is covered in PCR pipe
GreenⅠ；
(3) LAMP reaction condition: 65 DEG C of incubations 60 min, 85 DEG C of inactivation 10min terminate reaction on ice；
(4) it the measurement of reaction result: is measured using fluorescent dye visual observations method or agarose gel electrophoresis method；Using glimmering
Photoinitiator dye visual observations method, after reaction to LAMP, the centrifugation of SYBR Green I is arrived the bottom of PCR pipe, seen by brief centrifugation
Color change is examined, colour developing result observes that the judgement of green fluorescence is the positive, orange or orange there are succulent Pathogen
Color is judged as negative, and succulent Pathogen is not present；Using agarose gel electrophoresis method, 2.0 μ L LAMP amplification is taken to produce
Object is detected with 2% agarose gel electrophoresis, trapezoid-shaped strips is occurred and is judged as positive, there are succulent Pathogens, do not occur
Band is then judged as negative, and succulent Pathogen is not present.
4. primer as described in claim 1 combines answering in the early diagnosis, monitoring and identification of succulent Pathogen
With.
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| CN110982919A (en) * | 2019-11-13 | 2020-04-10 | 上海市农业科学院 | Method and primer for identifying trichoderma fungus by loop-mediated isothermal amplification method |
| CN111020057A (en) * | 2020-01-08 | 2020-04-17 | 北京林业大学 | LAMP primer and kit for detecting Botryosphaeria fabicciana |
| CN111041124A (en) * | 2020-01-09 | 2020-04-21 | 北京林业大学 | LAMP primer and kit for detecting Neofuscoccum algeriense |
| CN111041125A (en) * | 2020-01-09 | 2020-04-21 | 北京林业大学 | LAMP primer and kit for detecting Macrophomina vaccini |
| CN111041123A (en) * | 2020-01-08 | 2020-04-21 | 北京林业大学 | LAMP primer and kit for detecting Botryosphaeria sinensia |
| CN111041120A (en) * | 2020-01-07 | 2020-04-21 | 北京林业大学 | LAMP primer and kit for detecting Lasiodipia thailandica |
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| CN110982919A (en) * | 2019-11-13 | 2020-04-10 | 上海市农业科学院 | Method and primer for identifying trichoderma fungus by loop-mediated isothermal amplification method |
| CN111041120A (en) * | 2020-01-07 | 2020-04-21 | 北京林业大学 | LAMP primer and kit for detecting Lasiodipia thailandica |
| CN111020057A (en) * | 2020-01-08 | 2020-04-17 | 北京林业大学 | LAMP primer and kit for detecting Botryosphaeria fabicciana |
| CN111041123A (en) * | 2020-01-08 | 2020-04-21 | 北京林业大学 | LAMP primer and kit for detecting Botryosphaeria sinensia |
| CN111020057B (en) * | 2020-01-08 | 2022-07-05 | 北京林业大学 | LAMP primer and kit for detecting Botryosphaeria fabicciana |
| CN111041123B (en) * | 2020-01-08 | 2022-07-08 | 北京林业大学 | LAMP primer and kit for detecting Botryosphaeria sinensia |
| CN111041124A (en) * | 2020-01-09 | 2020-04-21 | 北京林业大学 | LAMP primer and kit for detecting Neofuscoccum algeriense |
| CN111041125A (en) * | 2020-01-09 | 2020-04-21 | 北京林业大学 | LAMP primer and kit for detecting Macrophomina vaccini |
| CN111088386A (en) * | 2020-01-09 | 2020-05-01 | 北京林业大学 | LAMP primer and kit for detecting Lasiodipia paraphysoides |
| CN111041124B (en) * | 2020-01-09 | 2022-07-05 | 北京林业大学 | LAMP primer and kit for detecting Neofusicoccum algeriense |
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Application publication date: 20181211 |