CN110093450A - A kind of LAMP detection primer and its application of sweet potato black rot pathogen - Google Patents
A kind of LAMP detection primer and its application of sweet potato black rot pathogen Download PDFInfo
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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 invention discloses a kind of LAMP primer of sweet potato black rot pathogen and its applications, belong to corps diseases detection, identification and Prevention Technique field.A kind of LAMP detection primer of sweet potato black rot pathogen of the invention includes 2 outer primer F3 and B3 and 2 inner primer FIP and BIP.LAMP detection primer chromogenic reaction or agarose gel electrophoresis detection after loop-mediated isothermal amplification, can be observed reaction solution display green or the characteristic trapezoid belt of LAMP occur.LAMP detection primer and its detection method of the invention can be used for the quick, sensitive of sweet potato black rot pathogen, accurate detection in the plant that sweet potato black rot pathogen infects in production practices and soil, it can be used for the early diagnosis of field diseases and the monitoring and identification of germ simultaneously, provide reliable technology and theoretical foundation for the microbial disease control of sweet potato black rot.
Description
Technical field
The invention belongs to corps diseases detection, identification and Prevention Technique fields, and in particular to a kind of sweet potato black rot pathogen
LAMP detection primer and its application.
A kind of sweet potato black rot pathogen LAMP detection primer and its rapid detection method, are exclusively used in the Gao Ling of sweet potato black rot pathogen
The detection of sensitivity rapid molecular, while can be used for the early diagnosis of field sweet potato black rot and the monitoring and identification of germ.
Background technique
Sweet potato is one of main cereal crops in China and importance light industry raw material and forage crop.China sweet potato
The form of consumption is other than producing starch, alcohol, and fresh food increased with, leaf vegetables with ratio, and National Development and Reform Committee selects sweet potato
It is set to the preferred non-grain raw material crop of fuel ethanol production.Sweet potato as new energy crop, ensure national food security and
It occupies an important position in energy security.China is that maximum sweet potato producing country, every annual planting area about 5,000,000 are public in the world
Hectare, account for about 53.5 % in the world；About 1.1 ten thousand tons of year output, account for about 82.2 % in the world.
Sweet potato black rot (sweep potato black rot) is very common in sweet potato cultivation and storage and harm
Extremely serious disease also known as black scar has generation in each sweet potato producing region in the world, is to seriously affect yield in sweet potato production
One of three major diseases.According to statistics, China causes the sweet potato of about 5%-10% to lose by the disease every year, even can achieve when serious
20%-50% or higher loss.Its pathogen be Ceratocystis fimbriata bacterium (Ceratocystis fimbriata Ellis et
Halsted), the bacterium is widely distributed, population differentiation is complicated, main harm seedling basal part of stem and root tuber tissue, causes dead seedling, rotten
Bed, rotten cellar are very big to sweet potato Influence of production.Black spot Resistant Difference is obvious between sweet potato variety, and highly resistance is of less types, and in height
In anti-kind, have both high yield, the kind that high level cadre leads not yet is found, become epidemic-stricken area output increased major limiting factors it
One.It can produce the furans terpene noxious material such as mould ketone of the black blister of sweet potato (ipomeamarone), family in sick potato with alternaria
It can cause poisoning symptom after poultry is edible, in addition, saccharomycete and carbohydrase bacterium can be poisoned by making fermentation raw material with sick potato, delay to ferment
Journey reduces alcohol output and quality, the serious development for restricting Sweet Potato Industry.Therefore, the early stage for developing sweet potato black rot is quick
Diagnostic method is simultaneously monitored in time, most important to the production of guarantee sweet potato health and food safety.
Defect inspection method mainly uses the technologies such as traditional pathogen separation detection method and Molecular Detection at present.Tradition is sweet
The taxonomic identification of potato alternaria is mainly based upon morphological feature, Pathogenicity, physio-biochemical characteristics etc., and program is cumbersome,
Required time is longer, interference when identification also vulnerable to factors such as artificial and environment, and cannot be directly from plant tissue
Detect pathogen, it is difficult to meet testing requirements quick, sensitive, stable in Disease management, it is easy to miss disease control
Best period.Therefore, it is not only very necessary to establish a set of quick, sensitive, accurate sweet potato black rot pathogen Examination and diagnosis, and
And it is very urgent.
Round pcr provides quick, sensitive, accurate advantage for pathogenic diagnosis, however PCR specific detection at present
There is still a need for the expensive special instrument such as PCR instrument, electrophoresis and gel imaging system and molecular biology reagents for technology, and need to divide
Sub- biology experimenter operation, limits the popularization and application of PCR detection method.Circulation constant temperature amplification technique (Loop-
Mediated isothermal amplification, abbreviation LAMP) it is 2000 by Japanese Rong Yan Co., Ltd. Notomi etc.
A kind of New Cycle constant temperature nucleic acid amplification technology of people's exploitation.LAMP reaction is designed 4 for 6 sites of target gene and is drawn
Object, using a kind of chain type substitute activity archaeal dna polymerase (BstDNA polymerase), under constant temperature conditions (60-65 DEG C)
Heat preservation 30-90 minutes, can be completed amplified reaction.The massive amplification of product is realized in this method short time, remolding sensitivity is conventional
PCR high, and operating procedure is easy, is not necessarily to special installation, testing result can be particularly well suited for by visually judging in production division, base
It promotes and applies.Due to features such as LAMP reaction is simple, quick, efficient, economy, thus there is extremely wide application prospect.Mesh
Preceding LAMP detection is mainly used in the detection of people and animals' pathogen, food safety and environmental sanitation, reports in phytopathogen detection
Road is less, and the detection of sweet potato black rot pathogen is not reported both at home and abroad.
Summary of the invention
The purpose of the present invention is for the length of period needed for the biological detection method of sweet potato black rot pathogen in the prior art, inspection
Survey method poor specificity, the low problem of sensitivity provide the LAMP detection primer and application of a kind of sweet potato black rot pathogen.The present invention
LAMP detection primer high specificity, high sensitivity, detection method is easy quickly, and result is reliable.
To achieve the above object, the present invention adopts the following technical scheme:
1. the design of sweet potato black rot pathogen LAMP detection primer
From Ceratocystis fimbriata bacterium (Ceratocystis fimbriataEllis et Halsted) genome in expand and survey
Sequence obtainsTsr1Gene order is drawn then according to the gene order using PrimerExplorer V5 software design LAMP detection
Object.
It is describedTsr1The nucleotide sequence of gene, as shown in SEQ ID NO.1.
By gained sweet potato black rot pathogenTsr1The sequence of the nucleotide sequence of gene and other known fungi carries out multiple sequence
Column compare, and choose sweet potato black rot pathogen distinguished sequence, utilize online software primer software PrimerExplorer V5
(http://primerexplorer.jp/lampv5e/index.html;Eiken Chemical Co., Japan) design
LAMP primer, outer primer F3/B3, positive inner primer (FIP) are made of the reverse complementary sequence of F1 plus F2, reversed inner primer
(BIP) (as shown in Figure 1) is constituted plus the reverse complementary sequence of B2 by B1.
Gained LAMP detection primer includes 2 outer primer F3 and B3 and 2 inner primer FIP and BIP, primer
Sequence is respectively as follows:
F3: 5’-CCAGGCACACGAGTCAGT-3’
B3: 5’-TCCTCCTTGGACTTGATCGA-3’
FIP: 5’- CGTCACAGGGTGCAGAGCAGGTGTACATCAGTGGCGCTAT-3’
BIP: 5’-TGCTGCGCCACGAACACAAGTGGGTATTCGGAGCTGAGAT-3’。
2. the method for carrying out LAMP reaction detection sweet potato black rot pathogen using LAMP detection primer, comprising the following steps:
(1) extraction of sample to be tested genomic DNA
There are when the detection of alternaria in organize for sweet potato, sweet potato black rot pathogen is extracted using NaOH rapid cleavage method
DNA, detailed process is as follows: (1) sweet potato disease stem or in spite of illness potato wedge cleaned, dried, clip site of pathological change；(2) 1 mg disease group is pressed
Knit 10 μ l(0.5 mol/L NaOH, 0.5 %PVP of middle addition) metering, tissue is sufficiently milled to paste, 12,000 rpm centrifugation 5
min；(3) Tris-HCl(pH8.0 of 20 μ l of supernatant with 0.1 isometric mol/L are taken) it mixes；(4) solution is diluted to 10
Times, 100 times, 1000 times of liquid, take respectively 1 μ l stoste, 10 times, 100 times, 1000 times of liquid are expanded as template.
Sweet potato black rot pathogen extracting genome DNA extracts genome using soil DNA extracts kit in morbidity soil
DNA。
(2) LAMP detects reaction system: the DNA extracted using step (1) utilizes F3/B3, FIP/BIP primer as template
It is expanded.Reaction system is 25 μ l, includes 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 8 mM
MgSO4, 0.8 mol/L of glycine betaine,BstPolymerase be 8 U, each 0.2 mmol/L of dNTPs 1.0 mmol/L, F3 and B3,
Each 1.6 mmol/L of FIP and BIP, 50 μm of ol/L of calcein, manganese chloride 500 μm of ol/L, TWeen-20 0.1%, template DNA
50 ~ 100 ng, aseptic double-distilled water complement to 25 μ l；
(3) LAMP reacts: the reaction system of step (2) is in 63-65 DEG C of incubation 45-60 min, 85 DEG C of inactivation 5-10min.
(4) result detects: being detected with fluorescent dye visual observations method and agarose gel electrophoresis method；
Fluorescent dye visual observations method: after LAMP reaction, reaction solution colour developing result observes that green fluorescence is judged as positive, orange
Color is judged as negative；
Agarose gel electrophoresis method: it takes 2 μ l LAMP reaction products to be detected with 2% agarose gel electrophoresis, LAMP characteristic occurs
Trapezoid-shaped strips be then judged as positive, there is not amplified band and be then judged as negative.
Application of the above-mentioned sweet potato black rot pathogen LAMP detection primer in the diagnosis, detection, identification of sweet potato black rot pathogen.
Above-mentioned LAMP detection primer carries out method the examining in sweet potato black rot pathogen of LAMP reaction detection sweet potato black rot pathogen
Application in disconnected, detection, identification.
The high sensitivity quickly detection for the plant and soil that this method can be used for carrying disease germs.Establish sweet potato black rot pathogen quickly,
Simplicity, high specificity, high sensitivity monitoring technology system, for sweet potato black rot pathogen cause disease show disease before early stage supervise
It surveys, determines that disease control best period has a very important significance.
Sweet potato black rot pathogen that the invention has the advantages that: the method for the present invention suitable for incidence tissue or soil it is fast and reliable
Detection and identification, disease control microbial for sweet potato black rot in agricultural production have important practical value.This hair
It is bright compared with prior art, have technical advantage below and good effect:
1, result is reliable: the designed LAMP detection primer out of the present invention, to the sweet potato derived from Fujian China, Sichuan and other places
Alternaria and the soil sample carried disease germs, plant tissue are tested verifying, therefore result reliability has adequately guarantee；
2, high specificity: LAMP primer of the present invention is for sweet potato black rot pathogenTsr16 differences in gene order
4 specific primers are designed in region, and any region and primer mismatch not can be carried out nucleic acid amplification, Gu Te in 6 regions
It is anisotropic high.
3, high sensitivity: LAMP can reach 1 pg to the detection sensitivity of sweet potato black rot pathogen on DNA level, than routine
PCR detection is 1000 times high.
4, practicability is good: the present invention it is designed go out LAMP primer, for the tissue with sweet potato black rot pathogen and soil
Highly sensitive quickly detection, this method it is practical, be able to satisfy to sweet potato black rot pathogen present in band hyphostroma and soil
The needs for being used for quickly detecting and identifying；
5, easy to operate quick: to apply the method for the present invention, tissue and soil to sweet potato black rot pathogen detect can be at 1 hour
Interior completion, and LAMP nucleic acid amplification is to carry out under isothermal conditions, only needs a water-bath, does not need complicated instrument and sets
Standby and expensive molecular agents, as a result naked eyes are directly visible.
Detailed description of the invention
The LAMP primer design diagram of Fig. 1 sweet potato black rot pathogen of the present invention.Yl moiety is expressed as outer primer F3, B3；
Positive inner primer (FIP) is made of the reverse complementary sequence of F1 plus F2, and rear reversed inner primer (BIP) adds B2 reverse mutual by B1
Complementary series is constituted.
The LAMP testing result figure of Fig. 2 sweet potato black rot pathogen of the present invention.A: colour developing result is indicated, in which: the 1st pipe is feminine gender
Control, the positive detection of the 2nd Guan Weihan sweet potato black rot pathogen DNA；B: agarose gel electrophoresis testing result is indicated, in which: swimming
Road M is 2000 DNA marker of DL, and swimming lane 1 is negative control (ddH2O)；Swimming lane 2 is the positive of the DNA containing sweet potato black rot pathogen
Control.
The LAMP specific detection result figure of Fig. 3 sweet potato black rot pathogen of the present invention.A: visualization colour developing result is indicated；B: table
Show agarose gel electrophoresis testing result, in which: swimming lane M is 2000 DNA marker of DL, and 1 is the sweet potato in In Fujian Province source
Alternaria DNA；2 be the sweet potato black rot pathogen DNA in Sichuan province source；3-26 is other fungal bacterial strains of 1 reference numeral of table
DNA。
The LAMP sensitivity testing result figure of Fig. 4 sweet potato black rot pathogen of the present invention.A: colour developing result is indicated, in which: the 1st pipe
For negative control, the 2nd -9 pipe template quantity is respectively 100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, and 100
Fg and 10 fg；B: agarose gel electrophoresis testing result is indicated, in which: swimming lane M is 2000 DNA marker of DL, the 1st swimming
Road is negative control, and the 2nd -9 swimming lane template quantity is respectively 100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg,
100 fg and 10 fg.
Testing result figure of Fig. 5 present invention to disease plant and soil.A: colour developing result is indicated, in which: the 1st pipe is the positive
Control, the 2nd, 3,4,5 pipes are morbidity soil, and the 6th, 7,8,9,10,11 pipes are incidence tissue, and the 12nd pipe is health tissues；B: table
Show agarose gel electrophoresis testing result, in which: swimming lane 1 is positive control, and swimming lane 2,3,4,5 is morbidity soil, swimming lane 6,7,
8,9,10,11 be incidence tissue, and swimming lane 12 is health tissues；Swimming lane M is 2000 DNA marker of DL.
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.
Main agents:BstDNA polymerase Large fragment is purchased from Britain NEB company；DNA marker is purchased from precious bioengineering
Dalian Co., Ltd；Remaining reagent is purchased from Sangon Biotech (Shanghai) Co., Ltd..Primer is by giving birth to work bioengineering
The synthesis of (Shanghai) limited liability company.
The design of 1 sweet potato black rot pathogen LAMP detection primer of embodiment
1. Ceratocystis fimbriata bacterium (Ceratocystis fimbriataEllis et Halsted)Tsr1The acquisition of gene
(1) extraction of sweet potato black rot pathogen genomic DNA:
Sweet potato black rot pathogen genomic DNA is extracted using CTAB method, detailed process is as follows: the mycelia after taking 50 mg to be freeze-dried
900 μ l, 2% CTAB(cetyl trimethylammonium bromide is added in 1.5 ml centrifuge tubes in powder) extracting solution (2% CTAB；100
Mmol/L Tris-HCl(Tri(Hydroxymethyl) Amino Methane Hydrochloride), pH 8.0；20 mmol/L EDTA(ethylenediamine tetra-acetic acids two
Sodium), pH 8.0；1.4 mol/L NaCl) and 90 μ l, 10% SDS(neopelex) mix afterwards, in 55~60 DEG C
1.5 h of water-bath, every 10 min oscillation mix once, (12,000rpm) 15 min are centrifuged after 1.5 h of water-bath, supernatant is taken to be added
Isometric phenol/chloroform/isoamyl alcohol (volume ratio of phenol, chloroform and isoamyl alcohol is 25:24:1), centrifugation (12,000 with supernatant
Rpm) 5 min takes supernatant (water phase), is added and the isometric chloroform of supernatant primary (12,000 rpm) centrifugation 5
Min, sucts clear (350 μ l), and the ice of the 3 mol/L NaAC solution and 2 volumes (700 μ l) that add 0.1 volume (35 μ l) is anhydrous
Ethyl alcohol precipitates 12,000 rpm after 30 min at -20 DEG C and is centrifuged 5 min, lightly removes supernatant, 700 μ l ice 70% of addition
Ethyl alcohol is washed and (is slightly centrifuged, incline and fall supernatant), with 1 × TE(10 after the alcohol-free taste of naturally dry on superclean bench
Mmol/L Tris-HCl, 0.1 mmol/L EDTA, pH 8.0) solution dissolved, DNA solution is obtained, with ultraviolet spectrometry light
Degree meter detection DNA concentration and to be diluted to 100 ng/ μ l stand-by.
(2) PCR reaction system: 50 μ l reaction systems are general to draw comprising 2 × Taq PCR Master Mix, 25 μ l
Object Tsr1_1453for/Tsr1_2308rev (10 μm of ol/L) each 2 μ l, the middle template DNA extracted of step (1)
100ng complements to 50 μ l with aseptic double-distilled water.For universal primer quoted from Schmitt et al. 2009, sequence is SEQ ID
NO.2 Tsr1_1453for:CCIGAYGARATYGARCTICAYCC；SEQ ID NO.3 Tsr1_2308rev:
CTTRAARTAICCRTGIGTICC。
(3) PCR reaction system: the reaction system of step (2), which is placed in following condition, reacts, 94 DEG C of 3 min of initial denaturation；
94 DEG C of 30 s of denaturation, 55 DEG C of 30 s of annealing, 72 DEG C of 1 min of extension, 30 recycle；10 min of last 72 DEG C of extensions.
(4) step (3) reaction products therefrom is sent to Sangon Biotech (Shanghai) Co., Ltd. and is sequenced, institute
?Tsr1The nucleotide sequence of gene, as shown in SEQ ID NO.1.
2. the design of sweet potato black rot pathogen LAMP detection primer
According to sequencing gainedTsr1The nucleotide sequence of gene utilizes online software primer software
PrimerExplorer V5 (http://primerexplorer.jp/lampv5e/index.html; Eiken
Chemical Co., Japan) design LAMP primer, outer primer F3/B3, positive inner primer (FIP) by F1 reverse complemental sequence
Column are constituted plus F2, and reversed inner primer (BIP) constitutes (as shown in Figure 1) plus the reverse complementary sequence of B2 by B1.
Gained LAMP detection primer includes 2 outer primer F3 and B3 and 2 inner primer FIP and BIP, primer
Sequence is respectively as follows:
F3: 5’-CCAGGCACACGAGTCAGT-3’
B3: 5’-TCCTCCTTGGACTTGATCGA-3’
FIP: 5’- CGTCACAGGGTGCAGAGCAGGTGTACATCAGTGGCGCTAT-3’
BIP: 5’-TGCTGCGCCACGAACACAAGTGGGTATTCGGAGCTGAGAT-3’。
The Visual retrieval of embodiment 2:LAMP primer pair sweet potato black rot pathogen
(1) extraction of sweet potato black rot pathogen genomic DNA:
Sweet potato black rot pathogen genomic DNA is extracted using CTAB method, detailed process is as follows: the mycelia after taking 50 mg to be freeze-dried
900 μ l, 2% CTAB(cetyl trimethylammonium bromide is added in 1.5 ml centrifuge tubes in powder) extracting solution (2% CTAB；100
Mmol/L Tris-HCl(Tri(Hydroxymethyl) Amino Methane Hydrochloride), pH 8.0；20 mmol/L EDTA(ethylenediamine tetra-acetic acids two
Sodium), pH 8.0；1.4 mol/L NaCl) and 90 μ l, 10% SDS(neopelex) mix afterwards, in 55~60 DEG C
1.5 h of water-bath, every 10 min oscillation mix once, (12,000 rpm) 15 min are centrifuged after 1.5 h of water-bath, supernatant is taken to be added
Isometric phenol/chloroform/isoamyl alcohol (volume ratio of phenol, chloroform and isoamyl alcohol is 25:24:1), centrifugation (12,000 with supernatant
Rpm) 5 min takes supernatant (water phase), is added and the isometric chloroform of supernatant primary (12,000 rpm) centrifugation 5
Min, sucts clear (350 μ l), and the ice of the 3 mol/L NaAC solution and 2 volumes (700 μ l) that add 0.1 volume (35 μ l) is anhydrous
Ethyl alcohol precipitates 12,000 rpm after 30 min at -20 DEG C and is centrifuged 5 min, lightly removes supernatant, 700 μ l ice 70% of addition
Ethyl alcohol is washed and (is slightly centrifuged, incline and fall supernatant), with 1 × TE(10 after the alcohol-free taste of naturally dry on superclean bench
Mmol/L Tris-HCl, 0.1 mmol/L EDTA, pH 8.0) solution dissolved, DNA solution is obtained, with ultraviolet spectrometry light
Degree meter detection DNA concentration and to be diluted to 100 ng/ μ l stand-by.
(2) LAMP reaction system: 25 μ l reaction systems include 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM
KCl, 8 mM MgSO4, 0.8 mol/L of glycine betaine, Bst polymerase is 8 U, dNTPs 1.0 mmol/L, F3 and B3 each 0.2
Each 1.6 mmol/L of mmol/L, FIP and BIP, 50 μm of ol/L of calcein, manganese chloride 500 μm of ol/L, TWeen-20 0.1%,
100 ng of template DNA that step (1) is extracted, complements to 25 μ l with aseptic double-distilled water；Template DNA is replaced with into equivalent
ddH2O does negative control.
(3) LAMP reacts: the reaction system of step (2) is placed in 64 DEG C of incubations 60 min, 85 DEG C of 10 min of inactivation；
(4) result detects: after LAMP reaction, colour developing result observes that green fluorescence is judged as positive, orange to be judged as negative；
Or 2 μ l amplified productions is taken to be detected with 2% agarose gel electrophoresis, there is the characteristic trapezoid belt of LAMP and be judged as positive, does not have
There is amplified band and is judged as negative.
The testing result of the present embodiment is shown in Fig. 2.Fig. 2A visual test result shows: experimental group is with sweet potato black rot pathogen base
Because group DNA is template, colour developing result observes green fluorescence, for the positive；And control group is with ddH2O is template, and colour developing result is seen
Observe it is orange, for feminine gender.Fig. 2 B agarose gel electrophoresis testing result shows: experimental group is with sweet potato black rot pathogen genomic DNA
For template, there is the characteristic trapezoid belt of LAMP, for the positive；And control group is with ddH2O is template, amplified band does not occur,
For feminine gender.Illustrate that LAMP detection primer of the invention can be used in the Visual retrieval of sweet potato black rot pathogen.
The specific amplification of embodiment 3:LAMP primer pair sweet potato black rot pathogen
Using China Fujian, 2 plants of sweet potato black rot pathogens in Sichuan and 24 kinds of other disease fungus and oomycetes as material to be tested, to detection
The specificity of primer carries out LAMP verifying.
(1) strains tested:
(2) extraction of strains tested genomic DNA:
Using CTAB method extract strains tested genomic DNA, detailed process is as follows: take 50 mg be freeze-dried after hypha powder in
In 1.5 ml centrifuge tubes, 900 μ l, 2% CTAB(cetyl trimethylammonium bromide is added) extracting solution (2% CTAB；100
Mmol/L Tris-HCl(Tri(Hydroxymethyl) Amino Methane Hydrochloride), PH 8.0；20 mmol/L EDTA(ethylenediamine tetra-acetic acids two
Sodium), pH 8.0；1.4 mol/L NaCl) and 90 μ l, 10% SDS(neopelex) mix afterwards, in 55~60 DEG C
1.5 h of water-bath, every 10 min oscillation mix once, (12,000rpm) 15 min are centrifuged after 1.5 h of water-bath, supernatant is taken to be added
Isometric phenol/chloroform/isoamyl alcohol (volume ratio of phenol, chloroform and isoamyl alcohol is 25:24:1), centrifugation (12,000 with supernatant
Rpm) 5 min takes supernatant (water phase), is added and the isometric chloroform of supernatant primary (12,000 rpm) centrifugation 5
Min, sucts clear (350 μ l), and the ice of the 3 mol/L NaAC solution and 2 volumes (700 μ l) that add 0.1 volume (35 μ l) is anhydrous
Ethyl alcohol precipitates 12,000 rpm after 30 min at -20 DEG C and is centrifuged 5 min, lightly removes supernatant, 700 μ l ice 70% of addition
Ethyl alcohol is washed and (is slightly centrifuged, incline and fall supernatant), with 1 × TE(10 after the alcohol-free taste of naturally dry on superclean bench
Mmol/L Tris-HCl, 0.1 mmol/L EDTA, pH 8.0) solution dissolved, DNA solution is obtained, with ultraviolet spectrometry light
Degree meter detection DNA concentration and to be diluted to 100 ng/ μ l stand-by.
(3) LAMP reaction system: 25 μ l reaction systems include 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM
KCl, 8 mM MgSO4, 0.8 mol/L of glycine betaine, Bst polymerase is 8 U, dNTPs 1.0 mmol/L, F3 and B3 each 0.2
Each 1.6 mmol/L of mmol/L, FIP and BIP, 50 μm of ol/L of calcein, manganese chloride 500 μm of ol/L, TWeen-20 0.1%,
100 ng of template DNA that step (1) is extracted, complements to 25 μ l with aseptic double-distilled water；Template DNA is replaced with into equivalent
ddH2O does negative control.
(3) LAMP reacts: the reaction system of step (2) is placed in 64 DEG C of incubations 60 min, 85 DEG C of 10 min of inactivation.
(4) result detects: after LAMP reaction, colour developing result observes that green fluorescence is judged as positive, orange to be judged as
It is negative；Or 2 μ l amplified productions is taken to be detected with 2% agarose gel electrophoresis, there is the characteristic trapezoid belt of LAMP and be judged as positive,
There is not amplified band and is judged as negative.
The testing result of the present embodiment is shown in Fig. 3.The specificity of detection: in addition to 2 plants of sweet potatoes from China Fujian, Sichuan are black
Green fluorescence can be observed for pinta bacterium colour developing result or having detected outside the characteristic trapezoid belt of LAMP occurs in agarose gel electrophoresis
Other 24 kinds of disease fungus bacterial strains colour developing results are that orange or agarose gel electrophoresis amplified band does not occur (result is shown in figure
3), illustrate that this primer has very strong specificity.
The sensitivity of embodiment 4:LAMP primer pair sweet potato black rot pathogen detects
(1) the sweet potato black rot pathogen DNA of extraction is diluted to 100 ng/ μ l, 10 ng/ μ l using 10 times of concentration series dilution methods,
1 ng/ μ l, 100 pg/ μ l, 10 pg/ μ l, 1 pg/ μ l, 100 fg/ μ l, 10 fg/ μ l DNA, totally 8 various concentration gradients.
(2) LAMP reaction system: 25 μ l reaction systems include 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM
KCl, 8 mM MgSO4, 0.8 mol/L of glycine betaine, Bst polymerase is 8 U, dNTPs 1.0 mmol/L, F3 and B3 each 0.2
Each 1.6 mmol/L of mmol/L, FIP and BIP, 50 μm of ol/L of calcein, manganese chloride 500 μm of ol/L, TWeen-20 0.1%,
100 ng of template DNA that step (1) is extracted, complements to 25 μ l with aseptic double-distilled water；Template DNA is replaced with into equivalent
ddH2O does negative control.
(3) LAMP reacts: the reaction system of step (2) is placed in 64 DEG C of incubations 60 min, 85 DEG C of 10 min of inactivation.
(4) result detects: after LAMP reaction, colour developing result observes that green fluorescence is judged as positive, orange to be judged as
It is negative；Or 2 μ l amplified productions is taken to be detected with 2% agarose gel electrophoresis, there is the characteristic trapezoid belt of LAMP and be judged as positive,
There is not amplified band and is judged as negative.
The testing result of the present embodiment is shown in Fig. 4.Fig. 4 the result shows that sweet potato black rot pathogen LAMP sensitivity detects, tie by colour developing
Green fluorescence can be observed in fruit or the characteristic trapezoid belt of LAMP occurs in agarose gel electrophoresis, and detection sensitivity is up to 1 pg/ μ
L has very high sensitivity.
Embodiment 5: the detection of sweet potato black rot pathogen in incidence tissue or soil.
1. sample acquires: plant tissue and pedotheque pick up from Zhangzhou, Fujian city Zhangpu County.
2. sweet potato black rot pathogen extracting genome DNA: morbidity plant tissue is mentioned using the NaOH rapid cleavage method of embodiment 1
Take sweet potato black rot pathogen genomic DNA；Sweet potato black rot pathogen extracting genome DNA extracts examination using soil DNA in morbidity soil
Agent box extracts genomic DNA.
25 μ l of LAMP reaction system: including 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 8 mM
MgSO4, 0.8 mol/L of glycine betaine, Bst polymerase is 8 U, dNTPs 1.0 mmol/L, F3 and B3 each 0.2 mmol/L, FIP
With each 1.6 mmol/L of BIP, 50 μm of ol/L of calcein, manganese chloride 500 μm of ol/L, TWeen-20 0.1%, step (1) is mentioned
100 ng of template DNA taken complements to 25 μ l with aseptic double-distilled water；Template DNA is replaced with to the ddH of equivalent2O does negative right
According to.LAMP reaction condition is in 65 DEG C of incubations 60 min, 82 DEG C of 10 min of heat preservation.
After LAMP reaction, colour developing result observes that green fluorescence is judged as positive, orange to be judged as negative；Or take 2 μ l
Amplified production is detected with 2% agarose gel electrophoresis, the characteristic trapezoid belt of LAMP is occurred and is judged as positive, does not expand
Band is judged as negative.
The testing result of the present embodiment is shown in Fig. 5；Fig. 5 the result shows that, infect sweet potato black rot pathogen in incidence tissue or soil,
Green fluorescence can be observed in colour developing result or the characteristic trapezoid belt of LAMP, health tissues colour developing knot occurs in agarose gel electrophoresis
Fruit then observes that fluorescent orange or agarose gel electrophoresis do not occur the characteristic trapezoid belt of LAMP.
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>LAMP detection primer and its application of a kind of sweet potato black rot pathogen
<130> 7
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 821
<212> DNA
<213>artificial sequence
<400> 1
attgaactct cacccaacgt cctagccagg gagcgtttgt ctcgataccg tggtctcaag 60
tccctgcgta ccagtgagtg ggttgtcgat gaggaccgtg cccacgagcc tgaagactgg 120
cgtcgtctat tgcgtgtctc tgactaccaa ggcacgagat cccgcgtgac tcgtgaggct 180
cttgttggcg gcgtggcccc aggcacacga gtcagtgtgt acatcagtgg cgctattcca 240
gagcatgtta agacgctccc gagcgctgct ctgcaccctg tgacgctctt ctcgctgctg 300
cgccacgaac acaagcagac ggtttccaat gtgctgatta atctcagctc cgaataccca 360
acttcgatca agtccaagga ggaactgatt atgcaatgcg gcgctcgccg ttttattatc 420
aagcctctat tctcacaacc aggcaacact cctaataacg tgcacaaata tgcccggtat 480
ctacatccag gacaaagcgc cgtggctacc ttcacagggc ctgtcacctg gggtcctgtg 540
ccagcattgt tctttaagcg cacggccgat gtagagaacg tggctgaaag ctcagctatc 600
cccagcagct cgggcctgac cctagtggca acaggcactt gcctaccacc atctacaaca 660
agagtcattg ccaagagagt aattctcact ggccacccgt accatattca caagcgagtg 720
gtaaccatcc gctacatgtt cttcaacaag gaggatgtgg agtggttcaa agccctgccg 780
ttgtggacta agagaggccg aactggtttc gtcaaggaaa c 821
<210> 2
<211> 21
<212> DNA
<213> Tsr1_1453for
<400> 2
ccigaygarat ygarcticayc c 23
<210> 3
<211> 18
<212> DNA
<213> Tsr1_2308rev
<400> 3
cttraartaic crtgigticc 21
<210> 4
<211> 18
<212> DNA
<213> F3
<400> 4
ccaggcacac gagtcagt 18
<210> 5
<211> 20
<212> DNA
<213> B3
<400> 5
tcctccttgg acttgatcga 20
<210> 6
<211> 40
<212> DNA
<213> FIP
<400> 6
cgtcacaggg tgcagagcag gtgtacatca gtggcgctat 40
<210> 7
<211> 40
<212> DNA
<213> BIP
<400> 7
tgctgcgcca cgaacacaag tgggtattcg gagctgagat 40
Claims (4)
1. a kind of LAMP detection primer of sweet potato black rot pathogen, it is characterised in that: the LAMP detection primer includes 2 outer primers
F3 and B3 and 2 inner primer FIP and BIP, primer sequence are respectively as follows:
F3:5'-CCAGGCACACGAGTCAGT-3',
B3:5'-TCCTCCTTGGACTTGATCGA-3'；
FIP:5'-CGTCACAGGGTGCAGAGCAGGTGTACATCAGTGGCGCTAT-3',
BIP:5'-TGCTGCGCCACGAACACAAGTGGGTATTCGGAGCTGAGAT-3'.
2. a kind of LAMP detection primer using sweet potato black rot pathogen described in claim 1 carries out LAMP reaction detection sweet potato blackspot
The method of germ, which comprises the following steps:
(1) extraction of sample to be tested genomic DNA；
(2) LAMP detects reaction system: the DNA extracted using step (1) is template, and using F3/B3, FIP/BIP primer is carried out
Amplification;
Reaction system is 25 μ l, includes 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 8 mM MgSO4, sweet tea
0.8 mol/L of dish alkali,BstPolymerase is 8 U, and dNTPs 1.0 mmol/L, F3 and B3 each 0.2 mmol/L, FIP and BIP is each
1.6 mmol/L, 50 μm of ol/L of calcein, manganese chloride 500 μm of ol/L, TWeen-20 0.1%, template DNA 50 ~ 100
Ng, aseptic double-distilled water complement to 25 μ l；
(3) LAMP reacts: the reaction system of step (2) is in 64 DEG C of incubations 60 min, 85 DEG C of 10 min of inactivation；
(4) result detects: being detected with fluorescent dye visual observations method and agarose gel electrophoresis method；
Fluorescent dye visual observations method: after LAMP reaction, reaction solution colour developing result observes that green fluorescence is judged as positive, orange
Color is judged as negative；
Agarose gel electrophoresis method: it takes 2 μ l LAMP reaction products to be detected with 2% agarose gel electrophoresis, LAMP characteristic occurs
Trapezoid-shaped strips be then judged as positive, there is not amplified band and be then judged as negative.
3. the LAMP detection primer of sweet potato black rot pathogen as described in claim 1 is in the diagnosis, detection, mirror of sweet potato black rot pathogen
Application in fixed.
4. the LAMP detection primer of sweet potato black rot pathogen as claimed in claim 2 carries out LAMP reaction detection sweet potato black rot pathogen
Application of the method in the diagnosis, detection, identification of sweet potato black rot pathogen.
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| CN111705159A (en) * | 2020-07-28 | 2020-09-25 | 河北省农林科学院植物保护研究所 | Real-time fluorescent RPA detection primer for sweet potato black spot germs and application thereof |
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