CN109207619A - The LAMP primer pair and its amplification method and purposes of detection chestnut Heisui River phytophthora - Google Patents
The LAMP primer pair and its amplification method and purposes of detection chestnut Heisui River phytophthora Download PDFInfo
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- CN109207619A CN109207619A CN201810890759.XA CN201810890759A CN109207619A CN 109207619 A CN109207619 A CN 109207619A CN 201810890759 A CN201810890759 A CN 201810890759A CN 109207619 A CN109207619 A CN 109207619A
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Abstract
The invention discloses a kind of LAMP primer pair for detecting chestnut Heisui River phytophthora and its amplification method and purposes, belong to plant pathogenic fungi detection field, designed, designed has synthesized 1 set of primer PC-F3, PC-B3, PC-FIP and PC-BIP, optimizing reaction system and amplification program, establish a kind of fast and convenient, high specificity, accurately and reliably plant and the Heisui River plant product Zhong Li phytophthora molecular detecting method, entire detection is completed in one working day, for the specific detection to chestnut Heisui River phytophthora genomic DNA.
Description
Technical field
The present invention relates to the methods for using LAMP technology detection plant and the Heisui River plant product Zhong Li phytophthora, belong to plant
Disease fungus detection field.
Background technique
Chestnut Heisui River phytophthora is the quarantine fungi that China's quarantine departments are attached great importance to.It in addition to chestnut black canker can be caused,
Root, stem rot disease can also be caused on many fruit trees.The germ of chestnut black canker usually infects butt and biggish, draws
Trunk and root rot are played, germ is infected initial stage, and symptom is unobvious；Later period, leaf become smaller, and turn yellow, fall off, or do not show obvious
Symptom, and trees are dead quickly.The germ and the typical root softening of other butt rot diseases, watery rotten symptom are not
Together, after infection, cardinal symptom is that butt rot makes the root browning infected, is hardened, becomes fragile, and harm is serious.
Epidemic disease caused by the phytophthora of chestnut Heisui River has become the problem that multiple national fruit industries face, and is that domestic each fruit enters the territory
One of the epidemic situation that port is paid close attention to.
Phytophthora conventional biology detection method is plant seed borne fungi at present, can be by naked eyes directly by being separately cultured
The morphological features such as bacterium colony, sporangium, egg spore are observed to be identified.The pathogen strain culturing time need to be up to 20 days or so ability
It lures to swash and generates sporangium, such situation not only time and effort consuming, morphological feature, cultural colony and the physiological property of phytophthora pathogen
Etc. very much like, it is difficult according to colonial morphology, the form of sporangium and deciduous, the generation for having sexual organ and form, thick wall spore
The presence or absence of son, mycelia the characteristics such as growth temperature come it is accurate, fast and effeciently identify chestnut Heisui River phytophthora, molecular biology method
Such as ELISA(Mohan. Evaluation of antisera raised against Phytophthora fragariae
for detecting the red core disease of strawberries by enzyme-linked
Immunosorbent assay (ELISA) Plant Pathology1988,37:206-216), monoclonal antibody
(Burns et al. The use of monoclonal antibodies for the detection of fungi.
Bulletin OEPP/EPPO Bulletin. 1995,25:31-38) etc. have application, but be only able to detect Phytophthora
It is horizontal.Current detection method is not able to satisfy the requirement of port detection " test fastly fastly put ", easily leads to disease and propagates in China.Cause
This, reinforce to port enter the territory plant product carry phytophthora fast and accurately Testing and appraisal seem particularly important.Ring mediated isothermal
Amplification technique (loop-mediated isothermal amplification, LAMP) is that Japanese scholars Notomi is equal to 2000
A kind of novel constant-temperature nucleic acid amplification method of year exploitation.The technology has high sensitivity, high specificity, facilitates simple and direct, detection cycle
Short, instrument and equipment requires low advantage, greatly reduces input cost, application value with higher has been widely used at present
In fields such as clinic, food safety, agriculturals.Application in terms of plant virus, bacterium is also quite mature, such as Fukuta is answered
Tomato yellow leaf curl virus (TYLCV) and tomato spotted wilf virus (TSWV) are successively detected with LAMP, Okuda etc. establishes yellow twig
LAMP detection method etc..But without the report that LAMP technology is applied to the detection of chestnut Heisui River phytophthora.
Summary of the invention
The technical problems to be solved by the present invention are: the LAMP primer of design chestnut Heisui River phytophthora, establishes chestnut Heisui River phytophthora
LAMP detection method.
In order to solve this problem, solution of the invention is to design the special LAMP primer of chestnut Heisui River phytophthora, is used for chestnut
Heisui River phytophthora genomic DNA specific detection.The LAMP special primer of chestnut Heisui River phytophthora, sequence are as follows:
PC-F3: TCT GTC CTA GGT CCA CCA T
PC-B3:CTA CAA TTC CGA ATA ATC ACA GTG T
PC-FIP:TGA TGG TCT TGC CGT CCA GTG ACC TCC AGG CTG ACG TTA T
PC-BIP:CCA GAT TGT GCG TGC ATT CCG TTA GCT CCA TGA AGC ACT TTG AA
After reaction, can observe by the naked eye whether reaction solution is white opacity (white opacity is the positive, is otherwise feminine gender)
To judge amplification；SYBR Green I fluorescent dye observation color change, reaction solution face can also be added in amplified production
Color is by the orange green that becomes for the positive, and otherwise color keep is constant, or using agarose gel electrophoresis observation scalariform band
Whether there is or not judge.
A kind of LAMP primer pair and its amplification method and purposes detecting chestnut Heisui River phytophthora, including following amplification systems
And amplification program:
(1) amplification system
The amplification system for establishing 25 μ L is, including 10 × ThermoPoL Buffer, 2.5 μ L(200 mmol/L Tris-HCl,
100 mmol/L KCl, 20 mmol/L MgSO4,100 mmol/L (NH4) 2SO4,1.0% Tritonx-100), 2.5
mmol/L dNTPs 2.5 μL、100 mmol/L MgSO4 1μL、10 mmol/L PC-F3 0.2 μL、10 mmol/L PC-
B3 0.2 μL、10 mmol/L PC-FIP 1.6 μL、10 mmol/L PC-BIP 1.6 μL、8 U/μL Bst DNA
1 μ L of Polymerase, 1 μ L of DNA profiling, surplus is distilled water；
(2) response procedures
60 min are reacted in 63 DEG C of water-baths, 5min is inactivated at 85 DEG C later, and reaction was completed.
It is a kind of detect chestnut Heisui River phytophthora LAMP primer pair and its amplification method purposes.
The beneficial effects of the present invention are: the LAMP specific primer of the chestnut Heisui River phytophthora of method simplicity is established, it can be by chestnut
Heisui River phytophthora is distinguished with other phytophthoras.Simplify and facilitate the detection to the phytophthora, has saved detection time, this method is fast
It is fast, reliable, it can be effectively applicable in the detection of port.
Detailed description of the invention
Scheme the amplification first is that the LAMP special primer of chestnut Heisui River phytophthora.
Specific embodiment
Chestnut Heisui River phytophthora LAMP specific primer of the invention, realizes the quick detection of chestnut Heisui River phytophthora.
The special LAMP primer of chestnut Heisui River phytophthora is designed, sequence is as follows:
PC-F3: TCT GTC CTA GGT CCA CCA T
PC-B3:CTA CAA TTC CGA ATA ATC ACA GTG T
PC-FIP:TGA TGG TCT TGC CGT CCA GTG ACC TCC AGG CTG ACG TTA T
PC-BIP:CCA GAT TGT GCG TGC ATT CCG TTA GCT CCA TGA AGC ACT TTG AA
After reaction, can observe by the naked eye whether reaction solution is white opacity (white opacity is the positive, is otherwise feminine gender)
To judge amplification；SYBR Green I fluorescent dye observation color change, reaction solution face can also be added in amplified production
For color by the orange green that becomes for the positive, otherwise color keep is constant, is used for chestnut Heisui River phytophthora genomic DNA specific detection.
The present invention establishes a kind of LAMP primer pair and its amplification method for detecting chestnut Heisui River phytophthora, including amplification system
It establishes and amplification program is set up:
(1) amplification system
The amplification system for establishing 25 μ L is that total volume is 25 μ L, including 10 × ThermoPoL Buffer, 2.5 μ L(200
mmol/L Tris-HCl、 100 mmol/L KCl、20 mmol/L MgSO4、100 mmol/L(NH4)2SO4、1.0%
Tritonx-100), 2.5 mmol/L dNTPs, 2.5 μ L, 100 mmol/L MgSO4,1 μ L, 10 mmol/L PC-F3 0.2
μL、10 mmol/L PC-B3 0.2 μL、10 mmol/L PC-FIP 1.6 μL、10 mmol/L PC-BIP 1.6 μL、8
1 μ L of U/ μ L Bst DNA Polymerase, 1 μ L of DNA profiling, surplus is distilled water；
(2) response procedures
60 min are reacted in 63 DEG C of water-baths, 5min is inactivated at 85 DEG C later, and reaction was completed.
The present invention designs the LAMP specific primer of synthesis chestnut Heisui River phytophthora, carries out matter to all experimental material mycelia DNA
Amount detection, avoids false negative.
The LAMP primer pair of the detection chestnut Heisui River phytophthora and its application of amplification method.
The present invention establishes easy quick, high specificity, high sensitivity, accurately and reliably chestnut Heisui River phytophthora LAMP detection
Chestnut Heisui River phytophthora and other phytophthoras can be distinguished by method.The quick detection for being established as chestnut Heisui River phytophthora of this method provides
Special feasible method, has saved the world and time, detection quickly, it is reliable, whole process is completed within a working day, can
Effectively promoted and applied in the detection of port.
Invention is further described in detail with specific embodiment with reference to the accompanying drawing:
Embodiment 1: the culture of experimental material mycelia
This experiment Phytophthora fungal bacterial strain moves herbivore center plant quarantine laboratory derived from Tianjin Entry-Exit Inspection and Quarantine Bureau.Total 15
Kind, relevant information is shown in Table 2.
Embodiment 2: the extraction of mycelia genomic DNA
Mycelia is chosen in 1.5mL centrifuge tube, liquid nitrogen grinding, is extracted according still further to the DNA extraction kit of QIAGEN company production
DNA, and DNA is dissolved in 100 μ L 1 × TE buffers, it is placed in -20 DEG C of preservations.
PCR amplification related reagent is precious biological (TaKaRa) engineering company product in Dalian.
The configuration of 3.1 reaction mixtures
The amplification system for establishing 25 μ L is, including 10 × ThermoPoL Buffer, 2.5 μ L(200 mmol/L Tris-HCl,
100 mmol/L KCl, 20 mmol/L MgSO4,100 mmol/L (NH4) 2SO4,1.0% Tritonx-100), 2.5
mmol/L dNTPs 2.5 μL、100 mmol/L MgSO4 1μL、10 mmol/L PC-F3 0.2 μL、10 mmol/L PC-
B3 0.2 μL、10 mmol/L PC-FIP 1.6 μL、10 mmol/L PC-BIP 1.6 μL、8 U/μL Bst DNA
1 μ L of Polymerase, 1 μ L of DNA profiling, surplus is distilled water.Using the mycelia of chestnut Heisui River phytophthora as positive control, sterile water
For negative control；
3.2 PCR response procedures
60 min are reacted in 63 DEG C of water-baths, 5min is inactivated at 85 DEG C later, and reaction was completed.2% agarose gel electrophoresis, EB
Dyeing and imaging scalariform purpose band, and in centrifuge tube, 1000 × SYBR Green I developing solution, observation knot is added
Fruit.
3.3 interpretation of result
The experimental results showed that LAMP product is glimmering using moment centrifugation observation magnesium pyrophosphate white precipitate and addition SYBR Green I
Photoinitiator dye colour developing determines that result is consistent with electroresis appraisal.
Embodiment described above is merely to illustrate technical idea and feature of the invention, in the art its object is to make
Technical staff it will be appreciated that the contents of the present invention and implement accordingly, patent model of the invention only cannot be limited with the present embodiment
It encloses, i.e., the same variation and modification that all disclosed spirit is done still are fallen in the scope of the patents of the invention.
Sequence table
<110>Animal-Plant and food Detecting Center, Tianjin Exit-Entery Inspection & Quarant
<120>LAMP primer pair of detection chestnut Heisui River phytophthora and its amplification method and purposes
<141> 2018-08-07
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>chestnut phytophthora Heisui River germ (P. cambivora (Petri) Buisman)
<400> 1
tctgtcctag gtccaccat 19
<210> 2
<211> 25
<212> DNA
<213>chestnut phytophthora Heisui River germ (P. cambivora (Petri) Buisman)
<400> 2
ctacaattcc gaataatcac agtgt 25
<210> 3
<211> 39
<212> DNA
<213>chestnut phytophthora Heisui River germ (P. cambivora (Petri) Buisman)
<400> 3
tgaggtcttg ccgtccagtg acctccaggc tgacgttat 39
<210> 4
<211> 44
<212> DNA
<213>chestnut phytophthora Heisui River germ (P. cambivora (Petri) Buisman)
<400> 4
ccagattgtg cgtgcattcc gttagctcca tgaagcactt tgaa 44
Claims (4)
1. a kind of LAMP primer pair for detecting chestnut Heisui River phytophthora and its amplification method and purposes, which is characterized in that designed, designed
Chestnut Heisui River phytophthora LAMP primer pair, realize the specific detection to chestnut Heisui River phytophthora.
Sequence is as follows:
PC-F3: TCT GTC CTA GGT CCA CCA T
PC-B3:CTA CAA TTC CGA ATA ATC ACA GTG T
PC-FIP:TGA TGG TCT TGC CGT CCA GTG ACC TCC AGG CTG ACG TTA T
PC-BIP:CCA GAT TGT GCG TGC ATT CCG TTA GCT CCA TGA AGC ACT TTG AA
The primer pair is for phytophthora hibernalis bacterium genomic DNA specific detection in plant and plant product.
2. a kind of LAMP amplification system for detecting chestnut Heisui River phytophthora: total volume is 25 μ L, including 10 × ThermoPoL
2.5 μ L(200 mmol/L Tris-HCl of Buffer, 100 mmol/L KCl, 20 mmol/L MgSO4,100 mmol/L
(NH4) 2SO4,1.0% Tritonx-100), 2.5 mmol/L dNTPs, 2.5 μ L, 100 mmol/L MgSO4,1 μ L, right
It is required that primer described in 1,10 mmol/L PC-F3,0.2 μ L, 10 mmol/L PC-B3,0.2 μ L, 10 mmol/L PC-
1.6 μ L of FIP, 10 mmol/L PC-BIP, 1.6 μ L, 8 U/ μ L Bst DNA Polymerase, 1 μ L, 1 μ of DNA profiling
L, surplus are distilled water.
3. a kind of LAMP primer pair and its amplification method for detecting chestnut Heisui River phytophthora, uses amplification body as claimed in claim 2
System, response procedures are as follows: react 70 min in 63 DEG C of water-baths, 5min is inactivated at 85 DEG C later, and reaction was completed.
4. described in claim 1 detect chestnut Heisui River phytophthora LAMP primer pair and claim 2,3 described in amplification system and
Application of the amplification method in terms of the phytophthora of specific detection chestnut Heisui River.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116254260A (en) * | 2023-03-16 | 2023-06-13 | 南京林业大学 | Specific detection target g7300 of phytophthora parasitica and application thereof |
| CN116463338A (en) * | 2023-03-16 | 2023-07-21 | 南京林业大学 | Specific detection target g2339 of phytophthora parasitica and application thereof |
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|---|---|---|---|---|
| CN104046698A (en) * | 2014-07-08 | 2014-09-17 | 天津出入境检验检疫局动植物与食品检测中心 | Quadruple PCR (Polymerase Chain Reaction) primers for synchronously detecting three quarantine phytophthoras on malus and amplification method thereof |
| CN106755342A (en) * | 2016-11-30 | 2017-05-31 | 中华人民共和国昆山出入境检验检疫局 | Based on the ring mediated isothermal amplification that color judges（LAMP）Technology for detection chestnut phytophthora Heisui River germ |
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2018
- 2018-08-07 CN CN201810890759.XA patent/CN109207619A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104046698A (en) * | 2014-07-08 | 2014-09-17 | 天津出入境检验检疫局动植物与食品检测中心 | Quadruple PCR (Polymerase Chain Reaction) primers for synchronously detecting three quarantine phytophthoras on malus and amplification method thereof |
| CN106755342A (en) * | 2016-11-30 | 2017-05-31 | 中华人民共和国昆山出入境检验检疫局 | Based on the ring mediated isothermal amplification that color judges（LAMP）Technology for detection chestnut phytophthora Heisui River germ |
Non-Patent Citations (1)
| Title |
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| 朱林慧: "进境水果及种苗检疫性疫霉分子检测方法的建立", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116254260A (en) * | 2023-03-16 | 2023-06-13 | 南京林业大学 | Specific detection target g7300 of phytophthora parasitica and application thereof |
| CN116463338A (en) * | 2023-03-16 | 2023-07-21 | 南京林业大学 | Specific detection target g2339 of phytophthora parasitica and application thereof |
| CN116463338B (en) * | 2023-03-16 | 2023-10-24 | 南京林业大学 | Specific detection target g2339 of phytophthora parasitica and application thereof |
| CN116254260B (en) * | 2023-03-16 | 2024-02-13 | 南京林业大学 | Specific detection target g7300 of phytophthora parasitica and application thereof |
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