CN106434989A - LAMP (Loop-Mediated Isothermal Amplification) quick detection method of alternaria alternata - Google Patents
LAMP (Loop-Mediated Isothermal Amplification) quick detection method of alternaria alternata Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
The invention provides a LAMP (Loop-Mediated Isothermal Amplification) quick detection method of alternaria alternate. By taking alternaria alternate ITS as a target gene, four LAMP specific primers are designed and comprise F3, B3, FIP and BIP; the sequences are shown in SEQ ID NO.1 to 4. According to the LAMP quick detection method provided by the invention, the defects of long detection period, low sensitivity, low specificity, complex operation, high requirement on instrument equipment and the like in the prior art are overcome, and the LAMP quick detection method with the characteristics of strong specificity, high sensitivity, simplicity and convenience in operation, low requirement on environmental equipment and low cost is provided; the LAMP quick detection method is particularly suitable for quickly detecting an alternaria alternate field sample and provides a new method for diagnosing, preventing and controlling tobacco brown spot disease.
Description
Technical field
The invention belongs to corps diseases detection, identification and Prevention Technique field, and in particular to a kind of tobacco brown spot pathogen
LAMP method for quick, can be used for quick, the sensitive and special Molecular Detection of tobacco brown spot pathogen, be simultaneously available for a kind cigarette
The monitoring of the early diagnosiss of careless rust and pathogenic bacteria and identification.
Background technology
Nicotiana tabacum L. is a kind of important leaf of China with industrial crops, is the important sources of country's income, job opportunity and tax revenue
One of.With the prolongation in tobacco planting time, on Nicotiana tabacum L., the species of disease also begins to increase, and these diseases have had a strong impact on China
The quality of Nicotiana tabacum L., wherein by Alternariaspp（Alternaria alternata）Infect the Alternaria alternate for causing（Tobacco
Brown Spot Disease）It is one of main fungal sexually transmitted disease (STD) evil in China's leaf tobacco production, in China, each cigarette district generally occurs.
It is reported that, equal in cigarette districts such as China Hunan, Anhui, Guizhou, Yunnan, Shaanxi, Liaoning, Zhejiang, Guangdong, Fujian, Heilungkiang, Jilin
There is rust to occur, become a kind of leaf spot of the maximum that causes harm, general time sickness rate is that 20%～30%, serious sickness rate reaches
90%, reduce the output value and more than 50% is reached, Yield and quality is affected larger.Alternaria alternate has short incubation period, breaks out fast feature,
Under conditions of humiture is suitable, pandemic by the short time is interior, bring about great losses to leaf tobacco production, be serious restriction
One class fungal disease of China's leaf tobacco production, and one of problem demanding prompt solution in tobacco industry sustainable development.Therefore build
Whether vertical tobacco brown spot pathogen rapid detection system, carry pathogenic bacteria in morbidity early stage to plant and fast and accurately detected, this
Accurate Prediction forecast for disease, formulate in good time effectively preventing measure, the control propagation of disease and popular（Spread）And
The economic loss that minimizing disease is caused all has important theoretical and practical significance.
At present the method for detection phytopathogen is a lot, mainly in the conventional way with molecular biology method based on, that is, divide
Include DNA probe, PCR technology and serum from culture, Pathogenicity, observation of symptoms etc. and advanced Protocols in Molecular Biology
Immunology detection etc., long the time required to traditional detection method, 5～7 days are generally needed, sometimes up to 10～15 days, very
Difficulty meets the needs of Rapid identification；The round pcr for growing up in recent years, is a kind of good technology of quick, sensitive, specificity,
Though round pcr can improve detection efficiency, however it is necessary that the expensive equipment such as special PCR instrument and professional operator and electrophoresis observation knot
Really, in the agricultural sector of basic unit, large-scale promotion application is difficult to；Quickly ELISA method detection, has as screening technique fast
Fast, sensitive feature, is the screening technique that is extensively welcome, but the reagent price for being used is higher, and needs to be equipped with
Its special expensive instrument.
Ring mediated isothermal amplification（Loop-mediated Isothermal Amplification, LAMP）It is Japanese Rong Yan
One kind based on detection hereditary material DNA that a kind of round pcr that continues that KCC was developed in 2000 grows up is new
Amplification technique.The technology designs 4-6 bar specific primers for 6 regions of target gene to be measured,BstArchaeal dna polymerase
Under effect, under isothermal conditions can efficiently, quick, high specifically expand target sequence；It is characterized in without the need for special, expensive inspection
Instrument is surveyed, simple thermostatic equipment or heat block is only needed, amplification can directly be observed magnesium pyrophosphate precipitation or add
Developer is judged, can meet the needs of Site Detection, is widely used in various Pathogen test in recent years.Have not yet to see
Application LAMP technology is used for detecting the correlation technique report of tobacco brown spot pathogen.
Content of the invention
It is an object of the invention to provide a kind of LAMP method for quick of tobacco brown spot pathogen, for right in prior art
Tobacco brown spot pathogen detection and identification are based primarily upon morphological feature, and time-consuming for method, program is loaded down with trivial details, empirical strong, accuracy
Low, it is difficult to the propagation of timely monitoring and control pathogen accomplishing disease, popular problem, and existing PCR molecule
The problems such as detecting needs the expensive instruments such as dependence amplification instrument, and detection time is longer, there is provided the new molecule of tobacco brown spot pathogen
Detection method, carries out LAMP detection to tobacco brown spot pathogen, and detection cycle is short, accuracy is high, susceptiveness is high, perusal detection
As a result.
Realize the purpose of the present invention to comprise the following steps（Technical scheme）：
1. tobacco brown spot pathogen LAMP detects the design of specific primer：By determining tobacco brown spot pathogen（Alternaria alternata）Ribosome transcribed spacer with other pathogen（ITS）Chain lattice are belonged to different inter-species ITS gene orders by gene
Compare, using online LAMP primer design software Primer software Explorer V4 (http://
primerexplorer.jp/elamp4.0.0/index.html;Eiken Chemical Co., Japan) design Nicotiana tabacum L. red
Star pathogenic bacteria specificity LAMP primer group, is made up of F3, B3, FIP and BIP, and primer sequence is as follows：F3：5’-
TCTCTTGGTTCTGGCATCGA-3 ', B3：5 '-GCG AGTCTCCAGCAAAGC-3 ', FIP：5’-
GGCGCAATGTGCGTTCAAAGAT-GAACG CAGCGAAATGCGATA-3 ', BIP：5’-
TGGTATTCCAAAGGGCATGCCT-GACAA GACGCCCAACACC- 3’.
2. the foundation of tobacco brown spot pathogen LAMP detection method, comprises the following steps：
（1）Extract testing sample genomic DNA.
During for pathogen pure culture is detected, extraction genomic DNA is carried out using CTAB method, concrete grammar is as follows：Take
A small amount of mycelium powder is in 1.5 mL centrifuge tubes（Mycelium powder had just covered semicircular base and had been advisable）, add 900 L 2%CTAB（16
Alkyl trimethyl ammonium bromide）Extracting solution（2% CTAB；100 mmol/L Tris-HCl, pH 8.0；20 mmol/L EDTA,
pH8.0；1.4 mol/L NaCl）With 90 L SDS（Dodecylbenzene sodium sulfonate）【Note：CTAB, SDS need 60 DEG C of preheatings】, make
Mixed with agitator vibration, 60 DEG C of water-bath 1h（During DNA is discharged to buffer）, 12000 r min-1It is centrifuged 15 min；Take supernatant
700 L of liquid, plus equal-volume phenol, chloroform, isoamyl alcohol（25:24:1）, gently vibration mixing, 12000 r min-1It is centrifuged 9 min；
500 L of supernatant is taken, adds equal-volume chloroform to extract again once, 12000 r min-1It is centrifuged 5 min；Take supernatant 350
L, adds 1/10 volume, 3 mol L-1NaAc and 2 times of volume dehydrated alcohol, -20 DEG C of precipitations 30 min, 12000 r min-1
It is centrifuged 5 min；Abandoning supernatant, adds 700 L ice, 70% ethanol to be washed（Slightly it is centrifuged；Incline and fall supernatant）, in ultra-clean work
Alcohol-free taste is dried in station, adds 30 ~ 60 L TE（10 mmol/L Tris-HCl, 0.1 mmol/L EDTA, pH 8.0）
Solution is dissolved, and obtains DNA solution, with UV spectrophotometer measuring DNA concentration and is diluted to 100 ng/ L and is treated
With.
When there is tobacco brown spot pathogen for detecting in plant tissue, DNA, concrete mistake are extracted using NaOH rapid cleavage method
Journey is as follows：10 L, 0.5 mol/L NaOH is added in per milligram of plant tissue, in mortar, tissue is fully milled to after paste
Proceed in 1.5mL centrifuge tube, 12,000 rpm are centrifuged 6 min, take 5 l of supernatant and add 495 L, 0.1 mol/L Tris-
HCl（pH=8.0）Mix homogeneously, takes 1.0 L and is expanded as pcr template；
When there is tobacco brown spot pathogen for detecting in pedotheque, using soil DNA extracts kit, DNA is extracted.
（2）The foundation of LAMP reaction system：With step（1）The DNA of extraction is template, using Outside primer F3/B3 and interior
Side primers F IP/BIP carries out LAMP amplification, and LAMP detection reaction system is 25 μ L, including 5 μM of primers F 3 and each 1.0 μ L of B3,
40 μM of primers F IP and each 1.0 μ L, the LAMP reaction mixture of BIP【40 mM Tris-HCl, 20 mM (NH4)2SO4, 20 mM
KCl, 16 mM MgSO4, 1.6 mol/L glycine betaines (Betaine), 2.0 mM dNTPs, 0.2wt.% Trion X-100】
12.5 μ L, 8 UBst1.0 μ L of polymerase, 1.0 μ L of DNA profiling, complement to 25 μ L with sterilizing ultra-pure water；
（3）LAMP reaction condition：64 DEG C of 60 min of incubation；
（4）The measure of reaction result：Using fluorescent dye visual observations method, after LAMP reaction terminates, in the expansion of LAMP reaction
I 1.0 μ L of developer SYBR green is added in volume increase thing, and the result that develops the color observes that the judgement of green fluorescence is the positive, there is cigarette
Careless brown spot pathogen, orange（Crocus）It is judged as feminine gender, there is no tobacco brown spot pathogen.
Beneficial effects of the present invention：The present invention establishes quick, the easy, high specificity of tobacco brown spot pathogen, sensitivity
High LAMP detection technique system, can be used for the detection of tobacco brown spot pathogen, or the morbidity early stage for Alternaria alternate, initial stage
Detection, is of great significance for the best period tool for determining disease control.
The present invention compared with prior art, with following technical advantage and good effect：
1st, high specificity：6 zoness of different of 4 specific primers recognition sequence altogether, in 6 regions, any region is not with primer
Coupling can not all carry out nucleic acid amplification, therefore specificity is high；
2nd, sensitivity height：The present invention can reach 10fg/ μ L on DNA level to the detection sensitivity of tobacco brown spot pathogen, have
Very high susceptiveness；
3rd, quick：Application detection method, can obtain testing result in 1～1.5h, and conventional PCR or nest-type PRC are examined
Surveying needs 4～6h that testing result is just obtained, and the method for the invention substantially reduces operating time, fast and easy；
4th, application is good：LMAP amplified reaction of the present invention is expanded at one temperature, it is not necessary to expensive PCR amplification instrument, is easy to
Basic unit promotes the use of；
5th, testing result is directly perceived, visually can determine whether：Amplified production of the present invention can be dyeed by adding developer, and green is sun
Property, i.e., contain tobacco brown spot pathogen in testing sample, orange for feminine gender, illustrate in testing sample, not containing tobacco brown spot pathogen, visually
Testing result is can determine whether, without the need for electrophoresis detection and gel imaging.
Description of the drawings
Fig. 1 is the LAMP specific detection of tobacco brown spot pathogen of the present invention.In figure 1-4 is tobacco brown spot pathogen, and 5-7 divides
Not Wei tomato early blight bacterium, melon alternaric bacteria, Radix Dauci Sativae alternaric bacteria, 8 be negative control, wherein 1-4 show green fluorescence.
Fig. 2 is tobacco brown spot pathogen LAMP detection sensitivity of the present invention.In figure 1-9 template DNA concentration respectively 1ng,
100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg, 100ag, 10 ag, 10 is that negative control, wherein 1-6 shows green
Fluorescence.
Fig. 3 is detection of the detection method to tobacco brown spot pathogen in incidence of leaf.In figure 1 is positive control, 2
For negative control, 3,5 is Alternaria alternate incidence of leaf, and 4 is health tobacco blade, wherein 1,3,5 display green fluorescences.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is further elaborated, but is not limited to the scope of the present invention.Below
Embodiment is all according to conventional laboratory conditions, or has delivered the operating technology code described in pertinent literature, or is built according to manufacturer
The experiment condition of view.
Embodiment 1：Tobacco brown spot pathogen ring mediated isothermal amplification（LAMP）The design of detection specific primer and primer spy
Opposite sex checking
1. the extraction of strains tested genomic DNA
Strains tested is extracted using CTAB method（Table 1）Genomic DNA, concrete grammar is as follows：A small amount of mycelium powder is taken in 1.5 mL
In centrifuge tube（Mycelium powder had just covered semicircular base and had been advisable）, add 900 L 2%CTAB（Cetyl trimethylammonium bromide）
Extracting solution（2% CTAB；100 mmol/L Tris-HCl, pH 8.0；20 mmol/L EDTA, pH8.0；1.4 mol/L
NaCl）With 90 L SDS（Dodecylbenzene sodium sulfonate）【Note：CTAB, SDS need 60 DEG C of preheatings】, mixed using agitator vibration,
60 DEG C of water-bath 1h（During DNA is discharged to buffer）, 12000 r min-1It is centrifuged 15 min；Take 700 L of supernatant, plus equal-volume
Phenol, chloroform, isoamyl alcohol（25:24:1）, gently vibration mixing, 12000 r min-1It is centrifuged 9 min；500 L of supernatant is taken, plus
Enter equal-volume chloroform to extract again once, 12000 r min-1It is centrifuged 5 min；350 L of supernatant is taken, adds 1/10 volume 3
mol·L-1NaAc and 2 times of volume dehydrated alcohol, -20 DEG C of precipitations 30 min, 12000 r min-1It is centrifuged 5 min；Discard
Clear liquid, adds 700 L ice, 70% ethanol to be washed（Slightly it is centrifuged；Incline and fall supernatant）, dry on superclean bench alcohol-free
Taste, adds 30 ~ 60 L TE（10 mmol/L Tris-HCl, 0.1 mmol/L EDTA, pH 8.0）Solution is dissolved, and is obtained
To DNA solution, with UV spectrophotometer measuring DNA concentration and to be diluted to 100 ng/ L stand-by.
1 strains tested of table
2. tobacco brown spot pathogen ring mediated isothermal amplification（LAMP）The design of special primer
By determining tobacco brown spot pathogen（Alternaria alternata）Ribosome transcribed spacer with other pathogen
（ITS）Chain lattice are belonged to different inter-species ITS gene orders and compare, using online LAMP primer design software by gene
Primer software Explorer V4 (http://primerexplorer.jp/elamp4.0.0/ index.html;
Eiken Chemical Co., Japan) design tobacco brown spot pathogen specificity LAMP primer group, by F3, B3, FIP and BIP group
Become, primer sequence is as follows：F3：5 '-TCTCTTGGTTCT GGCATCGA-3 ', B3：5 '-GCG AGTCTCCAGCAAAGC-3 ',
FIP：5 '-GGCGCAAT GTGCGTTCAAAGAT-GAACGCAGCGAAATGCGATA-3 ', BIP：5’-TGGTATTC
CAAAGGGCATGCCT-GACAA GACGCCCAACACC- 3’.
3. the foundation of tobacco brown spot pathogen LAMP detection method and primer specificity checking
With the DNA of 1 strains tested of table as template, LAMP amplification is carried out using F3, B3, FIP and BIP, LAMP detects reaction system
For 25 μ L, including 5 μM of primers F 3 and each 1.0 μ L of B3,40 μM of primers F IP and each 1.0 μ L, the LAMP reaction mixture of BIP
【40 mM Tris-HCl, 20 mM (NH4)2SO4, 20 mM KCl, 16 mM MgSO4, 1.6 mol/L glycine betaines
(Betaine), 2.0 mM dNTPs, 0.2% Trion X-100】12.5 μ L, 8 UBst1.0 μ L of polymerase, DNA profiling 1.0
μ L, complements to 25 μ L with sterilizing ultra-pure water；LAMP reaction condition：64 DEG C of 60 min of incubation；The measure of reaction result：Using glimmering
Photoinitiator dye visual observations method is measured.After LAMP reaction terminates, developer is added in the amplified production of LAMP reaction
I 1.0 μ L of SYBR green, the result that develops the color observes that the judgement of green fluorescence is the positive, there is tobacco brown spot pathogen, orange（Fructus Citri tangerinae
Yellow）It is judged as feminine gender, there is no tobacco brown spot pathogen.
4. primer specificity the result
LAMP amplification shows, green fluorescence can be observed for only tobacco brown spot pathogen colour developing result in the bacterial strain of examination, its
Remaining strains tested colour developing result is orange（Accompanying drawing 1）, designed tobacco brown spot pathogen primers F 3/B3 and primers F IP/ are described
Tobacco brown spot pathogen can be made a distinction by BIP with other pathogen, with the specificity that plants, can be used for tobacco brown spot pathogen fast
The reliable detection of speed and identification.
Embodiment 2：Tobacco brown spot pathogen ring mediated isothermal amplification（LAMP）Detection sensitivity is determined
1. the preparation of variable concentrations genomic DNA
With aseptic ultra-pure water, tobacco brown spot pathogen genomic DNA is diluted, the series concentration for being configured to 10 times of orders of magnitude is standby
With；
2. LAMP detection method sensitivity determination and result observation
With the tobacco brown spot pathogen genomic DNA of variable concentrations as template, carried out using primers F 3/B3 and primers F IP/BIP
LAMP is expanded, and LAMP detection reaction system is 25 μ L, including 5 μM of primers F 3 and each 1.0 μ L of B3,40 μM of primers F IP and BIP
Each 1.0 μ L, LAMP reaction mixture【40 mM Tris-HCl, 20 mM (NH4)2SO4, 20 mM KCl, 16 mM MgSO4,
1.6 mol/L glycine betaines (Betaine), 2.0 mM dNTPs, 0.2% Trion X-100】12.5 μ L, 8 UBstPolymerase
1.0 μ L, 1.0 μ L of variable concentrations DNA profiling, complement to 25 μ L with sterilizing ultra-pure water；LAMP reaction condition：64 DEG C incubate 60
min；The measure of reaction result：It is measured using fluorescent dye visual observations method, after LAMP reaction terminates, reacts in LAMP
Amplified production in add I 1.0 μ L of developer SYBR green, the result that develops the color observes that the judgement of green fluorescence is the positive, orange
Color（Crocus）It is judged as feminine gender.
3. LAMP expands sensitivity technique result
LAMP amplification sensitivity technique result shows, 1ng, 100pg, 10 pg, 1 pg, 100 fg, the Nicotiana tabacum L. of 10 fg/ μ L concentration
Brown spot pathogen genomic DNA colour developing result can be observed green fluorescence, and remaining concentration and negative control colour developing result are orange, say
Bright designed tobacco brown spot pathogen primers F 3, B3, FIP and BIP is expanded by LAMP, and the detection to tobacco brown spot pathogen is sensitive
Degree is up to 10 fg/ μ L（Accompanying drawing 2）.
Embodiment 3：The LAMP detection of tobacco brown spot pathogen in incidence of leaf
Sample collecting：From Nanping, Fujian, Sanming City, the typical blade of Longyan collection Alternaria alternate disease symptom and healthy leaves band
Return laboratory standby；
The extraction of plant tissue DNA：DNA is extracted using NaOH rapid cleavage method, detailed process is as follows：To per milligram of plant tissue
Tissue is fully milled to proceed in 1.5mL centrifuge tube after paste, 12 in mortar by middle addition 10 L, 0.5 mol/L NaOH,
000 rpm is centrifuged 6 min, takes 5 l of supernatant and adds 495 L, 0.1 mol/L Tris-HCl（pH=8.0）Mix homogeneously, takes
1.0 L are expanded as pcr template.
LAMP augmentation detection and observation：With the DNA of said extracted as template, carried out using primers F 3, B3, FIP and BIP
LAMP is expanded, and LAMP detection reaction system is 25 μ L, including 5 μM of primers F 3 and each 1.0 μ L of B3,40 μM of primers F IP and BIP
Each 1.0 μ L, LAMP reaction mixture【40 mM Tris-HCl, 20 mM (NH4)2SO4, 20 mM KCl, 16 mM MgSO4,
1.6 mol/L glycine betaines (Betaine), 2.0 mM dNTPs, 0.2% Trion X-100】12.5 μ L, 8 UBstPolymerase
1.0 μ L, 1.0 μ L of DNA profiling, complement to 25 μ L with sterilizing ultra-pure water；LAMP reaction condition：64 DEG C of 60 min of incubation；Reaction
As a result measure：It is measured using fluorescent dye visual observations method, after LAMP reaction terminates, produces in the amplification of LAMP reaction
I 1.0 μ L of developer SYBR green is added in thing, and the result that develops the color observes that the judgement of green fluorescence is the positive, orange（Orange
Color）It is judged as feminine gender.
Testing result：Testing result（Accompanying drawing 3）Show, the blade of Alternaria alternate morbidity is expanded by LAMP, colour developing knot
Fruit can be observed green fluorescence, illustrate there is tobacco brown spot pathogen, and healthy leaves and negative control colour developing result are orange, say
Bright do not have tobacco brown spot pathogen, and the set technology can be used for the rapid molecular detection of tobacco brown spot pathogen in plant tissue.
The foregoing is only presently preferred embodiments of the present invention, all impartial changes that is done according to scope of the present invention patent with
Modification, should all belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>Inst. of Plant Protection, fujian Academy of Agricultural Science
<120>The LAMP method for quick of tobacco brown spot pathogen
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
tctcttggtt ctggcatcga 20
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> 2
gcgagtctcc agcaaagc 18
<210> 3
<211> 42
<212> DNA
<213>Artificial sequence
<400> 3
ggcgcaatgt gcgttcaaag atgaacgcag cgaaatgcga ta 42
<210> 4
<211> 40
<212> DNA
<213>Artificial sequence
<400> 4
tggtattcca aagggcatgc ctgacaagac gcccaacacc 40
Claims (4)
1. a kind of LAMP detection primer of tobacco brown spot pathogen, it is characterised in that the primer includes as follows：
F3：5’-TCTCTTGGTTCTGGCATCGA-3’；
B3：5’-GCGAGTCTCCAGCAAAGC-3’；
FIP：5’-GGCGCAATGTGCGTTCAAAGAT-GAACGCAGCGAAATGCGA TA-3’；
BIP：5’-TGGTATTCCAAAGGGCATGCCT-GACAAGACGCCCAACACC- 3’ .
2. a kind of LAMP detection method of tobacco brown spot pathogen, it is characterised in that comprise the steps：
（1）Tobacco brown spot pathogen LAMP detection primer is designed：With tobacco brown spot pathogen ITS sequence as detecting target, design is suitable for
Specific primer F3, B3, FIP and BIP of LAMP detection, its nucleotide sequence is as follows：
F3：5’-TCTCTTGGTTCTGGCATCGA-3’；
B3：5’-GCGAGTCTCCAGCAAAGC-3’；
FIP：5’-GGCGCAATGTGCGTTCAAAGAT-GAACGCAGCGAAATGCGA TA-3’；
BIP：5’-TGGTATTCCAAAGGGCATGCCT-GACAAGACGCCCAACACC- 3’ ；
（2）The extraction of testing sample DNA：Testing sample genomic DNA is extracted according to CTAB method or using commercial kit；
（3）LAMP amplified reaction：With step（2）The testing sample DNA of extraction is template, uses step（1）Primer carry out LAMP
Reaction, LAMP detection reaction system is that 25 μ L, including 5 μM of primers F 3 and each 1.0 μ L of B3,40 μM of primers F IP and BIP are each
1.0 μ L, LAMP reaction mixture 12.5 μ L, 8 UBst1.0 μ L of polymerase, 1.0 μ L of DNA profiling, with sterilizing ultra-pure water benefit
Enough to 25 μ L；LAMP reaction condition：64 DEG C of 60 min of incubation；
（4）The measure of reaction result：It is measured using fluorescent dye visual observations method, after LAMP reaction terminates, in LAMP
I 1.0 μ L of developer SYBR green is added in the amplified production of reaction, and the result that develops the color observes that the judgement of green fluorescence is sun
Property, there is tobacco brown spot pathogen, orange or crocus are judged as feminine gender, there is no tobacco brown spot pathogen.
3. the LAMP detection method of a kind of tobacco brown spot pathogen according to claim 2, it is characterised in that LAMP reacts
Mixed liquor is composed of the following components：40 mM Tris-HCl, 20 mM (NH4)2SO4, 20 mM KCl, 16 mM MgSO4, 1.6
Mol/L glycine betaine, 2.0 mM dNTPs, 0.2wt.% Trion X-100.
4. application of the primer as claimed in claim 1 in the monitoring, identification of the early diagnosiss of Alternaria alternate and pathogenic bacteria.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108315472A (en) * | 2018-04-27 | 2018-07-24 | 安徽省农业科学院植物保护与农产品质量安全研究所 | It is a kind of to be used for the Primer composition and its application that alternaric bacteria LAMP is quickly detected |
| CN108330178A (en) * | 2017-10-26 | 2018-07-27 | 福建省农业科学院植物保护研究所 | It is a kind of detection tomato early blight bacterium loop-mediated isothermal amplification (LAMP) primer and application |
| CN111172314A (en) * | 2020-02-21 | 2020-05-19 | 福建省农业科学院植物保护研究所 | Method for detecting panax notoginseng black spot germs by LAMP |
| CN111197105A (en) * | 2020-03-18 | 2020-05-26 | 福建省农业科学院植物保护研究所 | Application of LAMP primer in detection of alternaria alternata |
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2016
- 2016-11-30 CN CN201611077015.3A patent/CN106434989B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| EIKEN CHEMICAL CO.,LTD,: "A guide to LAMP primer designing", 《百度文库》 * |
| 胡中会: "云南烟草赤星病菌及近似种rDNA-ITS 序列分析", 《云南农业大学学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108330178A (en) * | 2017-10-26 | 2018-07-27 | 福建省农业科学院植物保护研究所 | It is a kind of detection tomato early blight bacterium loop-mediated isothermal amplification (LAMP) primer and application |
| CN108315472A (en) * | 2018-04-27 | 2018-07-24 | 安徽省农业科学院植物保护与农产品质量安全研究所 | It is a kind of to be used for the Primer composition and its application that alternaric bacteria LAMP is quickly detected |
| CN111172314A (en) * | 2020-02-21 | 2020-05-19 | 福建省农业科学院植物保护研究所 | Method for detecting panax notoginseng black spot germs by LAMP |
| CN111197105A (en) * | 2020-03-18 | 2020-05-26 | 福建省农业科学院植物保护研究所 | Application of LAMP primer in detection of alternaria alternata |
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| Publication number | Publication date |
|---|---|
| CN106434989B (en) | 2019-08-27 |
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