CN107674924A - A kind of Phyllosticta musarum LAMP detection primer and its application - Google Patents
A kind of Phyllosticta musarum LAMP detection primer and its application Download PDFInfo
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Abstract
The invention provides a kind of Phyllosticta musarum LAMP detection primer and its application, is exclusively used in the specific detection of Phyllosticta musarum, belongs to corps diseases detection and biological technical field.The present invention devises a kind of Phyllosticta musarum LAMP detection primer, including outer primer F3 and B3, and inner primer FIP and BIP, sequence are shown in SEQ ID NO.1 4.The Phyllosticta musarum detection method established based on the primer, chromogenic reaction or agarose gel electrophoresis detection, can be observed green fluorescence or the trapezoid-shaped strips of LAMP characteristics occur after LAMP constant-temperature amplifications.The LAMP detection primer and its detection method invented can realize quick, sensitive, the accurate detection of Phyllosticta musarum in production practices, simultaneously available for the early diagnosis of field banana freckle and monitoring, the identification of germ, early warning and prevention and control for banana freckle provide reliable technology and theoretical foundation.
Description
Technical field
The present invention relates to a kind of Phyllosticta musarum LAMP detection primer and its detection method, Phyllosticta musarum is exclusively used in
Quick detection, while can realize field banana freckle early diagnosis and germ monitoring, identification, belong to corps diseases
Detection, identification, preventing and treating and biological technical field.
Background technology
Banana is the fruit crop that yield is maximum in the world, and the world-renowned torrid zone, subtropical fruit, and China
The main fruit of tropical and subtropical zone.It has unique taste and higher nutritive value to banana, always can be rated as a kind of superior
Health fruit.China is one of World banana main production country, has long cultivation history, and the banana planting in the world produces greatly
One of state, the cultivated area and yield of banana occupy the 4th of national fruit.
Banana freckle is also known as tar spot, ephelides, and can cause harm Leaf of banana, carpopodium and fruit, in each banana kind in China
Growing area generally existing.Blade and middle arteries produce many scattered or all living creatures the small blackspot of projection, diameter about l mm, its periphery during morbidity
Filbert, middle part is slightly sunk, and is gone up and is given birth to small black grain.The intensive blocking spot of scab, it is withered and yellow to finally result in blade, fruit morbidity, symptom
Occur within often 2~4 weeks after cutting off male flower cluster, refer to curved abdominal section in fruit more, there is full fruit, initial stage is rufous, peripheral dirty-green when serious
Water is swooned, as fruit meat degree increases, the increase of scab density, and the serious outward appearance and storage property that extend to full fruit, influence fruit, closely
Banana freckle generally occurs and the getting worse that causes harm in China banana producing region over year, it has also become the important disease in banana production
One of.
Phyllosticta musarum (Phyllosticta musarum) grown on the common culture mediums such as PDA, PSA and V8 it is very slow
Slowly, when being separated, bacterium colony is usually covered by miscellaneous bacteria, and pycnidia generation time evening, it is difficult in Early Identification.
Pure culture was not obtained also by 1972, and in addition the disease conventional diagnostic techniques based on symptom are, it is necessary to using Ke He Shi
Rule passes through pathogenicbacteria separation culture, pathogen identification, connects the steps such as bacterium, symptom analysis, and time-consuming, efficiency is low, accuracy is poor,
It is difficult to detection in time and the effectively propagation of control pathogen and plant disease epidemic, it is difficult to meet the reality of banana freckle diagnosis
Need, therefore there is an urgent need to establish a set of convenient and swift, reliable results, the quick diagnosis technology of high sensitivity.
In recent years, Protocols in Molecular Biology is quickly grown, as Protocols in Molecular Biology is constantly sent out in plant pathology subject
Exhibition and application, some molecular marking techniques provide new approach, PCR for the diagnosis detection of phytopathogen（polymerase
chain reaction）Technology with high specificity, high sensitivity, it is convenient and swift the features such as be used for the diagnosis of phytopathogen,
But it needs expensive instrument and equipment, it is difficult to realizes in department of basic unit and fast and accurately detects.Ring mediated isothermal amplification（loop-
mediated isothermal amplification, LAMP）Technology is by one kind of the exploitations such as Japanese Scientists Notomi
Easy, quick, accurately and efficiently nucleic acid constant-temperature amplification method.Short time interior realize largely expands the technology under isothermal conditions,
10 are realized in 30 min -60 min9-1010Amplification again, has very high sensitivity and specificity, and easy to operate, detection
As a result can visually judge.Compared to round pcr, LAMP technology whole process isothermal reaction, without PCR instrument, and the big high sensitivity of amplification amount.
LAMP detections at present have been successfully applied to the report of people and animals' pathogen, food security, Environmental security and various plants cause of disease analyte detection
Road, but there is not been reported for the research at present about Phyllosticta musarum LAMP detections.
The content of the invention
For the detection to Phyllosticta musarum and evaluation program are cumbersome, time-consuming in the prior art, will to identification experience
Ask height, the degree of accuracy low, PCR is detected the problem of needs by equipment such as amplification instruments, there is provided a kind of Phyllosticta musarum LAMP inspections
Survey primer and simplicity, quick, sensitive, special visible detection method.
To achieve the above object, this invention takes following technical scheme：
1. the design of Phyllosticta musarum LAMP detection primer
According to Phyllosticta musarum ribosomes the Internal Transcribed Spacer（rDNA-ITS）Well-conserved in fungi kind of sequence and
Section, which belongs to the design of the characteristics of inter-species changeability, has the LAMP detection primer that specific amplified acts on, including 2 to Phyllosticta musarum
Outer primer（F3 and B3）With 2 inner primers（FIP and BIP）, its nucleotide sequence is respectively：
F3：5’-CGGATCTCTTGGTTCTGGC-3’；
B3：5’-TACGCTCGAGGCTAGGAC-3’；
FIP: 5’-GGCGCAATGTGCGTTCAAAGATGAAGAACGCAGCGAAATGC-3’；
BIP: 5’-TGCCTGTTCGAGCGTCATTTCAAGGTCTTCAAGGCCCGTC-3’。
2. establishing Phyllosticta musarum LAMP detection method, comprise the following steps：
(1) DNA is extracted from detected sample；
During for detecting pathogen pure culture, strains tested genomic DNA is extracted using CTAB methods；For detecting banana
When plant tissue whether there is Phyllosticta musarum, banana plant tissue gene group DNA is extracted using NaOH rapid cleavages method.
(2) LAMP augmentation detections are carried out as template to extract testing sample DNA：The μ L of LAMP reaction systems 25, reactant
System includes 0.2 mmol/L F3 and B3, and 1.6 mmol/L FIP and BIP, Bst archaeal dna polymerase are 8 U, and DNA profiling 50~
100 ng, 12.5 uL LAMP reaction mixtures（40 mM Tris-HCl, 20 mM (NH4)2SO4, 20 mM KCl, 16 mM
MgSO4, 1.6mol/L glycine betaines, 2.0 mM dNTPs, 0.2% Trion X-100）, 25 uL are supplied with sterile ultra-pure water.
LAMP reaction conditions are 63-65 DEG C and incubate 45-60 min, 85 DEG C of inactivation 5-10 min.
(3) LAMP reaction results determine：Using fluorescent dye visual observations method or agarose gel electrophoresis method.Described is glimmering
Photoinitiator dye visual observations method：After LAMP reactions terminate, fluorescent dye developer is added in the amplified production of LAMP reactions
The uL of SYBR green I 1.0, colour developing result observe that the judgement of green fluorescence is judged as the moon for the positive, orange or crocus
Property.Described agarose gel electrophoresis method：Take 2.0 uL LAMP amplified productions to be detected with 2% agarose gel electrophoresis, ladder occur
Shape band is judged as the positive, trapezoid-shaped strips does not occur and is then judged as feminine gender.
The beneficial effects of the present invention are：
（1）High specificity, accuracy are high：The present invention is according to fungi ribosomes transcribed spacer（rDNA-ITS）Sequence is in fungi
The characteristics of well-conserved and section in kind belongs to inter-species changeability, it have chosen 6 specific regions and devise to Phyllosticta musarum
4 LAMP primers with specific amplified effect.To the Phyllosticta musarum of different geographic origins, Glorosprium musarum Cookeet Mass,
The Banana Tissue of Curvularia lunata bacterium, the plant tissue for carrying Phyllosticta musarum and health has carried out detection checking,
The positive is presented in the tissue of only Phyllosticta musarum and the carrying germ, illustrates that the primer designed by the present invention and detection method are used
In detection Phyllosticta musarum accurately and reliably, can the raw similar disease of symptom characteristic on banana of effective district distribution；
（2）High sensitivity：LAMP reachable 10 fg on DNA level to the detection sensitivity of Phyllosticta musarum, have very high
Sensitivity；
（3）Applicability is wide, practicality is good：The detection method of the Phyllosticta musarum of the present invention, can not only enter to germ mycelium
Row detection, can also be detected to susceptible Banana Tissue, and the early detection of Phyllosticta musarum can be achieved, i.e., show disease in disease
Before detected, prevent the eruption and prevalence of disease.
（4）It is easy to operate quick：LAMP is to carry out under isothermal conditions, only one water-bath of need, result visualization,
General whole detection process can be completed in 1.5 hours, simple and efficient to handle.
Brief description of the drawings
Fig. 1 is specific detection result of the LAMP detection method of the present invention to Phyllosticta musarum：Wherein upper figure is agar
Sugared detected through gel electrophoresis result, figure below are visualization colour developing result, and wherein swimming lane M is 2000bp DNA Marker, and swimming lane 2 is
Positive control, swimming lane 3-5 are Phyllosticta musarum, and swimming lane 6-11 is respectively：Glorosprium musarum Cookeet Mass, Curvularia lunata
Bacterium, Cercospora musae leaf spot fungi, banana blight bacteria, asparagus stem wilt bacteria, negative control.Wherein visualization colour developing result 2-5
Green fluorescence is shown, other do not show.
Fig. 2 is sensitivity testing result of the LAMP detection method of the present invention to Phyllosticta musarum：Wherein upper figure is agar
Sugared detected through gel electrophoresis result, figure below are visualization colour developing result, and wherein swimming lane M is 2000bp DNA Marker, swimming lane 2-11
Template DNA concentration be respectively：10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, negative control, positive control.
9,10 do not show green fluorescence wherein in visualization colour developing result, other display green fluorescences.
Fig. 3 is that LAMP detection method of the present invention is Ago-Gel to banana incidence tissue practicality testing result, upper figure
Electrophoresis detection result, figure below are visualization colour developing result, and wherein swimming lane M is that 2000bp DNA Marker, swimming lane 2-7 are respectively
Phyllosticta musarum, the Banana Tissue of banana freckle natural occurrence, artificial infection Phyllosticta musarum morbidity Banana Tissue,
Healthy Banana Tissue, positive control, negative control.Wherein visualization colour developing result 2-4,6 display green fluorescences, other do not show
Show.
Embodiment
In order that content of the present invention easily facilitates understanding, with reference to embodiment to of the present invention
Technical scheme is described further.
Test method used in following embodiments is conventional method unless otherwise specified.
Test material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
The Phyllosticta musarum LAMP primer of embodiment 1 designs
According to Phyllosticta musarum ribosomes the Internal Transcribed Spacer（rDNA-ITS）Well-conserved in fungi kind of sequence and
Section, which belongs to the design of the characteristics of inter-species changeability, has the LAMP detection primer that specific amplified acts on, including 2 to Phyllosticta musarum
Outer primer（F3 and B3）With 2 inner primers（FIP and BIP）, its nucleotide sequence is respectively：
F3：5’-CGGATCTCTTGGTTCTGGC-3’；
B3：5’-TACGCTCGAGGCTAGGAC-3’；
FIP: 5’-GGCGCAATGTGCGTTCAAAGATGAAGAACGCAGCGAAATGC-3’；
BIP: 5’-TGCCTGTTCGAGCGTCATTTCAAGGTCTTCAAGGCCCGTC-3’。
The foundation of the Phyllosticta musarum LAMP detection method of embodiment 2
1. testing sample DNA extraction：
1. for when detecting pathogen pure culture, extracting strains tested genomic DNA using CTAB methods, specific steps are such as
Under：
(1) 0.1 g hypha powders are taken in 1.5 mL centrifuge tubes, 900 μ L2wt.%CTAB extract solutions is added, is shaken using oscillator
Swing mixing, 60 DEG C of min of water-bath 60, under room temperature condition, 12000r/min centrifuges 15 min；
(2) the μ L of supernatant 700 are taken, add isometric phenol, chloroform, isoamyl alcohol mixed liquor（Each volume ratio is 25:24:1）, gently shake
Dynamic, under room temperature condition, 8000 r/min centrifuge 10 min；
(3) the μ L of supernatant 500 are taken, isometric chloroform is added and extracts again once, under room temperature condition, 8000 r/min centrifugations 10
min；
(4) the μ L of supernatant 350 are taken, add the mol/L NaAc of 1/10 volume 3 and 2 times of volume absolute ethyl alcohols, -20 DEG C of precipitations 60
Min, under the conditions of 4 DEG C, 8000 r/min centrifuge 5 min；
(5) abandoning supernatant, 700 μ L volumetric concentrations of addition are 70% ice ethanol, jog 10 sec, under the conditions of 4 DEG C, 8000 r/
Min centrifuges 10 sec, dries, and adds 50 μ L TE buffer solutions, -20 DEG C save backup.
2. whether there is Phyllosticta musarum for detecting banana plant tissue, extracted using NaOH rapid cleavages method
Banana plant tissue gene group DNA, is comprised the following steps that：
A. the g of plant tissue 0.1 to be detected is weighed, adds the μ L of 0.5 mol/L NaOH 30, tissue is fully milled to paste；
B. pasty state tissue is transferred in 1.5 mL centrifuge tubes, 12000r/min centrifuges 6 min, takes the μ l of supernatant 5；
C. the mol/L Tris-HCl of 495 μ L 0.1 are added in supernatant（pH=8.0）, it is well mixed, takes 1.0 μ L as PCR
Template is expanded；
2. carry out LAMP amplifications as template to extract testing sample DNA：The μ L of LAMP reaction systems 25, reaction system include 0.2
Mmol/L F3 and B3,1.6 mmol/L FIP and BIP, Bst archaeal dna polymerase are 8 U, DNA profiling 50~100 ng, 12.5
UL LAMP reaction mixtures（40 mM Tris-HCl, 20 mM(NH4)2SO4, 20 mM KCl, 16 mM MgSO4,1.6
Mol/L glycine betaines, 2.0 mM dNTPs, 0.2% Trion X-100）, 25 uL are supplied with sterile ultra-pure water.LAMP is anti-
Condition is answered to incubate 45-60 min, 85 DEG C of inactivation 5-10min for 63-65 DEG C.
3. LAMP reaction results determine：Using fluorescent dye visual observations method or agarose gel electrophoresis method.Described is glimmering
Photoinitiator dye visual observations method：After LAMP reactions terminate, fluorescent dye developer is added in the amplified production of LAMP reactions
The uL of SYBR green I 1.0, colour developing result observe that the judgement of green fluorescence is the positive, Phyllosticta musarum be present；It is orange
Or crocus is judged as feminine gender, in the absence of Phyllosticta musarum.Described agarose gel electrophoresis method：2.0 uL LAMP are taken to expand
Volume increase thing is detected with 2% agarose gel electrophoresis, trapezoid-shaped strips is such as occurred and is judged as the positive, Phyllosticta musarum be present；Do not go out
Existing band is then judged as feminine gender, in the absence of Phyllosticta musarum.
The Phyllosticta musarum LAMP of embodiment 3 detects specific assay
1. 3 plants of Phyllosticta musarums, Glorosprium musarum Cookeet Mass, Curvularia lunata bacterium, Cercospora musae are extracted using CTAB methods
Leaf spot fungi, banana blight bacteria, the genomic DNA of asparagus stem wilt bacteria.
2. LAMP amplifications are carried out as template using the DNA extracted for examination bacterium：The μ L of LAMP reaction systems 25, reaction system include
0.2 mmol/L F3 and B3,1.6 mmol/L FIP and BIP, Bst archaeal dna polymerase are 8 U, the ng of DNA profiling 50~100,
12.5 uL LAMP reaction mixtures（40 mM Tris-HCl, 20 mM (NH4)2SO4, 20 mM KCl, 16 mM MgSO4,
1.6 mol/L glycine betaines, 2.0 mM dNTPs, 0.2% Trion X-100）, 25 uL are supplied with sterile ultra-pure water.LAMP
Reaction condition is 63-65 DEG C and incubates 45-60 min, 85 DEG C of inactivation 5-10 min.
3. LAMP reaction results determine：Using fluorescent dye visual observations method or agarose gel electrophoresis method.Described is glimmering
Photoinitiator dye visual observations method：After LAMP reactions terminate, fluorescent dye developer is added in the amplified production of LAMP reactions
The uL of SYBR green I 1.0, colour developing result observe that the judgement of green fluorescence is the positive, Phyllosticta musarum be present；It is orange
Or crocus is judged as feminine gender, in the absence of Phyllosticta musarum.Described agarose gel electrophoresis method：2.0 uL LAMP are taken to expand
Volume increase thing is detected with 2% agarose gel electrophoresis, trapezoid-shaped strips is such as occurred and is judged as the positive, Phyllosticta musarum be present；Do not go out
Existing band is then judged as feminine gender, in the absence of Phyllosticta musarum.
4. specificity verification result
As shown in figure 1, green fluorescence can be observed in 3 plants of Phyllosticta musarum colour developing results, there is LAMP in agarose gel electrophoresis
Characteristic trapezoid-shaped strips, and it is orange to supply to try other crop pathogenses and negative control colour developing results, agarose gel electrophoresis is not
There are LAMP characteristic trapezoid-shaped strips, Phyllosticta musarum and other pathogens can be distinguished by showing the LAMP primer of the present invention
Come, there is very strong specificity, detection method of the invention can be used for specific detection and the identification of Phyllosticta musarum.
The Phyllosticta musarum LAMP detection sensitivities of embodiment 4 determine
1. using the genomic DNA of CTAB methods extraction Phyllosticta musarum；
It is 2. dilute with sterile ultra-pure water after spectrophotometric determination concentration by the genomic DNA of the Phyllosticta musarum of extraction
Release, be configured to series concentration, it is standby；
3. conventional LAMP amplifications are carried out as template into series concentration DNA using preparation：The μ L of LAMP reaction systems 25, reaction system
F3 and B3 including 0.2 mmol/L, 1.6 mmol/L FIP and BIP, Bst archaeal dna polymerase are 8 U, DNA profiling 50~100
Ng, 12.5 uL LAMP reaction mixtures（40 mM Tris-HCl, 20 mM (NH4)2SO4, 20 mM KCl, 16 mM
MgSO4, 1.6 mol/L glycine betaines, 2.0 mM dNTPs, 0.2% Trion X-100）, 25 are supplied with sterile ultra-pure water
uL.LAMP reaction conditions are 63-65 DEG C and incubate 45-60 min, 85 DEG C of inactivation 5-10 min.
4. LAMP reaction results determine：Using fluorescent dye visual observations method or agarose gel electrophoresis method.Described is glimmering
Photoinitiator dye visual observations method：After LAMP reactions terminate, fluorescent dye developer is added in the amplified production of LAMP reactions
The uL of SYBR green I 1.0, colour developing result observe that the judgement of green fluorescence is the positive, Phyllosticta musarum be present；It is orange
Or crocus is judged as feminine gender, in the absence of Phyllosticta musarum.Described agarose gel electrophoresis method：2.0 uL LAMP are taken to expand
Volume increase thing is detected with 2% agarose gel electrophoresis, trapezoid-shaped strips is such as occurred and is judged as the positive, Phyllosticta musarum be present；Do not go out
Existing band is then judged as feminine gender, in the absence of Phyllosticta musarum.
5. testing result
As shown in Fig. 2 green fluorescence can be observed in colour developing result, there is the trapezoid belt of LAMP characteristics in agarose gel electrophoresis,
Detection sensitivity is up to 10 fg.
The LAMP detections of Phyllosticta musarum in the morbidity banana plant of embodiment 5
1. using CTAB methods extraction Phyllosticta musarum genomic DNA；Banana plant group is extracted using NaOH rapid cleavages method
Knit genomic DNA.
2. LAMP amplifications are carried out as template using the DNA for extracting test sample：The μ L of LAMP reaction systems 25, reaction system bag
Include 0.2 mmol/L F3 and B3,1.6 mmol/L FIP and BIP, Bst archaeal dna polymerase are 8 U, DNA profiling 50~100
Ng, 12.5 uL LAMP reaction mixtures（40 mM Tris-HCl, 20 mM (NH4)2SO4, 20 mM KCl, 16 mM
MgSO4, 1.6 mol/L glycine betaines, 2.0 mM dNTPs, 0.2% Trion X-100）, 25 are supplied with sterile ultra-pure water
uL.LAMP reaction conditions are 63-65 DEG C and incubate 45-60 min, 85 DEG C of inactivation 5-10 min.
3. LAMP reaction results determine：Using fluorescent dye visual observations method or agarose gel electrophoresis method.Described is glimmering
Photoinitiator dye visual observations method：After LAMP reactions terminate, fluorescent dye developer is added in the amplified production of LAMP reactions
The uL of SYBR green I 1.0, colour developing result observe that the judgement of green fluorescence is the positive, Phyllosticta musarum be present；It is orange
Or crocus is judged as feminine gender, in the absence of Phyllosticta musarum.Described agarose gel electrophoresis method：2.0 uL LAMP are taken to expand
Volume increase thing is detected with 2% agarose gel electrophoresis, trapezoid-shaped strips is such as occurred and is judged as the positive, Phyllosticta musarum be present；Do not go out
Existing band is then judged as feminine gender, in the absence of Phyllosticta musarum.
4. testing result
As shown in figure 3, Phyllosticta musarum, the Banana Tissue of banana freckle natural occurrence, artificial infection Phyllosticta musarum
Green fluorescence can be observed in the Banana Tissue of morbidity, positive control colour developing result, and LAMP characteristics occurs in agarose gel electrophoresis
Trapezoid belt, and healthy Banana Tissue, negative control colour developing result is orange, and agarose gel electrophoresis does not occur LAMP characteristics
Trapezoid belt, show that LAMP primer and detection method of the present invention can be additionally used in the detection of field banana freckle disease plant.
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, it should all belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>Inst. of Plant Protection, fujian Academy of Agricultural Science
<120>A kind of Phyllosticta musarum LAMP detection primer and its application
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<400> 1
cggatctctt ggttctggc 19
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> 2
tacgctcgag gctaggac 18
<210> 3
<211> 41
<212> DNA
<213>Artificial sequence
<400> 3
ggcgcaatgt gcgttcaaag atgaagaacg cagcgaaatg c 41
<210> 4
<211> 40
<212> DNA
<213>Artificial sequence
<400> 4
tgcctgttcg agcgtcattt caaggtcttc aaggcccgtc 40
Claims (4)
- A kind of 1. Phyllosticta musarum LAMP detection primer, it is characterised in that：Described Phyllosticta musarum LAMP detection primer It is made up of positive outer primer F3, reverse outer primer B3, positive inner primer FIP and reverse inner primer BIP, each primer nucleotide sequences For：F3：5’-CGGATCTCTTGGTTCTGGC-3’；B3：5’-TACGCTCGAGGCTAGGAC-3’；FIP: 5’-GGCGCAATGTGCGTTCAAAGATGAAGAACGCAGCGAAATGC-3’；BIP: 5’-TGCCTGTTCGAGCGTCATTTCAAGGTCTTCAAGGCCCGTC-3’。
- A kind of 2. Phyllosticta musarum LAMP detection method, it is characterised in that：Utilize the banana freckle described in claim 1 Bacterium LAMP primer carries out LAMP reactions, and LAMP reaction systems are 25 uL, and reaction system includes 0.2 mmol/L F3 and B3, and 1.6 Mmol/L FIP and BIP, Bst archaeal dna polymerase is 8 U, and DNA profiling 50~100 ng, 12.5 uL LAMP reactions mix Liquid, 25 uL are supplied with sterile ultra-pure water；LAMP reaction conditions are 63-65 DEG C and incubate 45-60 min, 85 DEG C of inactivation 5-10 min；LAMP reaction mixtures contain 40 mM Tris-HCl, 20 mM (NH4)2SO4, 20 mM KCl, 16 mM MgSO4, 1.6mol/L glycine betaines, 2.0 mM dNTPs, 0.2% Trion X-100.
- A kind of 3. LAMP detection method of Phyllosticta musarum according to claim 2, it is characterised in that：Treat that LAMP reacts After end, the uL of developer SYBR green I 1.0 are added in the amplified production of LAMP reactions, colour developing result observes green The judgement of fluorescence is the positive, and orange or crocus is judged as feminine gender, or judges testing result using agarose gel electrophoresis method, takes 2.0 uL LAMP amplified productions are detected with 2% agarose gel electrophoresis, trapezoid-shaped strips are occurred and are judged as the positive, do not occur trapezoidal Band is then judged as feminine gender.
- 4. Phyllosticta musarum LAMP detection primer as claimed in claim 1 is supervised in the early diagnosis of banana freckle and germ Application in surveying and identifying.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711236365.4A CN107674924A (en) | 2017-11-30 | 2017-11-30 | A kind of Phyllosticta musarum LAMP detection primer and its application |
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| CN109652584A (en) * | 2019-01-16 | 2019-04-19 | 华中农业大学 | For detecting the LAMP primer and detection kit of the black star bacterium of peach |
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| CN102690887A (en) * | 2012-06-14 | 2012-09-26 | 福建省农业科学院植物保护研究所 | LAMP (loop-mediated isothermal amplification) detection primers of banana fusarium wilt bacteria No. 4 microspecies and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109652584A (en) * | 2019-01-16 | 2019-04-19 | 华中农业大学 | For detecting the LAMP primer and detection kit of the black star bacterium of peach |
| CN109652584B (en) * | 2019-01-16 | 2021-03-23 | 华中农业大学 | LAMP primer and detection kit for detecting alternaria persica |
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