CN106399529B - A kind of Phyllosticta musarum molecular detection primer and detection method - Google Patents
A kind of Phyllosticta musarum molecular detection primer and detection method Download PDFInfo
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Abstract
The present invention relates to a kind of Phyllosticta musarum molecular detection primer and its detection methods, belong to corps diseases detection and field of biotechnology.The specific primer includes upstream primer BMF:5 '-GCTACAACGCCGAAATGACC-3 ', downstream primer BMR:5 '-GACGTTGCCCAATACCAAGC-3 '.The detection primer and detection method invented can be used for the Testing and appraisal of Phyllosticta musarum pure culture, can also detect to Leaf of banana and fruit；Detection primer high specificity of the present invention, high sensitivity, detection method practicability is good, simple and efficient to handle；The present invention is able to achieve the early detection of banana freckle, similar anthracnose on banana, the mould leaf spot of curved spore, black spot, checkers spore leaf spot, dark double spore leaf spots can also effectively be distinguished, the diffusion sprawling of early warning and prevention and control to banana freckle, controlling disease is of great significance.
Description
Technical field
The present invention relates to a kind of Phyllosticta musarum molecular detection primer and its detection methods, are exclusively used in Phyllosticta musarum
Rapid molecular detection, while can realize field Phyllosticta musarum early diagnosis and germ monitoring identify, belong to farming
Object Defect inspection and field of biotechnology.
Background technique
Banana be in the world the maximum fruit crop of yield and it is world-renowned the torrid zone, subtropical fruit,.In being also
The main fruit of state's tropical and subtropical zone, banana its with unique taste and higher nutritive value, always can be rated as a kind of superior
Health fruit.China is one of World banana main production country, the banana planting production with long cultivation history and the world
One of big country, 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 is generally existing.Blade and middle arteries generate many scattered or all living creatures small blackspot of protrusion, diameter about l mm, periphery when 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, mostly refer to curved abdominal section in fruit, Quan Guojun has when serious, and initial stage is rufous, peripheral dirty-green
Water is dizzy, and as fruit meat degree increases, scab density increases, serious to extend to full fruit, influences the appearance and storage property of fruit, closely
Banana freckle generally occurs and causes harm to get worse 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, in addition to this disease conventional diagnostic techniques based on symptom, need using Ke He Shi
Rule by pathogenicbacteria separation culture, pathogen identification, connect bacterium, symptom analysis, time-consuming, low efficiency, accuracy are poor,
It is difficult to propagation and the plant disease epidemic for detecting and effectively controlling pathogen when disease occurs in time, it is difficult to meet banana freckle
The actual needs of diagnosis, therefore a set of convenient and efficient, result is reliable, quick diagnosis technology of high sensitivity there is an urgent need to establish.
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 for the diagnosis detection of phytopathogen, wherein PCR
(polymerase chain reaction) technology with high specificity, high sensitivity, it is convenient and efficient the features such as be used for phytopathy
The diagnosis of opportunistic pathogen.Guarantor in fungi ribosomes transcribed spacer (internal transcribed spacer, ITS) sequence kind
The characteristics of keeping property and section belong to inter-species changeability, design specific primer carry out PCR, are used for quickly detecting and identify to pathogen
Technology has been widely applied, and researchers at home and abroad successfully develop soybean phytophthora, Phytophthora capsici, citrus bacterial canker disease
The specific primer and detection method of a variety of pathogens such as bacterium, sweet potato black rot pathogen and corn south aecidium, realize quickly,
Sensitive and accurate identification.But there is not been reported for the current research in relation to Phyllosticta musarum Molecular Detection.
Summary of the invention
For pathogen morpha feature is based primarily upon to the detection and identification of Phyllosticta musarum in the prior art, banana is black
Star pathogen growth is slow, and separating difficulty is big, and separation program is cumbersome, the dead length of consumption, high to identification skill requirement, accuracy is low, it is difficult to
The actual needs problem for meeting banana freckle diagnosis provides a kind of Phyllosticta musarum molecular detection primer and its detection side
Method.
To achieve the above object, this invention takes following technical schemes:
Present invention firstly provides a kind of Phyllosticta musarum molecular detection primer, nucleotide sequences are as follows:
Upstream primer BMF:5 '-GCTACAACGCCGAAATGACC-3 '；
Downstream primer BMR:5 '-GACGTTGCCCAATACCAAGC-3 '.
The primer BMF and BMR goes out the product of 382bp to Phyllosticta musarum specific amplification.
The present invention also provides a kind of rapid detection methods of Phyllosticta musarum, comprising the following steps:
(1) DNA is extracted from Leaf of banana or fruit；
When for detecting pathogen pure culture, strains tested genomic DNA is extracted using CTAB method；For detecting
When Banana Tissue whether there is Phyllosticta musarum, banana plant tissue gene group DNA is extracted using NaOH rapid cleavage method；
(2) to extract the DNA of Leaf of banana or fruit as template progress PCR amplification: 25 μ L of PCR reaction system includes
2.5 μ 10 × PCR of L buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is 5U/ μ L's
Taq enzyme, each 0.5 μ L of the BMF and BMR of 10 μm of ol/L, 1 μ L of DNA profiling add ddH2O is to total volume up to 25 μ L；PCR reaction condition
Are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 45sec, 56 DEG C of annealing 45sec, 72 DEG C of extension 45sec, totally 35 recycle；72 DEG C are prolonged
Stretch 10min；
(3) resulting PCR product is walked on 1.0% Ago-Gel with the electrophoretic analysis of 0.5 × tbe buffer liquid, voltage is
4-5V/cm；Testing result is determined according to the size of amplified production, if energy specific amplification goes out the product of 382bp, can determine that
There are Phyllosticta musarums in the Leaf of banana or fruit, and otherwise there is no banana is black in the Leaf of banana or fruit
Star germ.
Positive beneficial effect of the invention is:
(1) accuracy is high: the present invention be according to fungi ribosomes transcribed spacer (rDNA-ITS) sequence in fungi kind
The characteristics of the belonging to inter-species changeability design of well-conserved and section there is the PCR of specific amplified effect to draw Phyllosticta musarum
Object.The plant leaf blade to the Phyllosticta musarum of different geographic origins, carrying Phyllosticta musarum, carrying banana freckle
The pericarp of bacterium and the Banana Tissue of health have carried out detection verifying, only Phyllosticta musarum and the Banana Tissue for carrying the germ
The electrophoretic band that 382 bp can specifically be amplified illustrates primer designed by the present invention for detecting the black star of banana
Germ is accurate and reliable；
(2) high specificity: primer pair Phyllosticta musarum designed by the present invention has very strong specificity, can be used in
Distinguish Phyllosticta musarum, banana anthracnose, Curvularia lunata bacterium, banana alternaria, banana checkers spore leaf spot
Bacterium and banana secretly pathogen common on the bananas such as double spore leaf spot fungis, so as to the raw symptom characteristic on banana of effective district distribution
Similar disease；
(3) high sensitivity: the present invention has combined the special primer of design with ITS gene universal primer (ITS1/ITS4)
After carrying out nest-type PRC amplification, 1fg can reach in DNA level to the detection sensitivity of Phyllosticta musarum；
(4) applicability is wide, practicability is good: the detection method of Phyllosticta musarum of the invention, can not only be to germ mycelia
Body is detected, and also can be detected the early detection, it can be achieved that Phyllosticta musarum to susceptible Leaf of banana and pericarp, i.e.,
It is detected before disease shows disease, it is significant to the eruption and prevalence of scab after prevention Banana Growth phase and picking.
(5), easy to operate quick: the present invention can sentence after need to only carrying out DNA extraction, PCR amplification and agarose electrophoresis
It is simple and efficient to handle calmly as a result, general entire detection process can be completed within a few hours.
Detailed description of the invention
Fig. 1 is primer pair Phyllosticta musarum specific amplification electrophoretogram of the present invention: wherein swimming lane M is 2000bp DNA
Marker, swimming lane 2, swimming lane 3 are Phyllosticta musarum, and swimming lane 4-11 is respectively as follows: Glorosprium musarum Cookeet Mass, Curvularia lunata
Bacterium, banana alternaria, banana checkers spore leaf spot fungi, banana secretly double spore leaf spot fungis, banana blight bacteria, Asparagus Stem Blight
Bacterium, negative control.
Fig. 2 is the sensitivity detection amplification electrophoretogram of primer Phyllosticta musarum of the present invention: A: regular-PCR sensitivity inspection
Survey, wherein swimming lane M be 2000bp DNA Marker, swimming lane 2-11 be respectively as follows: 100ng, 10ng, 1ng, 100pg, 10pg, 1pg,
100fg, 10fg, negative control, positive control；B is nest-type PRC sensitivity technique, and wherein swimming lane M is 2000bp DNA
Marker, swimming lane 2-13 are respectively as follows: 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, 100ag, feminine gender
Control, positive control.
Fig. 3 is that Leaf of banana of the present invention and the detection of Banana peel kind black star germ expand electrophoretogram, and wherein swimming lane M is
2000bp DNA Marker, swimming lane 2-10 are respectively natually morbid Leaf of banana, natural occurrence banana, artificial infection
Early stage banana after the Leaf of banana of morbidity, artificial infection, healthy Leaf of banana, the Banana peel of health, health
Banana carpopodium, negative control, positive control.
Specific embodiment
In order to make content of the present invention easily facilitate understanding, With reference to embodiment to of the present invention
Technical solution is described further.
Test method as used in the following examples is conventional method unless otherwise specified.
Test material as used in the following examples, reagent etc., unless otherwise specified, commercially obtain.
The design of 1 molecular detection primer of embodiment and the foundation of Phyllosticta musarum special molecular detection method
1. the extraction of Phyllosticta musarum genomic DNA:
The genomic DNA of 6 plants of Phyllosticta musarums of this laboratory preservation is extracted using CTAB method, specific steps are such as
Under:
(1) it takes 0.1 g hypha powder in 1.5 mL centrifuge tubes, 900 μ L2%CTAB extracting solutions is added, are shaken using oscillator
Mixing is swung, 60 DEG C of water-bath 60min, under room temperature, 12000r/min are centrifuged 15 min；
(2) 700 μ L of supernatant is taken, adds isometric phenol, chloroform, isoamyl alcohol mixed liquor (each volume ratio is 25:24:1), mildly
It shakes, under room temperature, 8000 r/min are centrifuged 10min；
(3) 500 μ L of supernatant is taken, isometric chloroform is added and extracts again once, under room temperature, 8000 r/min centrifugation
10min；
(4) 350 μ L of supernatant is taken, 1/10 volume, 3 mol/L NaAc and 2 times of volume dehydrated alcohols are added, -20 DEG C heavy
Form sediment 60 min, and under the conditions of 4 DEG C, 8000 r/min are centrifuged 5 min；
(5) liquid is discarded supernatant, 700 μ L volumetric concentrations of addition are 70% ice ethyl alcohol, jog 10sec, under the conditions of 4 DEG C, 8000
R/min is centrifuged 10sec, dries, and 50 μ L TE buffers is added, -20 DEG C save backup.
2. Phyllosticta musarum ITS sequence measures:
With fungi ribosomes internal gene transcribed spacers (rDNA-ITS) universal primer TS1:5'-
TCCGTAGGGAACCTGCGG-3' and ITS4:5'-TCCTCCGCTTATTGATATGC-3' is that primer pair extracts banana freckle
The DNA of bacterium carries out PCR amplification, amplification reaction system and response procedures are as follows: 25 μ L of PCR reaction system, comprising 2.5 μ L 10 ×
PCR buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is the Taq enzyme (Takara of 5U/ μ L
Dalian treasured bioengineering Co., Ltd), each 0.5 μ L of the TIS1 and ITS4 of 10 μm of ol/L, 1 μ L of DNA profiling add ddH2O is to totality
It accumulates up to 25 μ L；PCR reaction condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of 1 min of denaturation, 55 DEG C of annealing 45sec, 72 DEG C extend 1
Min, totally 35 recycle；72 DEG C of extension 10min；Gained PCR product send Dalian treasured bioengineering Co., Ltd to be sequenced, and submits
GenBank obtains Serial No. KX611161.
3. the design of Phyllosticta musarum special molecular detection primer:
According to Phyllosticta musarum ribosomes the Internal Transcribed Spacer (rDNA-ITS) sequence fungi inter-species height variation and
Kind internal stability will be sequenced in ITS sequence and the GenBank of 6 plants of obtained Phyllosticta musarums with Clustal X softwarePhyllostictaCategory ITS sequence not of the same race, Glorosprium musarum Cookeet Mass ITS sequence, Curvularia lunata bacterium ITS sequence
Column, banana alternaria ITS sequence, banana checkers spore leaf spot fungi ITS sequence, banana secretly double spore leaf spot fungi ITS sequences
Column, banana blight bacteria ITS sequence carry out homology analysis and difference site is compared, and is set with BMRimer BMRimer5 software
Having counted has one couple of PCR primers of specific amplification effect (by Shanghai Sheng Gong bioengineering Co., Ltd Phyllosticta musarum
Synthesis), i.e. the sequence of special molecular detection primer are as follows:
Upstream primer BMF:5 '-GCTACAACGCCGAAATGACC-3 '；
Downstream primer BMR:5 '-GACGTTGCCCAATACCAAGC-3 '
4. the foundation of Phyllosticta musarum rapid molecular detection method:
(1) DNA is extracted from Leaf of banana or fruit:
1. extracting strains tested genomic DNA using CTAB method for when detecting pathogen pure culture;
2. when for detecting banana plant tissue with the presence or absence of Phyllosticta musarum, being extracted using NaOH rapid cleavage method
Banana plant tissue gene group DNA, the specific steps are as follows:
A. 0.1 g of plant tissue to be detected is weighed, 0.5 mol/L NaOH, 30 μ L is added, tissue is sufficiently milled to
Paste；
B. paste tissue is transferred in 1.5 mL centrifuge tubes, 12000r/min is centrifuged 6 min, takes 5 μ l of supernatant；
C. 0.1 Tris-HCl(pH=8.0 mol/L 495 μ L are added in supernatant), it is uniformly mixed, takes 1.0 μ L conducts
PCR template is expanded；
(2) to extract Leaf of banana or banana DNA as template progress PCR amplification: 25 μ L of PCR reaction system includes
2.5 μ 10 × PCR of L buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is 5U/ μ L's
Taq enzyme, each 0.5 μ L of the BMF and BMR of 10 μm of ol/L, 1 μ L of DNA profiling add ddH2O is to total volume up to 25 μ L；PCR reaction condition
Are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 45sec, 56 DEG C of annealing 45sec, 72 DEG C of extension 45sec, totally 35 recycle；72 DEG C are prolonged
Stretch 10min；
(3) resulting PCR product is walked on 1.0% Ago-Gel with the electrophoretic analysis of 0.5 × tbe buffer liquid, voltage is
4-5V/cm；Testing result is determined according to the size of amplified production, if energy specific amplification goes out the product of 382bp, can determine that
There are Phyllosticta musarums in the Leaf of banana or banana, otherwise do not deposit in the Leaf of banana or banana
In Phyllosticta musarum.
2 Phyllosticta musarum specific amplification of embodiment
1. extracting 2 plants of Phyllosticta musarums, Glorosprium musarum Cookeet Mass, Curvularia lunata bacterium, banana using CTAB method
The base of alternaria, banana checkers spore leaf spot fungi, banana secretly double spore leaf spot fungis, banana blight bacteria and asparagus stem wilt bacteria
Because of a group DNA.
2. for the DNA for trying bacterium be that template carries out PCR amplification: 25 μ L of PCR reaction system to extract, include 2.5 μ L 10 ×
PCR buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is the Taq enzyme of 5U/ μ L, 10 μ
Each 0.5 μ L of the BMF and BMR of mol/L, 1 μ L of DNA profiling, adds ddH2O is to total volume up to 25 μ L；PCR reaction condition are as follows: 94 DEG C pre-
It is denaturalized 5min；94 DEG C of denaturation 45sec, 56 DEG C of annealing 45sec, 72 DEG C of extension 45sec, totally 35 recycle；72 DEG C of extension 10min；
Electrophoresis detection amplified production.
3. specific amplification result
As shown in Figure 1,2 plants of Phyllosticta musarums can go out the band of 382bp with specific amplification, and Glorosprium musarum Cookeet Mass,
Secretly double spore leaf spot fungis, banana are withered for Curvularia lunata bacterium, banana alternaria, banana checkers spore leaf spot fungi, banana
Germ, asparagus stem wilt bacteria and negative control wither without amplified band, shows that molecular detection primer of the invention can be by banana
Other pathogens of black star germ and banana of causing harm distinguish, and have very strong specificity, and detection method of the invention is available
In the specific amplification of Phyllosticta musarum.
The sensitivity of the primer pair Phyllosticta musarum of the present invention of embodiment 3 detects
1. extracting the genomic DNA of Phyllosticta musarum using CTAB method；
2. by the genomic DNA of the Phyllosticta musarum of extraction, after spectrophotometric determination concentration, with sterile ultrapure water
Dilution, is configured to series of concentrations, spare；
It include 2.5 3. carrying out standard PCR amplification: 25 μ L of PCR reaction system as template at series of concentrations DNA using preparation
μ 10 × PCR of L buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is the Taq of 5U/ μ L
Enzyme, each 0.5 μ L of the BMF and BMR of 10 μm of ol/L, 1 μ L of DNA profiling add ddH2O is to total volume up to 25 μ L；PCR reaction condition are as follows:
94 DEG C of initial denaturation 5min；94 DEG C of denaturation 45sec, 56 DEG C of annealing 45sec, 72 DEG C of extension 45sec, totally 35 recycle；72 DEG C of extensions
10min；Electrophoresis detection amplified production.
4. carrying out nested PCR amplification as template at series of concentrations DNA using preparation:
(1) first round PCR is expanded: with fungi ribosomes internal gene transcribed spacers (rDNA-ITS) universal primer TS1:
5'-TCCGTAGGGAACCTGCGG-3' and ITS4:5'-TCCTCCGCTTATTG ATATGC-3' be outer primer to preparation at
Series of concentrations DNA carries out first round PCR amplification, amplification reaction system and response procedures are as follows: 25 μ L of PCR reaction system includes
2.5 μ 10 × PCR of L buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is 5U/ μ L's
Taq enzyme (Dalian Takara treasured bioengineering Co., Ltd), each 0.5 μ L of the TIS1 and ITS4 of 10 μm of ol/L, 1 μ L of DNA profiling,
Add ddH2O is to total volume up to 25 μ L；PCR reaction condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of 1 min of denaturation, 55 DEG C of annealing
45sec, 72 DEG C of 1 min of extension, totally 35 recycle；72 DEG C of extension 10min；
(2) second wheel PCR amplifications: using the product of first round PCR amplification as template, second is carried out by primer of BMF/BMR
PCR amplification is taken turns, 25 μ L of PCR reaction system includes 2.5 μ 10 × PCR of L buffer, and 2.0 μ L concentration are the dNTP of 2.5mmol/L
Mixture, 0.15 μ L concentration are the Taq enzyme of 5U/ μ L, and each 0.5 μ L of the BMF and BMR of 10 μm of ol/L, 1 μ L of DNA profiling add ddH2O
To total volume up to 25 μ L；PCR reaction condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 45sec, 56 DEG C of annealing 45sec, 72 DEG C
Extend 45sec, totally 30 circulations；72 DEG C of extension 10min；Electrophoresis detection amplified production.
5. testing result
As shown in Fig. 2, when carrying out Standard PCR as primer using primer BMF/BMR of the present invention, in 25 μ L reaction systems,
The Phyllosticta musarum DNA of 10pg can obtain view strip band, and detection sensitivity can reach 10pg(Fig. 2-A)；And it is further
With the universal primer TS1:5'-TCCGTAGGGAACCTGCGG-3' of fungi ribosomes internal gene transcribed spacers (rDNA-ITS) and
ITS4:5'-TCCTCCGCTTATTGATATGC-3' is that the product of external primer amplification is template, with primer BMF/BMR of the present invention
When carrying out the second wheel amplification for primer, in 25 μ L reaction systems, the Phyllosticta musarum DNA of 1fg can obtain view strip band,
Its detection sensitivity can reach 1fg (Fig. 2-B).
The detection of Phyllosticta musarum in 4 Leaf of banana of embodiment and fruit
1. extracting banana plant tissue gene group DNA using NaOH rapid cleavage method.
It include 2.5 2. carrying out standard PCR amplification: 25 μ L of PCR reaction system as template at series of concentrations DNA using preparation
μ 10 × PCR of L buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is the Taq of 5U/ μ L
Enzyme, each 0.5 μ L of the BMF and BMR of 10 μm of ol/L, 1 μ L of DNA profiling add ddH2O is to total volume up to 25 μ L；PCR reaction condition are as follows:
94 DEG C of initial denaturation 5min；94 DEG C of denaturation 45sec, 56 DEG C of annealing 45sec, 72 DEG C of extension 45sec, totally 35 recycle；72 DEG C of extensions
10min；Electrophoresis detection amplified production.
3. testing result
As shown in figure 3, natually morbid Leaf of banana, natural occurrence banana, artificial infection morbidity Leaf of banana,
It can produce the view strip band of 382bp or so in artificial infection early stage banana and positive control, and healthy Banana Leaf
Piece, the Banana peel of health, the banana carpopodium of health, negative control occur without any band, show primer of the present invention and inspection
Survey method can also be used in the detection of Phyllosticta musarum in the banana plant of field.
SEQUENCE LISTING
<110>Inst. of Plant Protection, fujian Academy of Agricultural Science
<120>a kind of Phyllosticta musarum molecular detection primer and detection method
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
gctacaacgc cgaaatgacc 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
gacgttgccc aataccaagc 20
<210> 3
<211> 18
<212> DNA
<213>artificial sequence
<400> 3
tccgtaggga acctgcgg 18
<210> 4
<211> 20
<212> DNA
<213>artificial sequence
<400> 4
tcctccgctt attgatatgc 20
Claims (4)
1. a kind of Phyllosticta musarum molecular detection primer, it is characterised in that: the nucleotide sequence of primer are as follows:
Upstream primer BMF:5 '-GCTACAACGCCGAAATGACC-3 '
Downstream primer BMR:5 '-GACGTTGCCCAATACCAAGC-3 '.
2. Phyllosticta musarum molecular detection primer according to claim 1, it is characterised in that: the upstream primer BMF and
Downstream primer BMR goes out the product of 382bp to Phyllosticta musarum specific amplification.
3. a kind of rapid detection method of Phyllosticta musarum, it is characterised in that: the following steps are included:
(1) DNA of Leaf of banana or fruit is extracted；
(2) PCR amplification: 25 μ L of PCR reaction system is carried out by template of the DNA of the Leaf of banana of extraction or fruit, includes 2.5 μ
10 × PCR of L buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is the Taq enzyme of 5U/ μ L,
Each 0.5 μ L of BMF and BMR described in claim 1 of 10 μm of ol/L, 1 μ L of DNA profiling add ddH2O is to total volume up to 25 μ L；PCR
Reaction condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 45sec, 56 DEG C of annealing 45sec, 72 DEG C of extension 45sec, totally 35 are followed
Ring；72 DEG C of extension 10min；
(3) resulting PCR product is walked on 1.0% Ago-Gel with the electrophoretic analysis of 0.5 × tbe buffer liquid, voltage 4-
5V/cm；Testing result is determined according to the size of amplified production, if energy specific amplification goes out the product of 382bp, can determine that perfume (or spice)
There are Phyllosticta musarums for any of several broadleaf plants blade or fruit.
4. application of the Phyllosticta musarum molecular detection primer as described in claim 1 in detection Phyllosticta musarum.
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Non-Patent Citations (3)
| Title |
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| Identification and differentiation of Phyllosticta species causing freckle disease of banana using high resolution melting (HRM) analysis;M.-H. Wong等;《Plant Pathology》;20131231;第62卷;1285-1293 * |
| 瓜黑星病菌、枯萎病菌和蔓枯病菌的三重PCR检测;高永洋 等;《植物病理学报》;20101231;第40卷(第4期);343-350 * |
| 苹果黑星病菌的分子检测;商蓓 等;《西北农林科技大学学报（自然科学版）》;20100831;第38卷(第8期);104-110 * |
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