CN106244720B - A kind of anthracnose of peach bacterium molecule detection primer and detection method - Google Patents
A kind of anthracnose of peach bacterium molecule detection primer and detection method Download PDFInfo
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Abstract
The present invention relates to a kind of anthracnose of peach bacterium molecule detection primer and its detection methods, belong to corps diseases detection and field of biotechnology.The specific primer includes upstream primer PCGF:5 '-TACCTAACCGTTGCTTCGGC-3 ', downstream primer PCGR:5 '-CCCAGTGCGAGACGTTAGTT-3 '；Use above-mentioned primer that can specifically amplify fragment length in anthracnose of peach bacterium pure dna, the peach leaf piece and Peach fruits that carry disease germs through PCR amplification and agarose gel electrophoresis as the specific amplification products of 420bp.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 anthracnose of peach, moreover it is possible to effectively distinguish similar brown rot on peach, the diffusion sprawling of early warning and prevention and control to anthracnose of peach, controlling disease is of great significance.
Description
Technical field
The present invention relates to a kind of anthracnose of peach bacterium molecule detection primer and its detection methods, are exclusively used in the fast of anthracnose of peach bacterium
Fast Molecular Detection, while can realize that the early diagnosis of field anthracnose of peach bacterium and the monitoring of germ are identified, belong to corps diseases
Detection and field of biotechnology.
Background technique
Peach belongs to rosaceae Prunus deciduous fruit tree, and China is the centre of origin of world peach.Peach is known as " peach offered as a birthday present ", " flat peach "
Laudatory title, because of its delicious meat, and be known as " peerless fruit ".Peach meat containing protein, fat, carbohydrate, crude fibre,
Calcium, phosphorus, iron, carrotene, vitamin B1, sugar etc. are important one of the fruit in China.
Anthracnose of peach (Peach anthracnose) be peach [Prunus persica(L.) Batsch] it is a kind of in cultivation
Important disease, mainly cause harm peach branch, tender leaf and young fruit can lead to fruit rot, caducous.In the morbidity of young fruit initial stage,
The brown water stain shape spot in fruit face, also expands with Fruit scab, bronzing later, rounded or oval, and significant depressions.
Fruit is fallen ill when closely mature, and fruit face scab is significantly recessed, and is in apparent concentric ring, and it is small to will appear salmon pink when moist on concentric ring
Grain point, scab is gradually expanded or even entire fruit.After young sprout is infected, there is the slightly concave sunken oval scab of crineous.Peach charcoal
Subcutaneous ulcer disease pathogen asexual generation be Colletotrichum gloeosporiodes (Colletotrichum gloeosporioidsPenz), belong to and partly knowing
Bacterium subphylum.The pathogen is mainly overwintering on peach disease deadwood and mummy.Spring in next year, the spore generated in diseased tissues are passed by means of wind and rain
It is multicast on branch, blade and fruit, causes primary infection.Disease period of causing harm is long, influences entire growth period.Anthracnose of peach is initial
Phase is the best period of disease control, and, disease by symptom based on similar to peach brown rot symptom in its disease initial phase
Conventional diagnostic techniques, need to pass through using Koch's Postulates pathogenicbacteria separation culture, pathogen identification, connect bacterium, symptom analysis etc.
Step, time-consuming, low efficiency, accuracy are poor, it is difficult to accomplish to detect in time when disease occurs and the propagation of effectively control pathogen
And plant disease epidemic, it is difficult to meet the actual needs of anthracnose of peach diagnosis.Therefore, a kind of quick, sensitive detection method use is established
In the early diagnosis of anthracnose of peach, technical support is provided for the control approach of disease, while the propagation of controlling disease has
Significance.
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 research at present in relation to the detection of anthracnose of peach bacterium molecule.
Summary of the invention
It is based primarily upon pathogen morpha feature for the detection and identification in the prior art to anthracnose of peach bacterium, program is numerous
Trivial, the dead length of consumption, high to identification skill requirement, accuracy is low, it is difficult to which the actual needs problem for meeting anthracnose of peach diagnosis provides
A kind of anthracnose of peach bacterium molecule detection primer and its detection method.
To achieve the above object, this invention takes following technical schemes:
Present invention firstly provides a kind of anthracnose of peach bacterium molecule detection primer, nucleotide sequences are as follows:
Upstream primer PCGF:5 '-TACCTAACCGTTGCTTCGGC-3 '；
Downstream primer PCGR:5 '-CCCAGTGCGAGACGTTAGTT-3 '.
The primer PCGF and PCGR goes out the product of 420bp to anthracnose of peach bacterium specific amplification.
The present invention also provides a kind of rapid detection methods of anthracnose of peach bacterium, comprising the following steps:
(1) DNA is extracted from peach leaf piece or fruit；
When for detecting pathogen pure culture, strains tested genomic DNA is extracted using CTAB method；For detecting
When peach tissue is with the presence or absence of anthracnose of peach bacterium, peach plant tissue genomic DNA is extracted using NaOH rapid cleavage method.
(2) PCR amplification: 25 μ L of PCR reaction system is carried out as template to extract peach leaf piece or fruit DNA, includes 2.5 μ L
10 × PCR buffer, 2.0 μ L concentration be 2.5mmol/L dNTP Mixture, 0.15 μ L concentration be 5U/ μ L Taq enzyme, 10
Each 0.5 μ L of PCGF and PCGR of μm 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
Initial denaturation 5min；94 DEG C of denaturation 40sec, 55 DEG C of annealing 45sec, 72 DEG C of extension 45sec, totally 35 recycle；72 DEG C of extensions
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 420bp, can determine that
There are anthracnose of peach bacterium in the peach leaf piece or fruit, and anthracnose of peach bacterium otherwise is not present in the peach leaf piece or fruit.
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 primer of specific amplified effect to anthracnose of peach bacterium.
To the anthracnose of peach bacterium of different geographic origins, carry anthracnose of peach bacterium peach leaf piece, carry anthracnose of peach bacterium Peach fruits
Detection verifying, only energy in the blade and fruit of anthracnose of peach bacterium and the carrying germ have been carried out with the peach leaf piece and fruit of health
The electrophoretic band for specifically amplifying 420 bp illustrates primer designed by the present invention for detecting anthracnose of peach bacterium
Accurately and reliably；
(2) high specificity: primer pair anthracnose of peach bacterium designed by the present invention has very strong specificity, can be used in area
Other anthracnose of peach bacterium and Monilinia fructicola, so as to the raw similar disease of symptom characteristic on peach of effective district distribution；
(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 anthracnose of peach bacterium；
(4) applicability is wide, practicability is good: the detection method of anthracnose of peach bacterium of the invention, can not only be to germ mycelium
It is detected, the early detection, it can be achieved that anthracnose of peach bacterium can be also detected to susceptible peach leaf piece and fruit, i.e., in disease
It is detected before aobvious disease, the eruption and prevalence of controlling disease.
(5) easy to operate quick: the present invention can determine that after need to only carrying out DNA extraction, PCR amplification and agarose electrophoresis
As a result, general entire detection process can be completed within a few hours, it is simple and efficient to handle.
Detailed description of the invention
Fig. 1 is primer pair anthracnose of peach bacterium specific amplification electrophoretogram of the present invention: wherein swimming lane M is 2000bp DNA
Marker, swimming lane 2, swimming lane 3 are anthracnose of peach bacterium, and swimming lane 4-10 is respectively as follows: Monilinia fructicola, Colletotrichum truncatum, peach grey mold
Germ, dried peach maize ear rot bacterium, peach white mould germ, Pear black spot bacterium, negative control.
Fig. 2 is the sensitivity detection amplification electrophoretogram of primer anthracnose of peach bacterium of the present invention: figure A: regular-PCR sensitivity inspection
Survey, wherein swimming lane M be 2000bp DNA Marker, swimming lane 2-12 be respectively as follows: 100ng, 10ng, 1ng, 100pg, 10pg, 1pg,
100fg, 10fg, 1fg, negative control, positive control;Figure B is nest-type PRC sensitivity technique, and wherein swimming lane M is 2000bp
DNA Marker, swimming lane 2-13 be respectively as follows: 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, 100ag,
Negative control, positive control.
Fig. 3 is that anthracnose of peach bacterial examination surveys amplification electrophoretogram in peach leaf piece of the present invention and fruit, and wherein swimming lane M is 2000bp
DNA Marker, swimming lane 2-10 be respectively natually morbid peach leaf piece, natural occurrence Peach fruits, artificial infection morbidity peach leaf
Piece, artificial infection early stage Peach fruits, healthy peach leaf piece, healthy Peach fruits, healthy peach leaf handle, 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 anthracnose of peach bacterium special molecular detection method
1. the extraction of anthracnose of peach bacterium genomic DNA:
The genomic DNA of 8 plants of anthracnose of peach bacterium of this laboratory preservation is extracted using CTAB method, the specific steps are as follows:
(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, isometric phenol, chloroform, isoamyl alcohol mixed liquor (each volume ratio is 25:24:1), temperature are added
And shake, 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. anthracnose of peach bacterium 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 anthracnose of peach bacterium
DNA carry out PCR amplification, amplification reaction system and response procedures are as follows: 25 μ L of PCR reaction system includes 2.5 10 × PCR of μ L
Buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and (Takara is big for the Taq enzyme that 0.15 μ L concentration is 5U/ μ L
Lian Bao 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 extend 1
Min, totally 35 recycle；72 DEG C of extension 10min；Gained PCR product send Dalian treasured bioengineering Co., Ltd to be sequenced, sequencing result
GenBank is uploaded, acquisition accession number is KX611163.
3. the design of anthracnose of peach bacterium special molecular detection primer:
It makes a variation and plants in fungi inter-species height according to anthracnose of peach sclerotium sugar vivo transcription spacer region (rDNA-ITS) sequence
Internal stability will be sequenced in ITS sequence and the GenBank of 8 plants of obtained anthracnose of peach bacterium with Clustal X softwareColletotrichumCategory ITS sequence not of the same race, Monilinia fructicola ITS sequence, peach ash arrhizus bacteria ITS sequence carry out homologous
Property analysis and difference site compare, with Primer Primer5 software design to anthracnose of peach bacterium have specific amplification effect
One couple of PCR primers (Sheng Gong bioengineering Co., Ltd synthesizes by Shanghai), i.e. the sequence of special molecular detection primer are as follows:
Upstream primer PCGF:5 '-TACCTAACCGTTGCTTCGGC-3 '；
Downstream primer PCGR:5 '-CCCAGTGCGAGACGTTAGTT-3 '
4. the foundation of anthracnose of peach bacterium rapid molecular detection method:
(1) DNA is extracted from peach blade or fruit:
1. extracting strains tested genomic DNA using CTAB method for when detecting pathogen pure culture;
2. when for detecting peach blade or fruit tissue with the presence or absence of anthracnose of peach bacterium, using NaOH rapid cleavage method
Extract peach leaf piece or fruit tissue genomic 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) PCR amplification: 25 μ L of PCR reaction system is carried out as template to extract peach leaf piece or fruit DNA, includes 2.5 μ L
10 × PCR buffer, 2.0 μ L concentration be 2.5mmol/L dNTP Mixture, 0.15 μ L concentration be 5U/ μ L Taq enzyme, 10
Each 0.5 μ L of PCGF and PCGR of μm 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
Initial denaturation 5min；94 DEG C of denaturation 40sec, 55 DEG C of annealing 45sec, 72 DEG C of extension 45sec, totally 35 recycle；72 DEG C of extensions
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 420bp, can determine that
There are anthracnose of peach bacterium in the peach leaf piece or fruit, and anthracnose of peach bacterium otherwise is not present in the peach leaf piece or fruit.
2 anthracnose of peach bacterium specific amplification of embodiment
1. extracting 2 plants of anthracnose of peach bacterium, Colletotrichum truncatum, peach ash arrhizus bacteria, dried peach maize ear rot bacterium, peach using CTAB method
The genomic DNA of white mould germ, Pear black spot bacterium.
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 PCGF and PCGR 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
Initial denaturation 5min；94 DEG C of denaturation 40sec, 55 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. specific amplification result
As shown in Figure 1,2 plants of anthracnose of peach bacterium can go out the band of 420bp with specific amplification, and Colletotrichum truncatum, peach
Ash arrhizus bacteria, dried peach maize ear rot bacterium, peach white mould germ, Pear black spot bacterium and negative control show of the invention without amplified band
Molecular detection primer can distinguish anthracnose of peach bacterium and other pathogens, have very strong specificity, inspection of the invention
Survey method can be used for the specific amplification of anthracnose of peach bacterium.
The sensitivity of the primer pair anthracnose of peach bacterium of the present invention of embodiment 3 detects
1. extracting the genomic DNA of anthracnose of peach bacterium using CTAB method；
2. dilute with sterile ultrapure water after spectrophotometric determination concentration by the genomic DNA of the anthracnose of peach bacterium of extraction
It releases, is configured to series of concentrations, it is 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 PCGF and PCGR 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 40sec, 55 DEG C of annealing 45sec, 72 DEG C of extension 45sec, totally 35 recycle；72 DEG C are prolonged
Stretch 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: being primer progress the using PCGF/PCGR using the product of first round PCR amplification as template
Two wheel PCR amplifications, 25 μ L of PCR reaction system includes 2.5 μ 10 × PCR of L buffer, and 2.0 μ L concentration are 2.5mmol/L's
DNTP Mixture, Taq enzyme of the 0.15 μ L concentration for 5U/ μ L, each 0.5 μ L of the PCGF and PCGR 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 40sec, 55 DEG C of annealing
45sec, 72 DEG C of extension 40sec, totally 30 recycle；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 PCGF/PCGR of the present invention, in 25 μ L reaction systems,
The anthracnose of peach bacterium DNA of 10pg can obtain view strip band, and detection sensitivity can reach 10pg(Fig. 2-A)；And 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 PCGF/ of the present invention
When PCGR is that primer carries out the second wheel amplification, in 25 μ L reaction systems, the anthracnose of peach bacterium DNA of 1fg can obtain view strip
Band, detection sensitivity can reach 1fg (Fig. 2-B).
The detection of anthracnose of peach bacterium in 4 peach leaf piece of embodiment and Peach fruits
1. extracting peach leaf piece or fruit tissue genomic 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 PCGF and PCGR 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 40sec, 55 DEG C of annealing 45sec, 72 DEG C of extension 45sec, totally 35 recycle；72 DEG C are prolonged
Stretch 10min；Electrophoresis detection amplified production.
3. testing result
As shown in figure 3, natually morbid peach leaf piece, natural occurrence Peach fruits, artificial infection morbidity peach leaf piece, manually connect
It can produce the view strip band of 420bp or so in the Peach fruits of kind of early stage, positive control, and healthy peach leaf piece, healthy peach
Fruit, healthy peach leaf handle, negative control occur without any band, show that primer of the present invention and detection method can also be used in field
The detection of anthracnose of peach disease plant.
SEQUENCE LISTING
<110>Inst. of Plant Protection, fujian Academy of Agricultural Science
<120>a kind of anthracnose of peach bacterium molecule 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
tacctaaccg ttgcttcggc 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
cccagtgcga gacgttagtt 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 anthracnose of peach bacterium molecule detection primer, it is characterised in that: the nucleotide sequence of primer are as follows:
Upstream primer PCGF:5 '-TACCTAACCGTTGCTTCGGC-3 '
Downstream primer PCGR:5 '-CCCAGTGCGAGACGTTAGTT-3 '.
2. anthracnose of peach bacterium molecule detection primer according to claim 1, which is characterized in that the upstream primer PCGF is under
Trip primer PCGR goes out the product of 420bp to anthracnose of peach bacterium specific amplification.
3. a kind of rapid detection method of anthracnose of peach bacterium, it is characterised in that: the following steps are included:
(1) DNA of peach leaf piece or Peach fruits is extracted；
(2) PCR amplification: 25 μ L of PCR reaction system is carried out using the peach leaf piece of extraction or Peach fruits DNA as template, includes 2.5 μ L
10 × PCR buffer, 2.0 μ L concentration be 2.5mmol/L dNTP Mixture, 0.15 μ L concentration be 5U/ μ L Taq enzyme, 10
Each 0.5 μ L of PCGF and PCGR described in the claim 1 of μm 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 40sec, 55 DEG C of annealing 45sec, 72 DEG C of extension 45sec, totally 35
Circulation；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 420bp, can determine that peach
There are anthracnose of peach bacterium for blade or fruit.
4. a kind of primer as described in claim 1 answering in the early diagnosis of field anthracnose of peach bacterium and the monitoring identification of germ
With.
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| CN105063226A (en) * | 2015-09-08 | 2015-11-18 | 福建省农业科学院植物保护研究所 | Specific PCR detection primers and detection method for Colletotrichum truncatum of vegetable soybeans |
| CN105063219A (en) * | 2015-08-20 | 2015-11-18 | 福建省农业科学院植物保护研究所 | Guava colletotrichum orbiculare specificity PCR detecting primer and detecting method of guava colletotrichum orbiculare |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105063219A (en) * | 2015-08-20 | 2015-11-18 | 福建省农业科学院植物保护研究所 | Guava colletotrichum orbiculare specificity PCR detecting primer and detecting method of guava colletotrichum orbiculare |
| CN105063226A (en) * | 2015-09-08 | 2015-11-18 | 福建省农业科学院植物保护研究所 | Specific PCR detection primers and detection method for Colletotrichum truncatum of vegetable soybeans |
Non-Patent Citations (1)
| Title |
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| 桃炭疽病病原鉴定及木醋液防治研究;刘勇等;《湖北农业科学》;20141231;第53卷(第24期);第1.3、2.1节 * |
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