CN105039535A - Primers for detecting alternaria alternata and alternaria alternata detection method - Google Patents
Primers for detecting alternaria alternata and alternaria alternata detection method Download PDFInfo
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- CN105039535A CN105039535A CN201510396232.8A CN201510396232A CN105039535A CN 105039535 A CN105039535 A CN 105039535A CN 201510396232 A CN201510396232 A CN 201510396232A CN 105039535 A CN105039535 A CN 105039535A
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- primer
- black spot
- pear black
- pcr reaction
- spot bacterium
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
Abstract
The invention discloses primers for detecting alternaria alternata. The primers comprise an upstream primer sequence shown in HB1: TCACCCTTGTCTTTTGCGTA and a downstream primer sequence shown in HB2: ACCTTTGCTGATAGAGAGTG. The invention also discloses an alternaria alternata detection method utilizing the primers. Through design of a pair of the specific primers and use of a PCR amplification method, alternaria alternata in plant tissue can be effectively detected. The detection method has the advantages of accuracy, fastness, simpleness and operation easiness, can identify pathogens in a disease invasion initial stage, can be referentially used for field investigation and plant product examination, has an important meaning for controlling pear black spot large area outbreak and cross-regional propagation and provides technical guidance and theoretical basis for other pathogen detection.
Description
Technical field
The present invention relates to a kind of primer for detecting Pear black spot bacterium, and utilize described primer to detect the molecular detecting method of Pear black spot bacterium.
Background technology
Pear black spot germ (
alternariaalternata) main harm leaf, fruit and young sprout, cause tree body weak, and cause storage period fruit morbidity, causes heavy economic losses.This disease is in Japan, and France, all there are report in Korea S etc., all have generation, have a strong impact on the development of China's pears industry in the multiple province of China.But adopt traditional Plant diseases detection method directly to observe, the situation that their early stage does not show symptom can be omitted, and rely on the interference that morphologic detection method is vulnerable to human factor and envrionment conditions, in addition traditional sorting technique length consuming time, program is loaded down with trivial details, be not suitable for the requirement of rapid detection, be difficult to realize the disease timely monitoring occurred and the propagation and the plant disease epidemic that effectively control pathogenic bacteria.Along with the development of Protocols in Molecular Biology, employing molecular detecting method is that the diagnosis of this disease provides good detection means.
Summary of the invention
Technical problem to be solved by this invention is to provide detection method and the primer thereof of a kind of Pear black spot bacterium, and utilize this primer and detection method to detect Pear black spot bacterium, speed is fast, accurately, highly sensitive, reliable and stable.
Technical scheme provided by the invention is:
For detecting a primer for Pear black spot bacterium, comprising pair of primers, it is characterized in that: its upstream primer sequence as described in HB1, downstream primer sequence as described in HB2, wherein,
HB1 is: TCACCCTTGTCTTTTGCGTA
HB2 is: ACCTTTGCTGATAGAGAGTG.
A kind of method utilizing above-mentioned primer to detect Pear black spot bacterium, its process is: extract the DNA of testing sample as template, primer described in utilization carries out PCR reaction, get PCR reaction amplified production to detect, if there is the DNA band that molecular weight is about 400bp, then prove institute detect in sample contain Pear black spot bacterium.
The method of above-mentioned detection Pear black spot bacterium, the amplification system of described PCR reaction is: 10 × Taqbuffer2.5 μ L, 25mmol/LMgCl
22.0 μ L, 2.5mmol/LdNTP2.0 μ L, 10 μm of each 1.0 μ L of the forward and reverse primer of ol/L, 5U/LTagase0.2 μ L, 50ng/ μ LDNA template 2 μ L, moisturizing is to cumulative volume 25 μ L.
The method of described detection Pear black spot bacterium, the response procedures of described PCR reaction is: 94 DEG C of 4min; 94 DEG C of 30s, 55.8 DEG C of 30s, 72 DEG C of 30s, 35 circulations; 72 DEG C of 10min.4 DEG C of preservations.
The present invention also provides the another kind of method detecting Pear black spot bacterium, its process is: extract the DNA of testing sample as template, two pairs of primers are utilized to carry out sleeve type PCR reaction, the template that first round PCR reacts is extract the DNA of testing sample, primer is fungi ITS sequence universal primer ITS1 and ITS4, second template of taking turns PCR reaction is first round PCR reaction product, primer is described primer HB1 and HB2, get second take turns PCR react amplified production carry out detected through gel electrophoresis, if there is the DNA band that molecular weight is about 400bp, then prove in institute's sample product containing Pear black spot bacterium.
The present invention has following beneficial effect:
The present invention is by design a pair Auele Specific Primer, and the method in conjunction with pcr amplification can detect the Pear black spot bacterium in plant tissue effectively.The advantages such as it is accurate, quick, simple to operation that the inventive method has, pathogen can be identified at the disease infestation initial stage, can with reference to the inspection being applied to field investigation and plant prod, significant to the big area outburst and trans-regional propagation that control Pear black spot, meanwhile, the foundation of system of the present invention is also for the detection of other pathogenic bacterias provides technical director and theoretical foundation.
The present invention analyzes the rDNA-ITS sequence obtaining Pear black spot bacterium and other genus from GeneBank, devise a pair Auele Specific Primer, other pathogenic bacterias of this primer pair are utilized to carry out specific amplification, discovery can only be increased and be obtained the band of a 400bp from this genus, other bacterial strains all occur without amplified band, show that this has specificity among genus to primer.The Molecular Detection of the diagnosis and plant pathogenic fungi of carrying out disease with the rDNA-ITS sequences Design Auele Specific Primer of fungi is used widely.
Highly sensitive is an important indicator of detection of pathogens, and can it be related to and Fast Detection Technique be directly applied in the inspection and quarantine of plant prod.In the reaction system of 25 μ L, common PCR method can detect the genomic dna of 1ng, in the system that the present invention sets up, adopt sleeve type PCR technology that the sensitivity detecting genomic dna has been brought up to 100 times, only need the DNA of 10pg the existence of germ can be detected.
Accompanying drawing explanation
Fig. 1 is a pair Auele Specific Primer carrying out analysis and designation according to Pear black spot bacterium all in GeneBank and other rDNA-ITS sequences belonged to;
Fig. 2 is the checking of Auele Specific Primer, M:2000bpmarker; Swimming lane 1: Pear black spot bacterium; Swimming lane 2-23: other bacterial strains; Swimming lane 24: negative control;
Fig. 3 is the sensitivity technique of Standard PCR to Pear black spot bacterium, M:2000bpmarker; Swimming lane 1-9: template concentrations is respectively 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg; Swimming lane 10: negative control;
Fig. 4 is the sensitivity technique of sleeve type PCR to Pear black spot bacterium, M:2000bpmarker; Swimming lane 1-9: template concentrations is respectively 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg; Swimming lane 10: negative control;
Fig. 5 is that the PCR of Pear black spot bacterium in disease plant detects, M:2000bpmarker; Swimming lane 1: extract the PCR primer that DNA is template to be separated germ; Swimming lane 2-3: slightly extract the pcr amplification product that DNA is template with disease plant; Swimming lane 4: healthy plant contrasts; Swimming lane 5: negative control.
Embodiment
Detailed description below by embodiment illustrates the present invention further, but is not limitation of the present invention, only does example explanation.
Embodiment 1: design, synthetic primer set up the PCR reaction system of Pear black spot bacterium
One, design of primers and synthesis
The design of Auele Specific Primer: analyze Pear black spot bacterium in GeneBank and other rDNA-ITS sequences belonged to, devise a pair Auele Specific Primer, sequence is as follows:
HB1：TCACCCTTGTCTTTTGCGTA
HB2：ACCTTTGCTGATAGAGAGTG。
Primer its specificity of BLAST analysis verification in GeneBank again of design.
Also use a pair plant pathogenic fungi ITS universal primer ITS1 and ITS4 outer primer as sleeve type PCR, sequence is as follows simultaneously:
ITS1：TCCGTAGGTGAACCTGCGG
ITS4：TCCTCCGCTTATTGATATGC
All primers all entrust the raw work combining unit synthesis in Shanghai.
Two, Standard PCR reaction system is set up
Standard PCR amplification system is: 10 × Taqbuffer2.5 μ L, 25mmol/LMgCl
22.0 μ L, 2.5mmol/LdNTP2.0 μ L, 10 μm of each 1.0 μ L of the forward and reverse primer of ol/L, 5U/LTagase0.2 μ L, 20ng/ μ LDNA template 2 μ L, moisturizing is to cumulative volume 25 μ L, PCR amplification instrument carries out amplified reaction, and response procedures is: 94 DEG C of denaturation 4min; 94 DEG C of sex change 30s, 55.8 DEG C of annealing 30s, 72 DEG C extend 30s, totally 35 circulations; 72 DEG C extend 10min, 4 DEG C of preservations.
Three, sleeve type PCR reaction system is set up
For improving detection sensitivity, further established sleeve type PCR reaction system.With plant pathogenic fungi universal primer ITS1/ITS4 for the first round reacts primer, reaction system is as above-mentioned Standard PCR system.In response procedures, annealing temperature is 58 DEG C, and other parameters are with above-mentioned Standard PCR response procedures.Then with Auele Specific Primer, carry out second take turns pcr amplification with first round PCR primer for template, the primer is Auele Specific Primer HB1 and HB2 according to claim 1.System is the same, and annealing temperature is 55.8 DEG C, other parameter constants.All reagent is all purchased from the grand biological company limited of Hefei will.Get 5 μ L amplified productions electrophoresis in 1.0% sepharose, gel imaging system detects and takes pictures, if there is the DNA band that molecular weight is about 400bp, then prove that institute is detected in sample and contain Pear black spot bacterium.
Embodiment 2: prepare DNA profiling
The template that the DNA extracting all kinds of sample reacts as PCR, detailed process is as follows:
1. mycelial cultivation, collection and DNA extraction
PDA substratum: agar powder 20g, potato 200g, glucose 20g, adds water and is settled to 1L.
To go on PDA culture medium flat plate for examination germ, and cut 10 pieces of 2cm × 2cm bacterium colony blocks from colony edge after 20 DEG C of dark culturing 5d, go to PDA liquid nutrient medium, 28 DEG C of shaking culture 7 days, collecting by filtration mycelia, pulverizes through liquid nitrogen freezing, and-20 DEG C save backup.
The CTAB method that the extraction of genomic dna provides according to molecular cloning, concrete operations are as follows:
Take a morsel hypha powder, adds 900 μ L2%CTAB extracting solutions and 90 μ L10%SDS, and vortex mixes, and in 60 DEG C of water-bath 1h, period turns upside down several times every 10min, the centrifugal 10min of 12000rpm under normal temperature condition; Get supernatant, add equal-volume phenol/chloroform/primary isoamyl alcohol (25:24:1), put upside down mixing, the centrifugal 5min of 12000rpm under normal temperature condition; Supernatant is transferred in new EP pipe, adds equal-volume chloroform, put upside down mixing gently, the centrifugal 5min of 12000rpm under normal temperature condition; Supernatant is transferred in new EP pipe, adds 1 times to the Virahol of supernatant volume ,-20 DEG C of condition precipitation 15min; The centrifugal 5min of 12000rpm under normal temperature condition, incline supernatant, precipitation 70% washing with alcohol 2 times, treats that ethanol volatilization is done under 37 DEG C of conditions; Add appropriate sterilizing ultrapure water or TE(pH8.0) dissolution precipitation (containing 20 μ g/mlRNase), clear up 30min rear electrophoresis for 37 DEG C and detect ,-20 DEG C save backup.
2. the extraction of morbidity plant tissue DNA
To go on PDA culture medium flat plate for examination germ, cut 2cm × 2cm bacterium colony block from colony edge after 28 DEG C of dark culturing 5d, wound inoculation is on pomegranate plant.Cut incidence tissue after for some time and extract genomic dna, concrete operations: the plant tissue of getting some morbidities, every milligram of tissue adds 10 μ L, 0.5mol/LNaOH, be transferred in the centrifuge tube of 1.5ml after fully grinding in mortar, 12, the centrifugal 5min of 000g, gets 5 μ L supernatant liquors and adds 495 μ L, 0.1mmol/LTris (PH8.0), gets 1 μ L and be directly used in PCR reaction after mixing.
Embodiment 3: the specificity and the sensitivity that detect primer
One, specific detection
Bacterial strain uses therefor of the present invention and relevant information are in table 1.Adopt all strains tested genomic dnas in the Auele Specific Primer his-and-hers watches 1 designed by the present invention to carry out pcr amplification, from Pear black spot bacteria strain, only can amplify the band of a 400bp specifically, and other strains testeds and blank are all without amplified band.Show that this primer has species specificity, Pear black spot bacterium and other kinds can be distinguished
.
For screening the bacterial strain of primer specificity in table 1 the present embodiment
| Bacterial strain | Number of strains | Use HB1/HB2 amplification |
| Alternaria alternata | 1 | + |
| Botrytis cinerea | 1 | - |
| Botryosphaeria dothidea | 1 | - |
| Coniella granati | 1 | - |
| Coniothyrium diplodiella | 1 | - |
| Colletotrichum gloeosporioides | 1 | - |
| Cercospora circumscissa | 1 | - |
| Gymnosporangium yamadae | 1 | - |
| Gymnosporangium haraeanum | 1 | - |
| Glomerella acutata | 1 | - |
| Elsinoe fawcetti | 1 | - |
| Phyllosticta pirina | 1 | - |
| Pestalotiopsis theae | 1 | - |
| Fusicladium virescens | 1 | - |
| Marssonina coronaria | 1 | - |
| Podosphaera leucotricha | 1 | - |
| Podosphaera tridactyla | 1 | - |
| Penicillium italicum | 1 | - |
| Uncinula necator | 1 | - |
| Phytophthora capsici | 1 | - |
| Plasmopara viticola | 1 | - |
| Rhizopus stolonifer | 1 | - |
| Septoria piricola | 1 | - |
In upper table :+representing the specific amplification band with primer HB1/HB2, length is 400bp;-indicate without amplified production.
Two, sensitivity technique
In the reaction system of 25 μ L, determine the sensitivity of above-mentioned set up detection system, primer HB1 and HB2 Absorbable organic halogens ground amplification from the genomic templates of at least 1ng obtains the specific band of 400bp.And carry out the product of pcr amplification for template with the genomic dna of universal primer ITS1 and ITS4 of plant pathogenic fungi to Pear black spot bacteria strain different concns, re-use Auele Specific Primer HB1 and HB2 to carry out second and take turns shell type pcr amplification, the genomic dna of 10pg detected Absorbable organic halogens, make sensitivity improve 100 times.
Embodiment 4: the pathogen in Diseased Plant Tissues detects
Clip incidence tissue from the plant of artificial inoculation, extract DNA and carry out pcr amplification, morbidity sample has all amplified the specific band of 400bp, and healthy plant increase not go out band, illustrates that this cover technology can be used for the rapid molecular detection of Pear black spot bacterium in Diseased Plant Tissues.
Claims (5)
1. for detecting a primer for Pear black spot bacterium, comprising pair of primers, it is characterized in that: its upstream primer sequence is as described in HB1, downstream primer sequence is as described in HB2, wherein, HB1 is: TCACCCTTGTCTTTTGCGTA, HB2 are: ACCTTTGCTGATAGAGAGTG.
2. the method utilizing the primer described in claim 1 to detect Pear black spot bacterium, it is characterized in that: extract the DNA of testing sample as template, utilize above-mentioned design primer carry out PCR reaction, get PCR reaction amplified production to detect, if there is the DNA band that molecular weight is 400bp, then prove institute detect in sample contain Pear black spot bacterium.
3. the method for detection Pear black spot bacterium according to claim 2, is characterized in that: the amplification system of described PCR reaction is: 10 × Taqbuffer2.5 μ L, 25mmol/LMgCl
22.0 μ L, 2.5mmol/LdNTP2.0 μ L, 10 μm of each 1.0 μ L of the forward and reverse primer of ol/L, 5U/LTagase0.2 μ L, 20ng/ μ LDNA template 2 μ L, moisturizing is to cumulative volume 25 μ L.
4. the method for detection Pear black spot bacterium according to claim 2, is characterized in that: the response procedures of described PCR reaction is: 94 DEG C, 4min; 94 DEG C, 30s, 55.8 DEG C, 30s, 72 DEG C, 30s, 35 circulations; 72 DEG C, 10min, 4 DEG C of preservations.
5. the method utilizing the primer described in claim 1 to detect Pear black spot bacterium, it is characterized in that, concrete operations are as follows: extract the DNA of testing sample as template, two pairs of primers are utilized to carry out sleeve type PCR reaction, the template that first round PCR reacts is extract the DNA of testing sample, primer is fungi ITS sequence universal primer ITS1 and ITS4, second template of taking turns PCR reaction is first round PCR reaction product, primer is described primer HB1 and HB2, get second take turns PCR react amplified production carry out detected through gel electrophoresis, if there is the DNA band that molecular weight is about 400bp, then prove in institute's sample product containing Pear black spot bacterium.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105420372A (en) * | 2015-12-17 | 2016-03-23 | 安徽出入境检验检疫局检验检疫技术中心 | PCR primer for detecting alternaria kikuchiana and detection method using same |
| CN105543407A (en) * | 2016-03-16 | 2016-05-04 | 湖北省农业科学院果树茶叶研究所 | Method for species level classification of alternaria kikuchi-aria by means of multiple marker genes |
| CN105724138A (en) * | 2016-03-14 | 2016-07-06 | 阜阳市颍州区金湖丰种植农民专业合作社 | Method for reducing high-incidence season disease incidence of black spot of late-autumn yellow pears |
| CN106636341A (en) * | 2016-11-02 | 2017-05-10 | 白剑宇 | Detection primer and detection method for red date blackspot bacteria |
-
2015
- 2015-09-09 CN CN201510396232.8A patent/CN105039535B/en not_active Expired - Fee Related
Non-Patent Citations (3)
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| 李云飞等: "砀山梨黑斑病分子检测技术研究", 《中国农学通报》 * |
| 王凤军等: "实时荧光PCR检测香梨黑斑病菌", 《食品科学》 * |
| 马海波: "出口果园中库尔勒香梨黑斑病快速检测及疫情监测技术研究", 《中国优秀硕士学位论文全文数据库（农业科技辑）》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105420372A (en) * | 2015-12-17 | 2016-03-23 | 安徽出入境检验检疫局检验检疫技术中心 | PCR primer for detecting alternaria kikuchiana and detection method using same |
| CN105724138A (en) * | 2016-03-14 | 2016-07-06 | 阜阳市颍州区金湖丰种植农民专业合作社 | Method for reducing high-incidence season disease incidence of black spot of late-autumn yellow pears |
| CN105543407A (en) * | 2016-03-16 | 2016-05-04 | 湖北省农业科学院果树茶叶研究所 | Method for species level classification of alternaria kikuchi-aria by means of multiple marker genes |
| CN105543407B (en) * | 2016-03-16 | 2019-08-06 | 湖北省农业科学院果树茶叶研究所 | A method of kind of grade being carried out to Pear black spot bacterium using multiple marker gene and is classified |
| CN106636341A (en) * | 2016-11-02 | 2017-05-10 | 白剑宇 | Detection primer and detection method for red date blackspot bacteria |
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