CN105420372A - PCR primer for detecting alternaria kikuchiana and detection method using same - Google Patents
PCR primer for detecting alternaria kikuchiana and detection method using same Download PDFInfo
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Abstract
The invention discloses an alternaria kikuchiana molecular detection method and a primer thereof. The primer has specificity, a 254 bp specificity band can be specifically obtained from the genomic DNA of the alternaria kikuchiana through PCR amplification, but the band cannot be obtained from the genomic DNA of fungi of other categories through amplification, and thereby the rapid, efficient, stable and reliable alternaria kikuchiana molecular detection method is established by utilizing the specificity primer. The method has the advantages of being accurate, rapid, simple, easy to operate and the like, and pathogens can be identified at the initial stage of disease attack. The PCR primer for detecting the alternaria kikuchiana and the detection method using the same can be used for field investigation and plant product detection, great significance for controlling large-area burst and transregional spreading of pear black spot disease is achieved, and meanwhile, through system establishment of the primer and the detection method, technical guidance and theoretical support are also provided for detection of other types of pathogens.
Description
Technical field
The invention belongs to Molecular Detection field, be specifically related to detect the PCR primer of Pear black spot bacterium and use the detection method of this primer.
Background technology
Pear black spot has another name called pears cracked fruit, is one of international three large diseases of pear tree, and the Japan in Asia, Korea S and China occur very serious.At present, the rod method be separated to from pear fruit reported in the world has 9 groups, and named have 9 kinds.That has reported in China has 5 kinds, is pears blackspot rod method A.gaisenK.N.agano, rod method A.alternata (Fr.) Keissler respectively, Alternaria tenuissima A.tenuissima (Fr.) Wiltshire, pear infect rod method A.yaliinficiensR.G.Roberts and A.ventricosaR.G.Robert.Because this disease may be caused by the germ of multiple Alternaria, there is larger difference in the qualification of this disease pathogen bacterium, conventional disease screening technology is difficult to quick and precisely identify this 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 and a kind ofly detects the PCR primer of Pear black spot bacterium and use the detection method of this primer, by the Auele Specific Primer of design Pear black spot bacterium, utilize PCR method by the DNA cloning of measuring samples, detect this Pear black spot bacterium Auele Specific Primer and whether can amplify specific band from sample, whether can detect measuring samples rapidly and accurately containing Pear black spot bacterium, the method speed is fast, high specificity, highly sensitive, reliable and stable.
Technical scheme provided by the invention is:
1. design a kind of primer for detecting Pear black spot bacterium
The Alta1 gene order that the present invention belongs to according to Alternaria known in Genbank, devises a pair Auele Specific Primer, and its sequence is,
LiA-F:CCGCATCCTGCCCAGTTA,
LiA-R:GGTAACTTACTCGTCGCTA。
2. utilize above-mentioned primer to detect the method for Pear black spot bacterium
(1) testing sample DNA is extracted;
(2) described primer is utilized to carry out PCR reaction, the amplification system of reaction is: 10 × Taqbuffer5 μ L, 25mmol/LMgCl24 μ L, 2.5mmol/LdNTP2 μ L, 10 μm of each 2 μ L of the forward and reverse primer of ol/L, 5U/ μ LTaq enzyme 0.5 μ L, 50ng/ μ L (or proper concn) DNA profiling 1 μ L, sterilizing distilled water complements to cumulative volume 50 μ L.
(3) increase by PCR reaction conditions;
(4) get PCR react amplified production carry out electrophoresis detection, if there is the DNA band that size is 254bp, then prove institute detect in sample contain Pear black spot bacterium.
Preferably, PCR reaction conditions is: 94 DEG C of 5min; 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 has following beneficial effect:
The present invention, by design a pair Auele Specific Primer, in conjunction with the method for pcr amplification, can detect the Pear black spot bacterium in plant tissue effectively.The inventive method has following technical superiority:
(1) high specificity.Auele Specific Primer of the present invention obtains according to causing the Alternaria of Pear black spot to belong to the design of fungi Alta1 gene order, utilize this primer can obtain the band of a 254bp from causing the Alternaria of Pear black spot genus bacterial strain to increase specifically, other belong to other kinds that bacterial strains and Alternaria belong to and all occur without amplified band, show this to primer have genus plant between specificity.
(2) highly sensitive.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.Primer of the present invention and PCR method can detect the pathogenic bacteria gene group DNA of 10pg.
(3) applied range.Auele Specific Primer described in the present invention can identify pathogen at the disease infestation initial stage, can be widely used in the inspection of field investigation and plant prod, significant to the big area outburst and trans-regional propagation controlling Pear black spot.
(4) foundation of system of the present invention is also for the detection of other pathogens provides technical director and theories integration.
Accompanying drawing explanation
Fig. 1 primer specificity among genus is verified.Marker:DL2000; 1:A.gaisen; 2:A.alternata; 3:A.tenuissima; 4:A.ventricosa; 5:A.yaliinficiens; 6 ~ 23: other belong to bacterial strain (see table 1); 24:H
2o.
Specificity verification between Fig. 2 primer kind.Marker:DL2000; 1:A.gaisen; 2:A.alternata; 3:A.tenuissima; 4:A.ventricosa; 5:A.yaliinficiens; 6 ~ 37:Alternaria belongs to other kind of bacterial strain (see table 2); 38:H
2o.
Fig. 3 primer sensitivity technique.Marker:DL2000; 1-9: Pear black spot bacterium genomic dna, template content (25 μ L reaction system) is respectively 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg; 10:H
2o.
Fig. 4 PCR of alternaria in pear fruit that falls ill detects.Marker:DL2000; 1-2: morbidity pear fruit slightly extracts DNA; 3-6: healthy pear fruit slightly extracts DNA; 7: be separated germ and extract DNA; 8:H
2o.
Embodiment
Illustrate the present invention further below by detailed description embodiment, but be not limitation of the present invention, only do example explanation.
Embodiment 1: design, synthetic primer set up the PCR reaction system of Pear black spot bacterium
One, design of primers and synthesis
By belonging to the investigation of the Alta1 gene order of 38 kinds of bacterium to Alternaria, the gene order of investigation is carried out sequential analysis comparison, have chosen the fragment of several pears blackspot pathogenic bacterium disease-specific according to comparison result, devise a pair Auele Specific Primer, sequence is as follows:
LiA-F:CCGCATCCTGCCCAGTTA,
LiA-R:GGTAACTTACTCGTCGCTA。
After design of primers completes, again in GeneBank, carry out its specificity of BLAST analysis verification.Verify the raw work combining unit synthesis in rear trust Shanghai.
Two, the PCR reaction system of Pear black spot bacterium is set up
PCR amplification system is: 10 × Taqbuffer5 μ L, 25mmol/LMgCl
24 μ L, 2.5mmol/LdNTP2 μ L, the 10 μm of forward and reverse primer of ol/L each 2 μ L, 5U/ μ LTaq enzyme 0.5 μ L, 50ng/ μ L (or proper concn) DNA profiling 1 μ L, sterilizing distilled water complements to cumulative volume 50 μ L.
PCR amplification instrument carries out amplified reaction, and reaction conditions is: 94 DEG C of 5min; 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.
Three, interpretation of result
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 254bp, 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 will be gone to for examination germ, potato 200g, glucose 20g, add water and be settled to 1L) on flat board, cut 10 pieces of 2cm × 2cm bacterium colony blocks from colony edge after 28 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 is carried out, concrete operations are as follows: take a morsel hypha powder in 2mL centrifuge tube, add 900 μ L2%CTAB extracting solutions and 90 μ L10%SDS, vortex mixes, 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 centrifuge tube, adds equal-volume chloroform, turned upside down mixing gently, the centrifugal 5min of 12000rpm under normal temperature condition; Supernatant is transferred in new centrifuge tube, adds 1 times to the Virahol of supernatant volume ,-20 DEG C of condition precipitation 15min; Under normal temperature condition, the centrifugal 5min of 12000rpm, abandons 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), 37 DEG C of incubation 30min rear electrophoresis detect, and-20 DEG C save backup.
2. the thick extracting method 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 pear fruit.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 L0.5MNaOH, be transferred in 1.5mL centrifuge tube after fully grinding in mortar, the centrifugal 5min of 12000g, get 5 μ L supernatant liquors and add 495 μ L0.1mMTris (pH8.0), get 1 μ L after mixing and be directly used in PCR reaction.
Embodiment 3: primer specificity and sensitivity technique
One, specific detection
Bacterial strain uses therefor of the present invention and relevant information are in table 1 and table 2.Adopt the Auele Specific Primer designed by the present invention, according to the PCR reaction conditions of Pear black spot bacterium, strains tested genomic dnas all in his-and-hers watches 1 carries out pcr amplification, amplify the band of a treaty 254bp 5 kinds of pears blackspot pathogenic strainss that only can belong to from Alternaria specifically, and other strains testeds and blank are all without amplified band.Show that this primer has genus specificity, Alternaria genus can be belonged to other and distinguishing.
For verifying the bacterial strain of primer specificity in table 1 the present embodiment
| Sequence number | Bacterial strain | Number of strains | Use LiA-F/LiA-R amplification |
| 1 | A.gaisen | 1 | + |
| 2 | A.alternata | 1 | + |
| 3 | A.tenuissima | 1 | + |
| 4 | A.ventricosa | 1 | + |
| 5 | A.yaliinficiens | 1 | + |
| 6 | Colletotrichum gloeosporioides | 1 | - |
| 7 | Botryosphaeria dothidea | 1 | - |
| 8 | Phomopsis fukushii | 1 | - |
| 9 | Gymnosporangium haraeanum | 1 | - |
| 10 | Botrytis cinerea | 1 | - |
| 11 | Fusarium oxysporum | 1 | - |
| 12 | Pestalotiopsis theae | 1 | - |
| 13 | Pyricularia grisea | 1 | - |
| 14 | Ustilaginoidea virens | 1 | - |
| 15 | Rhizoctonia solani | 1 | - |
| 16 | Fusarium moniliforme | 1 | - |
| 17 | Aspergillus flavus | 1 | - |
| 18 | Ascochyta eriobotryae | 1 | - |
| 19 | Coniothyrium diplodiella | 1 | - |
| 20 | Podosphaera leucotricha | 1 | - |
| 21 | Glomerella acutata | 1 | - |
| 22 | Coniella granati | 1 | - |
| 23 | Sclerotinia sclerotiorum | 1 | - |
In upper table+and representing that LiA-F and LiA-R primer amplification obtains specific band, size is 254bp;-expression does not obtain amplified production.
Adopt the Auele Specific Primer designed by the present invention, according to the PCR reaction conditions of Pear black spot bacterium, strains tested genomic dnas all in his-and-hers watches 2 carries out pcr amplification, amplify the band of a 254bp 5 kinds of pears blackspot pathogenic strainss that only can belong to from Alternaria specifically, and other strains testeds and blank are all without amplified band.Show that this primer has genus species specificity, other bacterial strains that the pathogenic strains of pears blackspot and Alternaria belong to can be distinguished.
For verifying that the Alternaria of primer specificity belongs to bacterial strain in table 2 the present embodiment
| Sequence number | Bacterial strain | Number of strains | Use LiA-F/LiA-R amplification |
| 1 | A.gaisen | 1 | + |
| 2 | A.alternata | 1 | + |
| 3 | A.tenuissima | 1 | + |
| 4 | A.ventricosa | 1 | + |
| 5 | A.yaliinficiens | 1 | + |
| 6 | A.brassicae | 1 | - |
| 7 | A.brassicicola | 1 | - |
| 8 | A.capsici | 1 | - |
| 9 | A.carontiincultae | 1 | - |
| 10 | A.cetera | 1 | - |
| 11 | A.cheiranthi | 1 | - |
| 12 | A.cinerariae | 1 | - |
| 13 | A.conjuncta | 1 | - |
| 14 | A.crassa | 1 | - |
| 15 | A.cucumerina | 1 | - |
| 16 | A.dauci | 1 | - |
| 17 | A.dumosa | 1 | - |
| 18 | A.eryngii | 1 | - |
| 19 | A.ethzedia | 1 | - |
| 20 | A.euphorbiicola | 1 | - |
| 21 | A.japonica | 1 | - |
| 22 | A.limoniasperae | 1 | - |
| 23 | A.longipes | 1 | - |
| 24 | A.macrospora | 1 | - |
| 25 | A.metachromatica | 1 | - |
| 26 | A.mimiculata | 1 | - |
| 27 | A.mouchaccae | 1 | - |
| 28 | A.oregonensis | 1 | - |
| 29 | A.petroselini | 1 | - |
| 30 | A.photistica | 1 | - |
| 31 | A.porri | 1 | - |
| 32 | A.pseudorostrata | 1 | - |
| 33 | A.radicina | 1 | - |
| 34 | A.solani | 1 | - |
| 35 | A.sonchi | 1 | - |
| 36 | A.smyrniii | 1 | - |
| 37 | A.tagetica | 1 | - |
Two, sensitivity technique
Carry out sensitivity technique according to the PCR reaction system of Pear black spot bacterium and reaction conditions to Pear black spot bacterium genomic dna with LiA-F/R primer, in 25 μ L reaction systems, the content of template DNA is 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg.Result shows, and under described reaction system and reaction conditions, the amplification from least 10pg germ genomic dna of primer LiA-F/R Absorbable organic halogens ground obtains the specific band of 254bp.
Embodiment 4: the pathogen in Diseased Plant Tissues detects
According to the thick extracting method of plant tissue DNA of falling ill described in embodiment 2, from the pear fruit of this pathogenic bacteria of artificial inoculation, picking incidence tissue extracts DNA, pcr amplification is carried out by the PCR reaction system of Pear black spot bacterium and reaction conditions with LiA-F/R primer, morbidity fruit tissue all amplifies the specific band of 254bp, and healthy fruit tissue can not increase band, illustrate that the rapid molecular that this detection method may be used for Pear black spot bacterium in Diseased Plant Tissues detects.
Claims (3)
1. detect a PCR primer for Pear black spot bacterium, comprise upstream primer sequence LiA-F, downstream primer sequence LiA-R is as follows:
LiA-F:5’-CCGCATCCTGCCCAGTTA-3’,
LiA-R:5’-GGTAACTTACTCGTCGCTA-3’
The size of described primer is 254bp.
2. the PCR primer of detection Pear black spot bacterium according to claim 1 detects the purposes in Pear black spot bacterium at the nucleic acid samples obtained from organism by pcr amplification.
3. use the PCR primer described in claim 1 to detect a method for Pear black spot bacterium, it is characterized in that, comprise the following steps:
Step 1, extraction testing sample DNA, prepare DNA profiling;
Step 2, prepare primer: upstream primer sequence LiA-F, downstream primer sequence LiA-R is as follows:
LiA-F:5’-CCGCATCCTGCCCAGTTA-3’,
LiA-R:5’-GGTAACTTACTCGTCGCTA-3’；
Primer prepared by step 3, DNA profiling step 1 prepared and step 2 carries out pcr amplification, and amplification system is: 10 × Taqbuffer5 μ L, 25mmol/LMgCl
24 μ L, 2.5mmol/LdNTP2 μ L, the 10 μm of forward and reverse primer of ol/L each 2 μ L, 5U/ μ LTaq enzyme 0.5 μ L, 50ng/ μ LDNA template 1 μ L, sterilizing distilled water complements to cumulative volume 50 μ L;
Increase under the reaction conditions of pcr amplification;
Step 4, get pcr amplification product and carry out electrophoresis detection, the DNA band that molecular weight is about 254bp if exist, then prove that institute is detected containing Pear black spot bacterium in sample, otherwise namely proof detect in sample and do not contain Pear black spot bacterium.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111926105A (en) * | 2020-09-09 | 2020-11-13 | 江苏省农业科学院 | Primer and method for LAMP detection of alternaria brassicae |
| CN112125963A (en) * | 2019-06-24 | 2020-12-25 | 北京市农林科学院 | Phaseolus cactus LtALTA1 gene and application thereof |
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| CN105039535A (en) * | 2015-09-09 | 2015-11-11 | 安徽省农业科学院植物保护与农产品质量安全研究所 | Primers for detecting alternaria alternata and alternaria alternata detection method |
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2015
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2012120535A (en) * | 1999-05-28 | 2012-06-28 | Innogenetics Nv | Nucleic acid probe and method for detecting clinically important fungal pathogen |
| CN105039535A (en) * | 2015-09-09 | 2015-11-11 | 安徽省农业科学院植物保护与农产品质量安全研究所 | Primers for detecting alternaria alternata and alternaria alternata detection method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112125963A (en) * | 2019-06-24 | 2020-12-25 | 北京市农林科学院 | Phaseolus cactus LtALTA1 gene and application thereof |
| CN112125963B (en) * | 2019-06-24 | 2022-03-15 | 北京市农林科学院 | Phaseolus cactus LtALTA1 gene and application thereof |
| CN111926105A (en) * | 2020-09-09 | 2020-11-13 | 江苏省农业科学院 | Primer and method for LAMP detection of alternaria brassicae |
| CN111926105B (en) * | 2020-09-09 | 2022-06-24 | 江苏省农业科学院 | Primer and method for LAMP detection of alternaria brassicae |
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