CN106978482A - Primer and kit for detecting Tomato gray leaf spot opportunistic pathogen - Google Patents
Primer and kit for detecting Tomato gray leaf spot opportunistic pathogen Download PDFInfo
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- CN106978482A CN106978482A CN201710136558.6A CN201710136558A CN106978482A CN 106978482 A CN106978482 A CN 106978482A CN 201710136558 A CN201710136558 A CN 201710136558A CN 106978482 A CN106978482 A CN 106978482A
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- 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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Abstract
The invention discloses a kind of primer and kit for being used to detect Tomato gray leaf spot opportunistic pathogen, primer includes sense primer, and (sequence is shown in SEQ ID NO：1) (sequence is shown in SEQ ID NO with anti-sense primer：2), the present invention can quickly and accurately identify Tomato gray leaf spot opportunistic pathogen, and the preventing and treating for doing harm to early detection and disease to Tomato gray leaf spot is significant.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of primer and examination for being used to detect Tomato gray leaf spot opportunistic pathogen
Agent box.
Background technology
Tomato (Solanum esculentum L.) is one of topmost vegetables of China, deep by grower and consumer
Like, cultivated area constantly expands, can be planted with the anniversary.The factor for restricting tomato production removes the more difficult prediction of market conditions
Outside, it is topmost be exactly disease pest it is many, endanger it is heavier.Wherein, Tomato gray leaf spot (Tomato gray leaf spot
Disease, GLSD) it is worldwide disease.In recent years, Tomato gray leaf spot shows the even more serious trend of outbreak of epidemic, by mistake
Examine or prevent and treat not in time, often result in the tomato underproduction, the general underproduction 20% or so, the even underproduction more than 80% having, to planting household
Cause serious economic loss (Cao Shoujun etc., 2011；Liu Guohua, 2012).
Tomato gray leaf spot caused by Deuteromycotina Stemphylium (Stemphylium) fungi (Al-Mughrabi,
2003), mainly by seed dispersal, germ can be survived the winter on the invalid body in protecting field soil and seed, and next year humiture is fitted
Conidium is produced when preferably, First aggression is carried out, spore is propagated by wind and rain, then is infected.Tomato gray leaf spot has to tomato
Fatal harm, because even under normal temperature and damp condition, causing the pathogen of this disease to propagate quickly, it is weekly
Two spore circulations can be completed.The main blade of causing harm of the disease, early stage produces the grey brown of circular or irregular shape on blade
Color spot point, then scab gradually extended in irregular shape along vein, slow-growing is caused to tomato, stem, fruit and young leaves, plant
Strain is wilted, and the lighter's yield, quality reduction, severe one cause have no harvest (Chen Suiju, 2011；Du Baihai and Zhang Jianying, 2013).
Tomato gray leaf spot worldwide has a generation, warm moist area occur it is serious, be mainly distributed on the U.S.,
Italy, Australia, Israel, Canada, Britain, Mauritius, Senegal, Zambia, Angola, Nigeria,
The ground such as Seychelles, Thailand, Japan, Brazil, India, Venezuela, Libya, the Sudan, Florida, Malaysia
(Atherton＆Rudich, 1989；Rotem et al.,1966).In recent years, the disease is in TaiWan, China, Beijing, Liaoning, river
There is generation on the ground such as north, Chongqing, Guizhou, Hainan, Shandong, Wenzhou District of Zhejiang Province, Jiashan, Yuhang, Haining, Hangzhou, have a strong impact on tomato
Production, and prolong and aggravate progressively expanding.Therefore, the preventing and treating of Tomato gray leaf spot bacterium is strengthened for ensureing that tomato stable and high yields has
Important effect.Set up efficiently, fast and accurately Pathogen detection technology, these can be to formulating correct resistant breeding strategy
All there is extremely important reference value with rational deployment kind.
The content of the invention
It is an object of the invention to provide a kind of primer for being used to detect Tomato gray leaf spot opportunistic pathogen, high specificity, detection
Accuracy is high, has important practical value for disease control caused by Tomato gray leaf spot opportunistic pathogen in agricultural production.
Present invention also offers a kind of kit for including above-mentioned primer.
The technical solution adopted for the present invention to solve the technical problems is：
A kind of primer for being used to detect Tomato gray leaf spot opportunistic pathogen, including sense primer and anti-sense primer, particular sequence is such as
Under：
Sense primer：5’-CTGAACCCACCGCTTTTGCCATGTC-3’；
Anti-sense primer：5’-CGAACATGGGAGCGTCGGCGGAA-3’.
Above-mentioned primer specificity is strong, and detection accuracy is high, it is adaptable to which Tomato gray leaf spot opportunistic pathogen PCR is detected.Drawn with above-mentioned
Thing enters performing PCR amplification to testing sample, and amplified production has the band of 489bp sizes, then former containing Tomato gray leaf spot in sample
Bacterium.
A kind of kit for being used to detect Tomato gray leaf spot opportunistic pathogen, the kit includes box body and 4 PCR pipes, at 2
2 × Taq PCR MasterMix and ddH are respectively provided with PCR pipe2O；It is respectively provided with other 2 PCR pipes for detection kind
The sense primer of eggplant gray leaf spot opportunistic pathogen and the anti-sense primer for detecting Tomato gray leaf spot opportunistic pathogen.
Preferably, for detecting that the upstream primer sequence of Tomato gray leaf spot opportunistic pathogen is：5’-
CTGAACCCACCGCTTTTGCCATGTC-3’；For detecting that the downstream primer sequence of Tomato gray leaf spot opportunistic pathogen is：5’-
CGAACATGGGAGCGTCGGCGGAA-3’。
The beneficial effects of the invention are as follows：The quick detection of Tomato gray leaf spot opportunistic pathogen suitable for plant tissue, primer is special
Different in nature strong, detection accuracy is high, has important reality for disease control caused by Tomato gray leaf spot opportunistic pathogen in agricultural production
With value.
Brief description of the drawings
Fig. 1 is that primer specificity of the present invention detects the gel electrophoresis figure after sample DNA amplification.
Fig. 2 is the gel electrophoresis figure after isolated strains DNA cloning in Field diseased plant of the present invention.
Embodiment
Below by specific embodiment, technical scheme is described in further detail.
In the present invention, if not refering in particular to, raw material and equipment used etc. is commercially available or commonly used in the art.
Method in following embodiments, is the conventional method of this area unless otherwise instructed.
The primer of the present invention is synthesized by Shanghai Bioisystech Co., Ltd, and the work of all sequences measure is by upper marine growth skill
Art Co., Ltd completes.
Embodiment 1：
According to glyceraldehyde 3-phosphate dehydrogenase (glyceraldehyde-3-phosphate in GenBank
Dehydrogenase, gpd) conserved sequence (GenBank numberings KF921502.1, KU599919.1, KC796690.1,
AY278821.1, DQ000654.1, AF081398.1, AF443883.1, AF443886.1, KF479194.1), design primer
Stem-F and Stem-R, amplification length is 489bp, and its fragment is cloned, is sequenced and analyzed.Tomato gray leaf spot opportunistic pathogen is special
The primer sequence of the opposite sex is as follows：
Sense primer (Stem-F)：5 '-CTGAACCCACCGCTTTTGCCATGTC-3 ' (SEQ ID NO in sequence table：
1)；
Anti-sense primer (Stem-R)：5 '-CGAACATGGGAGCGTCGGCGGAA-3 ' (SEQ ID NO in sequence table：2).
SEQ ID NO in sequence table：1 by 25 base compositions, SEQ ID NO：2 by 23 base compositions, expand piece
Segment length is 489bp.
Embodiment 2：
A kind of kit for being used to detect Tomato gray leaf spot opportunistic pathogen, the kit includes box body and 4 PCR pipes, at 2
2 × Taq PCR MasterMix and ddH are respectively provided with PCR pipe2O；It is respectively provided with other 2 PCR pipes for detection kind
The sense primer of eggplant gray leaf spot opportunistic pathogen and the anti-sense primer for detecting Tomato gray leaf spot opportunistic pathogen.
Standard PCR detection is carried out with the primer pair Tomato gray leaf spot opportunistic pathogen of the present invention
Bacterial strain described in this example：Micro- provided Tomato gray leaf spot opportunistic pathogen is planted by academy of agricultural sciences of Zhejiang Province.
Detection method is carried out according to the following steps：
1st, mycelia DNA extraction：About 0.1 gram of mycelia is scraped from flat board with sterilizing toothpick, is placed in 2.0mL centrifuge tubes,
Routinely CTAB methods are extracted after DNA, are preserved respectively, standby；
2nd, PCR is expanded：After the μ L of each sample DNA 1 that step 1 extraction is separately added into each PCR pipe, 2 are sequentially added ×
The μ L of Taq PCR MasterMix 5 (commercially available, Tiangeng is biochemical), each 0.25 μ L of upstream and downstream primer (0.25 μM of final concentration), plus ddH2O
To 10 μ L, by PCR response procedures：94 DEG C of pre-degeneration 3min, 94 DEG C of 50seconds, 58 DEG C of annealing 45seconds, 72 DEG C
50seconds, 35 circulations are expanded, 72 DEG C of extension 10min, 4 DEG C of preservations of product；Upstream primer sequence is SEQ ID NO：
1, downstream primer sequence is SEQ ID NO：2.
3rd, the gel electrophoresis analysis of pcr amplification product：Each sample takes the μ L of amplified production 10 and by 0.25% bromjophenol blue respectively
Plus 40% μ L of sample-loading buffer 2 for being formulated of sucrose, mix, mixture is carried out into electrophoresis on 1.5% Ago-Gel divides
From, reached to bromjophenol blue and stop electrophoresis at 2/3 position of gel, gel observed after being dyed through EB with gel imaging system,
Photograph.
4th, the detection of Tomato gray leaf spot opportunistic pathogen：According to Gel electrophoresis results, analysis judges to be detected sample (see Fig. 1)：
Tomato gray leaf spot opportunistic pathogen amplifies 489bp bands, is consistent with expected results, shows that the primer of the present invention can be used for tomato bastard indigo
The Standard PCR detection of pinta opportunistic pathogen.
The bacterial strain separated with the primer pair of the present invention from Tomato gray leaf spot strain carries out Standard PCR detection
Bacterial strain described in this example：The bacterial strain of acquisition is separated in the strain of Zhejiang tomato Zhu Zai areas Tomato gray leaf spot.
Detection method is carried out according to the following steps：
1st, mycelia DNA extraction：About 0.1 gram of mycelia is scraped from flat board with sterilizing toothpick, is placed in 2.0mL centrifuge tubes,
Routinely CTAB methods are extracted after DNA, are preserved respectively, standby；
2nd, PCR is expanded：After the μ L of each sample DNA 1 that step (1) extraction is separately added into each PCR pipe, 2 are sequentially added
The μ L of × Taq PCR MasterMix 5, each 0.25 μ L of upstream and downstream primer, plus ddH2After O to 10 μ L, by PCR response procedures：94℃
Pre-degeneration 3min, 94 DEG C of 50seconds, 58 DEG C of annealing 45seconds, 72 DEG C of 50seconds, 35 circulations are expanded, and 72
DEG C extension 10min, the preservation of 4 DEG C of product；Upstream primer sequence is SEQ ID NO：1, downstream primer sequence is SEQ ID NO：2.
3rd, the gel electrophoresis analysis of pcr amplification product：Each sample takes the μ L of amplified production 10 and by 0.25% bromjophenol blue respectively
Plus 40% μ L of sample-loading buffer 2 for being formulated of sucrose, mix, mixture is carried out into electrophoresis on 1.5% Ago-Gel divides
From, reached to bromjophenol blue and stop electrophoresis at 2/3 position of gel, gel observed after being dyed through EB with gel imaging system,
Photograph.
4th, the detection of Tomato gray leaf spot opportunistic pathogen：According to Gel electrophoresis results, analysis judges to be detected sample (see Fig. 2)：
Tomato gray leaf spot opportunistic pathogen amplifies 489bp bands, shows that the primer of the present invention can be used for the routine of Tomato gray leaf spot opportunistic pathogen
PCR is detected.
Embodiment described above is a kind of preferably scheme of the present invention, not makees any formal to the present invention
Limitation, also has other variants and remodeling on the premise of without departing from the technical scheme described in claim.
SEQUENCE LISTING
<110>Zhejiang Academy of Agricultural Science
<120>Primer and kit for detecting Tomato gray leaf spot opportunistic pathogen
<130> 2017.03
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence
<400> 1
ctgaacccac cgcttttgcc atgtc 25
<210> 2
<211> 23
<212> DNA
<213>Artificial sequence
<400> 2
cgaacatggg agcgtcggcg gaa 23
Claims (3)
1. a kind of primer for being used to detect Tomato gray leaf spot opportunistic pathogen, it is characterised in that including sense primer and anti-sense primer, tool
Body sequence is as follows：
Sense primer：5’-CTGAACCCACCGCTTTTGCCATGTC-3’；
Anti-sense primer：5’-CGAACATGGGAGCGTCGGCGGAA-3’.
2. a kind of kit for being used to detect Tomato gray leaf spot opportunistic pathogen, it is characterised in that the kit includes box body and 4
PCR pipe, is respectively provided with 2 × Taq PCR MasterMix and ddH in 2 PCR pipes2O；Filled respectively in other 2 PCR pipes
There are the sense primer for detecting Tomato gray leaf spot opportunistic pathogen and the anti-sense primer for detecting Tomato gray leaf spot opportunistic pathogen.
3. kit according to claim 2, it is characterised in that the sense primer for detecting Tomato gray leaf spot opportunistic pathogen
Sequence is：5’-CTGAACCCACCGCTTTTGCCATGTC-3’；Downstream primer sequence for detecting Tomato gray leaf spot opportunistic pathogen
For：5’-CGAACATGGGAGCGTCGGCGGAA-3’.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710136558.6A CN106978482A (en) | 2017-03-08 | 2017-03-08 | Primer and kit for detecting Tomato gray leaf spot opportunistic pathogen |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710136558.6A CN106978482A (en) | 2017-03-08 | 2017-03-08 | Primer and kit for detecting Tomato gray leaf spot opportunistic pathogen |
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|---|---|
| CN106978482A true CN106978482A (en) | 2017-07-25 |
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| CN201710136558.6A Pending CN106978482A (en) | 2017-03-08 | 2017-03-08 | Primer and kit for detecting Tomato gray leaf spot opportunistic pathogen |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107974512A (en) * | 2017-12-12 | 2018-05-01 | 东北农业大学 | A kind of primer and method for detecting Tomato gray leaf spot bacterium |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998048276A1 (en) * | 1997-04-23 | 1998-10-29 | The Penn State Research Foundation | Non-obligate predator bacterium burkholderia casidae and uses thereof |
-
2017
- 2017-03-08 CN CN201710136558.6A patent/CN106978482A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998048276A1 (en) * | 1997-04-23 | 1998-10-29 | The Penn State Research Foundation | Non-obligate predator bacterium burkholderia casidae and uses thereof |
Non-Patent Citations (1)
| Title |
|---|
| 李戌清等: ""浙江番茄灰叶斑病病原菌"", 《菌物学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107974512A (en) * | 2017-12-12 | 2018-05-01 | 东北农业大学 | A kind of primer and method for detecting Tomato gray leaf spot bacterium |
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Application publication date: 20170725 |