CN106834488A - Tomato neckrot pine root fungus PCR detects primer special and its detection kit - Google Patents
Tomato neckrot pine root fungus PCR detects primer special and its detection kit Download PDFInfo
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- CN106834488A CN106834488A CN201710118643.XA CN201710118643A CN106834488A CN 106834488 A CN106834488 A CN 106834488A CN 201710118643 A CN201710118643 A CN 201710118643A CN 106834488 A CN106834488 A CN 106834488A
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Abstract
The invention discloses a kind of tomato neckrot pine root fungus PCR detection primer special and its detection kit, primer includes sense primer and anti-sense primer, and upstream primer sequence is shown in SEQ ID NO:Shown in 1, the sequence of anti-sense primer is shown in SEQ ID NO:Shown in 2.Quick, the precise Identification of present invention tomato neckrot pine root fungus suitable for plant tissue and pedotheque, have important practical value for the microbial disease control of tomato neckrot root rot in agricultural production.
Description
Technical field
The present invention relates to technical field of molecular biology, more particularly to a kind of tomato neckrot pine root fungus PCR detections are special
Primer and its detection kit.
Background technology
Tomato (Solanum esculentum L.) is one of important vegetable crop of China, nutritious, can be with the anniversary
Plantation, and yield more and more higher, variety design are more and more, widely used, deep to be liked by grower and consumer.According to 2014
Year Food and Agricultural Organization of the United Nations (FAO) counts, and tomato in China cultivated area and yield occupy first place in the world, and cultivated area is about
More than 1500 ten thousand mu, about 52,600,000 tons of yield.But tomato is long-term, in the production process of large area, its disease also develops therewith,
Harm is aggravated, especially the large area of tomato neckrot root rot (Fusarium crown and root rot, FCRR) in recent years
Break out, and the trend for making further progress.Tomato neckrot root rot is by Fusarium oxysporum (Fusarium oxysporum
F.sp.radicis-lycopersici, FORL) one of the most destructive tomato soil borne disease that causes (Roberts et
al.,2000).The disease generally occurs all over the world, first in 1974 Japan find, afterwards the U.S., Canada,
Many countries such as Mexico, Israel, South Korea, China are found (Scott and Jones, 2000 successively；Scott, 2005),
The disease causes great threat to the tomato production of majority state.In recent years, tomato neckrot root rot was in China Shandong, northeast, river
The tomato main producing region such as north, Beijing, Jiangsu, Zhejiang occurs on a large scale, mistaken diagnosis or preventing and treating not in time, the tomato incidence of disease up to 80% with
On, fatal rate causes Severe Reduction up to more than 30%, and serious economic loss (Cheng Lin etc., 2016) is caused to Planting household, because
This, strengthens the preventing and treating of tomato neckrot pine root fungus for ensureing that it is important that tomato stable and high yields plays the role of.Do not have in production at present
There are comparatively ideal prevention and controls, therefore, it is badly in need of carrying out it monitoring and warning research in production, tomato neckrot is in depth understood comprehensively
Pine root fungus cause a disease implementations, set up efficiently, fast and accurately Pathogen detection technology, these can be to formulating correct disease-resistant educate
Planting strategy and rational deployment kind all has extremely important reference value.
The content of the invention
It is an object of the invention to provide a kind of tomato neckrot pine root fungus PCR detection primer specials, high specificity, detection
Accuracy is high.
Present invention also offers the kit comprising above-mentioned primer, it is adaptable to tomato neckrot in plant tissue and pedotheque
Quick, the precise Identification of pine root fungus, have important for the microbial disease control of tomato neckrot root rot in agricultural production
Practical value.
The technical solution adopted for the present invention to solve the technical problems is：
A kind of tomato neckrot pine root fungus PCR detects primer special, including sense primer and anti-sense primer, sense primer and
The sequence of anti-sense primer is as follows：
Sense primer：5’-CCTTACTCGACATTTTTCAGA-3’(SEQ ID NO:Shown in 1)；
Anti-sense primer：5’-TGCCTGATGTCATTAGTA-3’(SEQ ID NO:Shown in 2).
A kind of tomato neckrot pine root fungus PCR detection kit, the kit includes box body and 7 PCR pipes, in 5 PCR
DNTP, MgCl are respectively provided with pipe2, PCR buffer solutions, Taq archaeal dna polymerases and ddH2O；It is respectively provided with other 2 PCR pipes
Detect sense primer, the anti-sense primer of detection tomato neckrot pine root fungus of tomato neckrot pine root fungus.
The invention provides for the detection primer and kit to tomato neckrot pine root fungus, by extracting testing sample
DNA, and combine PCR detection techniques, can reach identification testing sample in tomato neckrot pine root fungus purpose.
Preferably, the upstream primer sequence of detection tomato neckrot pine root fungus is：5’-
CCTTACTCGACATTTTTCAGA-3’。
Preferably, the downstream primer sequence of detection tomato neckrot pine root fungus is：5’-TGCCTGATGTCATTAGTA-
3’。
Preferably, as follows using the composition that the kit configures 10 μ L PCR amplification systems：
DNA profiling 20ng, detects 0.2 μM of the sense primer of tomato neckrot pine root fungus, detects tomato neckrot pine root fungus
0.2 μM of anti-sense primer, dNTP 0.25mM, MgCl22.0mM, 1 × PCR buffer solution, Taq archaeal dna polymerase 1U, plus ddH2O is mended
Enough to 10 μ L.
The beneficial effects of the invention are as follows：The present invention can quickly and accurately identify tomato neckrot pine root fungus, for agricultural
The microbial disease control of tomato neckrot root rot has important practical value in production.
Brief description of the drawings
Fig. 1 is the gel electrophoresis figure after primer specificity detection sample DNA amplification of the present invention.
Fig. 2 is the gel electrophoresis figure after isolated strains DNA cloning in Field diseased plant of the present invention.
Specific 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 for being used etc. is commercially available or commonly used in the art.
Method in following embodiments, unless otherwise instructed, is the conventional method of this area.
The primer of the present invention is synthesized by Dalian treasured biotech company, and the work of all sequences measure is by upper marine growth skill
Art Co., Ltd completes.
Embodiment
1st, the design of the primer for being detected to tomato neckrot pine root fungus
(GenBank is numbered conserved sequence according to polygalacturonase excision enzyme (pgx4) in GenBank
AB256798.1、AB256796.1、AB256797.1、AB256795.1、GU169176.1、AB256825.1)。
Design degenerate primer FL-F (5 '-GATGGTGGAACGGTATGACYC-3 ', SEQ ID NO:Shown in 3) and FL-R
(5 '-GATATCCTTRACACCATCACA-3 ', SEQ ID NO:Shown in 4), amplification length is 960bp, its fragment is carried out gram
Grand, sequencing and analysis, and design specific primer in 960bp interior sequences.The specific primer of tomato neckrot pine root fungus
Sequence is as follows：
Sense primer：5’-CCTTACTCGACATTTTTCAGA-3’(SEQ ID NO：1)；
Anti-sense primer：5’-TGCCTGATGTCATTAGTA-3’(SEQ ID NO：2)；
EQ ID NO：1 by 21 base compositions, SEQ ID NO：2 are by 18 base compositions, expanding fragment length
486bp。
2nd, Standard PCR detection is carried out with primer pair tomato neckrot pine root fungus of the invention
Bacterial strain described in this example：
Plant micro- provided tomato neckrot pine root fungus in academy of agricultural sciences of Zhejiang Province；
Detection method is carried out according to the following steps：
(1) extraction of mycelia DNA：About 0.1 gram of mycelia is scraped from flat board with sterilizing toothpick, is placed in 2.0mL centrifuge tubes,
After routinely CTAB methods extract DNA, preserve respectively, it is standby；
(2) PCR amplifications：After each sample DNA 20ng of step (1) extraction are separately added into each PCR pipe, then add successively
Enter to detect each 0.2 μM of the upstream and downstream primer of tomato neckrot pine root fungus, dNTP 0.25mM, MgCl22.0mM, 1 × PCR are buffered
Liquid (Dalian treasured biotech company), the unit of Taq archaeal dna polymerases 1, plus ddH2After the μ of O to 10 L, by PCR response procedures：94℃
Predegeneration 3min, 94 DEG C of 50seconds, 55 DEG C of annealing 50seconds, 72 DEG C of 60seconds, 35 circulations are expanded, and 72
DEG C extend 10min, the preservation of 4 DEG C of product；The upstream primer sequence of the detection tomato neckrot pine root fungus is SEQ ID NO：1,
Downstream primer sequence is SEQ ID NO：2.
(3) 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 electrophoresis point on 1.2% Ago-Gel
From, to bromjophenol blue reach gel 2/3 position at stop electrophoresis, gel through EB dye after observed with gel imaging system,
Photograph；
(4) detection of tomato neckrot pine root fungus：According to Gel electrophoresis results, analysis judges to be detected sample (see Fig. 1)：
Tomato neckrot pine root fungus amplify 486bp bands, are consistent with expected results, show that primer of the invention can be used for tomato neckrot
The Standard PCR detection of pine root fungus.
3rd, the bacterial strain from tomato neckrot root rot diseased plant separate with primer pair of the invention carries out Standard PCR detection
Bacterial strain described in this example：
The bacterial strain of acquisition is separated on Zhejiang tomato Zhu Zai areas tomato neckrot root rot diseased plant.
Detection method is carried out according to the following steps：
(1) extraction of mycelia DNA：About 0.1 gram of mycelia is scraped from flat board with sterilizing toothpick, is placed in 2.0mL centrifuge tubes,
After routinely CTAB methods extract DNA, preserve respectively, it is standby；
(2) PCR amplifications：After each sample DNA 20ng of step (1) extraction are separately added into each PCR pipe, then add successively
Enter to detect each 0.2 μM of the upstream and downstream primer of tomato neckrot pine root fungus, dNTP 0.25mM, MgCl22.0mM, 1 × PCR are buffered
Liquid, the unit of Taq archaeal dna polymerases 1, plus ddH2After the μ of O to 10 L, by PCR response procedures：94 DEG C of predegeneration 3min, 94 DEG C
50seconds, 55 DEG C of annealing 50seconds, 72 DEG C of 60seconds, 35 circulations are expanded, 72 DEG C of extension 10min, product
4 DEG C of preservations；The upstream primer sequence of the detection tomato neckrot pine root fungus is SEQ ID NO：1, downstream primer sequence is SEQ
ID NO：2；
(3) 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 electrophoresis point on 1.2% Ago-Gel
From, to bromjophenol blue reach gel 2/3 position at stop electrophoresis, gel through EB dye after observed with gel imaging system,
Photograph；
(4) detection of tomato neckrot pine root fungus：According to Gel electrophoresis results, analysis judges to be detected sample (see Fig. 2)：
Tomato neckrot pine root fungus amplify 486bp bands, show that primer of the invention can be used for the routine of tomato neckrot pine root fungus
PCR is detected.
A kind of tomato neckrot pine root fungus PCR detection kit, the kit includes box body and 7 PCR pipes, in 5 PCR
DNTP, MgCl are respectively provided with pipe2, PCR buffer solutions, Taq archaeal dna polymerases and ddH2O；It is respectively provided with other 2 PCR pipes
Detect sense primer (the SEQ ID NO of tomato neckrot pine root fungus：1) anti-sense primer of tomato neckrot pine root fungus, is detected
(SEQ ID NO：2).
Embodiment described above is a kind of preferably scheme of the 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>Tomato neckrot pine root fungus PCR detects primer special and its detection kit
<130> 2017.02
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
ccttactcga catttttcag a 21
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> 2
tgcctgatgt cattagta 18
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence
<400> 3
gatggtggaa cggtatgacy c 21
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence
<400> 4
gatatccttr acaccatcac a 21
Claims (5)
1. a kind of tomato neckrot pine root fungus PCR detects primer special, including sense primer and anti-sense primer, it is characterised in that
The sequence of sense primer and anti-sense primer is as follows：
Sense primer：5’-CCTTACTCGACATTTTTCAGA-3’；
Anti-sense primer：5’-TGCCTGATGTCATTAGTA-3’.
2. a kind of tomato neckrot pine root fungus PCR detection kit, it is characterised in that the kit includes box body and 7 PCR
Pipe, is respectively provided with dNTP, MgCl in 5 PCR pipes2, PCR buffer solutions, Taq archaeal dna polymerases and ddH2O；In other 2 PCR
Sense primer, the anti-sense primer of detection tomato neckrot pine root fungus of detection tomato neckrot pine root fungus are respectively provided with pipe.
3. tomato neckrot pine root fungus PCR detection kit according to claim 2, it is characterised in that detection tomato neck
The upstream primer sequence of rotten pine root fungus is：5’-CCTTACTCGACATTTTTCAGA-3’.
4. tomato neckrot pine root fungus PCR detection kit according to claim 2, it is characterised in that detection tomato neck
The downstream primer sequence of rotten pine root fungus is：5’-TGCCTGATGTCATTAGTA-3’.
5. the tomato neckrot pine root fungus PCR detection kit according to Claims 2 or 3 or 4, it is characterised in that use
The composition that the kit configures 10 μ L PCR amplification systems is as follows：
DNA profiling 20ng, detects 0.2 μM of the sense primer of tomato neckrot pine root fungus, under detection tomato neckrot pine root fungus
0.2 μM of primer of trip, dNTP 0.25mM, MgCl22.0mM, 1 × PCR buffer solution, Taq archaeal dna polymerase 1U, plus ddH2O is complemented to
10μL。
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|---|---|---|---|
| CN201710118643.XA CN106834488A (en) | 2017-03-01 | 2017-03-01 | Tomato neckrot pine root fungus PCR detects primer special and its detection kit |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002004651A2 (en) * | 2000-07-07 | 2002-01-17 | Clemson University | Jointless gene of tomato |
| US20090313716A1 (en) * | 2008-06-16 | 2009-12-17 | Frits Herlaar | Tomato Hybrid E33018 |
| CN101974650A (en) * | 2010-11-30 | 2011-02-16 | 中国农业科学院植物保护研究所 | Polymerase chain reaction (PCR) method for detecting fusarium oxysporum and kit |
| CN104263813A (en) * | 2013-07-18 | 2015-01-07 | 宁波市农业科学研究院 | Sequences of primer for identifying fusarium solani and/or fusarium oxysporum, kit and method thereof |
-
2017
- 2017-03-01 CN CN201710118643.XA patent/CN106834488A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002004651A2 (en) * | 2000-07-07 | 2002-01-17 | Clemson University | Jointless gene of tomato |
| US20090313716A1 (en) * | 2008-06-16 | 2009-12-17 | Frits Herlaar | Tomato Hybrid E33018 |
| CN101974650A (en) * | 2010-11-30 | 2011-02-16 | 中国农业科学院植物保护研究所 | Polymerase chain reaction (PCR) method for detecting fusarium oxysporum and kit |
| CN104263813A (en) * | 2013-07-18 | 2015-01-07 | 宁波市农业科学研究院 | Sequences of primer for identifying fusarium solani and/or fusarium oxysporum, kit and method thereof |
Non-Patent Citations (1)
| Title |
|---|
| YASUSHI HIRANO等: ""PCR-based differentiation of Fusarium oxysporum ff. sp. lycopersici and radicis-lycopersici and races of F. oxysporum f. sp. lycopersici"", 《JOURNAL OF GENERAL PLANT PATHOLOGY》 * |
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Application publication date: 20170613 |
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