CN103103255A - Primer and detection kit thereof for detecting phytophthora capsici - Google Patents
Primer and detection kit thereof for detecting phytophthora capsici Download PDFInfo
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Abstract
The invention discloses a primer and a detection kit of the primer for detecting phytophthora capsici. The primer is characterized by being a phytophthora capsici specific detection primer as follows: upstream (20bp) PcyktF: AGATGGTATGTCGCTATGAC, and downstream (20bp) PcyktR: ACATTTCGTAACGTCAACAT; a universal phytophthora primer is as follows: upstream (21bp) PyktF: AGAGTTGCTGCGCCAGGATGG, and downstream (21bp) PyktR: GTCTTGGACAACACGGCGGTG. The invention discloses a potential universal phytophthora detection primer, and tests show that the primer can amplify a stripe which is about 630bp (different species are different in size) for 14 different phytophthora species provided by the tests, so that a potential molecular target is provided for molecule detection of other phytophthora species.
Description
Technical field
The present invention relates generally to a kind of general molecular primer for detection of phytophthora, detects specific molecular primer and the detection kit thereof of Phytophthora capsici, belongs to biological technical field.
Background technology
Phytophthora capsici can cause the various crop morbidities such as capsicum, pumpkin, summer squash, is commonly called as " dead seedling is sick ", often causes the dead seedling of crop, the decayed fruits such as capsicum, general dead plant rate is 15%-30%, up to more than 60%, even ruin kind when serious, have a strong impact on the yield and quality of the crops such as capsicum.This disease found in the U.S. the earliest in 1918, Leonian in nineteen twenty-two separate and name into
Phytophthora capsiciLeon.China the beginning of the sixties first appeared in Xinjiang, occur the seventies once in a while, occured since the mid-80 year after year, and come through province's rapid spreads such as Gansu, Qinghai by Xinjiang very soon, the harm that the national most areas such as Shaanxi, Jiangxi, Shanghai, Hunan, Anhui should disease now is all very serious, particularly in recent years, adjustment along with China's agricultural structure, industrialized agriculture develops in a large number, and the pepper planting area constantly enlarges, and this disease has the trend that increases the weight of year by year in China.The detection method that the situation demands of this sternness is set up a cover rapid sensitive as early as possible is used for the early diagnosis of capsicum epidemic disease, for the control of capsicum epidemic disease provides foundation.
The method of traditional detection Plant diseases is the disease symptom by the direct viewing plant tissue, separates obtaining carrying out Morphological Identification after pathogen again.Yet direct viewing can be omitted the disease of incubation period or hidden disease, so that the control of incuring loss through delay disease, causes the outburst of disease.The microbial disease of Phytophthora capsici is easy to obscure mutually with pythium spp and the microbial disease symptom of reaping hook.Therefore for the Phytophthora capsici diagnosis, it is necessary separating the pure culture that obtains pathogenic bacteria.And the evaluation work that obtains after the pure culture of phytophthora blight of pepper is still difficult, and between Phytophthora capsici and the mould kind of other epidemic diseases, morphological differences is less, employing morphology is that traditional authentication method of classification foundation is difficult to phytophthora is identified accurately, also is difficult to carry out in time fast the diagnosis of disease.Waste time and energy, and require the operator to possess abundant phytophthora separation, Morphological Identification knowledge, the plant protection work person of basic unit is had certain difficulty.Therefore, develop rapid sensitive, easy to operate, easily universal detection method is of great significance by the microbial disease tool of Phytophthora capsici for controlling.
Since polymerase chain reaction (PCR) invention, just the characteristic with its rapid sensitive becomes the important method that the animals and plants pathogen detects.Compare with traditional detection method, round pcr has specificity and the sensitivity of height, and the advantage such as short consuming time, more can carry out detection by quantitative to plant virus, bacterium, fungi and oomycetes by Real-time quantitative PCR.The basis of developer molecule detection method is to seek suitable nosophyte numerator target, and it is ribosomal gene ITS sequence that the vegetable plague bacterium molecule that extensively adopts at present detects target, and this sequence lacks enough polymorphisms between the mould kind of many epidemic diseases.In addition, the elicitin gene also is used as the target that phytophthora detects, the relevant encoding gene of Ras family
Ypt1Be widely used in the Molecular Detection of phytophthora, mitochondrion-encoded genes
Cox1,
Cox2 and the storage protein of may encoding
LpvGene respectively as the Oak Tree epidemic disease mould (
P. ramorum) and the camphor tree epidemic disease mould (
P. cinnamomi) target that detects.
The present invention is intended to seek Molecular Detection target new in Phytophthora capsici.Ykt6p is the R-SNARE albumen that cell survival is necessary, Ykt6 is very conservative in Eukaryotic evolution, Ykt6 aminoacid sequence in different plant species is compared, result shows, nearer than fungi on sibship the mould and plant of epidemic disease on phyletic evolution and algae, the mould classification position of this and epidemic disease meets fully.We as a new Molecular Detection target, have designed Ykt6 the mould potential general molecular of a pair of epidemic disease by the sequence alignment analysis and have detected primer, and designed on this basis the specific molecular detection primer of Phytophthora capsici thus.
Summary of the invention
The purpose of this invention is to provide specific molecular detection primer and detection kit thereof that a kind of general molecular for detection of phytophthora detects primer, detects Phytophthora capsici.
The present invention is achieved by the following technical solutions:
A kind of molecule primer and detection kit thereof for detection of Phytophthora capsici, the Auele Specific Primer of described detection Phytophthora capsici is:
Upstream (20bp) PcyktF:AGATGGTATGTCGCTATGAC
Downstream (20bp) PcyktR:ACATTTCGTAACGTCAACAT
Described universal primer for detection of phytophthora is:
Upstream (21bp) PyktF:AGAGTTGCTGCGCCAGGATGG
Downstream (21bp) PyktR:GTCTTGGACAACACGGCGGTG
Described detection kit is:
Detecting the solution I comprises: 25mmol Tris.Cl (PH 8.3), 125mmol KCl, 3.75 mmol MgCl
2, universal primer PyktF/PyktR each 1 μ mol, 0.1mgBSA of 0.25mmoldNTPs, phytophthora, Taq archaeal dna polymerase 50U, add ultrapure water to be prepared into 1mL and detect solution.Storage life is 1 year.
Detecting the solution II comprises: 25mmol Tris.Cl (PH 8.3), 125mmol KCl, 3.75 mmol MgCl
2, universal primer PcyktF/PcyktR each 1 μ mol, 0.1mgBSA of 0.25mmoldNTPs, phytophthora, Taq archaeal dna polymerase 50U, add ultrapure water to be prepared into 1mL and detect solution.Storage life is 1 year.
The using method of described detection kit is:
When direct use Phytophthora capsici Auele Specific Primer detects, extract thing DNA to be detected, get 5 μ L solution as reaction template, get the solution 10 μ L in the test kit II, separately getting 10 μ L deionized waters adds in 200 μ L reaction tubules, pcr amplification after mixing, program are 94 ℃ of sex change 3 minutes, 94 ℃ of sex change 30 seconds; Annealed 30 seconds for 58 ℃; 72 ℃ were extended 30 seconds; 32 circulations, last 72 ℃ were extended 5 minutes;
When using sleeve type PCR to detect, extract thing DNA to be detected, get 5 μ L solution as reaction template, get the solution 10 μ L in the test kit I, separately get 10 μ L deionized waters and add in 200 μ L reaction tubules, pcr amplification after mixing, program is 94 ℃ of sex change 3 minutes, 94 ℃ of sex change 30 seconds; Annealed 30 seconds for 58 ℃; 72 ℃ were extended 30 seconds; 32 circulations, last 72 ℃ were extended 5 minutes.Get 5 μ L after 5 times of PCR product dilute with waters after reaction is finished, get the solution 10 μ L in the test kit II, separately get 10 μ L deionized waters and add in 200 μ L reaction tubules, pcr amplification after mixing, program is 94 ℃ of sex change 3 minutes, 94 ℃ of sex change 30 seconds; Annealed 30 seconds for 58 ℃; 72 ℃ were extended 30 seconds; 32 circulations, last 72 ℃ were extended 5 minutes;
The electrophoresis detection of amplified production: get 10 μ L pcr amplification products, at the 1%(mass volume ratio) carry out electrophoresis on sepharose, voltage is 120V, after 20 minutes under UV-light detected result.If exist molecular weight to be about the band of 282bp, the proof cause of disease of examining is Phytophthora capsici.
Advantage of the present invention is:
The general molecular that the invention provides phytophthora detects the Auele Specific Primer of primer and Phytophthora capsici and the test kit that contains this primer.The present invention passes through sequence alignment, found a general detection primer of potential phytophthora, checking by experiment, this primer can amplify in 16 mould kinds of different epidemic diseases that experiment provides the band of a treaty 630bp (size not of the same race is variant) size, for the Molecular Detection of the mould kind of other epidemic disease provides a potential molecular target.
A pair of Phytophthora capsici detection primer specificity designed in the present invention is high, when 14 mould kinds of different epidemic diseases and 12 other fungies are detected, only have the specific electrophoretic band that detects a 282bp of Phytophthora capsici energy, and the recall rate in the Phytophthora capsici bacterial strain reaches 100%.Can detect exactly Phytophthora capsici by complicated pathogenic bacteria environment from morbidity plant tissue and soil.Utilize Nested Polymerase Chain Reaction, the minimum sample that can detect the 1fg DNA content of the present invention namely is equivalent to detect a zoospore and an oospore, and detection sensitivity is high.
Description of drawings
Fig. 1. phytophthora universal primer PyktF, PyktR PCR the result, 1. soybean phytophthora (
Phytophthora sojae), 2. Phytophthora capsici (
Phytophthora capsici), 3. Phytophthora cactorum (
Phytophthora cactorum), 4. hidden ground epidemic disease mould (
Phytophthora cryptogea), 5. Phytophthora nicotianae (
Phytophthora nicotianae), 6. palm mould (
Phytophthora palmivora), 7. melon epidemic disease mould (
Phytophthora melonis), 8. the Jue Shi epidemic disease mould (
Phytophthora drechsleri), 9. the lichee epidemic disease mould (
Phytophthora litchii), 10. ramie mould (
Phytophthora boehmeriae), 11. phytophthora infestans (
Phytophthora infestans), 12. clover epidemic diseases mould (
Phytophthora medicaginis), 13. camphor tree epidemic diseases mould (
Phytophthora cinnamomi), 14. phytophthora parasiticas (
Phytophthora parasitica), the 15-18. pythium (
Phythium. spp), 19. Fusarium graminearums (
Fusarium graminearum), 20. Fusarium oxysporums (
Fusarium oxysporum), 21. Pyricularia oryzaes (Magnaporthe oryzae), 22. Colletotrichum capsicis (
Colletotrichum capsici), 23. Sclerotinia sclerotiorums (
Sclerotinia sclerotiorum), 24. the pathogen of Botrytis cinereas (
Botrytis cinerea).
Fig. 2. the checking of Phytophthora capsici Auele Specific Primer (PcyktF, PcyktR), 1-4, separate from the Phytophthora capsici bacterial strain of different areas, 5. soybean phytophthora, 6 Phytophthora cactorums, 7. hidden ground epidemic disease mould, 8. Phytophthora nicotianae, 9. palm mould, 10. melon epidemic disease is mould, 11. Jue Shi epidemic diseases are mould, 12. lichee epidemic diseases are mould, 13. ramie moulds, 14. phytophthora infestans, 15. clover epidemic diseases are mould, 16. camphor tree epidemic diseases are mould, 17. phytophthora parasiticas, 18-20. pythium, 19. Fusarium graminearums, 20. Fusarium oxysporums, 21. Pyricularia oryzaes, 22. Colletotrichum capsicis, 23. Sclerotinia sclerotiorums.
Fig. 3. use the mould universal primer of epidemic disease (PyktF, PyktR) and Phytophthora capsici Auele Specific Primer (PcyktF, PcyktR) to carry out the sleeve type PCR amplification and carry out the sensitivity checking.Corresponding Phytophthora capsici DNA concentration is respectively 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, 100ag, 10ag
Fig. 4. Phytophthora capsici Auele Specific Primer (PcyktF, PcyktR) detects the PCR of oospore in soil, 1. positive control, 2-3, the separating resulting of oospore in morbidity field soil.4, there is no field soil separating resulting 6. negative controls of falling ill
Fig. 5. Phytophthora capsici Auele Specific Primer (PcyktF, PcyktR) detects the PCR of disease plant, 1. positive control, 2-4. disease plant, 5. healthy plant, 6. negative control
Fig. 6. Phytophthora capsici Auele Specific Primer (PcyktF, PcyktR) detects the PCR of zoospore in polluted river water, 1. positive control, 2-3, the detected result in polluted river water.4, detected result 6. negative controls in unpolluted water.
Embodiment
(1) detected result of residual Phytophthora capsici oospore in soil:
The enrichment of oospore in soil: get pedotheque 20-100 gram to be checked, grind, successively adopt 200 eye mesh screens to remove larger grogs, then filter through 400,500,800 eye mesh screens, repeatedly rinse with the 3-10 premium on currency simultaneously, from 800 mesh sieve online collection oospore, use the 1ml aqueous suspension.Because oospore can not see through 800 eye mesh screens, processing can reach the effect that makes the oospore enrichment like this.
the extraction of oospore DNA: will transfer in the centrifuge tube of 1.5 mL with the oospore that sterilized water suspends, under 12000 r.min-1 rotating speeds centrifugal 5 minutes, the sucking-off supernatant liquor, suck in mortar after staying 15 μ L liquid suctions to beat mixing, get powder after liquid nitrogen grinding in tubule, the CTAB method is extracted genome, method is as follows, add 900 μ L 2% CTAB extracting solutions and 90 μ l 10% SDS, the whirlpool mixing, in 60 ℃ of water-bath 1 h, middle every 10 min turn upside down several times, centrifugal 10 min of 12000 rpm, get and reset and add equal-volume phenol/chloroform/primary isoamyl alcohol (25:24:1), put upside down mixing, centrifugal 10 min of 12000 rpm, supernatant is transferred to new pipe, adds the equal-volume chloroform, put upside down gently mixing, centrifugal 5 min of 12000 rpm.Supernatant is transferred in new pipe, adds the equal-volume methanol extraction, centrifugal 10 min of 12000 rpm, and the supernatant that inclines precipitates with 70% washing with alcohol once, and room temperature is dried.Add appropriate sterilization ultrapure water (containing 20 μ g/ml RNase), after 37 ℃ of processing 1 h, be used for the reserve experiment.
Pcr amplification and electrophoresis detection (Fig. 4) are referring to the test kit instruction manual.
(2) detected result of capsicum disease plant:
The extraction of disease plant DNA: after having the Pepper Leaves of water soaking mode scab or rhizome position with 70% alcohol disinfecting, with the powder that takes a morsel after liquid nitrogen grinding, adopt the CTAB method to extract genome (method see on).Also can use the alkaline lysis DNA rapid extraction, method is as follows, gets the plant tissue of one section morbidity, every milligram of tissue adds 10 μ l 0.5 M NaOH, be transferred in the EP pipe of 1.5 ml after fully grinding, centrifugal 5 min of 12000 rpm get supernatant and are used for follow-up pcr amplification experiment.
Pcr amplification and electrophoresis detection (Fig. 5) are referring to the test kit instruction manual.
(3) detected result of zoospore in the Phytophthora capsici polluted source:
The enrichment of zoospore and DNA extraction: Phytophthora capsici can form sporocyst and discharge a large amount of zoospores having under the environment of moisture film, be the important channel of infecting again.Get Phytophthora capsici polluted source 500mL, centrifugal 20min under the centrifugal force of 5000g, remove supernatant liquor, the zoospore of precipitation is with 100 μ L aqueous suspensions, change the 1.5mL centrifuge tube over to, add 0.05g quartz sand, the vortex concussion is after 10 seconds, and 2000r.min-1 gets supernatant and is used for follow-up pcr amplification after centrifugal 5 minutes.
Claims (1)
1. primer and detection kit thereof for detection of a Phytophthora capsici is characterized in that described Phytophthora capsici molecular detection primer is:
Upstream (20bp) PcyktF:AGATGGTATGTCGCTATGAC
Downstream (20bp) PcyktR:ACATTTCGTAACGTCAACAT,
Described phytophthora universal primer is:
Upstream (21bp) PyktF:AGAGTTGCTGCGCCAGGATGG
PyktR:GTCTTGGACAACACGGCGGTG described detection kit in downstream (21bp) is:
Detecting the solution I comprises: 25mmol Tris.Cl (PH 8.3), 125mmol KCl, 3.75 mmol MgCl
2, universal primer PyktF/PyktR each 1 μ mol, 0.1mgBSA of 0.25mmoldNTPs, phytophthora, Taq archaeal dna polymerase 50U, add ultrapure water to be prepared into 1mL and detect solution; Storage life is 1 year;
Detecting the solution II comprises: 25mmol Tris.Cl (PH 8.3), 125mmol KCl, 3.75 mmol MgCl
2, each 1 μ mol, 0.1mgBSA of 0.25mmoldNTPs, the mould Auele Specific Primer PcyktF/PcyktR of Phytophthora capsici epidemic disease, Taq archaeal dna polymerase 50U, add ultrapure water to be prepared into 1mL and detect solution; Storage life is 1 year;
The using method of described detection kit is:
When direct use Phytophthora capsici Auele Specific Primer detects, extract thing DNA to be detected, get 5 μ L solution as reaction template, get the solution 10 μ L in the test kit II, separately getting 10 μ L deionized waters adds in 200 μ L reaction tubules, pcr amplification after mixing, program are 94 ℃ of sex change 3 minutes, 94 ℃ of sex change 30 seconds; Annealed 30 seconds for 58 ℃; 72 ℃ were extended 30 seconds; 32 circulations, last 72 ℃ were extended 5 minutes;
When using sleeve type PCR to detect, extract thing DNA to be detected, get 5 μ L solution as reaction template, get the solution 10 μ L in the test kit I, separately get 10 μ L deionized waters and add in 200 μ L reaction tubules, pcr amplification after mixing, program is 94 ℃ of sex change 3 minutes, 94 ℃ of sex change 30 seconds; Annealed 30 seconds for 58 ℃; 72 ℃ were extended 30 seconds; 32 circulations, last 72 ℃ were extended 5 minutes; Get 5 μ L after 5 times of PCR product dilute with waters after reaction is finished, get the solution 10 μ L in the test kit II, separately get 10 μ L deionized waters and add in 200 μ L reaction tubules, pcr amplification after mixing, program is 94 ℃ of sex change 3 minutes, 94 ℃ of sex change 30 seconds; Annealed 30 seconds for 58 ℃; 72 ℃ were extended 30 seconds; 32 circulations, last 72 ℃ were extended 5 minutes;
The electrophoresis detection of amplified production: get 10 μ L pcr amplification products, at the 1%(mass volume ratio) carry out electrophoresis on sepharose, voltage is 120V, after 20 minutes under UV-light detected result; If exist molecular weight to be about the band of 282bp, the proof cause of disease of examining is Phytophthora capsici.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525913A (en) * | 2013-09-22 | 2014-01-22 | 福建省农业科学院植物保护研究所 | Phytophthora capsici LAMP (Loop-mediated isothermal Amplification) primer and rapid detection method thereof |
| CN103571946A (en) * | 2013-09-30 | 2014-02-12 | 安徽省农业科学院植物保护与农产品质量安全研究所 | Molecular detection primer specific for phytophthora sojae and application thereof |
| CN112501332A (en) * | 2020-09-11 | 2021-03-16 | 安徽省农业科学院植物保护与农产品质量安全研究所 | Primer for detecting specific molecules of wheat take-all germs as well as detection method and application thereof |
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| CN101974651A (en) * | 2010-12-02 | 2011-02-16 | 中国农业科学院植物保护研究所 | Fluorescence quantitative polymerase chain reaction (PCR) detection method and detection kit for phytophthora capsici leonian |
| CN102586469A (en) * | 2012-03-31 | 2012-07-18 | 中国科学院微生物研究所 | Loop-mediated isothermal amplification (LAMP) primers for detecting Phytophthora capsici and kit comprising primers |
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Patent Citations (2)
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| CN101974651A (en) * | 2010-12-02 | 2011-02-16 | 中国农业科学院植物保护研究所 | Fluorescence quantitative polymerase chain reaction (PCR) detection method and detection kit for phytophthora capsici leonian |
| CN102586469A (en) * | 2012-03-31 | 2012-07-18 | 中国科学院微生物研究所 | Loop-mediated isothermal amplification (LAMP) primers for detecting Phytophthora capsici and kit comprising primers |
Non-Patent Citations (1)
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| C. SILVAR等: "Development of specific PCR primers for identification and detection of Phytophthora capsici Leon.", 《EUROPEAN JOURNAL OF PLANT PATHOLOGY》, vol. 112, 31 December 2005 (2005-12-31), pages 43 - 52 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525913A (en) * | 2013-09-22 | 2014-01-22 | 福建省农业科学院植物保护研究所 | Phytophthora capsici LAMP (Loop-mediated isothermal Amplification) primer and rapid detection method thereof |
| CN103571946A (en) * | 2013-09-30 | 2014-02-12 | 安徽省农业科学院植物保护与农产品质量安全研究所 | Molecular detection primer specific for phytophthora sojae and application thereof |
| CN103571946B (en) * | 2013-09-30 | 2016-03-02 | 安徽省农业科学院植物保护与农产品质量安全研究所 | The specific molecular detection primer of soybean phytophthora and application thereof |
| CN112501332A (en) * | 2020-09-11 | 2021-03-16 | 安徽省农业科学院植物保护与农产品质量安全研究所 | Primer for detecting specific molecules of wheat take-all germs as well as detection method and application thereof |
| CN112501332B (en) * | 2020-09-11 | 2022-04-12 | 安徽省农业科学院植物保护与农产品质量安全研究所 | Primer for detecting specific molecules of wheat take-all germs as well as detection method and application thereof |
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