CN105755122A - Sweet potato fusarium wilt germ molecule detecting primer and rapid detection method - Google Patents
Sweet potato fusarium wilt germ molecule detecting primer and rapid detection method Download PDFInfo
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- CN105755122A CN105755122A CN201610155504.XA CN201610155504A CN105755122A CN 105755122 A CN105755122 A CN 105755122A CN 201610155504 A CN201610155504 A CN 201610155504A CN 105755122 A CN105755122 A CN 105755122A
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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
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention relates to a sweet potato fusarium wilt germ molecule detecting primer and a rapid detection method and belongs to the technical field of crop disease detection and biology. The specific primer comprises an upstream primer FUSF:5'-GCCAGAGGACCCCTAAACTC-3' and a downstream primer FUSR:5'-GAAGTTGGGGTTTAACGGCG-3'. The detecting primer has high specificity and high sensitivity, the detection method is good in practicability and is simple, convenient and rapid to operate; fusarium wilt germs in sweet potato cultivation soil can be detected, the sweet potato fusarium wilt germ molecule detecting primer and the rapid detection method have great significance in prevention of sweet potato fusarium wilt, early detection of the sweet potato fusarium wilt can be realized, sweet potato acute communicable diseases, sweet potato black rot and similar diseases can be effectively distinguished, and the primer and the method have great significance in early control of the sweet potato fusarium wilt and diffusion and spreading of diseases.
Description
Technical field
The present invention relates to a kind of stem rot of sweet potato bacterium molecule detection primer and detection method thereof, it is exclusively used in the rapid molecular detection of stem rot of sweet potato bacterium, the monitoring that simultaneously can realize the detection of field sweet potato cultivation soil and the early diagnosis of stem rot of sweet potato and germ is identified, belongs to corps diseases detection and biological technical field.
Background technology
Sweet potato has another name called Ipomoea batatas, sweet potato, pachyrhizus etc., the cultivation history of existing more than 400 year.Yield of sweet potato is high, purposes is wide, strong adaptability, is important grain, feed and insutrial crop, and stem apex blade also can use vegetables as, is deemed likely to be to break away from grain and energy crisis " last trump " in the future.China is that sweet potato maximum in the world produces country, and in China cereal crops produce, sweet potato is only second to paddy rice, wheat and corn, occupies the 4th.Sweet potato has the features such as lean, yield is high, wide adaptability cold-resistant, resistance to, is still the first-selected crop of China's dry-land cultivation.China resident remains the traditional habit eating sweet potato raw, along with nutrient sweet potato value is re-recognized by people, and the understanding of idea wholesome to edible coarse food grain and acceptance, sweet potato market demand market also will expanding day.
Stem rot of sweet potato, also known as droop, blight dis-ease, stem rot etc., all has generation in a lot of countries and regions.Stem rot of sweet potato is mainly distributed on Fujian, Zhejiang, Sichuan, Guangdong, Taiwan, Shandong, Liaoning, Henan, Jiangsu etc. in China and saves.Stem rot of sweet potato is typical soil-borne disease, by sickle-like bacteria (Fusarium oxysporum f. spbatatas) infect and cause, cause plant generation complete stool property withered, dead, be one of most important disease in south China potato district, cause harm greatly.It is particularly acute in each good quality and high output potato district of China, especially hilly upland and basin is many produces with rotation systems such as Spring Peanut or soybean-evening potato, the potato of potato-early, early rice-evening potatos, cause dead arm bacterium radix in soil big, cause stem rot of sweet potato occurring and damage serious, general underproduction 10%-20%, the underproduction of serious plot reaches more than 50%.
Fungus in soil and potato seed of carrying disease germs, seedling are the source of infection causing seedling field and land for growing field crops that stem rot of sweet potato occurs, germ has soil to pass through the wound of rice shoot base portion or root, or invaded rice shoot by potato seed of carrying disease germs by conduit, breed in tracheal tissue, causing ill sweet potato plant generation complete stool property withered and dead, variable rate technology is that aerial part blade turns yellow from bottom to top and comes off, cauline bundle browning look, last stem cracking, complete stool is dead.Stem rot of sweet potato bacterium (Fusarium oxysporum f. spbatatas) it is a kind of thryptophytism soil inhabitant, chlamydospore can be at soil long-term survival, and the chemical prevention before therefore the process of lesion soil-fumigating and sweet potato are transplanted is main prophylactico-therapeutic measures.Therefore, set up that a set of stem rot of sweet potato bacterium is convenient and swift, reliable results, highly sensitive quick diagnosis technology, realize stem rot of sweet potato early warning and alert, the propagation effectively controlling pathogen and plant disease epidemic, realize the prophylactico-therapeutic measures of " keeping away disease ", effectively control the generation of stem rot of sweet potato, support Sweet Potato Industry develops in a healthy way, and will effectively lower bactericide simultaneously and use, and have significant economy and ecological benefits.
In recent years, Protocols in Molecular Biology quickly grows, wherein PCR(polymerase chain reaction) technology has high specificity, highly sensitive, conveniently advantage, is widely used in diagnosis and the qualification of phytopathogen in Plant Pathology.Fungi ribosomes transcribed spacer ITS(internal transcribed spacer) conservative in sequence kind and section belong to kind between variable feature, PCR is carried out according to its this characteristics design specific primer, and then Rapid identification and diagnosis of plant pathogen are achieved with extensively application, specific primer and the detection method of various plants pathogen are developed, achieve and identify fast and accurately, such as cymbidium anthracnose bacterium, tomato early blight bacterium, citrus processing, sweet potato black rot pathogen etc..There is not been reported in the current research about the detection of stem rot of sweet potato bacterium rapid molecular.
Summary of the invention
It is difficult to prevent and treat for after stem rot of sweet potato is fallen ill by prior art, in sweet potato cultivation soil dead arm bacterium be difficult to detect, detection and the evaluation program of stem rot of sweet potato bacterium are loaded down with trivial details, to identifying that skill requirement is high, the degree of accuracy is low, it is difficult to the difficult problem such as prediction and warning of stem rot of sweet potato, it is provided that a kind of stem rot of sweet potato bacterium molecule detection primer and detection method thereof.
For achieving the above object, this invention takes techniques below scheme:
Present invention firstly provides a kind of stem rot of sweet potato bacterium molecule detection primer, its nucleotides sequence is classified as:
Upstream primer FUSF:5 '-GCCAGAGGACCCCTAAACTC-3 '
Downstream primer FUSR:5 '-GAAGTTGGGGTTTAACGGCG-3 '.
Described primers F USF and FUSR go out the product of 434bp to stem rot of sweet potato bacterium specific amplification.
Present invention also offers the method for quick of a kind of stem rot of sweet potato bacterium, comprise the following steps:
(1) from sweet potato plant or potato block or cultivating soil, DNA is extracted；
For detect pathogen pure culture whether there is dead arm bacterium time, use CTAB method extract strains tested genomic DNA；When whether there is dead arm bacterium in sweet potato, NaOH rapid cleavage method is used to extract Asparagus Plants tissue gene group DNA；When whether sweet potato cultivation soil existing dead arm bacterium for detecting, soil DNA rapid extraction kit is used to extract.
(2) carry out PCR amplification for template extracting DNA in sweet potato plant or potato block or cultivating soil: PCR reaction system 25 μ L, comprise 2.5 μ L 10 × PCR
Buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is the Taq enzyme of 5U/ μ L, FUSF and FUSR each 0.5 μ L, the DNA profiling 1 μ L of 10 μm ol/L, add ddH2O reaches 25 μ L to cumulative volume；PCR reaction condition is: 94 DEG C of denaturations 5min；94 DEG C of sex change 45sec, 57 DEG C of annealing 45sec, 72 DEG C extend 1min, totally 35 circulations；72 DEG C extend 10min；
(3) PCR primer of upper step gained is with the liquid electrophoretic analysis of 0.5 × tbe buffer on 1.0% Ago-Gel, and voltage is 4-5V/cm；Size according to amplified production judges testing result, if energy specific amplification goes out the product of 434bp, then can determine that and there is dead arm bacterium in described sweet potato or cultivating soil, there is not stem rot of sweet potato bacterium in otherwise described sweet potato or cultivating soil.
The beneficial effects of the present invention is:
(1) high specificity: stem rot of sweet potato bacterium is had the strongest specific by the primer designed by the present invention, it is possible to be used for distinguishing sweet potato pest, sweet potato black rot pathogen and other soil inhabitants, it is thus possible to effectively dead arm bacterium on detection sweet potato and in soil；
(2) accuracy is good: the present invention be according to fungi ribosomes transcribed spacer (rDNA-ITS) sequence well-conserved and section in fungi kind belong to kind between the design of variable feature stem rot of sweet potato bacterium had the PCR primer of specific amplified effect.The sweet potato to the different stem rot of sweet potato bacterium of geographic origin, the sweet potato tissue of morbidity, the soil carrying stem rot of sweet potato bacterium and health has carried out detection checking, only stem rot of sweet potato bacterium, the sweet potato tissue of morbidity and can specifically amplify the electrophoretic band of 434 bp in carrying the soil of stem rot of sweet potato bacterium, illustrates that the primer designed by the present invention is used for detecting stem rot of sweet potato bacterium accurately and reliably；
(3) highly sensitive: after the special primer of design is joined together to carry out nest-type PRC amplification by the present invention with ITS gene universal primer (ITS1/ITS4), the detection sensitivity of stem rot of sweet potato bacterium to be can reach 10fg in DNA level；
(4) applicability is wide, practicality good: the detection method of the stem rot of sweet potato bacterium of the present invention; germ mycelium can not only be detected; also the sweet potato for sweet potato cultivation soil and infection dead arm bacterium can be detected, thus " keeping away disease " measure important in Plant Protection can be realized.
(5) easy and simple to handle quickly: the present invention only need to carry out DNA extraction, PCR amplification and agarose electrophoresis after get final product result of determination, general whole detection process can complete in 5 hours, simple and efficient to handle.
Accompanying drawing explanation
Fig. 1 be primer of the present invention to stem rot of sweet potato bacterium specific amplification electrophoretogram: wherein swimming lane M is 2000bp DNA Marker, swimming lane 2, swimming lane 3 are stem rot of sweet potato bacterium, and swimming lane 4-10 is respectively as follows: sweet potato black rot pathogen (Ceratocystis fimbriata bacterium), sweet potato pest (Pseudomonas solanacearum), Sclerotinia sclerotiorum (sclerotinite), Rhizoctonia solani Kahn (Rhizoctonia solani Kuhn), Didymella bryoniae (melon spherical cavity bacterium), wet bubble bacterium (wart spore is mould), negative control.
Fig. 2 is the sensitivity detection amplification electrophoretogram of primer stem rot of sweet potato bacterium of the present invention: figure A: substance PCR, figure B is nest-type PRC, wherein figure A swimming lane M is 2000bp DNA Marker, swimming lane 2-12 and is respectively as follows: 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, negative control, positive control；Figure B swimming lane M is 2000bp DNA Marker, swimming lane 2-13 and is respectively as follows: 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, 100ag, negative control, positive control.
Fig. 3 is the sweet potato that falls ill of the present invention and cultivating soil amplification electrophoretogram, wherein swimming lane 1 is that 2000bp DNA Marker, swimming lane 2-10 are respectively the sweet potato plant of natural occurrence, natural occurrence sweet potato cultivation soil, the sweet potato plant of artificial infection morbidity, artificial infection morbidity sweet potato cultivation soil, healthy sweet potato potato block, healthy Sweet potato stem, the soil of sterilizing, negative control, positive control.
Detailed description of the invention
In order to make content of the present invention easily facilitate understanding, below in conjunction with detailed description of the invention, technical solutions according to the invention are described further.
Test method used in following embodiment if no special instructions, is conventional method.
Test material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Embodiment 1 Molecular detection primer design and the foundation of stem rot of sweet potato bacterium special molecular detection method
1. the extraction of stem rot of sweet potato bacterium genomic DNA:
Use CTAB method to extract the genomic DNA of the 10 strain stem rot of sweet potato bacterium that this laboratory preserves, specifically comprise the following steps that
(1) taking 0.1 g hypha powder in 1.5 mL centrifuge tubes, add 900 μ L2%CTAB extracts, use oscillator vibration mixing, 60 DEG C of water-bath 60min, under room temperature condition, 12000r/min is centrifuged 15
min；
(2) taking supernatant 700 μ L, add equal-volume phenol, chloroform, isoamyl alcohol mixed liquor (each volume ratio is 25:24:1), gentle shake, under room temperature condition, 8000 r/min are centrifuged 10min
；
(3) take supernatant 500 μ L, add equal-volume chloroform and extract again once, under room temperature condition, 8000
R/min is centrifuged 10min；
(4) taking supernatant 350 μ L, add 1/10 volume 3 mol/L NaAc and 2 times of volume absolute ethyl alcohols ,-20 DEG C of precipitation 60 min, under the conditions of 4 DEG C, 8000 r/min are centrifuged 5 min
；
(5) abandoning supernatant, adding 700 μ L volumetric concentrations is 70% ice ethanol, jog 10sec, and under the conditions of 4 DEG C, 8000 r/min are centrifuged 10sec, dry, and adds 50 μ L TE buffer solutions, and-20 DEG C save backup.
2. stem rot of sweet potato bacterium ITS sequence measures:
It is that primer carries out PCR amplification to the DNA extracting stem rot of sweet potato bacterium with fungi Internal Transcribed Spacer (rDNA-ITS) universal primer TS1:5'-TCCGTAGGGAACCTGCGG-3' and ITS4:5'-TCCTCCGCTTATTGATATGC-3', amplification reaction system and response procedures be: PCR reaction system 25 μ L, comprise 2.5 μ L 10 × PCR buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is the Taq enzyme (Takara of 5U/ μ L
Dalian treasured bioengineering Co., Ltd), TIS1 and ITS4 each 0.5 μ L, the DNA profiling 1 μ L of 10 μm ol/L, add ddH2O reaches 25 μ L to cumulative volume；PCR reaction condition is: 94 DEG C of denaturations 5min；94 DEG C of sex change 1 min, 55 DEG C of annealing 45sec, 72 DEG C extend 1 min, totally 35 circulations；72 DEG C extend 10min；Gained PCR primer send Dalian treasured bioengineering Co., Ltd to check order.
3. the design of stem rot of sweet potato bacterium special molecular detection primer:
According to the height variation and plant internal stability between fungi kind of stem rot of sweet potato sclerotium sugar vivo transcription spacer region (rDNA-ITS) sequence, the ITS sequence of 10 strain stem rot of sweet potato bacterium order-checking obtained with Clustal X software with in GenBankFusariumBelong to ITS sequence, sweet potato brown patch germ ITS sequence the most of the same race, sweet potato soft rot bacterium ITS sequence carries out homology analysis and difference site is compared, have the one couple of PCR primers (being synthesized by Shanghai Sheng Gong bioengineering Co., Ltd) of specific amplification effect to stem rot of sweet potato bacterium with Primer Primer5 Software for Design, i.e. the sequence of special molecular detection primer is:
Upstream primer FUSF:5 '-GCCAGAGGACCCCTAAACTC-3 '；
Downstream primer FUSR:5 '-GAAGTTGGGGTTTAACGGCG-3 '.
4. the foundation of stem rot of sweet potato bacterium molecule detection method:
(1) from stem rot of sweet potato bacterium or sweet potato plant tissue or sweet potato cultivation soil, DNA is extracted:
When being 1. used for detecting pathogen pure culture, use CTAB
Method extracts strains tested genomic DNA；
2. it is used for detecting sweet potato plant tissue when whether there is stem rot of sweet potato bacterium, uses NaOH rapid cleavage method to extract sweet potato plant tissue genomic DNA, specifically comprise the following steps that
A. weigh plant tissue 0.1 g to be detected, add 0.5 mol/L NaOH 30 μ L, be fully milled to tissue stick with paste；
B. being proceeded in 1.5 mL centrifuge tubes by pasty state tissue, 12000r/min is centrifuged 6 min, takes supernatant 5 μ L；
C., supernatant adds 495 μ L 0.1 mol/L Tris-HCl(pH=8.0), mix, take 1.0 μ L and expand as PCR template；
3. it is used for detecting time whether cultivating soil exists stem rot of sweet potato bacterium, uses soil DNA rapid extraction kit to extract.
(2) PCR amplification is carried out with the DNA extracted for template: PCR reaction system 25 μ L, comprise 2.5 μ L 10 × PCR buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, 0.15 μ L concentration is the Taq enzyme of 5U/ μ L, the each 0.5 μ L of FUSF and FUSR of 10 μm ol/L, DNA profiling 1 μ L, adds ddH2O reaches 25 μ L to cumulative volume；PCR reaction condition is: 94 DEG C of denaturations 5min；94 DEG C of sex change 45sec, 57 DEG C of annealing 45sec, 72 DEG C extend 1min, totally 35 circulations；72 DEG C extend 10min；
(3) PCR primer of upper step gained is with the liquid electrophoretic analysis of 0.5 × tbe buffer on 1.0% Ago-Gel, and voltage is 4-5V/cm；Size according to amplified production judges testing result, if energy specific amplification goes out the product of 434bp, then can determine that and described sweet potato plant or cultivating soil exist stem rot of sweet potato bacterium, otherwise described sweet potato plant or cultivating soil do not exist stem rot of sweet potato bacterium.
Embodiment 2Stem rot of sweet potato bacterium specific amplification
1. use CTAB method to extract 2 strain stem rot of sweet potato bacterium, sweet potato black rot pathogen (Ceratocystis fimbriata bacterium), sweet potato pest (Pseudomonas solanacearum), Sclerotinia sclerotiorum (sclerotinite), Rhizoctonia solani Kahn (Rhizoctonia solani Kuhn), Didymella bryoniae (melon spherical cavity bacterium) and the genomic DNA of wet bubble bacterium (wart spore is mould).
2. carry out PCR amplification for template extracting the DNA for examination bacterium: PCR reaction system 25 μ L, comprise 2.5 μ L 10 × PCR
Buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is the Taq enzyme of 5U/ μ L, FUSF and FUSR each 0.5 μ L, the DNA profiling 1 μ L of 10 μm ol/L, add ddH2O reaches 25 μ L to cumulative volume；PCR reaction condition is: 94 DEG C of denaturations 5min；94 DEG C of sex change 45sec, 57 DEG C of annealing 45sec, 72 DEG C extend 1min, totally 35 circulations；72 DEG C extend 10min；Electrophoresis detection amplified production.
3. specific amplification result
As shown in Figure 1, 2 strain stem rot of sweet potato bacterium can go out the band of 434bp with specific amplification, and sweet potato black rot pathogen (Ceratocystis fimbriata bacterium), sweet potato pest (Pseudomonas solanacearum), Sclerotinia sclerotiorum (sclerotinite), Rhizoctonia solani Kahn (Rhizoctonia solani Kuhn), Didymella bryoniae (melon spherical cavity bacterium), wet bubble bacterium (wart spore is mould) and negative control are all without amplified band, show that stem rot of sweet potato bacterium can be made a distinction by the molecular detection primer of the present invention with other crop pathogenses, have the strongest specific, the detection method of the present invention can be used for the specific amplification of stem rot of sweet potato bacterium.
Embodiment 3The sensitivity of stem rot of sweet potato bacterium is detected by primer of the present invention
1. use CTAB method to extract the genomic DNA of sweet potato germ；
2., by the genomic DNA of the sweet potato germ of extraction, after spectrophotometric determination concentration, dilute with aseptic ultra-pure water, be configured to series concentration, standby；
3. carrying out standard PCR amplification with one-tenth series concentration DNA of preparation for template: PCR reaction system 25 μ L, comprise 2.5 μ L 10 × PCR buffer, 2.0 μ L concentration are the dNTP of 2.5mmol/L
Mixture, 0.15 μ L concentration is the Taq enzyme of 5U/ μ L, FUSF and FUSR each 0.5 μ L, the DNA profiling 1 μ L of 10 μm ol/L, adds ddH2O reaches 25 μ L to cumulative volume；PCR reaction condition is: 94 DEG C of denaturations 5min；94 DEG C of sex change 45sec, 57 DEG C of annealing 45sec, 72 DEG C extend 1min, totally 35 circulations；72 DEG C extend 10min；Electrophoresis detection amplified production.
4. carry out nested PCR amplification with one-tenth series concentration DNA of preparation for template:
(1) first round PCR amplification: be that preparation is become series concentration DNA to carry out the amplification of first round PCR by outer primer with fungi Internal Transcribed Spacer (rDNA-ITS) universal primer TS1:5'-TCCGTAGGGAACCTGCGG-3' and ITS4:5'-TCCTCCGCTTATTG ATATGC-3', amplification reaction system and response procedures be: PCR reaction system 25 μ L, comprise 2.5 μ L 10 × PCR buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is the Taq enzyme (Takara of 5U/ μ L
Dalian treasured bioengineering Co., Ltd), TIS1 and ITS4 each 0.5 μ L, the DNA profiling 1 μ L of 10 μm ol/L, add ddH2O reaches 25 μ L to cumulative volume；PCR reaction condition is: 94 DEG C of denaturations 5min；94 DEG C of sex change 1 min, 55 DEG C of annealing 45sec, 72 DEG C extend 1 min, totally 35 circulations；72 DEG C extend 10min；
(2) second take turns PCR amplification: with the first round PCR amplification product as template, carry out second with FUSF/FUSR for primer and take turns PCR amplification, PCR reaction system 25 μ L, comprise 2.5 μ L 10 × PCR buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, and 0.15 μ L concentration is the Taq enzyme of 5U/ μ L, each 0.5 μ L of FUSF and FUSR of 10 μm ol/L, DNA profiling 1 μ L, adds ddH2O reaches 25 μ L to cumulative volume；PCR reaction condition is: 94 DEG C of denaturations 5min；94 DEG C of sex change 45sec, 57 DEG C of annealing 45sec, 72 DEG C extend 1min, totally 35 circulations；72 DEG C extend 10min；Electrophoresis detection amplified production.
5. testing result
As in figure 2 it is shown, with primers F USF/FUSR of the present invention for primer carry out Standard PCR time, in 25 μ L reaction systems, the stem rot of sweet potato bacterium DNA of 10pg can obtain visual band, and its detection sensitivity can reach 10pg(Fig. 2-A)；And further with product as external primer amplification of universal primer TS1:5'-TCCGTAGGGAACCTGCGG-3' and ITS4:5'-TCCTCCGCTTATTGATATGC-3' of fungi Internal Transcribed Spacer (rDNA-ITS) as template, with primers F USF/FUSR of the present invention for primer carry out second take turns amplification time, in 25 μ L reaction systems, the stem rot of sweet potato bacterium DNA of 1fg can obtain visual band, and its detection sensitivity can reach 1fg (Fig. 2-B).
Embodiment 4The detection of stem rot of sweet potato bacterium in morbidity sweet potato plant tissue and sweet potato cultivation soil
1. use NaOH rapid cleavage method to extract sweet potato plant tissue genomic DNA；Soil DNA rapid extraction kit is used to extract soil genomic DNA to be measured.
2. carrying out standard PCR amplification with one-tenth series concentration DNA of preparation for template: PCR reaction system 25 μ L, comprise 2.5 μ L 10 × PCR buffer, 2.0 μ L concentration are the dNTP of 2.5mmol/L
Mixture, 0.15 μ L concentration is the Taq enzyme of 5U/ μ L, FUSF and FUSR each 0.5 μ L, the DNA profiling 1 μ L of 10 μm ol/L, adds ddH2O reaches 25 μ L to cumulative volume；PCR reaction condition is: 94 DEG C of denaturations 5min；94 DEG C of sex change 45sec, 57 DEG C of annealing 1min, 72 DEG C extend 45sec, totally 35 circulations；72 DEG C extend 10min；Electrophoresis detection amplified production.
3. testing result
As shown in Figure 3, the sweet potato plant of natural occurrence, natural occurrence sweet potato cultivation soil, the sweet potato plant of artificial infection morbidity, artificial infection morbidity sweet potato cultivation soil, positive control all can produce the visual band of about 434bp, and health sweet potato potato block, healthy Sweet potato stem, the soil of sterilizing, negative control all occur without any band, show that primer of the present invention and detection method can be additionally used in field stem rot of sweet potato disease plant and the detection of cultivating soil.
The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent and modification, all should belong to the covering scope of the present invention.
Sequence table
<110>Inst. of Plant Protection, fujian Academy of Agricultural Science
<120>a kind of stem rot of sweet potato bacterium molecule detection primer and method for quick
<160>4
<170>PatentIn version 3.5
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<211>18
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gccagaggacccctaaactc 20
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gaagttggggtttaacggcg 20
Claims (4)
1. a stem rot of sweet potato bacterium molecule detection primer, it is characterised in that: the nucleotides sequence of primer is classified as:
Upstream primer FUSF:5 '-GCCAGAGGACCCCTAAACTC-3 '
Downstream primer FUSR:5 '-GAAGTTGGGGTTTAACGGCG-3 '.
Stem rot of sweet potato bacterium molecule detection primer the most according to claim 1, it is characterised in that described upstream primer FUSF and downstream primer FUSR goes out the product of 434bp to stem rot of sweet potato bacterium specific amplification.
3. the method for quick of a stem rot of sweet potato bacterium, it is characterised in that: comprise the following steps:
(1) from the sweet potato infecting dead arm bacterium or sweet potato cultivation soil, DNA is extracted；
(2) to carry out PCR amplification for template from infecting the DNA that extracts in the sweet potato of dead arm bacterium or sweet potato cultivation soil: PCR reaction system 25 μ L, comprise 2.5 μ L 10 × PCR buffer, 2.0 μ L concentration are the dNTP Mixture of 2.5mmol/L, 0.15 μ L concentration is the Taq enzyme of 5U/ μ L, the each 0.5 μ L of FUSF and FUSR of 10 μm ol/L, DNA profiling 1 μ L, adds ddH2O reaches 25 μ L to cumulative volume；PCR reaction condition is: 94 DEG C of denaturations 5min；94 DEG C of sex change 45sec, 57 DEG C of annealing 45sec, 72 DEG C extend 1min, totally 35 circulations；72 DEG C extend 10min；
(3) PCR primer of upper step gained is with the liquid electrophoretic analysis of 0.5 × tbe buffer on 1.0% Ago-Gel, and voltage is 4-5V/cm；Size according to amplified production judges testing result, if energy specific amplification goes out the product of 434bp, then judges to there is dead arm bacterium in sweet potato or sweet potato cultivation soil.
Application in a kind of stem rot of sweet potato bacterium molecule detection primer the most as claimed in claim 1 dead arm bacterium on detection sweet potato and in soil.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636401A (en) * | 2016-12-22 | 2017-05-10 | 福建省农业科学院植物保护研究所 | Anoectochilus roxburghii stalk rot pathogen molecular detection primers and detection method thereof |
| CN107723380A (en) * | 2017-11-30 | 2018-02-23 | 福建省农业科学院植物保护研究所 | A kind of stem rot of sweet potato bacterium LAMP detection primer and its application |
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| CN106636401A (en) * | 2016-12-22 | 2017-05-10 | 福建省农业科学院植物保护研究所 | Anoectochilus roxburghii stalk rot pathogen molecular detection primers and detection method thereof |
| CN107723380A (en) * | 2017-11-30 | 2018-02-23 | 福建省农业科学院植物保护研究所 | A kind of stem rot of sweet potato bacterium LAMP detection primer and its application |
| CN107868845A (en) * | 2017-12-18 | 2018-04-03 | 福建省农业科学院植物保护研究所 | A kind of sweet potato blackspot bacterium nest-type PRC detection primer and its molecular detecting method |
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