CN106978505A - Detect primer pair and its application of eggplant stemphylium botryosum - Google Patents
Detect primer pair and its application of eggplant stemphylium botryosum Download PDFInfo
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Abstract
The invention discloses the primer pair of detection eggplant stemphylium botryosum and its application.The single stranded DNA that the primer pair of detection eggplant stemphylium botryosum disclosed by the invention is respectively Stem g7F and Stem g7R by title is constituted；Stem g7F be nucleotide sequence be SEQ ID No.1 in sequence table single stranded DNA；Stem g7R be nucleotide sequence be SEQ ID No.2 in sequence table single stranded DNA.1000 times are improved using the sensitivity of the primer pair detection eggplant stemphylium botryosum of the present invention, up to 4.285 × 102fg·μL‑1, and accurate quantitative analysis can be realized.It can quantitatively be detected before not showing disease at disease early period of origination, can apply to field soil seed borne fungi and Seed-associated fungi with the primer pair of the present invention.
Description
Technical field
The present invention relates to primer pair and its application in biological technical field, detecting eggplant stemphylium botryosum.
Background technology
Tomato (Lycopersicon esculentum Mill), is Solanaceae tomato genus 1 year or herbaceos perennial,
South America is originated in, is the vegetable crop cultivated extensively in the world, the southern and northern cultivation extensively of China.Tomato disease species is complicated, main
To include all kinds of fungal diseases, bacteriosis and virosis, wherein fungal disease species is relatively more, early blight, late blight, grey mold
Disease, leaf mold etc. are tomato diseases relatively common in producing, and more rare for the report of Tomato gray leaf spot generation.
Tomato gray leaf spot worldwide has generation, occurs in warm moist area serious.The U.S., Israel, New Zealand etc.
The leaf spot of tomato as caused by stemphylium botryosum was reported on ground, and huge economic loss is caused to local tomato production.In recent years
Come being widely applied at home with import tomato variety, Tomato gray leaf spot occurs increasingly to weigh.2002, in Shandong fish
In the Tomato in Greenhouse of platform plantation, sick canopy rate has reached 43%, and has arrived the sick canopy rate of Second Year and reach more than 90%, and tomato subtracts
Yield reaches 20% or so, endangers the serious canopy room underproduction more than 80%.
Tomato gray leaf spot belongs to fungi (Stemphylium) by handle of crawling and caused, and the disease may occur in which on spire and old leaf.
Early stage blade face is covered with circular or irregular shape dot, and scab center is light brown, surrounding dark brown, later stage easily rupturable perforation.
Blade edge can also fall ill, and be in irregular shape scab along leaf margin, with extension, scab Liancheng sheet, dark brown becomes with blade
Dry, spot is gradually taken off to be greyish white to taupe, and symptom changes quickly, causes complete stool blade to turn yellow when endangering serious.
It is the thread class Common fungi of conidium one that handle of crawling, which belongs to fungi (Stemphylium), can cause various plants
Disease, up to the present, in the world it has been reported that about 150 kinds, China record more than 40 kinds.Cause the grey tikka of tomato in China
The handle of mainly crawling of disease belongs to two kinds of fungi, eggplant stemphylium botryosum (Stemphylium solani) and S. lycopersici bacterium
(Stemphylium lycopersici), in recent years investigation finds that eggplant stemphylium botryosum turns into the essentialspecies of harm tomato,
Its mycelia branch, separation, taupe, conidiophore differentiation is not obvious, and single life or 2-3 roots Shu Sheng, cylindric, brown are smooth.
Conidium basidixed, many Dan Sheng, club-like, brown is smooth, and muriform separates, and typically has a 1-5 tabula, several mediastinum films,
Hung at conidium medial septum contracting.
Conventional Tomato gray leaf spot identification detection method is mainly Morphological Identification method, by way of microscopy, observation point
Sporogenic form, determines the species of disease hazard, and this results contrast is accurate, but also there is also some problems, in disease hair
At raw initial stage, show before disease, spore is not produced, the species of disease can not accurately be judged by traditional microscopy mode, and be now
One critical period of chemical control, this period medication can play a multiplier effect.Being badly in need of one kind at present can be quick
The method that eggplant stemphylium botryosum especially can detect eggplant stemphylium botryosum initial stage in infection pathogen.
The content of the invention
The technical problems to be solved by the invention are how to detect eggplant stemphylium botryosum (Stemphylium solani).
In order to solve the above technical problems, present invention firstly provides can be used for detecting or aid in detection eggplant stemphylium botryosum or
Disease primer pair caused by detection or auxiliary detection eggplant stemphylium botryosum, the primer pair is respectively Stem-g7F and Stem- by title
G7R single stranded DNA composition；
The Stem-g7F be nucleotide sequence be SEQ ID No.1 in sequence table single stranded DNA；The Stem-g7R is
Nucleotide sequence is the single stranded DNA of SEQ ID No.2 in sequence table.
Institute Stem-g7F and the Stem-g7R mol ratio can be 1:1.Institute Stem-g7F and the Stem-g7 can be independent
Packaging, can also be packaged together.
In order to solve the above technical problems, present invention also offers can be used for detecting or aid in detection eggplant stemphylium botryosum or inspection
The system of disease caused by surveying or aiding in detection eggplant stemphylium botryosum, the system includes the primer pair.
Said system may also include the reagent and/or instrument carried out needed for quantitative fluorescent PCR.The carry out fluorescent quantitation
Reagent needed for PCR can be 2 × SYBR Mix of TIANGEN Biotech (Beijing) Co., Ltd..The carry out quantitative fluorescent PCR
Required instrument can be Real-time PCR instruments, and such as Applied Biosystems 7500Real Time PCRSystem are (beautiful
State, ABI).
Said system can be made up of the primer pair with the reagent and/or instrument needed for the progress quantitative fluorescent PCR.
The system can be to include the reagent or kit of related reagent.
In order to solve the above technical problems, present invention also offers following any applications of the primer pair：
X1, the application in detection or auxiliary detection eggplant stemphylium botryosum product is prepared；
X2, the application in disease product caused by preparing detection or auxiliary detection eggplant stemphylium botryosum.
X3, the application in detecting or aiding in detection eggplant stemphylium botryosum；
X4, the application in disease caused by detecting or aiding in detection eggplant stemphylium botryosum.
In order to solve the above technical problems, present invention also offers following any applications of the system：
X1, the application in detection or auxiliary detection eggplant stemphylium botryosum product is prepared；
X2, the application in disease product caused by preparing detection or auxiliary detection eggplant stemphylium botryosum.
X3, the application in detecting or aiding in detection eggplant stemphylium botryosum；
X4, the application in disease caused by detecting or aiding in detection eggplant stemphylium botryosum.
The product can be reagent or kit.
In order to solve the above technical problems, present invention also offers the preparation method of the primer pair, methods described includes：Will
Two single stranded DNAs of the primer pair are packed respectively.
In order to solve the above technical problems, present invention also offers detection or the method for auxiliary detection eggplant stemphylium botryosum, it is described
Method includes：Using the genomic DNA of testing sample as template, quantitative fluorescent PCR is carried out with the primer pair, according to fluorescent quantitation
The change of material determines whether the testing sample contains eggplant stemphylium botryosum or whether be eggplant stemphylium botryosum in PCR reaction systems.
The change of material can be determined by amplification curve in the quantitative fluorescent PCR reaction system：The quantitative fluorescent PCR
The amplification curve of reaction system is S type amplification curves, and the testing sample contains or candidate contains eggplant stemphylium botryosum or is or waits
Elect eggplant stemphylium botryosum as；The non-S types amplification curve of amplification curve of the quantitative fluorescent PCR reaction system, the testing sample is not
Contain or candidate does not contain eggplant stemphylium botryosum or the non-eggplant stemphylium botryosum of non-or candidate.
The change of material also can be by detecting that the size of reaction product is determined in the quantitative fluorescent PCR reaction system：Institute
The DNA fragmentation that quantitative fluorescent PCR reaction product contains 150bp is stated, the testing sample contains or candidate contains eggplant stemphylium botryosum
Or be or candidate is eggplant stemphylium botryosum；The quantitative fluorescent PCR reaction product does not contain 150bp DNA fragmentation, described to treat test sample
Product are not contained or candidate does not contain eggplant stemphylium botryosum or the non-eggplant stemphylium botryosum of non-or candidate.
The big I of the reaction product passes through electrophoresis detection.The DNA fragmentation of the 150bp may be embodied in 100-200bp
Between have a band.
In order to solve the above technical problems, present invention also offers the side of disease caused by detection or auxiliary detection eggplant stemphylium botryosum
Method, methods described includes：Using the genomic DNA of testing sample as template, quantitative fluorescent PCR is carried out with the primer pair, according to
The change of material determines whether the disease of the testing sample is caused a disease by eggplant stemphylium botryosum in quantitative fluorescent PCR reaction system
Evil.
The change of material can be determined by amplification curve in the quantitative fluorescent PCR reaction system：The quantitative fluorescent PCR
The amplification curve of reaction system is S type amplification curves, and the disease of the testing sample is or candidate is caused a disease by eggplant stemphylium botryosum
Evil；The non-S types amplification curve of amplification curve of the quantitative fluorescent PCR reaction system, the disease of the testing sample is not or waited
Choosing is not disease caused by eggplant stemphylium botryosum.
The change of material also can be by detecting that the size of reaction product is determined in the quantitative fluorescent PCR reaction system：Institute
State the DNA fragmentation that quantitative fluorescent PCR reaction product contains 150bp, the disease of the testing sample is or candidate is that eggplant handle of crawling is mould
Disease caused by bacterium；The quantitative fluorescent PCR reaction product does not contain 150bp DNA fragmentation, and the disease of the testing sample is not
Or candidate is not disease caused by eggplant stemphylium botryosum.
The big I of the reaction product passes through electrophoresis detection.The DNA fragmentation of the 150bp may be embodied in 100-200bp
Between have a band.
Above, concentration of two chains of the primer pair in the quantitative fluorescent PCR reaction system can be 0.2 μ
M。
The annealing temperature of the quantitative fluorescent PCR reaction can be 60 DEG C.The annealing conditions concretely 60 DEG C of annealing
32s.The reaction condition of quantitative fluorescent PCR reaction can be：95 DEG C of pre-degenerations 15min, 95 DEG C of denaturation 10s, 60 DEG C of annealing
32s, 40 circulations.
Detection eggplant stemphylium botryosum primer pair specificity of the invention is good, identification is quick：In environment, especially heliogreenhouse
Interior, microbe species are various, and eggplant stemphylium botryosum does not produce spore at the initial stage for infecting tomato, and this is just added to early stage early warning and monitoring
Difficulty.And eggplant stemphylium botryosum is difficult production spore under artificial isolated culture condition, which increases the difficulty of separation identification.This hair
Bright primer pair overcomes microexamination and the traditional plant pathology such as is separately cultured time-consuming and can not realize early warning and monitoring
Shortcoming.Compared with normal PCR detection technique, fluorescence quantitative PCR detection eggplant stemphylium botryosum is carried out using the primer pair of the present invention
Sensitivity improves 1000 times, up to 4.285 × 102fg·μL-1, and accurate quantitative analysis can be realized.With the primer pair of the present invention
Quantitative fluorescent PCR is carried out can quantitatively to be detected before not showing disease, while because compared with traditional PCR method in disease early period of origination
Sensitivity is greatly improved, and can apply to field soil seed borne fungi and Seed-associated fungi.
Brief description of the drawings
Fig. 1 is the specific detection result of primer pair.Wherein, N is negative control, and 1-9 is respectively eggplant stemphylium botryosum
(YQZ11020901), ash arrhizus bacteria (HG09021201), sclerotinite (HG09021301), alternaric bacteria (FQ15051201), eggplant
Alternaric bacteria (QZ08092301), sporulation (JGFQ15080715), melon alternaric bacteria (HG09042301), capsicum thorn disk
Spore bacterium (LJ10013001) and how main rod spore bacterium (SD21), M are DNA molecular amount standard.
Fig. 2 is the standard curve using construction of recombinant plasmid real-time fluorescence quantitative PCR.
Fig. 3 is the solubility curve for building standard curve process.
Fig. 4 is the amplification curve for building standard curve process.
Fig. 5 is regular-PCR sensitivity technique result.
Fig. 6 is real-time PCR sensitivity technique results.
Fig. 7 is phenotype of the tomato leaf in the different vaccination time of eggplant stemphylium botryosum inoculation.
Fig. 8 is the primer pair of embodiment 1 and primer pair Stem-g0 amplification curve.Wherein, Stem-g7 represents embodiment 1
Primer pair.
Fig. 9 is the primer pair of embodiment 1 and primer pair Stem-g0 solubility curve.Wherein, Stem-g7 represents embodiment 1
Primer pair.
Embodiment
The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the scope being not intended to be limiting of the invention.Experimental method in following embodiments, unless otherwise specified, be
Conventional method.Material, reagent, instrument used etc., unless otherwise specified, are commercially obtained in following embodiments.
Quantitative test in following examples, is respectively provided with three repetition experiments, results averaged.
2 × Taq PCR Master Mix in following embodiments are Beijing Bo Maide Bioisystech Co., Ltd product, 2
× SYBR Mix are TIANGEN Biotech's product.
The preparation of embodiment 1, the primer pair of detection eggplant stemphylium botryosum
The primer pair of detection eggplant stemphylium botryosum provided by the present invention is respectively Stem-g7F and Stem-g7R by title
Single stranded DNA constitute, Stem-g7F be nucleotide sequence be SEQ ID No.1 in sequence table single stranded DNA；Stem-g7R is nucleosides
Acid sequence is the single stranded DNA of SEQ ID No.2 in sequence table.
Two equal independent packagings of single stranded DNA in the primer pair, Stem-g7F and Stem-g7R mol ratio are 1:1.
The specific detection of embodiment 2, the primer pair of detection eggplant stemphylium botryosum
Strains tested is：1 plant of strain of eggplant stemphylium botryosum, 1 plant of the pathogen of Botrytis cinerea, 1 plant of phytophthora, 2 plants of alternaric bacterias, 3 plants of eggplants
Rod method and main rod spore (table 1) more than 1 plant, it is comprehensive that strains tested is stored in Vegetable ＆ Flower Inst., Chinese Academy of Agriculture Science's vegetable disease
Close preventing and treating seminar.
The strains tested information of table 1
| Sequence number | Numbering | Classification system | Chinese |
| 1 | YQZ11020901 | Stemphylium solani | Eggplant stemphylium botryosum |
| 2 | HG09021201 | Botrytis cinerea | Ash arrhizus bacteria |
| 3 | HG09021301 | Sclerotinia sclerotiotum | Sclerotinite |
| 4 | FQ15051201 | Alternaria alternata | Alternaric bacteria |
| 5 | QZ08092301 | Alternaria solani | Sporulation |
| 6 | JGFQ15080715 | Alternaria solani | Sporulation |
| 7 | HG09042301 | Alternaria cucumerina | Melon alternaric bacteria |
| 8 | LJ10013001 | Colletotrichum capsici | Capsicum colletotrichum |
| 9 | SD21 | Corynespora cassiicola | More main rod spore bacterium |
In table 1, the eggplant stemphylium botryosum (Stemphylium solani) that numbering is YQZ11020901 is documented in document (Xie
et al.,First report of Stemphylium solani causing leaf spot on wild eggplant
In China, Can.J.Plant Pathol., 2016, Vol.38, No.4,517-521) in；
The ash arrhizus bacteria (Botrytis cinerea) that numbering is HG09021201 is documented in document (Tang Ming etc., pyridine acyl bacterium
Amine is to the overall merit of gray mold of cucumber prevention effect, China's Vegetable, 2016 (2)：In 51-55)；
Sclerotinite (Sclerotinia sclerotiotum) that numbering is HG09021301, numbering are QZ08092301's
Capsicum colletotrichum (the Colletotrichum that sporulation (Alternaria solani), numbering are LJ10013001
Capsici the melon alternaric bacteria (Alternaria cucumerina) and numbering that), numbering is HG09042301 are many of SD21
Main rod spore bacterium (Corynespora cassiicola) is documented in document (high reed etc., the Dot-ELISA of cucumber rod spore leaf spot
Detection technique research, plant protection, 2013,39 (6)：In 69-73).
2nd, specific detection
The genomic DNA of each strains tested of extraction step 1, the primer pair of embodiment 1 is utilized to each strain gene group DNA
Regular-PCR and real-time PCR amplifications are carried out respectively, and the specificity of detection primer utilizes deionized water replacement gene group
DNA is used as negative control.
1st, common PCR reaction
Common PCR reaction system：
Common PCR reaction program：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, altogether
35 circulations；72 DEG C of supplement extension 5min.After PCR primer is detected through 2% agarose gel electrophoresis, gel imaging system analysis knot
Really.
2nd, Real-time PCR react
Real-time PCR reaction systems：
Real-time PCR reaction conditions：95 DEG C of pre-degenerations 15min, 95 DEG C of denaturation 10s, 60 DEG C of annealing 32s, 40 are followed
Ring, system automatically generates solubility curve.Real-time PCR reactions are using in the Real of Applied Biosystems 7500
Time PCR System (U.S., ABI) are carried out.By non-specific caused by the whether single exclusion dimer of melt curve analysis peak value
Property amplification, amplified production is subjected to electrophoresis detection, for the purpose of judging whether judge.
Real-time PCR reaction results only have eggplant stemphylium botryosum to amplify about 150bp band, and without dimerization
Body, other bacterial strains do not expand with negative control and obtain purpose product (Fig. 1), and common PCR reaction has also obtained identical knot
Really.Real-time PCR reaction results are shown, Real-time is carried out only by template of the genomic DNA of eggplant stemphylium botryosum
PCR has S type amplification curves when reacting, other bacterial strains, without S type amplification curves, show that the primer pair can be special with negative control
Opposite sex detection eggplant stemphylium botryosum.
When carrying out Real-time PCR reaction detection testing samples using the primer pair of embodiment 1, it can be expanded by it
Curve determines that sample to be tested, also can be by detecting Real-time whether containing eggplant stemphylium botryosum or whether be eggplant stemphylium botryosum
PCR reaction products determine whether sample to be tested contains eggplant stemphylium botryosum or whether be eggplant stemphylium botryosum：As Real-time PCR are anti-
The curve that should be obtained is S type amplification curves, or Real-time PCR reaction products contain 150bp purpose band, then production to be measured
Thing contains eggplant stemphylium botryosum or is eggplant stemphylium botryosum；Such as Real-time PCR react the obtained non-S types amplification curve of curve, or
Real-time PCR reaction products do not contain 150bp purpose band, then product to be measured does not contain eggplant stemphylium botryosum or is not eggplant
Stemphylium botryosum.
The standard curve of embodiment 3, the primer pair of detection eggplant stemphylium botryosum
The structure of recombinant plasmid：Genomic DNA using the eggplant stemphylium botryosum of embodiment 2 utilizes drawing for embodiment 1 as template
Thing is to entering performing PCR amplification, by the correct PCR primer of obtained sequence and pEASY-TI Cloning Vector (Beijing Quan Shi gold
Bioisystech Co., Ltd, catalog number (Cat.No.) CT101-02) connection, recombinant plasmid is obtained, the recombinant plasmid is named as pEASY-TI-
Stem7。
PEASY-TI-Stem7 is dissolved in deionized water and obtains plasmid concentration for 1.57 × 102ng/μL(3.7×1010Copy
Shellfish/μ L) pEASY-TI-Stem7 solution, by the pEASY-TI-Stem7 solution carry out 10 times of gradient dilutions, respectively obtain
PEASY-TI-Stem7 concentration is 1.57 × 101ng/μL(3.7×109Copy/μ L), 1.57 × 100ng/μL(3.7×108Copy
Shellfish/μ L), 1.57 × 10-1ng/μL(3.7×107Copy/μ L), 1.57 × 10-2ng/μL(3.7×106Copy/μ L), 1.57 ×
10-3ng/μL(3.7×105Copy/μ L), 1.57 × 10-4ng/μL(3.7×104Copy/μ L), 1.57 × 10-5ng/μL(3.7
×103Copy/μ L) and 1.57 × 10-6μg/μL(3.7×102Copy/μ L) pEASY-TI-Stem7 solution.
According to the reaction system of the step 2 of embodiment 2, strain gene group DNA is replaced with to above-mentioned each pEASY-TI- respectively
Stem7 solution, carries out Real-time PCR according to the reaction condition of the step 2 of embodiment 2, is respectively provided with three repetitions.
Using the logarithm value of template concentrations as abscissa, the Ct values using Real-time PCR build real- as ordinate
Time PCR standard curves are：Y=30.789-3.2989x, coefficient correlation (R2) it is 0.9919, quantitative fluorescent PCR system is automatic
The amplification efficiency of generation is 100.974% (Fig. 2), and solubility curve is unimodal (Fig. 3), is expanded without the non-specificity such as dimer
Increase (Fig. 4).
Embodiment 4, the detection of the sensitivity of the primer pair of detection eggplant stemphylium botryosum
Strain number is dissolved in deionized water for YQZ11020901 eggplant stemphylium botryosum genomic DNA and obtains DNA concentration
For 4.285 × 108fg·μL-1(1.12×1010Copy/μ L Genomic DNA solution, carries out 10 times of gradients dilute by the DNA solution
Release, it is 4.285 × 10 to respectively obtain genomic DNA concentration7fg·μL-1(1.12×109Genomic DNA copy/μ L), 4.285
×106fg·μL-1(1.12×108Genomic DNA copy/μ L), 4.285 × 105fg·μL-1(1.12×107Genomic DNA is copied
Shellfish/μ L), 4.285 × 104fg·μL-1(1.12×106Genomic DNA copy/μ L), 4.285 × 103fg·μL-1(1.12×
105Genomic DNA copy/μ L), 4.285 × 102fg·μL-1(1.12×104Genomic DNA copy/μ L), 4.285 ×
101fg·μL-1(1.12×103Genomic DNA copy/μ L), 4.285 × 100fg·μL-1(1.12×102Genomic DNA is copied
Shellfish/μ L) and 4.285 × 10-1fg·μL-1(1.12×101Genomic DNA copy/μ L) Genomic DNA solution.
According to the reaction system of the step 2 of embodiment 2, strain gene group DNA is replaced with to above-mentioned each eggplant stemphylium botryosum respectively
Genomic DNA solution, carry out the primer that Real-time PCR detect embodiment 1 according to the reaction condition of the step 2 of embodiment 2
To sensitivity, using agarose gel electrophoresis detect PCR primer, be used as negative control by the use of deionized water.
According to the reaction system of the step 1 of embodiment 2, strain gene group DNA is replaced with to above-mentioned each eggplant stemphylium botryosum respectively
Genomic DNA solution, according to the step 1 of embodiment 2 reaction condition carry out regular-PCR detection embodiment 1 primer pair spirit
Sensitivity, negative control is used as by the use of deionized water.The calibration curve coefficient correlation R of target gene and reference gene2More than 0.99,
PCR amplification efficiencies E is between 0.9~1.05, and both amplification efficiency differences are no more than 5%.
As a result show, when carrying out regular-PCR amplification, the concentration of eggplant stemphylium botryosum Genomic DNA solution 4.285 ×
105fg·μL-1And its purposeful band during the above, and the concentration of eggplant stemphylium botryosum genomic DNA less than 4.285 ×
105fg·μL-1When and negative control without purpose band (Fig. 5), show, regular-PCR amplification embodiment 1 primer pair spirit
Sensitivity is 4.285 × 105fg·μL-1；When carrying out real-time PCR, the concentration of eggplant stemphylium botryosum Genomic DNA solution exists
4.285×102fg·μL-1And its have amplification curve during the above, and the concentration of eggplant stemphylium botryosum Genomic DNA solution less than
4.285×105fg·μL-1When and negative control without amplification curve (Fig. 6), show, real-time PCR embodiment 1
The sensitivity of primer pair is 4.285 × 102fg·μL-1。
In Fig. 5 and Fig. 6,1-10 is respectively that eggplant stemphylium botryosum genomic DNA concentration is 4.285 × 108fg·μL-1、4.285
×107fg·μL-1、4.285×106fg·μL-1、4.285×105fg·μL-1、4.285×104fg·μL-1、4.285×
103fg·μL-1、4.285×102fg·μL-1、4.285×101fg·μL-1、4.285×100fg·μL-1With 4.285 × 10- 1fg·μL-1Eggplant stemphylium botryosum Genomic DNA solution result, N is negative control, and M is DNA molecular amount standard.
Embodiment 5, the tomato leaf Real-time PCR detections of eggplant stemphylium botryosum inoculation
By low temperature, 4 DEG C preserve eggplant stemphylium botryosum bacterial strain of the strain number for YQZ11020901 using PDA plate activation, turn
It is connected to after PD fluid nutrient mediums, 28 DEG C of culture 7d, is smashed with soy bean milk making machine after mycelia, with spray bacteria suspension method inoculating tomato blade inoculation
The tomato plant of 3-4 leaf phases, heat and moisture preserving, respectively at inoculation 6h, 12h, 24h, 48h, 72h, 96h, 108h period to tomato
Blade carries out sampling (Fig. 7) of taking pictures.Extract after each tomato leaf genomic DNA (concentration is 86.7ng/ μ l), according to embodiment
The reaction system of 2 steps 2, strain gene group DNA is replaced with respectively each above-mentioned tomato leaf genomic DNA, according to implementation
The reaction condition of the step 2 of example 2 carries out Real-timePCR, and negative control is used as by the use of deionized water.
As a result show, eggplant stemphylium botryosum inoculating tomato blade 6h can detect eggplant stemphylium botryosum in tomato leaf, and
Bacterium amount, which is increased over time, to be continuously increased；During eggplant stemphylium botryosum inoculating tomato blade 108h, whole leaves infected eggplant handle of crawling is mould
Bacterium, the genome of eggplant stemphylium botryosum in the genomic DNA that now tomato leaf is obtained is calculated according to the standard curve of embodiment 3
DNA content is 1.16 × 10-3ng/μl。
The eggplant stemphylium botryosum content dynamic change on plants of table 2
The comparison of comparative example 1, different primers to detection eggplant stemphylium botryosum
Strain number is crawled for YQZ11020901 eggplant respectively using the primer pair and primer pair Stem-g0 of embodiment 1
The genomic DNA of handle mould carries out Real-time PCR, reaction system and the equal step 2 of be the same as Example 2 of reaction condition.Stem-g0
Primer sequence be shown in Table 3, be used as negative control by the use of deionized water.
The Stem-g0 of table 3 primer sequence
As a result show, the primer pair positive amplification of embodiment 1 is good, and negative control is not expanded, and solubility curve is single
Peak value, without primer dimer；Primer pair Stem-g0 positive amplifications are good, and there is amplification in the negative control later stage, and solubility curve is double
, there is primer dimer (Fig. 8 and Fig. 9) at peak, illustrates that effect of the primer pair of embodiment 1 when detecting eggplant stemphylium botryosum is better than
Stem-g0。
<110>Vegetable ＆ Flower Inst., Chinese Academy of Agriculture Science
<120>Detect primer pair and its application of eggplant stemphylium botryosum
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 1
agacaatctt cccgaaaatc cgctg 25
<210> 2
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 2
ccttgatctc acccttgaac tggcc 25
Claims (9)
1. primer pair, the single stranded DNA for being respectively Stem-g7F and Stem-g7R by title is constituted；
The Stem-g7F be nucleotide sequence be SEQ ID No.1 in sequence table single stranded DNA；The Stem-g7R is nucleosides
Acid sequence is the single stranded DNA of SEQ ID No.2 in sequence table.
2. the system for detecting or aiding in disease caused by detecting eggplant stemphylium botryosum or detection or auxiliary detection eggplant stemphylium botryosum, bag
Include the primer pair described in claim 1.
3. system according to claim 2, it is characterised in that：The system is also included needed for progress quantitative fluorescent PCR
Reagent and/or instrument.
4. following any applications of primer pair described in claim 1：
X1, the application in detection or auxiliary detection eggplant stemphylium botryosum product is prepared；
X2, the application in disease product caused by preparing detection or auxiliary detection eggplant stemphylium botryosum.
X3, the application in detecting or aiding in detection eggplant stemphylium botryosum；
X4, the application in disease caused by detecting or aiding in detection eggplant stemphylium botryosum.
5. following any applications of the system described in Claims 2 or 3：
X1, the application in detection or auxiliary detection eggplant stemphylium botryosum product is prepared；
X2, the application in disease product caused by preparing detection or auxiliary detection eggplant stemphylium botryosum.
X3, the application in detecting or aiding in detection eggplant stemphylium botryosum；
X4, the application in disease caused by detecting or aiding in detection eggplant stemphylium botryosum.
6. the preparation method of primer pair described in claim 1, including：Two single stranded DNAs of the primer pair are wrapped respectively
Dress.
7. the preparation method of system described in Claims 2 or 3, including：Each single stranded DNA of the primer pair and/or progress is glimmering
Reagent needed for Fluorescent Quantitative PCR is individually packed.
8. detection or the method for auxiliary detection eggplant stemphylium botryosum, including：Using the genomic DNA of testing sample as template, right is used
It is required that primer pair described in 1 carries out quantitative fluorescent PCR, treated according to being determined the change of material in quantitative fluorescent PCR reaction system
Whether whether test sample product contain eggplant stemphylium botryosum or be eggplant stemphylium botryosum.
9. the method for disease caused by detection or auxiliary detection eggplant stemphylium botryosum, including：Using the genomic DNA of testing sample as mould
Plate, quantitative fluorescent PCR is carried out with primer pair described in claim 1, true according to the change of material in quantitative fluorescent PCR reaction system
Whether the disease of the fixed testing sample is disease caused by eggplant stemphylium botryosum.
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| CN201710307083.2A CN106978505B (en) | 2017-05-04 | 2017-05-04 | Primer pair for detecting pedunculosis solani and application thereof |
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| CN201710307083.2A CN106978505B (en) | 2017-05-04 | 2017-05-04 | Primer pair for detecting pedunculosis solani and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112725494A (en) * | 2020-12-31 | 2021-04-30 | 中国农业科学院蔬菜花卉研究所 | Method and primer for detecting 17 tomato pathogens by using micro-fluidic chip |
Citations (2)
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|---|---|---|---|---|
| CN105754926A (en) * | 2016-05-19 | 2016-07-13 | 中国农业科学院蔬菜花卉研究所 | Method for quickly inducing spore production of stemphylium botryosum |
| CN106591418A (en) * | 2016-12-20 | 2017-04-26 | 天津市农业生物技术研究中心 | Identification method for identifying resistance of tomatoes on gray leaf spot by utilizing in-vitro leaves |
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2017
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105754926A (en) * | 2016-05-19 | 2016-07-13 | 中国农业科学院蔬菜花卉研究所 | Method for quickly inducing spore production of stemphylium botryosum |
| CN106591418A (en) * | 2016-12-20 | 2017-04-26 | 天津市农业生物技术研究中心 | Identification method for identifying resistance of tomatoes on gray leaf spot by utilizing in-vitro leaves |
Non-Patent Citations (3)
| Title |
|---|
| GENBANK: "GenBank:KC796643.1", 《GENBANK》 * |
| XUE-WEN XIE ET AL: "First report of Stemphylium solani causing leaf spot on wild eggplant in China", 《CAN. J. PLANT PATHOL.》 * |
| 杜公福等: "海南省冬季北运蔬菜匍柄霉叶斑病病原的鉴定", 《植物保护》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112725494A (en) * | 2020-12-31 | 2021-04-30 | 中国农业科学院蔬菜花卉研究所 | Method and primer for detecting 17 tomato pathogens by using micro-fluidic chip |
| CN112725494B (en) * | 2020-12-31 | 2022-06-14 | 中国农业科学院蔬菜花卉研究所 | Method and primer for detecting 17 tomato pathogens by using micro-fluidic chip |
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| CN106978505B (en) | 2020-03-31 |
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