CN108384881A - The fluorescent quantitative PCR detection method of coal dirt vacation tail spore - Google Patents
The fluorescent quantitative PCR detection method of coal dirt vacation tail spore Download PDFInfo
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- CN108384881A CN108384881A CN201810473961.2A CN201810473961A CN108384881A CN 108384881 A CN108384881 A CN 108384881A CN 201810473961 A CN201810473961 A CN 201810473961A CN 108384881 A CN108384881 A CN 108384881A
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Abstract
The invention discloses a kind of fluorescent quantitative PCR detection methods of coal dirt vacation tail spore.The present invention provides the primer pairs for detecting coal dirt vacation tail spore, are following any：A1) the primer pair shown in SEQ ID No.1 and SEQ ID No.2；A2) by by SEQ ID No.1 and SEQ ID No.2, two single strand dnas shown in gained sequence are formed by the substitution of one or several nucleotide and/or after lacking and oring add, and with a1) described in the identical primer pair of primer pair function.Coal dirt vacation tail spore leaf spot fungi fluorescent quantitative PCR detection method provided by the invention is easy to operate, quickly, high specificity, high sensitivity；And can Quantitative Monitoring directly be carried out to coal dirt vacation tail spore on tomato field plant, good basis is provided for the early prediction forecast of disease.Therefore, this method is suitable in plant disease diagnostic field wide popularization and application.
Description
Technical field
The present invention relates to biotechnologies, and in particular to a kind of fluorescent quantitative PCR detection method of coal dirt vacation tail spore.
Background technology
Tomato coal dirt vacation tail spore leaf spot is a kind of important fungal disease in tomato production, and pathogen is the dirty false tail of coal
Spore (Pseudocercospora fuligena) belongs to Ascomycota (Ascomycota), seat capsule bacterium in classification of fungi system
Guiding principle (Dothideomycetes), smoke from burbning coal mesh (Capnodiales), spherical cavity Cordycepps (Mycosphaerellaceae) pseudo-cercospora
(Pseudocercospora) fungi.Tomato coal dirt vacation tail spore leaf spot main harm tomato leaf, when plant falls ill, inferior leads
Piece shows symptom first, forms the chlorisis spot of 1-5mm wide in face of blade and the back side, becomes light brown later, eventually become
Black, leaf back forms brown to the mould layer of black when humidity is big.When falling ill serious, infected blade is withered and falls off from plant,
It can cause about 32% production loss.Tomato coal dirt vacation tail spore leaf spot belongs to high temperature and humidity type disease, is distributed mainly on the torrid zone
Or subtropical zone, there are different journeys in Asia, Africa, Oceania, North America, South America and northern China and southern area
The generation of degree.
Currently, people are mainly by the culture form of Symptom Observation and pathogen come to tomato coal dirt vacation tail spore germ
Carry out identification detection.Symptom Observation method needs testing staff to have very sturdy professional knowledge, but due to coal dirt vacation tail spore leaf
Pinta and leaf mold disease symptoms are more similar, therefore easily lead to the mistaken diagnosis of disease；Bacteria culturing Morphological Identification also needs to enrich
Professional knowledge, and the cultivation cycle of pathogen is longer and situations such as be susceptible to pollution.Two kinds of detection methods all need to wait planting
Pathogen could be identified after strain performance manifest symptom, this hysteresis quality frequently can lead to miss best prevention opportunity.Cause
This, develops a kind of technology of i.e. detectable coal dirt vacation tail spore germ before Symptoms, can control the sprawling of disease in time.
The application of serological technique provides a kind of new method for the detection of plant epiphyte, but the sensitivity compared with
It is low and be susceptible to false positive reaction, and cannot directly detect the pathogen in vegetable material.Relative to serological technique,
The birth of round pcr, not only increases sensitivity, the pathogen in acceptable directly qualitative detection vegetable material.Real-time fluorescence
Quantitative PCR technique is a kind of technology of the real time monitoring target gene amplification to grow up on the basis of regular-PCR technology, can
With accurate quantification target nucleic acid.Its cardinal principle is that fluorophor is added in PCR reaction systems, using fluorescence signal accumulation come real
When testing goal gene amplification procedure, finally according to Ct values and standard curve quantitative analysis target nucleic acid.Compared with other technologies,
Real-time fluorescence quantitative PCR detection technique has following features：1) susceptibility is high：The target gene of low-copy, sensitivity can be detected
It is usually 100 times higher than regular-PCR；2) quantitation capabilities are strong：Simple flow can obtain the quantitative data of high quality；3) dynamics
Range is wide：About 9 orders of magnitude；4) time-consuming short, do not need electrophoresis detection；5) safety：Without EB or radioactive substance.
Invention content
The object of the present invention is to provide a kind of fluorescent quantitative PCR detection methods of coal dirt vacation tail spore.
In a first aspect, a kind of claimed primer pair for detecting coal dirt vacation tail spore.
Primer pair provided by the present invention for detecting coal dirt vacation tail spore can be following any：
(a1) primer pair that single stranded DNA shown in single stranded DNA and SEQ ID No.2 shown in SEQ ID No.1 forms；
(a2) by by SEQ ID No.1 and SEQ ID No.2 by one or several nucleotide substitution and/or missing
And/or two single strand dnas composition shown in gained sequence after addition, and it is identical with primer pair function described in (a1)
Primer pair.
Wherein, two single stranded DNAs in the primer pair both can be packed individually, can also equimolar mixing packet
Dress.
Second aspect, a kind of claimed reagent for detecting coal dirt vacation tail spore.
Reagent provided by the present invention for detecting coal dirt vacation tail spore, it may include previously described primer pair, SYBR
Green I real-time fluorescence quantitative PCRs amplification buffers and dd H2O。
Further, the SYBR Green I real-time fluorescence quantitative PCR amplification buffers, it may include dNTPs, Mg2+、
SYBR Green I and Taq archaeal dna polymerases.
Further, reference dye can also be contained in the reagent.
In the specific implementation mode of the present invention, the reference dye is specially ROX reference dyes (ROX Reference
Dye)。
The third aspect, a kind of claimed kit for detecting coal dirt vacation tail spore.
Kit provided by the present invention for detecting coal dirt vacation tail spore can contain following (b1) and (b2)：
(b1) coal dirt vacation tail spore positive plasmid；
(b2) primer pair described previously or previously described reagent.
Further, the coal dirt vacation tail spore positive plasmid can be the plasmid containing DNA fragmentation shown in SEQ ID No.3.
In the specific implementation mode of the present invention, the coal dirt vacation tail spore positive plasmid is specially by SEQ ID No.3 institutes
Show the recombinant plasmid obtained after DNA fragmentation is connected with pEASY-T1 carriers.
Fourth aspect, in a kind of claimed detection sample to be tested whether the method containing coal dirt vacation tail spore.
In detection sample to be tested provided by the present invention whether the method containing coal dirt vacation tail spore, can be following (c1) or
(c2)：
(c1) it may include following steps：Total DNA is extracted from sample to be tested, as template, primer pair described earlier
PCR amplification is carried out, amplified production is obtained；Then according to as follows determine the sample to be tested in whether contain coal dirt vacation tail spore：Such as
DNA fragmentation containing 232bp in amplified production described in fruit then contains in the sample to be tested or candidate contains coal dirt vacation tail spore；
If not containing the DNA fragmentation of 232bp in the amplified production, do not contained in the sample to be tested or candidate dirty without containing coal
False tail spore.
Further, the DNA fragmentation of the 232bp is DNA fragmentation shown in SEQ ID No.3.
(c2) it may include following steps：Total DNA is extracted from sample to be tested, as template, primer pair described earlier
Carry out fluorescent quantitative PCR；Then whether dirty false containing coal in the sample to be tested according to determining as follows according to amplification curve
Tail spore：If gained amplification curve is S types or J-type curve, contain in the sample to be tested or candidate dirty false containing tomato coal
Tail spore bacterium；If gained amplification curve is straight line, do not contained in the sample to be tested or candidate without containing the dirty false tail of coal
Spore.
5th aspect, a kind of claimed method detecting coal dirt vacation tail spore target DNA content in sample to be tested.
The method of coal dirt vacation tail spore Genome DNA content, specifically may include in detection sample to be tested provided by the present invention
Following steps：
(d1) the coal dirt vacation tail spore positive plasmid in the kit is diluted to serial known concentration, respectively as mould
Plate, primer pair described earlier carry out fluorescent quantitative PCR, and drawing quantitative fluorescent PCR standard curve, (abscissa is that coal is dirty
The logarithm of false tail spore positive plasmid copy number, ordinate are Ct values)；
(d2) total DNA is extracted from sample to be tested, as template, primer pair described earlier carries out quantitative fluorescent PCR
It expands (with step (d1) same to condition)；Then the quantitative fluorescent PCR standard curve drawn according to step (d1) obtains described wait for
The content of coal dirt vacation tail spore genomic DNA in test sample sheet.
Wherein, in step (d2), coal dirt vacation tail spore genome in the sample to be tested is specifically obtained as follows
The content of DNA：The Ct values of the sample to be tested are substituted into the quantitative fluorescent PCR standard curve that step (d1) is drawn, to
Obtain the content of coal dirt vacation tail spore genomic DNA described in the sample to be tested.
In the specific implementation mode of the present invention, the quantitative fluorescent PCR is specially that SYBR Green I real-time fluorescences are fixed
Measure PCR.
In fourth aspect and the 5th aspect, the annealing temperature of the PCR amplification and the fluorescent quantitative PCR has
Body can be 64 DEG C.
In fourth aspect and the 5th aspect, the sample to be tested can be the plant containing or without containing coal dirt vacation tail spore
Sample, such as the tissue or seed of tomato.
6th aspect, claimed following any application：
(e1) application of the previously described primer pair in preparing reagent described previously or the kit.
(e2) application of previously described primer pair or the reagent or the kit in detecting coal dirt vacation tail spore, or
Application of the person in preparing the product for detecting coal dirt vacation tail spore.
(e3) previously described primer pair or the reagent or the kit or the method are to the dirty false tail of tomato coal
Spore leaf spot carries out the application in early prediction forecast.
Wherein, the detection coal dirt vacation tail spore can be qualitative detection, or quantitative detection.
Coal dirt vacation tail spore fluorescent quantificationally PCR detecting kit provided by the invention, for coal dirt vacation tail spore genome Avr4
The target sequence design primer of gene specific, by the optimization of reaction condition (primer concentration, annealing temperature etc.), using quantitative detection
System (including ABI Prism systems, PE Applied Bioasystems, Bio-Rad), can be quick, sensitive, accurately quantitative
Detect the coal dirt vacation tail spore on tomato plant.This fluorescent quantitative PCR detection method is tested through being repeated several times, and repdocutbility is good, draws
The detection limit of object is 67 copy numbers, and 100 times are higher by than regular-PCR sensitivity.
Compared with the prior art, the present invention has the following advantages and effect：
1) high specific, Rapid identification
The specific sequence of primer amplification coal dirt vacation tail spore, PCR detection specificity are high.Traditional tomato coal dirt vacation tail spore leaf
The diagnostic method of pinta is mainly identified by Symptom Observation, pathogenicbacteria separation culture and micromorphology, and disease at the initial stage of a disease
Shape is not easy to discover, and pathogen is slow-growing in tablet culture, therefore can lead to the lag and detection time of detection
Extend.The present invention overcomes this shortcomings, can be quickly detected at the initial stage of a disease to pathogen.
2) it precisely quantifies, high sensitivity
Compared with regular-PCR, the present invention can detect object bacteria with accurate quantitative analysis.In 30.6ng/ μ L-0.306fg/ μ L targets
In the concentration range of DNA, good linear relationship is all had, detection sensitivity is higher by 100 times than regular-PCR；In addition, the detection
The repdocutbility of method is good.
3) practicability is good, has a wide range of application
The coal dirt vacation tail spore on detection infected seed and tomato field plant can be quantified, morbidity threshold is passed in conjunction with the disease kind
Value can be that the early prediction of disease forecasts providing method foundation.
Description of the drawings
Fig. 1 is the PCR amplification result of tomato coal dirt vacation tail spore specific primer pair in embodiment 2.1,2 template is that coal is dirty
The template of false tail spore genomic DNA, 3-11 is respectively how main stick spore (Corynespora cassiicola) DNA, Botrytis cinerea
Bacterium (Botrytis cinerea) DNA, Fusarium oxysporum (Fusarium oxysporum) DNA, Fusarinm solani (Fusarium
Solani) DNA, eggplant are crawled mould (Stemphylium solani) DNA of handle, sclerotinite (Sclerotinia sclerotiorum)
DNA, Rhizoctonia solani Kuhn (Rhizoctonia solani) DNA, sporulation (Alternaria solani) DNA, capsicum epidemic disease
Mould (Phytophthora capsici) DNA；N is negative control；M is BM 5000DNA Marker (TIANGEN companies).
Fig. 2 is quantitative fluorescent PCR specific primer JWB-9F/JWB-7R amplification curves in embodiment 3.The longitudinal axis is Delta
Rn(ΔRn)；Horizontal axis is recurring number (cycle number), and the template of curve 1,2,3,4,5,6,7,8,9,10 is positive criteria
Product, DNA content is respectively 30.6ng, 3.06ng, 306pg, 30.6pg, 3.06pg, 306fg, 30.6fg in reaction system,
3.06fg；0.306fg；0.0306fg；11 be negative control.
Fig. 3 is the solubility curve of quantitative fluorescent PCR specific primer JWB-9F/JWB-7R amplifications in embodiment 3.The longitudinal axis is
Derivative Reporter (- Δ Rn ＇), horizontal axis are temperature (Temperature), curve 1,2,3,4,5,6,7,8,9,10
Template be positive criteria product, DNA content is respectively 30.6ng, 3.06ng, 306pg, 30.6pg, 3.06pg in reaction system,
306fg, 30.6fg, 3.06fg；0.306fg；0.0306fg；11 be negative control.
Fig. 4 is the standard curve of quantitative fluorescent PCR specific primer JWB-9F/JWB-7R amplifications in embodiment 3.The longitudinal axis is
Ct values (Ct Value), horizontal axis are copy number logarithm (Log copies)；Linear relationship is y=-3.522x+40.242, mark
Standard misses R2It is 0.976.
Fig. 5 is to expand obtained by the fluorescence quantitative PCR detection of tomato field coal dirt vacation tail spore leaf spot tissue samples in embodiment 4
Increase curve.1 is positive control, 2-8 be respectively to number is FQ16070301, FQ16070302, FQ16070303,
The tomato coal dirt vacation tail spore leaf spot tissue samples DNA of FQ16070304, FQ16070305, FQ16070306, FQ16070307
Amplification curve, 9 be negative control.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1, the design of primer and synthesis
To avoid primer from occurring false positive results during PCR amplification or being carried out to other non-purpose bacterial strains non-specific
Amplification, the present invention Avr4 DNA homolog sequence not of the same race to pseudo-cercospora (Pseudocercospora) have carried out system point
Analysis, compares the sequence using MEGA5, determines that coal dirt vacation tail spore (Pseudocercospora fuligena) is special
Property sequence coal dirt vacation tail spore specificity is designed with software Primer Primer 5.0 according to specific sequence difference site
Fluorescence quantification PCR primer JWB-9F/JWB-7R；In the websites NCBI, primer is passed through into BLAST program and other biological kind
Tetraploid rice is carried out, does not as a result have homology between display and other biological kind, and not of the same race under pseudo-cercospora
Between show high specificity.
Preceding primer JWB-9F：5’-TCGCCAGCAGATG ACTCCTAC-3’(SEQ ID No.1)；
Primer JWB-7R afterwards：5’-TATTTGCCTGCGCAGATGGTCAG-3’(SEQ ID No.2).
Theoretical amplification fragment length is 232bp, as shown in SEQ ID No.3.
Embodiment 2, the detection of the primer specificity of coal dirt vacation tail spore
Using tomato coal dirt vacation tail spore leaf spot fungi --- coal dirt vacation tail spore (Pseudocercospora fuligena),
And other tomato Common mycotic pathogens are material (table 1), the coal dirt vacation tail spore primer pair JWB- designed embodiment 1
9F/JWB-7R carries out specific detection.After all bacterial strains are activated by PDA solid mediums, it is transferred to PD fluid nutrient medium cultures, 28
DEG C culture after a week, collects mycelia, extraction pathogen DNA.The extraction of DNA is using improvement CTAB extraction methods.
Using coal dirt vacation tail spore bacterium and other tomato encountered pathogenic fungal genomic DNAs template, primer pair JWB-9F/ is utilized
JWB-7R carries out regular-PCR amplification to the above fungi, to detect the primer specificity.Common PCR reaction system is as follows：Overall reaction
20 μ L, 2 × Taq Master PCR Mix of volume 10 μ L, primer (10 μm of ol/L) each 0.5 μ L, 1 μ L, dd H of template DNA2O is mended
Enough to 20 μ L.PCR response procedures are 94 DEG C of pre-degeneration 4min；94 DEG C of denaturation 30s, 64 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 35
A cycle；72 DEG C of extension 10min.PCR product carries out electrophoresis detection with 1.5% agarose is solidifying.
Primer JWB-9F/JWB-7R is to the PCR amplification of coal dirt vacation tail spore and other tomato disease fungus as a result, showing that this draws
Object has good specificity.Under 64 DEG C of annealing conditions, only coal dirt vacation tail spore amplifies product (the SEQ ID of 232bp
No.3), other disease fungus and negative control are without any amplified production.It is specific as shown in Figure 1.
1 primer specificity of table detects bacterial strain
| Number | Strain number | Latin name | Chinese | Source | Disease title |
| 1 | FQ10013003 | Pseudocercospora fuligena | The dirty false tail of coal Spore | Chinese Academy of Agricultural Sciences's vegetable or flower Research institute | Tomato coal dirt vacation tail spore leaf Pinta |
| 2 | HG15030802 | Corynespora cassiicola | Mostly main stick spore Bacterium | Chinese Academy of Agricultural Sciences's vegetable or flower Research institute | Tomato stick spore leaf spot |
| 3 | FQ130010701 | Botrytis cinerea | Botrytis cinerea Bacterium | Chinese Academy of Agricultural Sciences's vegetable or flower Research institute | Graw mold of tomato |
| 4 | HG15060516 | Fusarium oxysporum | Sharp spore reaping hook Bacterium | Chinese Academy of Agricultural Sciences's vegetable or flower Research institute | Tomato wilt |
| 5 | FQ14111912 | Stemphylium solani | Eggplant stemphylium | Chinese Academy of Agricultural Sciences's vegetable or flower Research institute | Tomato gray leaf spot |
| 6 | DBC16070604 | Rhizoctonia solani | Miliary damping-off Bacterium | Chinese Academy of Agricultural Sciences's vegetable or flower Research institute | Tomato seedling damping-off |
| 7 | HG13060501 | Fusarium solani | Eggplant disease reaping hook Bacterium | Chinese Academy of Agricultural Sciences's vegetable or flower Research institute | Tomato root rot |
| 8 | JGFQ1508070 2 | Alternaria solani | Alternaria solani sorauer Bacterium | Chinese Academy of Agricultural Sciences's vegetable or flower Research institute | Early blight of tomato |
| 9 | LJ08042301 | Sclerotinia sclerotiorum | Sclerotinite | Chinese Academy of Agricultural Sciences's vegetable or flower Research institute | Sclerotinia of tomato |
| 10 | LJ12010805 | Phytophthora capsici | Phytophthora capsici Bacterium | Chinese Academy of Agricultural Sciences's vegetable or flower Research institute | Tomato cotton disease |
The structure of the quantitative fluorescent PCR standard curve of embodiment 3, coal dirt vacation tail spore
The subregion of coal dirt vacation tail spore Avr4 genes is expanded using primer JWB-9F/JWB-7R, is cut after PCR product electrophoresis
Under the gel containing target fragment, target fragment is recycled using gel reagents box, target fragment and pEASY-T1 carriers are at room temperature
Be attached, after be transferred to Escherichia coli Trans-1.Converted product is coated on the LB tablets containing ampicillin and stays overnight for 38 DEG C
Culture, picking hickie carry out PCR identifications using primer pair JWB-9F/JWB-7R.Sequence analysis is carried out to positive colony, with true
Recognize the accuracy of result (DNA fragmentation shown in SEQ ID No.3 is connected with pEASY-T1 carriers).Picking PCR and sequence analysis
It is the colonies of the positive, is inoculated into the LB culture mediums containing ampicillin shake culture overnight.It is carried using plasmid is small
Kit extracts Plasmid DNA as positive criteria product.
DNA is quantitative through ultraviolet specrophotometer, is 30.6ng/ μ L-0.0306fg/ μ L through 10 times of gradient dilutions.With the positive
Standard items and RNase-Free dd H2O is that template carries out fluorescent quantitative PCR, after reaction, according to each concentration ladder
The cycle threshold (Ct) of degree, drawing quantitative fluorescent PCR standard curve, (abscissa is pair of coal dirt vacation tail spore positive plasmid copy number
Numerical value, ordinate are Ct values).
Real-time fluorescence quantitative PCR reaction system is：Quantitative fluorescent PCR reaction system total volume is 20 μ L, wherein 2 ×
Super Real PreMix Plus (include dNTP Mixture, Mg2+, SYBR Green I and Taq archaeal dna polymerases) and 10 μ L,
Each 0.2 μ L, 50 × ROX Reference Dye, 0.4 μ of preceding primer JWB-9F and rear primer JWB-7R of a concentration of 10 μm of ol/L
L, 10 times of gradient dilution liquid of standard items, 1 μ L, RNase-Free dd H2O polishings are to 20 μ L.Response procedures are：PCR uses two steps
Method, 95 DEG C of 15min, then 95 DEG C of 10s, 64 DEG C of 32s are a cycle, are recycled 40 times altogether.0.1 DEG C of s is pressed after the completion of PCR-1Heating
Rate rises to the analysis verification of 95 DEG C of progress solubility curves from 72 DEG C.
It is bent that primer pair JWB-9F/JWB-7R makes amplification curve, solubility curve and standard to the amplification of positive criteria
Line, as shown in Figure 2, Figure 3 and Figure 4.In Fig. 2, negative control shown in positive criteria product shown in 10 and 11 is equal in amplification curve
For straight line, and the positive criteria product of various concentration shown in 1-9 is S types or J-type curve.In Fig. 3 solubility curves shown in 1-9 not
Positive criteria product with concentration is simple spike.The good y=-3.522x+40.242 of standard curve linear relationship shown in Fig. 4,
Standard error R2=0.976, amplification efficiency 112.455%.In Fig. 2 " 9 " shown in Plasmid DNA corresponding to amplification curve it is dense
Degree is 0.306fg μ L-1, the minimal detectable concentration of a concentration of quantitative fluorescent PCR.According to plasmid copy number calculation formula：Matter
Grain copy number=plasmid concentration (ng μ L-1) × plasmid volume (μ L) × 6.02 × 1023/ { [carrier lengths (bp)+fragment length
(bp)]×660g·mol-1Show that plasmid copy number is about 67, therefore the sensitivity of the fluorescent quantitative PCR detection method is 67
A copy.
If, can be according to expansion using whether coal dirt vacation tail spore DNA is contained in the method detection sample to be tested of quantitative fluorescent PCR
Increase curve to be judged：If amplification curve is S types or J-type curve, then it is assumed that contain coal dirt vacation tail spore DNA in sample to be tested；
If amplification curve is straight line, then it is assumed that do not contain coal dirt vacation tail spore DNA in sample to be tested.
Pathogen quantitatively detects in embodiment 4, field acquisition tomato coal dirt vacation tail spore leaf spot tissue samples
In July, 2016, Shandong Shouguang acquire tomato coal dirt vacation tail spore leaf spot tissue samples, by Morphological Identification, really
Fixed infected by coal dirt vacation tail spore causes.Incidence tissue's sample DNA is extracted as template, according to embodiment 3 using modified CTAB method
In reaction system and program carry out coal dirt vacation tail spore bacterium quantitative fluorescent PCR in diseased tissues using primer JWB-9F/JWB-7R
Detection, after reaction, by the cycle threshold (Ct) and standard curve control of DNA sample, obtains Avr4 genes piece in DNA sample
Section copy number, and then obtain the concentration of coal dirt vacation tail spore in original sample.Positive control is coal dirt vacation tail spore DNA, negative control
For healthy tomato tissue DNA.
Primer pair JWB-9F/JWB-7R to tomato field disease plant sample disease be good for intersection tissue DNA, positive control with
And the fluorescent quantitative PCR result of negative control.It is all for trying coal dirt vacation tail spore leaf spot sample group under 64 DEG C of annealing conditions
Knit DNA and positive control recycled at 40 in produce amplification, and negative control is without any amplification.As a result such as table 2 and Fig. 5
It is shown.Test result shows that the fluorescence quantitative PCR detection technique can be used for tomato field coal dirt vacation tail spore leaf spot disease sample
This quantitative detection.
The fluorescence quantitative PCR detection of germ in 2 tomato field coal dirt vacation tail spore leaf spot disease sample of table
| Number | Latin name | Ct values | Copy number/μ L |
| FQ16070301 | Pseudocercospora fuligena | 26.93 | 104.48 |
| FQ16070302 | Pseudocercospora fuligena | 29.65 | 103.78 |
| FQ16070303 | Pseudocercospora fuligena | 30.92 | 103.01 |
| FQ16070304 | Pseudocercospora fuligena | 30.94 | 102.65 |
| FQ16070305 | Pseudocercospora fuligena | 30.27 | 102.64 |
| FQ16070306 | Pseudocercospora fuligena | 30.93 | 102.83 |
| FQ16070307 | Pseudocercospora fuligena | 30.92 | 103.01 |
Embodiment 5, the detection of manual simulation seed belt coal dirt vacation tail spore bacterium
In September, 2017, in progress manual simulation's seed belt in Vegetable ＆ Flower Inst., Chinese Academy of Agriculture Science's experiment greenhouse
The preparation of bacterium sample.15min is handled with hypochlorous acid again after sterilizing 30s to healthy tomato seeds with 75% alcohol, then uses nothing
Bacterium water cleans.Prepare the spore suspension (1.0 × 10 of 8 kinds of coal dirt vacation tail spores containing various concentration8, 1.0 × 107, 1.0 × 106,
1.0×105, 1.0 × 104, 1.0 × 103, 1.0 × 102, 1.0 × 101A spore/mL).50, healthy seed is taken respectively, is placed in
It is stirred evenly in 20mL various concentration spore suspensions, each processing is impregnated for 24 hours, and experiment is in triplicate.Seed after immersion is placed in
It is dried up in superclean bench spare.It when Seed-associated fungi, respectively repeats to take 10 seeds at random, be placed in 1mL aseptic water washings,
Using modified CTAB method extraction leachate DNA.With reference to described in embodiment 3 and 4 quantitative fluorescent PCR reaction system and program into
Row detection.Calculate the content of coal dirt vacation tail spore bacterium DNA in seed to be checked.
The results are shown in Table 3.Using the coal dirt vacation tail spore bacterium quantitative fluorescent PCR reaction system of foundation, manual simulation is detected
Cause of disease bacterial content in infected seed, the results showed that there is the technology higher reliability and sensitivity, detection tomato seeds to carry disease germs
Minimum be limited to 101.98The DNA of copy number, corresponding content of molds are 1000 spores/gram seed.
Germ DNA content in infected seed sample after 3 various concentration spore suspension of table impregnates
Comparative example：
The present invention devises 18 pairs of primers (table 4) altogether in actual mechanical process, based on coal dirt vacation tail spore bacterium Avr4 genes
And the specificity of all primer pairs is screened (concrete operations are referring to embodiment 2).As a result it shows：Serial number 1-17 in table 4
Shown in primer can amplify target fragment from coal dirt vacation tail spore, but can also to one in control strain (table 1) or
Multiple pathogens generate amplification (table 5), therefore without specificity.JWB-9F/JWB-7R primer pairs shown in table 4 can be from
The target fragment of 232bp is amplified in coal dirt vacation tail spore genome, and to other control strains without any amplification, therefore this draws
Object is good to specificity, can be used for the specificity to coal dirt vacation tail spore and quantitative detection.
The primer information designed in 4 present invention of table
5 primer specificity testing result of table
Note："+" represents testing result as the positive；"-" represents testing result as feminine gender.
<110>Vegetable ＆ Flower Inst., Chinese Academy of Agriculture Science
<120>The fluorescent quantitative PCR detection method of coal dirt vacation tail spore
<130> GNCLN180893
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence
<400> 1
tcgccagcag atgactccta c 21
<210> 2
<211> 23
<212> DNA
<213> Artificial sequence
<400> 2
tatttgcctg cgcagatggt cag 23
<210> 3
<211> 232
<212> DNA
<213> Pseudocercospora fuligena
<400> 3
tcgccagcag atgactccta cttgacaggt atcattcacg agatgccttg tccaagcggg 60
cttttgtgga atgacaacaa gaagtggtgt gattggcctg agaacacgac ctgtggattg 120
gttaagagtg ccggtgcgac tgaagttgat gcaacgaagc aggggccaac tccaggtatg 180
ctatcgcttt ctccaggtag agccttgtac tgaccatctg cgcaggcaaa ta 232
Claims (10)
1. the primer pair for detecting coal dirt vacation tail spore is following any：
(a1) primer pair that single stranded DNA shown in single stranded DNA and SEQ ID No.2 shown in SEQ ID No.1 forms；
(a2) by by SEQ ID No.1 and SEQ ID No.2 by one or several nucleotide substitution and/or missing and/or
Two single strand dnas shown in gained sequence form after addition, and primer identical with primer pair function described in (a1)
It is right.
2. the reagent for detecting coal dirt vacation tail spore, including primer pair described in claim 1, SYBR Green I real-time fluorescences
Quantitative pcr amplification buffer solution and dd H2O。
3. reagent according to claim 2, it is characterised in that：The SYBR Green I real-time fluorescence quantitative PCRs amplification
Buffer solution, including dNTPs, Mg2+, SYBR Green I, Taq archaeal dna polymerases.
4. reagent according to claim 3, it is characterised in that：Also contain reference dye in the reagent；
Specifically, the reference dye is ROX reference dyes.
5. the kit for detecting coal dirt vacation tail spore, contains following (b1) and (b2)：
(b1) coal dirt vacation tail spore positive plasmid；
(b2) any reagent in primer pair described in claim 1 or claim 2-4.
6. kit according to claim 5, it is characterised in that：The coal dirt vacation tail spore positive plasmid is to contain SEQ ID
The plasmid of DNA fragmentation shown in No.3.
7. in a kind of detection sample to be tested whether the method containing coal dirt vacation tail spore, for following (c1) or (c2)：
(c1) include the following steps：Total DNA is extracted from sample to be tested, as template, with primer pair described in claim 1 into
Row PCR amplification, obtains amplified production；Then according to as follows determine the sample to be tested in whether contain coal dirt vacation tail spore：If
DNA fragmentation containing 232bp in the amplified production then contains in the sample to be tested or candidate contains coal dirt vacation tail spore；Such as
The DNA fragmentation of 232bp is not contained in amplified production described in fruit, then is not contained in the sample to be tested or candidate is dirty false without containing coal
Tail spore；
(c2) include the following steps：Total DNA is extracted from sample to be tested, as template, with primer pair described in claim 1 into
Row fluorescent quantitative PCR；Then whether the dirty false tail of coal is contained according in the determining sample to be tested as follows according to amplification curve
Spore：If gained amplification curve is S types or J-type curve, contains in the sample to be tested or candidate contains coal dirt vacation tail spore；Such as
Amplification curve obtained by fruit is straight line, then is not contained in the sample to be tested or candidate does not contain coal dirt vacation tail spore.
8. the method for coal dirt vacation tail spore Genome DNA content, includes the following steps in a kind of detection sample to be tested：
(d1) the coal dirt vacation tail spore positive plasmid in the kit of claim 5 or 6 is diluted to serial known concentration, respectively
As template, fluorescent quantitative PCR is carried out with primer pair described in claim 1, draws quantitative fluorescent PCR standard curve；
(d2) total DNA is extracted from sample to be tested, and as template, quantitative fluorescent PCR is carried out with primer pair described in claim 1
Amplification；Then the quantitative fluorescent PCR standard curve drawn according to step (d1) obtains coal dirt vacation tail spore in the sample to be tested
The content of genomic DNA.
9. following any application：
(e1) primer pair described in claim 1 any reagent or claim 5 or 6 institutes in preparing claim 2-4
State the application in kit；
(e2) in primer pair or claim 2-4 described in claim 1 described in any reagent or claim 5 or 6
Application of the kit in detecting coal dirt vacation tail spore, or the application in preparing the product for detecting coal dirt vacation tail spore；
(e3) in primer pair or claim 2-4 described in claim 1 described in any reagent or claim 5 or 6
Kit or claim 7 or 8 described in method to tomato coal dirt vacation tail spore leaf spot carry out early prediction forecast in
Using.
10. being existed according to any primer pair or reagent or kit or method or application, feature in claim 1-9
In：The DNA fragmentation of the 232bp is DNA fragmentation shown in SEQ ID No.3.
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| 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 |
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| CN108977584A (en) * | 2018-09-05 | 2018-12-11 | 中国农业科学院蔬菜花卉研究所 | A kind of tomato mottle mosaic poison real-time fluorescence quantitative PCR detection method |
| 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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