CN105200122B - Quantitative detection kit for wheat stripe rust and application thereof - Google Patents
Quantitative detection kit for wheat stripe rust and application thereof Download PDFInfo
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Abstract
The invention provides a quantitative detection kit for wheat stripe rust and application thereof, wherein the kit comprises a conventional reagent for fluorescent quantitative PCR amplification, and is characterized by also comprising a primer for detecting stripe rust; the forward and reverse nucleotide sequences of the primers are as follows: osgQ607/osgQ 608: GGATGCCTCGAAGGGTTATACC/TGCTAGATACAATGGCACATCTGA. The invention also discloses a method for quantitatively detecting the copy number of the puccinia striiformis in wheat by using the kit. The kit and the method provided by the invention can relatively accurately and quantitatively determine the infection amount of the wheat stripe rust in the submerged state, have the characteristics of low detection limit, good sensitivity, strong specificity, high amplification efficiency and the like, and provide a reliable detection means for early prediction and prevention of the wheat stripe rust.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of immue quantitative detection reagent box of wheat stripe rust and its answers
With.
Background technique
The early monitoring of stripe rust of wheat and quantitative analysis are always one of important link of the disease management.Especially exist
When Wheat Seedling stripe rust is in the incobation stage, fast and accurately measuring method, prediction and prevention and treatment to the disease are established
Have great importance.The occurrence degree of disease can be determined and predict as early as possible in the incubation period of disease, and in the not big face of disease
Before product is popular, chemical control measure is formulated in time to reduce initial bacterium source amount by fast and accurately early monitoring, inhibits or subtracts
The development of slow disease.
For a long time, existing monitoring method, such as manual research, leaf culture, remote sensing monitoring still can not achieve to disease
Opportunistic pathogen infects the accurately and rapidly quantitative analysis that degree is infected when rear wheat leaf blade is in incobation state.In recent years, molecular biosciences
The development of technology makes it possible the quick detection to disease, and starts the early detection applied to stripe rust of wheat.
Zhao etc., Cao etc. design the specific primer of wheat stripe rust, realize the molecule inspection to stripe rust using regular-PCR method
It surveys.Hereafter, the method that Wang etc. establishes the strip rust bacteria in PCR method detection intrusion wheat leaf blade, Wang etc. further develop
The detection method of nest-type PRC.Real-time PCR be grew up in recent years can be with quantitative detection phytopathogen
Method.Winton etc. is detected simultaneously by a variety of pathogens using fluorescence Real-time PCR in a pipe.In addition, Gachon etc.,
Bohm etc., Mercado-Blanco etc. realize that dynamic change is chased after in host plant to pathogen using identical method
Track.Fraaije etc. has carried out the research work of wheat stripe rust using Real-time PCR, but primer is minimum detectable
Cause of disease bacterium amount is only 40pg.
The qualitative detection research for having carried out some pairs of stripe rust of wheat pathogens both at home and abroad at present, there is not yet stripe rust of wheat
The report of early stage Quantitative Monitoring method.
Summary of the invention
This research is excellent in terms of early detection stripe rust of wheat by comparing regular-PCR and Real-time PCR method
Disadvantage establishes SYBR Green I method, to quantitative determine infect amount of the puccinia striiformis under incobation state, for the disease
Early prediction and control decision making provide foundation.
Technical scheme is as follows:
For the kit of quantitative detection wheat stripe rust, the conventional reagent including fluorescent quantitative PCR, feature exists
In, further include detect strip rust bacteria primer；The nucleotide sequence of the forward and reverse of the primer is as follows:
OsgQ607/osgQ608:
GGATGCCTCGAAGGGTTATACC/TGCTAGATACAATGGCACATCTGA。
The conventional reagent of the fluorescent quantitative PCR includes: 2 × SG PCR Master Mix and/or 2 × SG
Green qPCR Mix and distilled water.
A kind of method of quantitative detection wheat stripe rust, which comprises the steps of:
(1) calibration curve equation is obtained:
The calibration curve equation is using the gradient of serial DNA standard items copy Particle density as abscissa, to use primer
OsgQ607/osgQ608 carries out the resulting Ct value of fluorescent quantitative PCR to the series DNA standard items respectively and draws for ordinate
The equation that the standard curve fit of system obtains；
(2) fluorescent quantitative PCR is carried out to the gDNA for extracting from sample to be tested using primer osgQ607/osgQ608；
(3) fluorescence signal for collecting the fluorescent quantitative PCR of sample to be tested DNA, determines according to the threshold value to test sample
The Ct value of product DNA；
(4) the Ct value of sample to be tested DNA is substituted into the copy Particle density that standard curve obtains sample to be tested DNA；
The DNA standard items are the regular-PCR amplified production of strip rust bacteria gDNA；The sample to be tested is wheat gDNA；Institute
The sequence for stating primer osgQ607/osgQ608 is as follows: GGATGCCTCGAAGGGTTATACC/
TGCTAGATACAATGGCACATCTGA。
The reaction system of the fluorescent quantitative PCR are as follows: 2 × SG Green qPCR Mix:0.5 μ l/ μ l, it is described to draw
Object forward and reverse is each are as follows: 0.25 μM, DNA profiling: 0.05 μ l/ μ l, remaining is distilled water.
The response procedures of the fluorescent quantitative PCR are as follows: 95 DEG C initial denaturation 10 minutes；95 DEG C are denaturalized 20 seconds, and 60 DEG C are moved back
Fire extends 30 seconds, 45 circulations.
The DNA profiling is gDNA or using gDNA as the regular-PCR amplified production of template.
The regular-PCR amplification refers to using gDNA as template, is carried out with the primer osgQ607/osgQ608 common
Pcr amplification reaction；The reaction system of regular-PCR amplification are as follows: 2 × SG PCR Master Mix:0.5 μ l/ μ l, it is described to draw
Object forward and reverse is each are as follows: 0.25 μM, regular-PCR amplified production: 0.05 μ l/ μ l, remaining is distilled water；Response procedures are as follows: 94
DEG C initial denaturation 10 minutes；94 DEG C are denaturalized 20 seconds, and 60 DEG C of annealing extend 30 seconds, 40 circulations.
This research use " Glass and Donaldson, the fungi conservative gene primer Bt2a/Bt2b of 1995. " reports,
Its nucleotide sequence are as follows: the ACCCTCAGTGTAGTGACCCTTGGC-3 ' of 5 '-GGTAACCAAATCGGTGCTGCTTTC-3 '/5 '.
Using the genomic DNA of the pathogens such as wheat stripe rust, puccinia triticinia, red rust as template, carries out PCR and compare amplification.
Amplification and sequencing result show that the primer amplifies the segment of 361bp length in the DNA of wheat stripe rust, in wheat leaf rust
Bacterium, red rust DNA in the band that amplifies all be 495bp, be specifically shown in Fig. 6,7,8.In order to obtain can specifically by
Strip rust bacteria becomes rusty from leaf, identifies in puccinia graminis and other similar wheat disease fungus, needs further to design special primer.
The present invention is according to the gene of the specific fragment 362bp size of wheat stripe rust (Puccinia striiformis)
Sequence design has the primer osgQ607/osgQ608 of specificity to the pathogen species, and respectively in regular-PCR and Real-
The specificity of the primer and sensitivity are determined when time PCR amplification.The result shows that the primer pair wheat stripe rust is special
It is anisotropic high, the target stripe for amplifying 82bp can be stablized.Using this specific primer, Real-time PCR measurement system is established
System has quantitative determined DNA of the strip rust bacteria after wheat leaf blade inoculation in tissue and has changed with time.Utilize reagent of the present invention
The minimum detectability of the primer in box, regular-PCR and Real-time PCR amplification is respectively 10pg and 1fg.Real-
The sensitivity of time PCR is 100 times of regular-PCR.The present invention has also set up Real-time PCR standard curve, for quantitative
The DNA of the stripe rust pathogen in wheat leaf blade is infected in measurement.The present invention is based on ABI7500 fluorescent quantitation amplification instrument, optimizations
Real-Time PCR reaction system is successfully set up SYBR Green I fluorescent dye determination quantitative PCR detection system.
Primer designed by the present invention is more suitable for compared with other primers for being applied to stripe rust Molecular Detection at present
The detection of Real-time PCR.Object bacteria is shown as single product in the solubility curve of Real-time PCR in the present invention
Peak, the non-specific band interference such as primer free dimer.It is found during primer is screened in design, the too long primer of amplified fragments
Although showing specificity in regular-PCR detection, amplified production is single segment, may in Real-time PCR detection
And single product is not appeared as；And the selected primer of the present invention, eliminate other primers lacking in Real-time detection
Point.
Kit provided by the invention and method can relatively accurately quantitative determine wheat stripe rust under incobation state
The amount of infecting has the characteristics that detection limit is low, sensitivity is good, high specificity, amplification efficiency are high, is early prediction and prevention and treatment wheat item
Rust provides a kind of reliable detection means.
Detailed description of the invention
Fig. 1 is using the regular-PCR amplified production of the strip rust bacteria gDNA of gradient concentration as series DNA standard items as template,
The amplification curve diagram and threshold value of fluorescent quantitative PCR acquisition are carried out using kit of the present invention.
Fig. 2 is using the product of the regular-PCR of the strip rust bacteria gDNA of gradient concentration amplification as series DNA standard items as mould
Plate carries out the melting curve figure of fluorescent quantitative PCR acquisition using kit of the present invention.
Fig. 3 is using the product of the regular-PCR of the strip rust bacteria gDNA of gradient concentration amplification as series DNA standard items as mould
Plate carries out the standard curve of fluorescent quantitative PCR acquisition using kit of the present invention.
Fig. 4 is the amplification curve that using kit of the present invention wheat samples are carried out with fluorescent quantitative PCR acquisition
Figure.
Fig. 5 is the melting curve that using kit of the present invention wheat samples are carried out with fluorescent quantitative PCR acquisition
Figure.
The PCR of the gDNA of Fig. 6 wheat stripe rust, leaf rust and puccinia graminis compares amplification,
Wherein, the sample in loading wells is followed successively by 2 item rust, 2 leaf rust, 2 stem rusts from left to right；Marker band is big
It is small to be followed successively by 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp from the bottom up.
The DNA fragment specific comparison result of Fig. 7 red rust and leaf rust,
Wherein, 6-1 segment is the DNA fragment specific of red rust, and 3-1 segment is the specificity of puccinia triticinia
DNA fragmentation, homology 100%.
The DNA fragment specific comparison result of Fig. 8 wheat stripe rust and leaf rust,
Wherein, 1-5 segment is the DNA fragment specific of wheat stripe rust, and 3-1 segment is the specificity of puccinia triticinia
DNA fragmentation, homology 31.23%.
The common pcr of Fig. 9 verifies the specificity of specific primer of the present invention:
Wherein, swimming lane 1-5: wheat stripe rust CY33, CY32, CY17, CY18, CY19；
6-8, puccinia triticinia PHT, THT, THD；
9-11, red rust 34C1, MFM；
12-13, wheat powdery mildew 1,10；
14-15, gibberella saubinetii PH-1, PH-2；
16-17, TCK；18, TFL；19；TCT；20；ddH2O；Marker is the Low MW DNA Ladder of NEB.
Specific embodiment
The present invention is described in further detail With reference to embodiment, but is not intended to limit model of the invention
It encloses.Unless otherwise specified, operation used in following embodiments is conventional method, and used reagent commercially available can obtain
?.
The source and record source of biomaterial
Strip rust bacteria CY33 can be commercially available as known bacterial strain；
The wheat subject material that the present invention uses is known kind bright virtuous 169, by the Chinese Academy of Agricultural Sciences's Plant Protection Institute
Wheat disease group saves, and can be provided in 20 years to the public from the applying date and be used for experimental study.
Wheat Pathogenic Fungi strain and source: Wheat rust fungus strain CY33, CY32, CY17, CY18, CY19；Wheat leaf rust
Bacterium strain PHT, THT, THD；Red rust strain 34C1, MFM；Wheat powdery mildew 1,10；Gibberella saubinetii strain PH-1,
PH-2；T. contraversa TCK；Light Tilletia foetida t bacteria FL；Net fungus tilletia t bacteria CT is bacterium known to the public
Kind, there is preservation in Plant Protection institute, Chinese Academy of Agricultral Sciences, and promising to undertake can be used for from the applying date in 20 years to public's granting
Experimental study.
Main agents
2 × SG PCR Master Mix is purchased from SinoGene, product type #33-10201)；2×SG Green
QPCR Mix (with ROX) is purchased from SinoGene, product type #22-10102；Extraction of plasmid DNA kit；Plant gene
Group DNA extraction kit；Distilled water.
Key instrument equipment
Spectrophotometer (Biophotometer, Eppendorf)；Quantitative PCR apparatus (StepOnePLUS, Applied
Biosystems)。
Embodiment 1, using fungi conservative gene Primer Analysis Wheat rust fungus
Referring to " its nucleotide sequence of Glass and Donaldson, the Bt2a/Bt2b of 1995. " reports are as follows:
Bt2a:5 '-GGTAACCAAATCGGTGCTGCTTTC-3 ' (Seq ID No.1),
Bt2b:5 '-ACCCTCAGTGTAGTGACCCTTGGC-3 ' (Seq ID No.2).
Carry out PCR analysis to wheat stripe rust, leaf rust and puccinia graminis gDNA, analysis in M J Research,
Inc. it is carried out in PTC2220 type PCR instrument.
Amplification system is 25 μ L, (each including 10 × PCR Buffer (Mg2+Plus), 2 μ L, dNTP Mixture
2.5mmol/L) 0.3 μ L, Bt2a/Bt2b (10 μm of ol/L) 1 μ L, 1.0 μ L, TaKaRa Taq (5U/ μ of template DNA (20ng/ μ L)
L) 0.3 16.9 μ L of μ L, ddH2O.
PCR reaction condition are as follows: 94 DEG C of 2min, 1 circulation；94 DEG C of 15s, 55 DEG C of 30s, 72 DEG C of 90s, 30 circulations；72℃
10min, 1 circulation.
Amplified production mass fraction be 1.5 Ago-Gel, 0.5 × TBE electrophoretic buffer electrophoretic separation, with
ULTRA-URLET PRODUCTS gel imaging system records and analyzes Electrophoretic pattern.Wheat stripe rust is quickly cut in the UV lamp
Specific PCR band, with Shenzhen Yi Nuojin biology Co., Ltd Catcher Sp in GEK2050 gel reclaims kit return
Purifying is received, Hai Sheng work technology company bidirectional sequencing is handed in.Sequencing result is with the BasicLocalAlignment Search of NCBI
Tool carries out sequence assembly, obtains the sequence of Specific PCR fragments, then with BLASTN program and Gen2Bank, EMBL,
Sequence in DDBJ, PDB carries out sequence analysis.
Experimental result: PCR is carried out to the gDNA of wheat stripe rust, leaf rust and puccinia graminis with primer Bt2a and Bt2b combination
Amplification, it has unexpectedly been found that, which can distinguish wheat item rust and leaf rust and puccinia graminis.That is, wheat stripe rust has length
For the DNA fragment specific (Seq ID No.3) of 361bp, and it is 495bp's that puccinia triticinia and red rust, which have length,
DNA fragment specific (Fig. 6).The study found that DNA fragmentation (the Seq ID that puccinia triticinia and red rust amplify
No.4) homology is 100% (Fig. 7), and wheat stripe rust and its homology are 31.23% (Fig. 8).
The preparation of embodiment 2, kit of the present invention
1. the acquisition of primer
According to the strip rust bacteria specific sequence Seq ID No.3 that embodiment 1 obtains, the design of software Primer 5.0 one is utilized
Series is used for the primer of fluorescence quantitative PCR detection.
By primer screening and verifying, the specific primer in kit of the present invention is finally obtained, it is positive and negative
Nucleotide sequence to primer is as follows:
OsgQ607/osgQ608:
GGATGCCTCGAAGGGTTATACC(Seq ID No.5)/TGCTAGATACAATGGCACATCTGA(Seq ID
No.6)。
2. kit assembles
By the primer, 2 × SG PCR Master Mix, 2 × SG Green qPCR Mix (with ROX) and double steamings
Water is assembled into kit of the present invention by the cooperation of certain usage ratio.
Embodiment 3, kit of the present invention detect wheat samples
Step:
A. standard curve is obtained
The preparation of 1.DNA standard items: strip rust bacteria CY33 bacterial strain is extracted according to the operating instruction of extraction of plasmid DNA kit
Genomic DNA (Plasmid DNA), and using Plasmid DNA as template, primer osgQ607/osgQ608 is added, regular-PCR amplification is commonly used
Reagent (such as table 1) carries out PCR amplification；The reaction system of the PCR amplification is as shown in table 1；Response procedures are as shown in table 2；Amplification
Strip rust bacteria PCR product out is DNA standard items.
Table 1
Table 2
2. the concentration with the above-mentioned PCR product of spectrophotometric determination is 498ng/ μ l, gradient is carried out in 1/10th ratios
Serial DNA standard dilutions are diluted to, and serial dilutions are calculated separately out according to gradient concentration and PCR product molecular weight
It copies Particle density (copies/ μ l).
3. a pair above-mentioned series DNA standard dilutions carry out fluorescent quantitative PCR, the amplification system is as shown in table 3,
Amplified reaction program is as shown in table 4, forward and reverse primer: osgQ607/osgQ608.
Table 3
| Ingredient | Dosage (μ l) |
| 2×SG Green qPCR Mix | 10 |
| PCR product | 1 |
| Forward primer (10 μM) | 0.5 |
| Reverse primer (10 μM) | 0.5 |
| Distilled water | 8 |
| Total reaction volume | 20 |
Table 4
4. utilizing the included software set threshold value of quantitative PCR apparatus.
5. the fluorescence signal of the product of fluorescent quantitative PCR series DNA standard dilutions is collected, according to the threshold value
Determine the serial Ct value of series DNA standard dilutions.
6. using above-mentioned series Ct value as ordinate, the copy Particle density (copies/ μ l) of serial DNA standard dilutions is
Abscissa draws standard curve.
B. the detection of 28 parts of wheat samples to be tested
The processing of wheat samples:
The blade of wheat is infected using strip rust bacteria CY33, and respectively in processing 1h, 2h, 3h, 5h, 6h, 7h, 9h, 12h, for 24 hours
When be sampled, the leaf sample of each period does 3 respectively and parallel repeats；The wheat leaf blade not infected is also carried out simultaneously
Sampling is used as negative control.
The detection of wheat samples:
1. using plant genome DNA extracts kit, to above-mentioned wheat sample to be tested, totally 28 parts of progress gDNA are mentioned respectively
It takes, see Table 5 for details for the corresponding relationship of wheat samples number and handling duration.Measure the first of 28 parts of gDNA respectively using spectrophotometer
Beginning concentration, while all 28 parts of gDNA are uniformly diluted to consistent detectable concentration 100ng/ μ l.
2. carrying out fluorescent quantitation respectively to the gDNA that 28 parts of detectable concentrations are 100ng/ μ l using kit of the present invention
PCR amplification, the amplification system is as shown in table 5, and amplified reaction program is as shown in table 4；Each sample does 3 parallel reactions.
Table 5
| Ingredient | Dosage (μ l) |
| 2×SG Green qPCR Mix | 10 |
| gDNA | 1 |
| Forward primer (10 μM) | 0.5 |
| Reverse primer (10 μM) | 0.5 |
| Distilled water | 8 |
| Total reaction volume | 20 |
3. collecting the signal of fluorescent quantitative PCR product, substituting into standard curve according to the corresponding Ct value of threshold value be can get
Copy number containing strip rust bacteria in every microlitre of gDNA sample can also obtain 1ng gDNA by the conversion of detectable concentration and volume
The copy number of the strip rust bacteria contained in sample.
As a result:
A. standard curve
Serial DNA standard dilution obtains amplification curve as shown in Figure 1 after fluorescent quantitative PCR, and threshold value is set
It is set to 11426.020655；According to the amplification curve, corresponding recurring number i.e. Ct value under the threshold value is found out；PCR product
Molecular weight MW are as follows: 82bp；The gradient concentration of serial DNA standard dilutions, the corresponding relationship such as table for copying Particle density X and Ct value
6, thus obtained standard curve is as shown in figure 3, corresponding linear regression equation 1 is Ct=-3.479log (X)+43.755.
Table 6
B. sample to be tested testing result
The gDNA concentration of 28 parts of wheat samples to be measured substitutes into the item rust contained in every μ l sample that standard curve obtains respectively
See Table 7 for details for the strip rust bacteria copy number statistical result contained in bacterium copy number and every ng sample.
Table 7
By above-mentioned table 7 it is found that can accurately be calculated in wheat samples using kit of the present invention in incobation
The strip rust bacteria of phase day part accurately infects bacterium amount；From the data in table can be seen that the same period parallel duplicate sample it
Between bacterium amount difference it is little, more accurately.And the minimum bacterium amount that kit of the present invention can detect is that every ng is mentioned to be measured small
The strip rust bacteria copied in the DNA of wheat sample containing 2.92, it was confirmed that kit provided by the invention has detection limit low, sensitive
The advantages that degree is high, accuracy is good.
The quality of the kit of the present invention of embodiment 4 controls
The present invention passes through regular-PCR and Real-time PCR respectively and is tested the specificity and sensitivity of the primer
Card.
Step:
A. regular-PCR detects
Detailed step is referring to the step 1 in embodiment 3 " step " portion A.
B. fluorescence quantitative PCR detection
Detailed step referring to embodiment 3 " step " part B.
As a result:
(1) regular-PCR detection is carried out using the primer pair wheat stripe rust, the target item for amplifying 82bp can be stablized
Band, and any band can not be expanded in the wheat leaf blade of negative control；And the primer can only amplify in strip rust bacteria
Band, and not shaping band is expanded in the approximate strain of strip rust bacteria, such as leaf rust, puccinia graminis, illustrate that the primer is used for
Regular-PCR, which expands strip rust bacteria, has very high specificity, sees Fig. 9；Either strip rust bacteria DNA standard items or wheat sample to be tested
DNA is illustrated as single product peak (as schemed using the melting curve that the above-mentioned DNA of the primer pair is obtained through Real-time PCR
2 and Fig. 5), the interference of the nonspecific products such as primer free dimer illustrates in terms of using fluorescence quantitative PCR detection strip rust bacteria, this
Invent primer specificity equally with higher.
(2) wheat item is detected respectively for regular-PCR and Real-time quantitative fluorescent PCR using primer of the present invention
Rest fungus, high sensitivity, detection limit is low, and the minimum detectability of regular-PCR is 10pg, and Real-time quantitative fluorescent PCR is most
Low detection limits are 1fg.
(3) amplification efficiency that the quantitative PCR of standard curve is obtained using the regular-PCR product of gDNA is 94%, shows benefit
The amplification efficiency for carrying out real-time quantitative PCR amplification with the primer of kit of the present invention is higher.
Claims (6)
1. being used for the kit of quantitative detection wheat stripe rust, the conventional reagent including fluorescent quantitative PCR, feature exists
In, further include detect strip rust bacteria primer；The nucleotide sequence of the forward and reverse of the primer is as follows:
OsgQ607/osgQ608:
GGATGCCTCGAAGGGTTATACC/TGCTAGATACAATGGCACATCTGA；
The minimum bacterium amount that the kit can detect is that every ng is mentioned from the DNA of wheat samples to be measured containing 2.92 copies
Strip rust bacteria.
2. kit according to claim 1, wherein the conventional reagent of the fluorescent quantitative PCR includes: 2 × SG
PCR Master Mix and/or 2 × SG Green qPCR Mix and distilled water.
3. a kind of method of quantitative detection wheat stripe rust, which comprises the steps of:
(1) standard curve is obtained with DNA standard items:
A. the DNA standard dilutions of graded series concentration will be diluted to after DNA standard items measurement concentration and molecular weight, and according to
Gradient concentration and molecular weight calculate separately out the copy Particle density (copies/ μ l) of series DNA standard dilutions；
B. fluorescent quantitative PCR is carried out to the DNA in serial DNA standard dilutions using primer osgQ607/osgQ608；
C. given threshold；
D. the fluorescence signal for collecting fluorescent quantitative PCR product determines series DNA standard dilutions according to the threshold value
Ct value；
E. using Ct value as ordinate, the copy Particle density of DNA standard dilutions is abscissa, draws standard curve；
(2) fluorescence is carried out using regular-PCR amplified production of the primer osgQ607/osgQ608 to the gDNA for extracting from sample to be tested
Quantitative pcr amplification；
(3) fluorescence signal for collecting the fluorescent quantitative PCR of sample to be tested DNA, determines sample to be tested DNA according to the threshold value
Ct value；
(4) the Ct value of sample to be tested DNA is substituted into the copy Particle density that standard curve obtains sample to be tested DNA；
The DNA standard items are the regular-PCR amplified production of strip rust bacteria gDNA；The sample to be tested is wheat gDNA；It is described to draw
The sequence of object osgQ607/osgQ608 is as follows: GGATGCCTCGAAGGGTTATACC/TGCTAGATACAATGGCACATCTGA.
4. according to the method described in claim 3, the wherein reaction system of the fluorescent quantitative PCR are as follows: 2 × SG Green
QPCR Mix:0.5 μ l/ μ l, the primer forward and reverse are each are as follows: and 0.25 μM, regular-PCR amplified production: 0.05 μ l/ μ l,
Remaining is distilled water.
5. the method according to claim 3 or 4, wherein the response procedures of the fluorescent quantitative PCR are as follows: 95 DEG C of pre- changes
Property 10 minutes；95 DEG C are denaturalized 20 seconds, and 60 DEG C of annealing extend 30 seconds, 45 circulations.
6. according to the method described in claim 3, wherein the regular-PCR amplification refers to using gDNA as template, with the primer
The regular-PCR amplified reaction that osgQ607/osgQ608 is carried out；The reaction system of the regular-PCR amplification are as follows: 2 × SG PCR
Master Mix:0.5 μ l/ μ l, the primer forward and reverse are each are as follows: and 0.25 μM, regular-PCR amplified production: 0.05 μ l/ μ l,
Remaining is distilled water；Response procedures are as follows: 94 DEG C initial denaturation 10 minutes；94 DEG C are denaturalized 20 seconds, and 60 DEG C of annealing extend 30 seconds, and 40 are followed
Ring.
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| CN110205400B (en) * | 2019-06-27 | 2022-08-02 | 河北农业大学 | Kit and method for detecting puccinia triticina |
| CN113881804A (en) * | 2021-11-11 | 2022-01-04 | 青海省农林科学院 | Primer pair for wheat stripe rust detection, probe and application |
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