CN102399896A - Method for detecting phytophthora parasitica in soil - Google Patents
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Abstract
The invention belongs to the field of plant disease and pest quarantine, and provides a method for detecting phytophthora parasitica in soil. According to the method for DNA extraction and molecular detection of the phytophthora parasitica in the soil, the conventional isolating culture method is eliminated, and the detection period is greatly shortened; compared with a common polymerase chain reaction (PCR) detection method, the method has the advantages that: a subsequent treatment process is not needed, and a result can be monitored in real time; primers are designed on the basis of a changeful area of pathogen ITS, and corresponding bands are not produced when similar species are amplified; the method is high in specificity and suitable for fluorescent quantitative PCR conditions; because 2 percent of polyvinyl pyrrolidone (PVP) is added into the total DNA extraction buffer solution of the soil, the pollution of partial impurities in the soil is effectively removed, the influence of the impurities on the PCR effect is reduced, and the detection sensitivity of fluorescent quantitative PCR is improved; meanwhile, the fluorescent quantitative PCR system and the reaction conditions are obtained according to the size study conditions of the designed primers, so the detection sensitivity is high and can reach 2fg/mu l.
Description
Technical field
The invention belongs to plant pest quarantine field, relate in particular to a kind of method that is used for detecting the soil tobacco black shank bacterium.
Background technology
Tobacco is the annual herb plant, belongs to Solanaceae (Solanaceae) Nicotiana (Nicotiana), distributes in the world extensively, and the Nicotiana of finding at present has 66 kinds.2010, national tobacco industry realized that taxes and profits are above 6,000 hundred million yuan.Black shank is one of destructive infectious disease of tool on tobacco produces, and maintains 10-15% at the long-term sickness rate of China, and the about 76372hm2 of onset area loses the about 2869.26Kg of output in the year of causing, and financial loss reaches more than 1.23 hundred million yuans.Present research shows that the generation of quantity of balck shank pathogenic bacteria and black shank is closely related in the soil.Therefore, detect the quantity of tobacco black shank bacterium in the soil, can prevent early, reduce the generation of balck shank.
The detection method of black shank bacterium mainly is through yeast culture and common PCR method in the soil at present commonly used, and these two kinds of methods have shortcomings such as low, the time-consuming and poor repeatability of accuracy.And quantitative fluorescent PCR (Real time fluorescent quantitative PCR; Real-time Q-PCR) method is a kind of highly sensitive, good stability, timesaving detection technique; And have characteristics of robotization; Detected result can at home and abroad have in the detection of pathogenic micro-organism widely and use in 10～1012 copy scopes.Through this method the tobacco black shank bacterium in the soil is carried out fluorescence quantitative PCR detection, reference standard curve simultaneously can detect the quantity of flue black shank bacterium in the soil, for the prevention black shank prerequisite and guarantee is provided.
Summary of the invention
The invention provides a kind of method that is used for detecting the soil tobacco black shank bacterium; The detection method that is intended to solve balck shank germ in the soil commonly used at present mainly is through yeast culture and common PCR method, the problem of low, the time-consuming and poor repeatability of these two kinds of method accuracys.
The object of the present invention is to provide a kind of method that is used for detecting the soil tobacco black shank bacterium, this method may further comprise the steps:
This method may further comprise the steps:
Get the black shank germ of cryopreservation and cultivate, and utilize the CTAB method to extract genomic dna with other fungies;
Extract total DNA of black shank germ soil;
According to the gene order design primer of black shank germ in the gene pool, and carry out the checking of primer specificity pcr amplification;
Detect, analyze the sensitivity of primer and make the quantitative fluorescent PCR typical curve;
Utilize the quantitative fluorescent PCR typical curve, the DNA of different black shank occurring degree soil is carried out quantitative analysis.
The method that is used for detecting the soil tobacco black shank bacterium provided by the invention is carried out DNA extraction and Molecular Detection to tobacco black shank bacterium in the soil, has saved the method for traditional separation and Culture, has shortened sense cycle in time greatly; Whole detection can be accomplished in 0.5 day, compared with the regular-PCR detection method, need not follow-up treating processes; And can monitor in real time the result, designed primer is based on the variable zone design of pathogenic bacteria ITS, does not have respective strap during close species amplification; The size of products therefrom is 172bp, warp and approximate bacterial classification amplification checking, and specificity is good; Be suitable for the quantitative fluorescent PCR reaction conditions, increased the process for extracting of total DNA in the soil, added 2% PVP at soil total DNA extraction damping fluid; Removed the pollution of partial impurities in the soil effectively, reduced influence, improved the detection sensitivity of quantitative fluorescent PCR greatly the PCR effect; The system and the reaction conditions of quantitative fluorescent PCR are groped the condition gained according to design primer size simultaneously, and be simple to operate, and detection sensitivity is high; Can reach 2fg/ μ l, have only the existence of a cause of disease spore in the soil, can detect.
Description of drawings
Fig. 1 is the realization flow figure that detects the embodiment of the invention being used for of providing the method for soil tobacco black shank bacterium;
Fig. 2 is that the CTAB method of utilizing that the embodiment of the invention provides is extracted the schema of the implementation method of genomic dna;
Fig. 3 is the schema of implementation method of total DNA of the extraction black shank germ soil that provides of the embodiment of the invention.
Embodiment
In order to make the object of the invention, technical scheme and advantage clearer,, the present invention is further specified below in conjunction with accompanying drawing and embodiment.Should be appreciated that specific embodiment described herein only in order to explanation the present invention, and be not used in the qualification invention.
Fig. 1 shows that the embodiment of the invention provides is used for detecting the realization flow of the method for soil tobacco black shank bacterium.
This method may further comprise the steps:
In step S101, get the black shank germ of cryopreservation and cultivate, and utilize the CTAB method to extract genomic dna with other fungies;
In step S102, extract total DNA of black shank germ soil;
In step S103,, and carry out the checking of primer specificity pcr amplification according to the gene order design primer of black shank germ in the gene pool;
In step S104, detect, analyze the sensitivity of primer and make the quantitative fluorescent PCR typical curve;
In step S105, utilize the quantitative fluorescent PCR typical curve, the DNA of different black shank occurring degree soil is carried out quantitative analysis.
The black shank germ of in embodiments of the present invention, getting cryopreservation is cultivated further with other fungies and comprises:
After 28 ℃ of incubated overnight of PDA liquid nutrient medium, get 1.5mL bacterium liquid, collect mycelium, distilled water washing 2 times.
As shown in Figure 2, in embodiments of the present invention, the implementation method of utilizing the CTAB method to extract genomic dna is:
In step S201, add the CTAB solution of 500 μ l, cool off 30s in the liquid nitrogen, put into 65 ℃ of water-bath 30s immediately, repeat 3 times;
In step S202, add granulated glass sphere vortex oscillation 5min, 65 ℃ of insulation 20min, during shake once;
In step S203, add phenol, chloroform, primary isoamyl alcohol, volume ratio 25: 24: 1, the centrifugal 10min of 12000r/min adds chloroform, primary isoamyl alcohol in the supernatant, volume ratio 24: 1, the centrifugal 10min of 12000r/min;
In step S204, get supernatant, add-20 ℃ of precooling isopropanol precipitatings of 2/3 times of volume, leave standstill about 30min;
In step S205, the slow speed of revolution is centrifugal, and deposition is used the absolute ethyl alcohol rinsing 1 time again with 70% ethanol rinsing 2 times, dries up, and is resuspended in the 100ul distilled water, and-20 ℃ of preservations are subsequent use.
As shown in Figure 3, in embodiments of the present invention, the implementation method of extracting total DNA of black shank germ soil is:
In step S301, the preparation conidial suspension is also inoculated soil, 3 repetitions of every part of soil;
In step S302, add the DNA extraction damping fluid in the pedotheque, 37 ℃ of temperature are bathed 30min, every 5min mixing;
In step S303, add 20%SDS, final concentration is 2%, 65 ℃ of water-bath 2h, every 10min puts upside down mixing, the centrifugal 10min of 6000g room temperature;
In step S304, add phenol, chloroform, primary isoamyl alcohol, three's volume ratio is 25: 24: 1, and the centrifugal 10min of 12000r/min adds chloroform, primary isoamyl alcohol in the supernatant, and the two volume ratio is 24: 1, the centrifugal 10min of 12000r/min;
In step S305, get supernatant, add-20 ℃ of precooling isopropanol precipitatings of 2/3 times of volume, leave standstill about 30min;
In step S306, the slow speed of revolution is centrifugal, and deposition is used the absolute ethyl alcohol rinsing 1 time again with 70% ethanol rinsing 2 times, dries up, and is resuspended in the 100ul distilled water, and-20 ℃ of preservations are subsequent use.In embodiments of the present invention, added 2% PVP in the DNA extraction damping fluid, be used for removing the soil partial impurities pollution, reduce influence, improve the detection sensitivity of quantitative fluorescent PCR the PCR effect.
In embodiments of the present invention, consisting of of soil total DNA extraction damping fluid:
The pH value is 8.0 100mmol/L Tris-HCl, 100mmol/L EDTA-Na2,2%PVP, 1.5mol/L NaCl, 1%CTAB, 125 μ g/mL Proteinase Ks.
In embodiments of the present invention, according to the gene order of black shank germ in gene pool design primer, and the implementation method of carrying out the checking of primer specificity pcr amplification is:
18S rDNA gene order according to black shank germ in the ncbi database; Utilize Primer5.0 design quantitative fluorescent PCR special primer SP:5 '-TGAAGAACGCTGCGAACTGC-3 '; AP:5 '-CTGACATCTCCTCCACCGACTA-3 ', amplification purpose fragment length is 172bp;
The genomic dna of the black shank pathogenic bacteria gene group DNA that extracts and other pathomycetes is carried out the primer specificity detection.
In embodiments of the present invention, the design primer specificity is good in this method, and close species do not have respective strap when increasing, and the size of products therefrom is 172bp, is suitable for the quantitative fluorescent PCR reaction conditions.
In embodiments of the present invention, the amplification system of quantitative fluorescent PCR and reaction conditions are groped the condition gained according to design primer size in this method, adapt with the institute designed primer; Wherein, the amplification system of quantitative fluorescent PCR is:
1 * SYBR Green
I Master Mix (TaKaRa), 10 μ l; Primer respectively is 400nM; Template 2 μ L, amplification system are 25 μ L;
The response procedures of quantitative fluorescent PCR is:
94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 20s, 65 ℃ of annealing 40s, 72 ℃ are extended 40s, 72 ℃ of 5min, 40 circulations.
In embodiments of the present invention, the implementation method that detects, analyzes the sensitivity of primer and make the quantitative fluorescent PCR typical curve is:
With the conidial soil DNA of inoculation different concns germ is template, checks this primer to conidial sensitivity in the soil;
10 times of gradient dilution liquid with black shank germ genomic dna are template, and check primer SP/AP is to the sensitivity of genomic dna, and it is limited to 2fg/ μ l genomic dna under detecting, and explains that this primer has very high sensitivity;
The DNA that chooses different dilution gradients is a template, and fluorescent quantitative PCR is made the quantitative fluorescent PCR typical curve.
Below in conjunction with accompanying drawing and specific embodiment application principle of the present invention is further described.
The method that is used for detecting the soil tobacco black shank bacterium as shown in Figure 1, that the embodiment of the invention provides, its step is following:
(1) fungus culture and extracting genome DNA: get cryopreservation the former bacterium of black shank and other pathomycete activation culture, after 28 ℃ of incubated overnight of PDA liquid nutrient medium, get 1.5mL bacterium liquid, collect mycelium, distilled water washing 2 times;
As shown in Figure 2, the CTAB method is extracted genomic dna: 1. add the CTAB solution of 500 μ l, cool off 30s in the liquid nitrogen, put into 65 ℃ of water-bath 30s immediately, repeat 3 times; 2. add granulated glass sphere vortex oscillation 5min, 65 ℃ the insulation 20min, during shake once; 3. add phenol, chloroform, primary isoamyl alcohol, three's volume ratio is 25: 24: 1, and the centrifugal 10min of 12000r/min adds chloroform, primary isoamyl alcohol in the supernatant, and the two volume ratio is 24: 1, the centrifugal 10min of 12000r/min; 4. get supernatant, add-20 ℃ of precooling isopropanol precipitatings of 2/3 times of volume, leave standstill about 30min; 5. the slow speed of revolution is centrifugal, and deposition is used the absolute ethyl alcohol rinsing 1 time again with 70% ethanol rinsing 2 times, dries up, and is resuspended in the 100ul distilled water, and-20 ℃ of preservations are subsequent use.
(2) extraction of the total DNA of black shank bacterium soil: as shown in Figure 3, the preparation conidial suspension is also inoculated soil, 3 repetitions of every part of soil; Add the DNA extraction damping fluid in the pedotheque, 37 ℃ of temperature are bathed 30min, every 5min mixing; Add 20%SDS (final concentration is 2%) then, 65 ℃ of water-bath 2h, every 10min puts upside down mixing, and the centrifugal 10min of 6000g room temperature, following treatment step are with reference to the step in above-mentioned (1) method 3.～5..The OD260/OD280 of this method gained DNA is 1.58～1.81, and the DNA purity of extracting is higher, can be in order to pcr amplification.Consisting of of soil total DNA extraction damping fluid: 100mmol/L Tris-HCl (pH8.0), 100mmol/L EDTA-Na2,2%PVP, 1.5mol/L NaCl, 1%CTAB, 125 μ g/mL Proteinase Ks.
(3) primer design and specific PCR amplification checking: according to the 18S rDNA gene order of tobacco black shank bacterium in the ncbi database; Utilize Primer5.0 design quantitative fluorescent PCR special primer SP:5 '-TGAAGAACGCTGCGAACTGC-3 '; AP:5 ' CTGACATCTCCTCCACCGACTA-3 '; Amplification purpose fragment length is 172bp, and it is synthetic that primer is given birth to the worker by Shanghai;
The genomic dna of the black shank pathogenic bacteria gene group DNA that extracts and other pathomycetes is carried out the primer specificity detection; Conventional pcr amplification system (25 μ l) is: the dNTP2 μ l of 10mM, the MgCl22 μ l of 20mM, pH8.6 damping fluid 2.5 μ l; Distilled water 16 μ l; Each 0.5 μ l of the upstream and downstream primer of 10 μ M, 2u/deTaq enzyme 1 μ l, dna profiling 0.5 μ l.The PCR reaction conditions is: 95 ℃ of preparatory sex change 30s, 95 ℃ of sex change 5s, 60 ℃ of annealing 30s, 35 circulations.The PCR product identifies to have only black shank pathogenic bacteria gene group to detect the band of about 170bp with 1.2% agarose gel electrophoresis, shows that the design of primers specificity is good.
(4) making of detection sensitivity analysis and typical curve: with the conidial soil DNA of inoculation different concns germ is template, checks this primer to conidial sensitivity in the soil; 10 times of gradient dilution liquid with black shank germ genomic dna are template, and check primer SP/AP is to the sensitivity of genomic dna, and it is limited to 2fg/ μ l genomic dna under detecting, and explains that this primer has very high sensitivity.Simultaneously, the DNA that chooses different dilution gradients is a template, fluorescent quantitative PCR, production standard curve.The fluorescent quantitative PCR system is: 1 * SYBR Green
I Master Mix (TaKaRa), 10 μ l; Primer respectively is 400nM; Template 2 μ L, amplification system are 25 μ L.Response procedures is: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 20s, 65 ℃ of annealing 40s, 72 ℃ are extended 40s, 72 ℃ of 10min, 40 circulations.
(5) application of fluorescent quantitative PCR technique: extract the DNA of different black shank occurring degree soil, carry out quantitative analysis, find that the quantity of tobacco black shank bacterium in the soil is consistent with the occurring degree of corresponding plot tobacco with the quantitative fluorescent PCR typical curve.
The method that is used for detecting the soil tobacco black shank bacterium provided by the invention; Tobacco black shank bacterium in the soil is carried out DNA extraction and Molecular Detection; Saved the method for traditional separation and Culture, shortened sense cycle in time greatly, whole detection can be accomplished in 0.5 day; Compare with the regular-PCR detection method, need not follow-up treating processes, and can monitor in real time the result; Designed primer of the present invention is based on the variable zone design of pathogenic bacteria ITS, warp and approximate bacterial classification amplification checking, and specificity is good.Detection sensitivity of the present invention is high, can detect the DNA of 2fg/ μ l, has only the existence of a cause of disease spore in the soil, can detect.In a word, the present invention has advantages such as operation is fairly simple, sensitivity height.
Positively effect of the present invention and advantage are: (1) has increased the process for extracting of total DNA in the soil; Soil total DNA extraction damping fluid has added 2% PVP; Can remove the pollution of partial impurities in the soil (like type materials such as humic acidss); Reduced influence, improved the detection sensitivity of quantitative fluorescent PCR greatly the PCR effect; (2) the design primer specificity is good, and close species do not have respective strap when increasing, and the size of products therefrom is 172bp, is suitable for the quantitative fluorescent PCR reaction conditions; (3) system of quantitative fluorescent PCR and reaction conditions are groped the condition gained according to design primer size, adapt with the institute designed primer.The fluorescent quantitative PCR system is: 1 * SYBR Green
I Master Mix (TaKaRa), 10 μ l; Primer respectively is 400nM; Template 2 μ L, amplification system are 25 μ L; Response procedures is: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 20s, 65 ℃ of annealing 40s, 72 ℃ are extended 40s, 72 ℃ of 5min, 40 circulations.Comprehensive above-mentioned condition, the method that is used for detecting the soil tobacco black shank bacterium provided by the invention, detection sensitivity is higher, can reach 2fg/ μ l, is higher than the detection line of the 0.610fg/ μ l that prior art provides.
The method that is used for detecting the soil tobacco black shank bacterium that the embodiment of the invention provides, the method that is used for detecting the soil tobacco black shank bacterium provided by the invention is carried out DNA extraction and Molecular Detection to tobacco black shank bacterium in the soil; Saved the method for traditional separation and Culture, shortened sense cycle in time greatly, whole detection can be accomplished in 0.5 day; Compare with the regular-PCR detection method, need not follow-up treating processes, and can monitor in real time the result; Designed primer is based on the variable zone design of pathogenic bacteria ITS, and not having a respective strap, the size of products therefrom during close species amplification is 172bp; Warp and approximate bacterial classification amplification checking, specificity is good, is suitable for the quantitative fluorescent PCR reaction conditions; Increased the process for extracting of total DNA in the soil, added 2% PVP, removed the pollution of partial impurities in the soil effectively at soil total DNA extraction damping fluid; Reduced the influence to the PCR effect, improved the detection sensitivity of quantitative fluorescent PCR greatly, the system and the reaction conditions of quantitative fluorescent PCR are groped the condition gained according to design primer size simultaneously; Simple to operate, detection sensitivity is high, can reach 2fg/ μ l; Have only the existence of a cause of disease spore in the soil, can detect.
The above is merely preferred embodiment of the present invention, not in order to restriction the present invention, all any modifications of within spirit of the present invention and principle, being done, is equal to and replaces and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a method that is used for detecting the soil tobacco black shank bacterium is characterized in that, this method may further comprise the steps:
Get the black shank germ of cryopreservation and cultivate, and utilize the CTAB method to extract genomic dna with other fungies;
Extract total DNA of black shank germ soil;
According to the gene order design primer of black shank germ in the gene pool, and carry out the checking of primer specificity pcr amplification;
Detect, analyze the sensitivity of primer and make the quantitative fluorescent PCR typical curve;
Utilize the quantitative fluorescent PCR typical curve, the DNA of different black shank occurring degree soil is carried out quantitative analysis.
2. the method for claim 1 is characterized in that, said black shank germ of getting cryopreservation is cultivated further with other fungies and comprises:
After 28 ℃ of incubated overnight of PDA liquid nutrient medium, get 1.5mL bacterium liquid, collect mycelium, distilled water washing 2 times.
3. the method for claim 1 is characterized in that, the implementation method that the said CTAB of utilization method is extracted genomic dna is:
Add the CTAB solution of 500 μ l, cool off 30s in the liquid nitrogen, put into 65 ℃ of water-bath 30s immediately, repeat 3 times;
Add granulated glass sphere vortex oscillation 5min, 65 ℃ of insulation 20min, during shake once;
Add phenol, chloroform, primary isoamyl alcohol, volume ratio 25: 24: 1, the centrifugal 10min of 12000r/min adds chloroform, primary isoamyl alcohol in the supernatant, volume ratio 24: 1, the centrifugal 10min of 12000r/min;
Get supernatant, add-20 ℃ of precooling isopropanol precipitatings of 2/3 times of volume, leave standstill about 30min;
The slow speed of revolution is centrifugal, and deposition is used the absolute ethyl alcohol rinsing 1 time again with 70% ethanol rinsing 2 times, dries up, and is resuspended in the 100ul distilled water, and-20 ℃ of preservations are subsequent use.
4. the method for claim 1 is characterized in that, the implementation method of total DNA of said extraction black shank germ soil is:
The preparation conidial suspension is also inoculated soil, 3 repetitions of every part of soil;
Add the DNA extraction damping fluid in the pedotheque, 37 ℃ of temperature are bathed 30min, every 5min mixing;
Add 20%SDS, final concentration is 2%, 65 ℃ of water-bath 2h, and every 10min puts upside down mixing, the centrifugal 10min of 6000g room temperature;
Add phenol, chloroform, primary isoamyl alcohol, three's volume ratio is 25: 24: 1, and the centrifugal 10min of 12000r/min adds chloroform, primary isoamyl alcohol in the supernatant, and the two volume ratio is 24: 1, the centrifugal 10min of 12000r/min;
Get supernatant, add-20 ℃ of precooling isopropanol precipitatings of 2/3 times of volume, leave standstill about 30min;
The slow speed of revolution is centrifugal, and deposition is used the absolute ethyl alcohol rinsing 1 time again with 70% ethanol rinsing 2 times, dries up, and is resuspended in the 100ul distilled water, and-20 ℃ of preservations are subsequent use.
5. like claim 1 or 4 described methods, it is characterized in that, added 2% PVP in the said DNA extraction damping fluid, be used for removing the soil partial impurities pollution, reduce influence, improve the detection sensitivity of quantitative fluorescent PCR the PCR effect.
6. like claim 1 or 4 described methods, it is characterized in that the consisting of of said soil total DNA extraction damping fluid:
The pH value is 8.0 100mmol/L Tris-HCl, 100mmol/L EDTA-Na2,2%PVP, 1.5mol/L NaCl, 1%CTAB, 125 μ g/mL Proteinase Ks.
7. the method for claim 1 is characterized in that, said gene order design primer according to black shank germ in the gene pool, and the implementation method of carrying out the checking of primer specificity pcr amplification is:
18S rDNA gene order according to black shank germ in the ncbi database; Utilize Primer5.0 design quantitative fluorescent PCR special primer SP:5 '-TGAAGAACGCTGCGAACTGC-3 '; AP:5 '-CTGACATCTCCTCCACCGACTA-3 ', amplification purpose fragment length is 172bp;
The genomic dna of the black shank pathogenic bacteria gene group DNA that extracts and other pathomycetes is carried out the primer specificity detection.
8. like claim 1 or 7 described methods, it is characterized in that the design primer specificity is good in this method, close species do not have respective strap when increasing, and the size of products therefrom is 172bp, is suitable for the quantitative fluorescent PCR reaction conditions.
9. like claim 1 or 7 described methods, it is characterized in that the amplification system of quantitative fluorescent PCR and reaction conditions are groped the condition gained according to design primer size in this method, adapt with the institute designed primer; Wherein, the amplification system of quantitative fluorescent PCR is:
1 * SYBR Green
I Master Mix (TaKaRa), 10 μ l; Primer respectively is 400nM; Template 2 μ L, amplification system are 25 μ L;
The response procedures of quantitative fluorescent PCR is:
94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 20s, 65 ℃ of annealing 40s, 72 ℃ are extended 40s, 72 ℃ of 5min, 40 circulations.
10. the method for claim 1 is characterized in that, said detection, the implementation method of analyzing the sensitivity of primer and making the quantitative fluorescent PCR typical curve are:
With the conidial soil DNA of inoculation different concns germ is template, checks this primer to conidial sensitivity in the soil;
10 times of gradient dilution liquid with black shank germ genomic dna are template, and check primer SP/AP is to the sensitivity of genomic dna, and it is limited to 2fg/ μ l genomic dna under detecting, and explains that this primer has very high sensitivity;
The DNA that chooses different dilution gradients is a template, and fluorescent quantitative PCR is made the quantitative fluorescent PCR typical curve.
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| CN110257366A (en) * | 2019-07-05 | 2019-09-20 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | It is a kind of based on the bacillus subtilis of charcoal to the control method of tobacco diseases |
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| CN113430290A (en) * | 2021-06-15 | 2021-09-24 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Probe gene, primer pair, kit and application for detecting tobacco black shank |
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| CN104232748B (en) * | 2014-03-10 | 2016-05-18 | 浙江省农业科学院 | Whether a kind of red bayberry nursery stock carries the rapid molecular detection method of wilting germ |
| CN105256047A (en) * | 2015-11-06 | 2016-01-20 | 中国烟草总公司郑州烟草研究院 | Locked nucleotide sensitized real-time fluorescent quantitative PCR (polymerase chain reaction) detection method for detecting colonization quantity of phytophthora parasitica in tobacco planting soil |
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