CN107164462A - The quantitative detecting method of withered germ of water-melon in a kind of soil - Google Patents
The quantitative detecting method of withered germ of water-melon in a kind of soil Download PDFInfo
- Publication number
- CN107164462A CN107164462A CN201710270280.1A CN201710270280A CN107164462A CN 107164462 A CN107164462 A CN 107164462A CN 201710270280 A CN201710270280 A CN 201710270280A CN 107164462 A CN107164462 A CN 107164462A
- Authority
- CN
- China
- Prior art keywords
- soil sample
- soil
- water
- melon
- withered germ
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of quantitative detecting method of withered germ of water-melon in soil, comprise the following steps：The graded series standard soil sample of the withered germ of water-melon containing various concentrations is prepared first；Extract graded series standard soil sample and soil sample STb gene to be measured；Quantitative fluorescent PCR reaction is carried out to graded series standard soil sample STb gene and soil sample STb gene to be measured using specific primer；Standard curve is made according to watermelon blight bacteria concentration in graded series standard soil sample and corresponding Ct values, watermelon blight bacteria concentration in soil sample to be measured is calculated.The present invention makes standard curve with standard soil sample known to watermelon blight bacteria concentration and then quantitatively detects watermelon blight bacteria concentration in soil sample to be measured, acquired results unit is every gram of soil of bacterium number, operating procedure efficiently facilitates, watermelon blight bacteria concentration in soil can be quickly and accurately detected, is suitable for large-scale promotion application.
Description
Technical field
The invention belongs to biological technical field, it is related to microorganism detection, and in particular to withered germ of water-melon in a kind of soil
Quantitative detecting method.
Background technology
Watermelon blight is by withered germ of water-melon i.e. Fusarium oxysporum f. sp. niveum (Fusarium oxysporum
F.sp.niveum a kind of fibrovascular system disease caused by) infecting, is a kind of destructive soil-borne disease, equal all over the world
There is different degrees of generation.In recent years, due to the change (such as high yield continuous cropping) of planting system, the disease occurs serious, it has also become system
The about major obstacle of China's watermelon industry development.Therefore, withered germ of water-melon is accurately and rapidly detected, for area west
Cucurbit wilt integrated control is significant.
The separation that the quantitative detecting method of withered germ of water-melon generally carries out germ using selective medium is trained at present
Support and colony counting.This method accuracy is low, time-consuming, and is easily influenceed by miscellaneous bacteria.As Protocols in Molecular Biology is increasingly mature,
Fluorescent quantitative PCR technique is widely used in phytopathogen dynamic detection and early warning.
Both at home and abroad studies have reported that molecular detecting method on withered germ of water-melon.Such as Cao of the Chinese Academy of Agricultural Sciences
Month rosy clouds etc., Lin Yinghong of Chung Hsing University etc., and establish can by the Zhang Zhenggang etc. of Agricultural University Of Nanjing
With the regular-PCR Molecular Detection system of specificity identification withered germ of water-melon.Zhang Zhenggang etc., which have been also set up, to be determined
The fluorescence quantifying PCR method of amount detection withered germ of water-melon, but this method is to be measured with the quantization of withered germ of water-melon DNA concentration
Sample, acquired results unit is every gram of soil of ng DNA, rather than every gram of soil of bacterium number, it is impossible to get information about watermelon blight in soil
The concentration of bacterium.In addition, the country there is no the patent of withered germ of water-melon quantitative detecting method.
The content of the invention
Goal of the invention：Withered germ of water-melon determines in the problem of existing for prior art, a kind of soil of present invention offer
Quantity measuring method, this method can be used for quickly detecting quantitative to watermelon blight bacterium number amount in soil.
Technical scheme：To achieve these goals, in a kind of soil of the present invention withered germ of water-melon quantitative detection
Method, comprises the following steps：
(1) the graded series standard soil sample of the withered germ of water-melon containing various concentrations is prepared；
(2) graded series standard soil sample and soil sample STb gene to be measured are extracted respectively using soil DNA extracts kit；
(3) specific primer detected available for withered germ of water-melon is selected and synthesized, is extracted respectively with step (2)
Graded series standard soil sample STb gene and soil sample STb gene to be measured are template, carry out quantitative fluorescent PCR reaction, respectively obtain and are
The Ct values of row gradient standard soil sample and soil sample to be measured；
(4) standard curve, root are made according to watermelon blight bacteria concentration in graded series standard soil sample and corresponding Ct values
Ct values and standard curve are reacted according to soil sample quantitative fluorescent PCR to be measured, it can obtain watermelon blight in soil sample to be measured by calculating
Bacteria concentration.
Wherein, the preparation method of the graded series standard soil sample of the withered germ of water-melon containing various concentrations described in step (1) is：
Take arable soil to be sterilized after air-drying and obtain soil sample；By withered germ of water-melon inoculated by hypha block to three equipped with mung bean fluid nutrient medium
In the bottle of angle, culture obtains withered germ of water-melon spore liquid, dilutes spore liquid successively after counting, takes the spore liquid after dilution to add
Air-dried into soil sample, that is, obtain the graded series standard soil sample of the withered germ of water-melon containing various concentrations.
Watermelon blight bacteria concentration is followed successively by 2 × 10 in the graded series standard soil sample8、2×107、2×106、 2×
105、2×104、2×103With 2 × 102Individual/g soil.
Wherein, step (3) specific primer is to for Fon-1/Fon-2.Can be from west by Fon-1/Fon-2 primer pairs
Specific amplification goes out 174bp product in cucurbit wilt bacterium.
Further, the quantitative fluorescent PCR reaction system is：The μ L of 2 × SYBR Premix Ex Taq 10, upstream and downstream
Each 0.4 μ L of primer (concentration is 10 μm of ol/L), the μ L of 0.4 μ L, DNA templates of ROX Reference Dye II 2, sterilizing are ultrapure
Water complements to 20 μ L.Quantitative fluorescent PCR response procedures are 95 DEG C of 30s；95 DEG C of 5s, 60 DEG C of 34s, 40 circulations.
The quantitative detecting method of withered germ of water-melon in soil of the present invention, is to use quantitative fluorescent PCR technology,
Mark song is drawn with standard soil sample known to watermelon blight bacteria concentration, so as to quantitatively detect the withered germ of water-melon in soil sample to be measured
The detection technique of concentration.Tracing detection available for withered germ of water-melon.
Beneficial effect：Compared with prior art, advantage of the present invention is as follows：
The detection method of the present invention can directly detect watermelon blight bacterium number amount in soil.The inventive method is with watermelon
Standard soil sample STb gene known to wilt concentration is dense according to withered germ of water-melon in graded series standard soil sample as template
Degree and corresponding Ct values make quantitative PCR standard curve, can directly calculate watermelon blight bacteria concentration, gained in soil sample to be measured
As a result unit is every gram of soil of bacterium number, rather than result unit is every gram of soil of every gram of soil of copy number or ng DNA in the prior art, can
Directly to detect the quantity of withered germ of water-melon in soil.The inventive method had both overcome classic flat-plate coating counting method result
Unreliability, the not intuitive of general measure PCR results is solved again, can substitute continue to use always flat board coating counting method
With conventional fluorescent quantifying PCR method.
Brief description of the drawings
Fig. 1 is that specific primer is expanded to withered germ of water-melon and cucumber fusarium axysporum and the PCR of common soil bacteria
Electrophoresis result；Swimming lane 1-2 is withered germ of water-melon, and 3-4 is cucumber fusarium axysporum, and 5-6 is bacillus；
Fig. 2 is the withered germ of water-melon fluorescent quantitative PCR solubility curve figure of standard soil sample and pedotheque to be measured, molten
It is 81.2 DEG C to solve temperature；
Fig. 3 is the withered germ of water-melon fluorescent quantitative PCR curve map of standard soil sample and pedotheque to be measured；
Fig. 4 is the quantitative fluorescent PCR canonical plotting drawn with standard soil sample known to watermelon blight bacteria concentration.
Embodiment
Below in conjunction with drawings and examples, the invention will be further described.
Bacterial strain and culture medium that embodiment is related to：
The bacterial strain being related in embodiment respectively withered germ of water-melon；Cucumber fusarium axysporum；Bacillus subtilis.(bacterial strain
Derive from Jiangsu Province Agriculture Science Institute)
Beef extract-peptone fluid nutrient medium：Beef extract 5g, peptone 10g, NaCl 5g, water 1L, pH 7.2-7.4.
Potato dextrose agar：Potato (is cleaned peeling chopping to boil by glucose 20g, potato 200g
30min, four layers of filtered through gauze, takes filtrate), agar 20g, water 1L, pH=7.0.
Mung bean fluid nutrient medium：Mung bean 20g, 30min is boiled by mung bean, and four layers of filtered through gauze take filtrate plus pure water to mend to 1L.
The primer sequence that embodiment is related to：
Fon-1：CGATTAGCGAAGACATTCACAAGACT
Fon-2：ACGGTCAAGAAGATGCAGGGTAAAGGT
Fon-1/Fon-2 primer pairs are synthesized by Jin Sirui bio tech ltd.
Reagent and instrument that embodiment is related to：
Soil DNA extracts kit is MP soil DNA extracts kits, purchased from Nanjing Shou De bio tech ltd,
Article No. is 116560200；
SYBR premix Ex Taq kits (include 2 × SYBR Premix Ex Taq and ROX Reference Dye
II) purchased from the precious bioengineering Co., Ltd in Dalian, article No. is RR420A；
2 × Taq PCR Master Mix are purchased from Nanjing Ji Tian biotechnologys Co., Ltd；
Quantitative real time PCR Instrument model ABI7500 in embodiment, data analysis by ABI7500 quantitative real time PCR Instruments from
The analysis software of band is completed, after the completion of quantitative fluorescent PCR reaction, and software is carried out to each fluorescence signal being collected into that reacts automatically
Analysis, selects suitable fluorescence threshold, the period then undergone using each sample at fluorescence threshold (Ct) and standard
The watermelon blight bacteria concentration of pedotheque sets up standard curve, and pedotheque to be measured can be according to its Ct value and standard curve meter
Calculate corresponding watermelon blight bacteria concentration.
Embodiment 1
Withered germ of water-melon primer specificity is detected.
(1) prepared by each bacterial strain STb gene：
It is special with potato dextrose agar culture Fusarium oxysporum f. sp. niveum FW0 and Fusarium oxysporum cucumber
Change type FOC, then extract STb gene with OMEGA fungal DNA extraction kits；With beef extract-peptone fluid nutrient medium culture withered grass
Bacillus BS211, then extract STb gene with OMEGA DNA of bacteria extracts kit.
(2) special primer is synthesized：
Fon-1：CGATTAGCGAAGACATTCACAAGACT
Fon-2：ACGGTCAAGAAGATGCAGGGTAAAGGT
Synthesized by Jin Sirui bio tech ltd.
(3) the PCR amplifications of withered germ of water-melon, cucumber fusarium axysporum, the specific fragment of bacillus subtilis：
Respectively using each bacterial strain DNA as template, Fon-1 and Fon-2 are upstream and downstream primer, enter performing PCR amplification.
PCR reaction systems are as follows：
2 × Taq PCR Master Mix 12.5 μ L, 20 μm of ol/L each 0.5 μ L of primer, 1 μ L concentration are 40-60ng/
μ L DNA profiling, sterilizing ultra-pure water complements to 25 μ L.
PCR response procedures are as follows：
95℃5min；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 30 circulations；72℃10min.
(4) PCR primer is identified：
6 μ L PCR primers are taken to carry out 1.5% agarose gel electrophoresis detection, according to the presence or absence of DNA bands and size to west
The specificity of cucurbit wilt bacterium primer special is verified.
(5) result of the test：
PCR amplifications are as shown in figure 1, in figure：Swimming lane 1-2 is withered germ of water-melon, and 3-4 is cucumber fusarium axysporum, 5-6
For bacillus subtilis.As seen from Figure 1, Fon-1/Fon-2 primer pairs can only specificity amplification from withered germ of water-melon STb gene
Go out 174bp band, and without amplified band in cucumber fusarium axysporum and bacillus subtilis.Therefore this pair of primer pair watermelon is withered
The germ that withers has good specificity.
Embodiment 2
Prepare standard soil sample known to watermelon blight bacteria concentration.
(1) preparation of withered germ of water-melon spore liquid：
By the inoculated by hypha block of withered germ of water-melon into the triangular flask equipped with 500mL mung bean fluid nutrient mediums, 30 DEG C,
170r/min shaking table cultures produce withered germ of water-melon spore liquid after 4 days, blood counting chamber is standby after counting.
(2) preparation of standard soil sample：
Topsoil soils are taken to air-dry rear 121 DEG C of sterilizings 30min in the rice terrace of Jiangsu Province Agriculture Science Institute six directions base, i.e.,
Obtain soil sample；It is 2 × 10 that withered germ of water-melon spore liquid is diluted into concentration successively with sterilized water8、 2×107、2×106、2×
105、2×104、2×103With 2 × 102Individual/mL；The spore liquid after 10mL dilutions is taken to be added in the soil sample of 10g acquisitions simultaneously respectively
Quick-air-drying, that is, obtain watermelon blight bacteria concentration and be followed successively by 2 × 108、2×107、2×106、2×105、2×104、2×103
With 2 × 102The graded series standard soil sample of individual/g soil.
Embodiment 3
The quantitative detection of withered germ of water-melon in soil sample to be measured.
(1) preparation of standard soil sample and soil sample STb gene to be measured：
Soil sample to be measured is derived from the watermelon facility plastic greenhouse of Jiangsu Province Agriculture Science Institute six directions base plants 1 batch and 2 batches of west respectively
The topsoil soils of melon, soil sample STb gene to be measured is extracted using MP soil DNAs extracts kit；Using MP soil DNA extracts reagents
It is respectively 2 × 10 that box, which extracts watermelon blight bacteria concentration,8、2×107、2×106、 2×105、2×104、2×103With 2 × 102
The graded series standard soil sample STb gene of individual/g soil.
(2) it is used for the PCR reaction systems for quantitatively detecting withered germ of water-melon：
20 μ L quantitative PCR reaction systems are as follows：2 × SYBR Premix Ex Taq10 μ L, 10 μm of ol/L Fon-1/
Each 0.4 μ L of Fon-2 primers (concentration is 10 μm of ol/L), the μ L of ROX Reference Dye II 0.4, concentration are 40-60ng/ μ
The μ L of L DNA profilings 2, sterilizing ultra-pure water complements to 20 μ L.
(3) it is used for the PCR response procedures for quantitatively detecting withered germ of water-melon：
95℃30s；95 DEG C of 5s, 60 DEG C of 34s, 40 circulations.
(4) fluorescent quantitative PCR：
It is respectively 2 × 10 by watermelon blight bacteria concentration8、2×107、2×106、2×105、2×104、2×103With 2 ×
102The standard soil sample and soil sample STb gene to be measured of individual/g soil carry out fluorescent quantitative PCR simultaneously, and with watermelon in standard soil sample
Wilt concentration and the mapping of corresponding Ct values, and carry out linear fit and obtain standard curve.According to soil sample Ct values to be measured and mark
Directrix curve, calculates watermelon blight bacteria concentration in soil sample to be measured.
(5) result of the test：
Watermelon blight bacteria concentration in watermelon facility plastic greenhouse soil is determined using the above method.Solubility curve is shown in Fig. 2, molten
It is 81.2 DEG C to solve temperature, and standard sample and testing sample solubility curve are in unimodal.Amplification curve is shown in Fig. 3, and amplification efficiency is
106.912。
Standard curve is shown in Fig. 4, and linear equation is y=36.958-3.167x, R2=0.99.
According to soil sample Ct values to be measured and linear equation, watermelon blight bacteria concentration in soil sample to be measured can be calculated, knot is determined
Fruit is as shown in table 1：
Watermelon blight bacteria concentration is determined in the soil of a kind of plant different stubble watermelon of table
As can be seen from Table 1, detect that withered germ of water-melon can be straight in growth of watermelon soil using the inventive method
The watermelon blight bacterium number amount in soil that draws is connect, the copy number obtained with general measure PCR /g soil or the native phases of ng DNA/g
Than as a result more directly perceived.
Claims (6)
1. the quantitative detecting method of withered germ of water-melon in a kind of soil, it is characterised in that comprise the following steps：
(1) the graded series standard soil sample of the withered germ of water-melon containing various concentrations is prepared；
(2) graded series standard soil sample and soil sample STb gene to be measured are extracted respectively using soil DNA extracts kit；
(3) select and synthesize the specific primer quantitatively detected available for withered germ of water-melon, be with what step (2) was extracted respectively
Row gradient standard soil sample STb gene and soil sample STb gene to be measured are that template carries out quantitative fluorescent PCR reaction, respectively obtain serial ladder
Spend the Ct values of standard soil sample and soil sample to be measured；
(4) standard curve is made according to watermelon blight bacteria concentration in graded series standard soil sample and corresponding Ct values, according to treating
Survey soil sample quantitative fluorescent PCR reaction Ct values and standard curve, you can calculate watermelon blight bacteria concentration in soil sample to be measured.
2. the quantitative detecting method of withered germ of water-melon in soil according to claim 1, it is characterised in that step (1)
The preparation method of the graded series standard soil sample of the withered germ of water-melon containing various concentrations is：Arable soil is taken to be sterilized after air-drying
Obtain soil sample；By withered germ of water-melon inoculated by hypha block into the triangular flask equipped with mung bean fluid nutrient medium, culture obtains watermelon
Wilt spore liquid, dilutes spore liquid after counting successively, takes the spore liquid after dilution to be added in soil sample and air-dries, that is, obtains
The graded series standard soil sample of the withered germ of water-melon containing various concentrations.
3. the quantitative detecting method of withered germ of water-melon in soil according to claim 1 or 2, it is characterised in that described
Watermelon blight bacteria concentration in graded series standard soil sample is followed successively by 2 × 108、2×107、2×106、2×105、2×104、2
×103With 2 × 102Individual/g soil.
4. the quantitative detecting method of withered germ of water-melon in soil according to claim 1, it is characterised in that step (3)
The specific primer is to for Fon-1/Fon-2.
5. the quantitative detecting method of withered germ of water-melon in soil according to claim 1, it is characterised in that step (3)
The quantitative fluorescent PCR reaction system is：The μ L of 2 × SYBR Premix Ex Taq 10, each 0.4 μ L, ROX of upstream and downstream primer
The μ L of Reference Dye II 0.4, the μ L of DNA profiling 2, sterilizing ultra-pure water complement to 20 μ L.
6. the quantitative detecting method of withered germ of water-melon in soil according to claim 1, it is characterised in that step (3)
The quantitative fluorescent PCR response procedures are 95 DEG C of 30s；95 DEG C of 5s, 60 DEG C of 34s, 40 circulations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710270280.1A CN107164462A (en) | 2017-04-24 | 2017-04-24 | The quantitative detecting method of withered germ of water-melon in a kind of soil |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710270280.1A CN107164462A (en) | 2017-04-24 | 2017-04-24 | The quantitative detecting method of withered germ of water-melon in a kind of soil |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107164462A true CN107164462A (en) | 2017-09-15 |
Family
ID=59812222
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710270280.1A Pending CN107164462A (en) | 2017-04-24 | 2017-04-24 | The quantitative detecting method of withered germ of water-melon in a kind of soil |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107164462A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103740806A (en) * | 2013-11-06 | 2014-04-23 | 中国热带农业科学院环境与植物保护研究所 | Method for quantitatively detecting Fusarium oxysporium f.sp.cubense Tripical race 4 from soil |
| CN104561278A (en) * | 2014-12-19 | 2015-04-29 | 中国农业科学院蔬菜花卉研究所 | Detection primer, detection kit and detection method for watermelon wilt disease fungi |
| CN105441543A (en) * | 2015-12-04 | 2016-03-30 | 江苏省农业科学院 | Primers for identifying fusarium oxysporum watermelon forma specialis physiological races and application thereof |
| CN106399511A (en) * | 2016-09-28 | 2017-02-15 | 湖南省西瓜甜瓜研究所 | Method and primer for detecting soil watermelon fusarium oxysporum through fluorescent quantitative real-time PCR |
| CN106520969A (en) * | 2016-11-24 | 2017-03-22 | 江苏省农业科学院 | Quantitative detection method for pseudomonadaceae in soil on basis of standard soil sample |
-
2017
- 2017-04-24 CN CN201710270280.1A patent/CN107164462A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103740806A (en) * | 2013-11-06 | 2014-04-23 | 中国热带农业科学院环境与植物保护研究所 | Method for quantitatively detecting Fusarium oxysporium f.sp.cubense Tripical race 4 from soil |
| CN104561278A (en) * | 2014-12-19 | 2015-04-29 | 中国农业科学院蔬菜花卉研究所 | Detection primer, detection kit and detection method for watermelon wilt disease fungi |
| CN105441543A (en) * | 2015-12-04 | 2016-03-30 | 江苏省农业科学院 | Primers for identifying fusarium oxysporum watermelon forma specialis physiological races and application thereof |
| CN106399511A (en) * | 2016-09-28 | 2017-02-15 | 湖南省西瓜甜瓜研究所 | Method and primer for detecting soil watermelon fusarium oxysporum through fluorescent quantitative real-time PCR |
| CN106520969A (en) * | 2016-11-24 | 2017-03-22 | 江苏省农业科学院 | Quantitative detection method for pseudomonadaceae in soil on basis of standard soil sample |
Non-Patent Citations (2)
| Title |
|---|
| ZHANG Z ET AL: "Molecular Detection of Fusarium Oxysporum F. Sp. Niveum and Mycosphaerella Melonis in Infected Plant Tissues and Soil", 《FEMS MICROBIOL LETT.》 * |
| 赵爽 等: "基因宏阵列和荧光定量PCR方法对西瓜枯萎病害土壤中尖孢镰刀菌的快速检测和定量", 《土壤学报》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103146691B (en) | SNP loci linked with blight resistant gene Fon-1 in watermelon, and markers thereof | |
| CN105441543B (en) | It is a kind of identification Fusarium oxysporum f. sp. niveum biological strain primer and its application | |
| CN103397099B (en) | Method for detecting quantity of pseudomonas fluorescent in rhizospheric soil during growth period of transgenic wheat by virtue of fluorescent quantitative PCR (Polymerase Chain Reaction) | |
| CN104830698A (en) | Pokkah boeng disease pathogen separating and identifying method | |
| CN104232748B (en) | Whether a kind of red bayberry nursery stock carries the rapid molecular detection method of wilting germ | |
| CN105316427A (en) | Fluorescent quantitative PCR detection kit for quickly detecting pythium aphanidermatum | |
| CN104278028B (en) | It is positioned at haynaldia villosa 6VS DNA and penetrates into wheat anti-powdery mildew NIL sequence and application | |
| CN101812517B (en) | Molecular marker of wheat scab ear rot and seedling rot resistance | |
| CN103882032B (en) | Cucumber fusarium axysporum disease-resistant gene Foc 4 and its encoding proteins and application | |
| CN107164462A (en) | The quantitative detecting method of withered germ of water-melon in a kind of soil | |
| CN103333886A (en) | Functional marker for analyzing rice blast resistance gene by using DNA (Deoxyribose Nucleic Acid) melting temperature | |
| CN103131755A (en) | Method for rapidly detecting brassica oleracea var. italic hybrid purity by using SRAP mark | |
| CN112522254B (en) | Molecular marker for detecting tomato bacterial wilt resistance locus Bwr12 and application | |
| CN110862930B (en) | White agrocybe cylindracea and molecular marker identification method thereof | |
| CN113789405A (en) | Method for detecting content of gibberella zeae in soil based on real-time fluorescent quantitative PCR | |
| CN103882017B (en) | The molecule marker of cucumber fusarium axysporum disease-resistant gene Foc-4 and primer special thereof and application | |
| CN106399529A (en) | Molecular detection primer for banana cladosporium cucumerinum and detection method | |
| CN106868145B (en) | Molecular marker, primer and probe for identifying tricholoma plumbum | |
| CN106755541B (en) | Molecular marker, primer and probe for identifying lyophyllum decastes | |
| CN102424851B (en) | Molecular detection method for Edrus deodara (Roxb) Lobd blight and primers therefor | |
| CN104975015A (en) | Method for assisting in screening stripe-rust-resistant wheat and special primers thereof | |
| CN106244721B (en) | A kind of capsicum stain anthrax bacteria molecular detection primer and detection method | |
| CN101086019A (en) | Mushroom 'Shenxiang'93 bacteria molecular specific mark, and method and uses | |
| CN104195223A (en) | Method for identifying iron deficiency-resistant apple rootstocks | |
| CN103525833B (en) | Gene, method used for identifying pathogenicity of Verticillium dahliae and kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170915 |