CN101805793B - PCR detection method of L.maculans and primer used for detection - Google Patents
PCR detection method of L.maculans and primer used for detection Download PDFInfo
- Publication number
- CN101805793B CN101805793B CN 201010142440 CN201010142440A CN101805793B CN 101805793 B CN101805793 B CN 101805793B CN 201010142440 CN201010142440 CN 201010142440 CN 201010142440 A CN201010142440 A CN 201010142440A CN 101805793 B CN101805793 B CN 101805793B
- Authority
- CN
- China
- Prior art keywords
- primer
- pcr
- maculans
- dna
- rape
- 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.)
- Expired - Fee Related
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a PCR detection method of L.maculans and a primer used for detection. According to the difference of the sequences of the L.maculans and a similar species of ITS thereof, a specific primer Lmb3(5'-caccaattggatccccta-3')/Lmb4(5'-aggcgagtcccaagtggaaca-3') for detecting the L.maculans is designed, and the PCR detection method of the L.maculans is constructed. The detection sensitivity of the method is 4pg hypha DNA. The detection experiment of a rapeseed sample shows that the PCR method can be used for the quick detection of the L.maculans in the imported rapeseed sample.
Description
Technical field
The present invention relates to agricultural and Plant Quarantine technical field, be specifically related to a kind of detection method of rape stem foot ulcer bacteria, relate in particular to a kind of PCR detection method of rape stem foot ulcer bacteria.In addition, the invention still further relates to the primer that detects rape stem foot ulcer bacteria.
Background technology
Rape stem foot ulcer bacteria Leptosphaeria maculans (being L.maculans) and rape black shank bacterium Leptosphaeria biglobosa (being L.biglobosa) are two kinds of pathogenic bacterias that cause the rape balck shank in the world wide.The morphological specificity of these two kinds of germs is close, but it is different with the ability of base portion ulcer to cause that oily stem infects.Rape stem foot ulcer bacteria L.maculans virulence is strong, causes the basal part of stem morbidity.Rape black shank bacterium L.biglobosa virulence a little less than, cause that generally the stem middle and upper part causes harm, L.maculans causes the serious popular major cause with the yield of rape loss of rape balck shank.At present; China only has L.biglobosa to distribute; But the international trade of rape exchanges very frequent with seed resource; The inward a large amount of Semen Brassicae campestris of state all will take place from rape stem foot Peptic Ulcerss such as Australia, Ukraine, Canada every year in China, and China entered the territory the rape total amount above 3,200,000 tons in 2009.Therefore, the enter the territory rapid detection of rape stem foot ulcer bacteria in the Semen Brassicae campestris has become the new problem of port quarantine department.
At present rape stem foot ulcer bacteria detection technique comprises filter paper culture method and PCR method, PCR with its fast, accurately and sensitive characteristics receive widespread use.Taylor (Taylor andBorgman, 1993) etc. have set up the PCR method that detects strong virulence bacterial strain according to one section Tumor-necrosis factor glycoproteins design special primer; Mahuku et al. (1995) detects the L.maculans of strong virus force from ITS sequences Design primer; Nineteen ninety-five; The special primer HV17S/HV26C that Mahuku etc. had once designed the ITS district detects strong virus force bacterial strain (being rape stem foot ulcer bacteria) (Mahuku et al.1995), and the sequence of part rape stem foot ulcer bacteria bacterial strain can not be mated fully among sequential analysis discovery downstream primer 3 ' end and the GenBank.2006, Liu etc. according to the ITS sequences Design detect the primer LmacF/LmacR (Liu et al.2006) of L.maculans, use this primer in the test and detect when entering the territory the Semen Brassicae campestris sample, false-positive result appears in the amplification of sample segment easily.In addition, the annealing temperature of PCR reaction also influences the specificity of amplification.
Domestic rape balck shank is to be caused by L.biglobosa, mainly is distributed in rape main product provinces and regions such as Zhejiang, Anhui, Hubei, Hunan, Sichuan and the Inner Mongol.Rape stem foot Peptic Ulcers does not take place in China as yet, and its pathogenic bacteria is quarantine harmful organisms L.maculans.Two kinds of germs are all got over the summer and survive the winter strong stress resistance with perithecium and mycelia in invalid strain.Main through air, invalid body and seed dispersal, invalid body and infected seed are the main modes of long-distance communications.At present, under the macroclimate of Global warming, rape stem foot Peptic Ulcers also spreads on Europe, America, Australia and other places, comes tremendous loss for the rape industrial zone.China is that rape the biggest in the world is produced country, and cultivated area reaches more than 700 ten thousand hectares, and YO is more than 1,000 ten thousand tons.And domestic rapeseed cultivation kind lacks disease resistance to rape stem foot Peptic Ulcers, and rape producing region weather is with abroad should the popular area of disease similar.If rape stem foot ulcer bacteria imports China into, bring destructive strike will for the rape production of China.Since in August, 2009, rape stem foot ulcer bacteria and rape black shank bacterium are are intercepted and captured in the port, Shanghai from the multiple batches of Semen Brassicae campestris that states such as Australia, Ukraine and Canada enter the territory.The various places sanitary authority should be strengthened prevention awareness, the active and effective quarantine of carrying out the Semen Brassicae campestris that enters the territory.Relevant department can take preventive measures and monitor and prevention and control, in addition, actively develops the resistance resource screening of China's rape and cress, cultivates disease-resistant variety, with reply Biosafety hidden danger, and protection China rape production safety.
Summary of the invention
The technical problem that the present invention will solve is to provide a kind of PCR detection method of rape stem foot ulcer bacteria; For this reason, the present invention also is provided for the detection primer of aforesaid method.
In order to solve the problems of the technologies described above, the present invention realizes through following technical scheme:
In one aspect of the invention, a kind of PCR detection method of rape stem foot ulcer bacteria is provided, comprises the steps:
(1) design primer, the sequence of this primer is:
Lmb3:5 '-caccaattggatccccta-3 ' (SEQ ID NO.1) or its complementary strand;
Lmb4:5 '-aggcgagtcccaagtggaaca-3 ' (SEQ ID NO.2) or its complementary strand;
(2) collect mycelia and extract DNA;
(3) adopting step (1) designed primer to carry out PCR respectively detects.
In the step (2), said collection mycelia is also extracted DNA and is specially: bacterial strain is collected mycelia with PDA substratum breeding back, gets hypha powder after the liquid nitrogen grinding, extracts test kit with Plant Genome and extracts DNA.
In the step (3), the reaction system that said PCR detects is 30 μ L, comprises 3 μ L, 10 * PCRBuffer, 3 μ L dNTP, 3 μ L MgCl
2, each 0.5 μ L of step (1) upstream and downstream primer, 1 μ L template DNA, 1U Taq polysaccharase, add sterilized water and mend to 30 μ L.
In the step (3), the response procedures that said PCR detects is: 94 ℃ of preparatory sex change 3min; Then with 94 ℃ of 30s, 56 ℃ of 30s, 30 circulations of 72 ℃ of 40s amplifications; Last 72 ℃ are extended 5min.
In another aspect of this invention, a kind of primer that is used to detect rape stem foot ulcer bacteria is provided, its sequence is:
Lmb3:5 '-caccaattggatccccta-3 ' (SEQ ID NO.1) or its complementary strand;
Lmb4:5 '-aggcgagtcccaagtggaaca-3 ' (SEQ ID NO.2) or its complementary strand.
At present; Different according to the host's specialization of germ and geographic origin; Be divided into two subspecies or group under the L.maculans kind, comprise L.maculans subsp.brassicae and L.maculans subsp.lepidii, the former host belongs to Brassica spp. for the cultivation rape.For cultivation rapes such as aimed detection rape belong to the L.maculans subsp.brassicae on the host; The present invention is according to the ITS sequence of L.maculans among the Genbank; Design the harmful organism L.maculans subsp.brassicae in the primer Lmb3/4 specific detection Semen Brassicae campestris sample, set up the PCR detection method of L.maculans.58 bacterial strains to supplying examination detect, and test-results shows, but primer Lmb3/4 specific detection L.maculans, and the sensitivity that detects is very high.Primer Lmb3/4 can increase and supply 22 L.maculans bacterial strains of examination, obtains the expection band of 276bp, supplies 30 L.biglobosa bacterial strains and 6 relevant kind of bacterial strains of examination not to have amplified production.Detection sensitivity is 4pg mycelia DNA, and the detection test of Semen Brassicae campestris sample shows that this PCR method can be used for the rapid detection of L.maculans in the inward Semen Brassicae campestris sample.
Description of drawings
Fig. 1 is the detection sensitivity synoptic diagram as a result of PCR in the embodiment of the invention;
Fig. 2 is that the PCR of Semen Brassicae campestris sample in the embodiment of the invention detects electrophorogram.
Embodiment
Following examples only be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions among the embodiment usually by normal condition, or is pressed the condition that manufacturer advises.
1 material method
1.1 strains tested
Test with L.maculans and L.biglobosa part bacterial strain by this laboratory (Shanghai Entry-Exit Inspection and Quarantine bureau) separation and purification from the inward Semen Brassicae campestris samples of state such as Canadian, Australia and Ukraine; The part bacterial strain is provided by Shenzhen, Jiangsu and Ningbo Entry-Exit Inspection and Quarantine Bureau technique center; Domestic L.biglobosa bacterial strain is given birth to teacher by the Li Qiang of academy of agricultural sciences, Anhui and is provided, and table 1 is seen in strain number and source.
Table 1 test strain
In the table 1 ,+expression positive reaction (positive reaction);-expression negative reaction (negative reaction); A: the Wu Cuiping of Jiangsu Entry-Exit Inspection and Quarantine Bureau provides; B: clever the providing of Shenzhen Entry-Exit Inspection and Quarantine Bureau journey; C: the Zhang Jiancheng of Ningbo Entry-Exit Inspection and Quarantine Bureau provides; D: the Li Qiang of academy of agricultural sciences, Anhui gives birth to and provides.
1.2 supply the examination primer
According to L.maculans among the GenBank and sibling species ITS sequence difference thereof; Designed Auele Specific Primer Lmb3 (5 '-caccaattggatccccta-3 ')/Lmb4 (5 '-aggcgagtcccaagtggaaca-3 ') of L.maculans, the amplified production clip size is respectively 276bp.Primer entrusts Shanghai living worker's biotechnology Services Co., Ltd synthetic.
1.3 mycelia is collected and DNA extraction
Strains tested is transferred to PDA substratum (potato dextrose agar; Every liter of substratum contains yam 200g; Sucrose 20g and agar 17g) breeding back collection mycelia; Get the 0.1g hypha powder after the liquid nitrogen grinding, (TIANGEN BIOTECH company DP305-02) extracts DNA to extract test kit with Plant Genome.
1.4 primer specific property testing
PCR reagent is all available from precious biotechnology (Dalian) ltd (Takara).Supply the bacterial strain DNA of examination with primer Lmb3/4 amplification, the PCR reaction system is 30 μ L (Takara), comprises 3 μ L, 10 * PCRBuffer, 3 μ L dNTP (each 250 μ M), 3 μ L MgCl
2(25mM), each 0.5 μ L of upstream and downstream primer (5 μ M), 1 μ L template DNA, 1U Taq polysaccharase, add sterilized water and mend to 30 μ L.The PCR response procedures is: 94 ℃ of preparatory sex change 3min; Then with 94 ℃ of 30s, 56 ℃ of 30s, 30 circulations of 72 ℃ of 40s amplifications; Last 72 ℃ are extended 5min.Amplified production is electrophoresis in 1.5% sepharose, 1 * TAE damping fluid, and EB dyeing back is analyzed with gel imaging system.
1.5 primer sensitivity test
With difference serial dilution 10,10 behind the DNA mensuration nucleic acid concentration of the L.maculan bacterial strain that is numbered 8129-5
2, 10
3, 10
4Doubly, carry out pcr amplification, the detection sensitivity of test PCR with primer Lmb3/4.
The detection of Semen Brassicae campestris sample 1.6 enter the territory
Picking variable color from the inward Semen Brassicae campestris sample (sample number into spectrum 333) of Ukraine, deformity or wrinkled seed are a sample with 50,10,5 and 1 respectively, and each prepares 30 duplicate samples.Extract DNA after the sample liquid nitrogen grinding, every duplicate samples DNA gets 1 μ L and carries out the PCR detection.
2 results and analysis
2.1 primer specific property testing
Supply 58 bacterial strain DNA of examination to carry out pcr amplification with primer Lmb3/4, primer Lmb3/4 22 strains of can increasing supply the L.maculans bacterial strain DNA of examination, obtain the expection amplified production of 276bp, and other bacterial strains DNA and the blank that supply to try do not have amplified band (table 1).Test-results shows, but primer Lmb3/4 specific detection L.maculans.
2.2 primer sensitivity test
Primer Lmb3/4 amplification different series extent of dilution is numbered the mycelia DNA of the L.maculan bacterial strain of 8129-5, and the DNA of 4ng, 400pg and 40pg can increase and obtain expecting band, and band (Fig. 1) does not appear expecting in the DNA of 4pg; Among Fig. 1, M. is Markers DL2000 (Takara) 100,250,500,750,1000,2000bp; 1～4 expression is numbered the 4ng of the L.maculan bacterial strain of 8129-5,400pg, 40pg, 4pg DNA; Test-results shows that the detection sensitivity of primer Lmb3/4 is 4pg mycelia DNA.
2.3 the pcr amplification of Semen Brassicae campestris sample
The doubtful seed of catching an illness in the inward Semen Brassicae campestris sample is a test sample with 10,5,1 respectively, extracts the laggard performing PCR of DNA and detects.As shown in table 2, detected result shows that PCR method is respectively 10%, 23.3% and 40% to the positive rate of 1,5 and 10 Semen Brassicae campestris sample.It is as shown in Figure 2 that the PCR of Semen Brassicae campestris sample detects electrophoresis result, and among Fig. 2, M representes Markers DL2000 (Takara) 100,250,500,750,1000,2000bp; 1～3 representes PCR to 10,5, and 1 detection; The DNA of 4 expression L.maculans; The DNA of 5 expression L.biglobosa; The no dna profiling of 6 expressions.
The PCR of table 2 Semen Brassicae campestris sample detects
3 discuss
Different according to the host's specialization of germ and geographic origin are thought to comprise six subspecies (Mendes-Pereira et al.2003 under the rape black shank bacterium L.biglobosa kind at present; Voigt et al.2005; Vincenot et al.2008), comprise L.biglobosa subsp.brassicae, the host belongs to Brassica spp. for rape; L.biglobosa subsp.thlaspii, host Wei Thlaspi (penny cress) Thlaspiarvense; L.biglobosa subsp.erysimii, host are Erysimum Erysimum sp.; L.biglobosa subsp.canadensis; L.biglobosa subsp.australiensis and L.biglobosasubsp.occiaustralensis.Comprise two subspecies under the rape stem foot ulcer bacteria L.maculans kind, comprise L.maculans subsp.brassicae, the host belongs to Brassica spp. for the cultivation rape; L.maculans subsp.lepidii, host are separate row Vegetable spp Lepidium spp. (Mendes-Pereira et al.2003; Voigt et al.2005; Vincenot et al.2008)..In the test from inward Semen Brassicae campestris samples such as Australia, Ukraine and Canada surplus in the of isolating 100 a rape stem foot ulcer bacteria isolate through order-checking and sequential analysis; All isolates are all similar with the sequence height of L.maculans subsp.brassicae, and similarity is 99.8%.A rape black shank bacterium isolate shape has comprised three subspecies such as L.biglobosa subsp.brassicae, L.biglobosa subsp.canadensis and L.biglobosasubsp.australiensis surplus in the of isolating 100 simultaneously.The L.maculans Auele Specific Primer that test is designed is aimed detection subspecies L.maculans subsp.brassicae; These subspecies mainly are distributed in ground such as Europe, Canada, Australia and New Zealand; Host plant comprises swede type rape Brassica napus, wild cabbage B.oleracea, leaf mustard B.juncea and beet Beta vulgaris (Mendes-Pereira et al.2003).
The separation of pathogenic bacteria is to cultivate after the unusual seed-coat sterilization such as picking variable color from PCR initial survey male Semen Brassicae campestris sample, deformity or shrinkage in the test; The possibility that is separated to germ is higher than random sampling, and the PCR detected result of picking seed sample and chance sample has also confirmed this possibility; On the other hand, seed sample is effectively killed inactivation attached to germ mycelia of seed-coat etc. in the process of surface sterilization, and therefore, the probability that is separated to germ in the picking sample can not represent truly that seed carries the probability of germ in the primary sample.See that from the result who separates germ the inward Semen Brassicae campestris sample separation success ratio of Australia is 2.0-2.6%; The inward Semen Brassicae campestris sample separation success ratio of Ukraine is 0-2.6%; The inward Semen Brassicae campestris sample separation success ratio of Canada is 0-0.3%.Generally, the probability that seed carries rape stem foot ulcer bacteria in the inward Semen Brassicae campestris sample of Canada is less than Australia and Ukraine's sample.Because test sample is through preliminary screening, the actual bacterial bearing rate of Semen Brassicae campestris should be lower than this value in the sample.The content of molds of each test sample is different, and is in lower level, even 1 Semen Brassicae campestris seed carries disease germs; Content is also very limited, and conventional PCR is difficult for detecting, and has detected the sample of 30 parts of 1 seeds in the test; Only can detect 6 positives, and a little less than the amplified band.
Sequence table
< 110>Shanghai Bureau of Emigration &. Engression Examination &. Quarantine People's R
< 120>primer is used in the PCR detection method and the detection of rape stem foot ulcer bacteria
<130>NP-10-14073
<160>2
<170>PatentIn?version?3.3
<210>1
<211>18
<212>DNA
< 213>artificial sequence
<220>
<221>misc_feature
< 223>primer
<400>1
caccaattgg?atccccta 18
<210>2
<211>21
<212>DNA
< 213>artificial sequence
<220>
<221>misc_feature
< 223>primer
<400>2
aggcgagtcc?caagtggaac?a 21
Claims (3)
1. the PCR detection method of a rape stem foot ulcer bacteria is characterized in that, comprises the steps:
(1) design primer, the sequence of this primer is:
Lmb3：5’-caccaattggatccccta-3’(SEQ?ID?NO.1)；
Lmb4：5’-aggcgagtcccaagtggaaca-3’(SEQ?ID?NO.2)；
(2) collect mycelia and extract DNA;
(3) adopting step (1) designed primer to carry out PCR detects; The reaction system that said PCR detects is 30 μ L, comprises 3 μ L10 * PCR Buffer, 3 μ L dNTP, 3 μ L MgCl
2, step
(1) each 0.5 μ L of upstream and downstream primer, 1 μ L template DNA, 1U Taq polysaccharase add sterilized water and mend to 30 μ L; The response procedures that said PCR detects is: 94 ℃ of preparatory sex change 3min; Then with 94 ℃ of 30s, 56 ℃ of 30s, 30 circulations of 72 ℃ of 40s amplifications; Last 72 ℃ are extended 5min.
2. the PCR detection method of rape stem foot ulcer bacteria as claimed in claim 1; It is characterized in that in the step (2), said collection mycelia is also extracted DNA and is specially: bacterial strain is collected mycelia with PDA substratum breeding back; Get hypha powder after the liquid nitrogen grinding, extract test kit with Plant Genome and extract DNA.
3. a primer that is used to detect rape stem foot ulcer bacteria is characterized in that, its sequence is:
Lmb3：5’-caccaattggatccccta-3’(SEQ?ID?NO.1)；
Lmb4：5’-aggcgagtcccaagtggaaca-3’(SEQ?ID?NO.2)。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010142440 CN101805793B (en) | 2010-04-08 | 2010-04-08 | PCR detection method of L.maculans and primer used for detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010142440 CN101805793B (en) | 2010-04-08 | 2010-04-08 | PCR detection method of L.maculans and primer used for detection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101805793A CN101805793A (en) | 2010-08-18 |
| CN101805793B true CN101805793B (en) | 2012-04-18 |
Family
ID=42607747
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010142440 Expired - Fee Related CN101805793B (en) | 2010-04-08 | 2010-04-08 | PCR detection method of L.maculans and primer used for detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101805793B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277430A (en) * | 2011-08-02 | 2011-12-14 | 李鑫 | Rape stem base botryosphaeria dothidea molecule standard sample and preparation method thereof |
| CN103255207A (en) * | 2013-01-15 | 2013-08-21 | 内蒙古农牧业科学院 | Molecular detection method for brassica campestris seeds carrying brassica campestris leptosphaeria maculans pathogenic bacterial |
| CN103131778A (en) * | 2013-02-05 | 2013-06-05 | 内蒙古农牧业科学院 | Molecular detection method of rapeseed-carried rapeseed blackleg pathogen |
| CN104818339B (en) * | 2015-05-29 | 2017-12-08 | 舟山出入境检验检疫局综合技术服务中心 | A kind of detection method of real-time fluorescent RCR ulcer bacteria |
| CN107099613A (en) * | 2017-06-27 | 2017-08-29 | 中国检验检疫科学研究院 | A kind of primer combination of probe and detection method for being used to detect real-time fluorescent RCR ulcer bacteria |
| CN107641660A (en) * | 2017-10-18 | 2018-01-30 | 湛江出入境检验检疫局检验检疫技术中心 | A kind of LAMP HNB primer sets, kit and method for being used to detect real-time fluorescent RCR ulcer bacteria |
| CN109371164B (en) * | 2018-12-21 | 2020-04-10 | 华中农业大学 | Application of specific sequence of rape black shank bacterium subspecies in rape black shank bacterium detection |
| CN112680538B (en) * | 2020-12-25 | 2023-08-18 | 重庆海关技术中心 | Primer, kit and detection method for real-time fluorescence PCR detection of cruciferous vegetable black shank single-row vegetable variety |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2092115C (en) * | 1993-03-22 | 1998-12-15 | Janet L. Taylor | Testing for infestation of rapeseed and other cruciferae by the fungus leptosphaeria maculans (blackleg infestation) |
-
2010
- 2010-04-08 CN CN 201010142440 patent/CN101805793B/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| 周国梁等.进境油菜籽中黑胫病菌和茎基溃疡病菌的检测.《植物保护学报》.2010,第37卷(第4期), * |
| 易建平等.进境澳大利亚油菜籽中茎基溃疡病菌的检测.《植物病理学报》.2010,第40卷(第6期), * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101805793A (en) | 2010-08-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101805793B (en) | PCR detection method of L.maculans and primer used for detection | |
| CN101974650B (en) | Polymerase chain reaction (PCR) method for detecting fusarium oxysporum and kit | |
| Datta et al. | Application of molecular markers for genetic discrimination of fusarium wilt pathogen races affecting chickpea and pigeonpea in major regions of India | |
| Agarkova et al. | Genetic diversity among Curtobacterium flaccumfaciens pv. flaccumfaciens populations in the American high plains | |
| Quazi et al. | Characterization of Fusarium proliferatum through species specific primers and its virulence on rice seeds | |
| CN104232782B (en) | A kind of detect tobacco soil-borne fungus pathogen PCR primer and application and method | |
| CN101805794B (en) | Real-time fluorescent PCR detection method of L.maculans as well as primer and probe used for detection | |
| CN101974651B (en) | Fluorescence quantitative polymerase chain reaction (PCR) detection method and detection kit for phytophthora capsici leonian | |
| CN103725782A (en) | Method, kit and primer for detecting pathogenic bacteria of lotus fungal rot disease carried by lotus seeds | |
| Shimano et al. | Application of nested PCR-DGGE (denaturing gradient gel electrophoresis) for the analysis of ciliate communities in soils | |
| CN105177148A (en) | Dual PCR primer for detecting two types of pathogens of grape canker at same time and application of dual PCR primer | |
| Li et al. | Incidence of sugarcane ratoon stunting disease in the major cane-growing regions of China | |
| CN101928779B (en) | Real-time fluorescent RCR molecular detection kit for leptosphaeria maculans and detection method thereof | |
| CN103276061A (en) | Kit having LAMP nucleic acid test strips and used for detecting brucella spp., and application thereof | |
| CN103388026A (en) | Phomopsis longicolla Hobbs detection target, PCR primer composition thereof and applications of the detection target and the PCR primer composition | |
| CN105316427A (en) | Fluorescent quantitative PCR detection kit for quickly detecting pythium aphanidermatum | |
| CN104099408A (en) | Qualitative detection method for potato scab germs | |
| CN101805795A (en) | Detection reagent kit and detection method of soybean phytophthora | |
| Yang et al. | Characterization of the mycelial compatibility groups and mating type alleles in populations of Sclerotinia minor in central China | |
| CN109136394A (en) | One kind proposing Dick's Salmonella TaqMan fluorescent quantitative PCR detection method and application until dawn | |
| Armenta-Cárdenas et al. | Epidemiological etiology of Erysiphe sp. and putative viral and phytoplasma-like symptoms in Ayocote bean (Phaseolus coccineus). | |
| SAVAzzINI et al. | Phenotypic and genotypic characterization of Italian Phytophthora infestans isolates | |
| CN104212896B (en) | The Molecular Identification primer of a kind of Tobacco Angular Leaf Spot Disease bacterium and authentication method | |
| CN102154462A (en) | Method and applications for fast detecting pathogenic bacteria molecules of bacterial soft rotting disease for banana | |
| CN103290138B (en) | The PCR in real time method for quick of abaca bunchy top virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120418 Termination date: 20180408 |