CN103060448A - Method for high sensitivity detection of Phytophthora hibernalis Carne - Google Patents
Method for high sensitivity detection of Phytophthora hibernalis Carne Download PDFInfo
- Publication number
- CN103060448A CN103060448A CN201210583427XA CN201210583427A CN103060448A CN 103060448 A CN103060448 A CN 103060448A CN 201210583427X A CN201210583427X A CN 201210583427XA CN 201210583427 A CN201210583427 A CN 201210583427A CN 103060448 A CN103060448 A CN 103060448A
- Authority
- CN
- China
- Prior art keywords
- concentration
- centrifuge tube
- pcr
- dna
- reaction product
- 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.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for high sensitivity detection of Phytophthora hibernalis Carne. The method comprises the following steps of orderly adding sterile deionized water, a phi29 DNA polymerase buffer solution, a forward primer PHIB1, a reverse primer PHIB2 and an object DNA into a sterile centrifugation tube, uniformly mixing, carrying out centrifugation of the mixed reaction liquid to tube bottom, heating the sterile centrifugation tube, fast cooling in an ice-bath, orderly adding a dNTPs mixture, BSA and a phi29 DNA polymerase into the sterile centrifugation tube to obtain an RCA reaction product, utilizing a novel sterile centrifugation tube, carrying out PCR amplification to obtain a PCR reaction product, and detecting the PCR reaction product. The method can realize fast and sensitive detection of Phytophthora hibernalis Carne. The sensitivity of the method provided by the invention is 10*4<9> times higher than that of the traditional PCR amplification detection method.
Description
Technical field
The present invention relates to a kind of method that detects the mould brown rot pathogenic bacteria of living epidemic disease of oranges and tangerines winter, relate in particular to the method for a kind of mould brown rot pathogenic bacteria of living epidemic disease of application RCA-PCR technology for detection oranges and tangerines winter of high sensitivity.
Background technology
It is wide that the mould brown rot pathogenic bacteria of living epidemic disease of oranges and tangerines winter Phytophthora hibernalis Carne has host range, and the Orange Producing of causing harm is serious, the characteristics that distributional region is wide, and fruit, tissue or asexual propagation material carry out long-distance communications in spite of illness.China's oranges and tangerines planting area is widely distributed, and these pathogenic bacterias are along with the probability of the seed nursery stock successor China that introduces high-quality is increasing in recent years.In case this disease is imported into, will cause very big harm to China's Citrus Industry.It is to find first on the citrusfruit of western australia that the oranges and tangerines winter is given birth to the mould brown rot disease of living epidemic disease of microbial oranges and tangerines winter of the mould brown rot cause of disease of epidemic disease, also finds successively in other countries and area subsequently.This epidemic disease is that what to infect that oranges and tangerines cause is a kind of destructive disease, infects the initial stage, and light brown variable color appears in fruit surface, and infection site cortex is hard, and humidity grows the white hypha body when being fit to.Be injured fruit bitter, tool rotten odor.The oranges and tangerines planting area of China is extensive, and geography and climate is various, and the area that has suitable disease to occur is so it is large surely to grow possibility.Closely circulation way has the routes of transmission such as wind and rain, instrument, and long-distance communications are mainly propagated with soil, fruit, reproductive material.This disease continues perplexing the Orange Producing on the ground such as Italy, Portugal always.
For the existing part Study of the phylogeny of phytophthora fungi, the report of PCR detection is arranged also for the molecular detection technology of the mould brown rot pathogenic bacteria of living epidemic disease of oranges and tangerines winter at present.2008, Zhang Haifeng etc. have relatively analyzed the winter and have given birth to the mould and mould ITS sequence of other epidemic disease of epidemic disease, design on this basis 1 pair and detected the mould special primer 751F/752R of living epidemic disease of winter, this can give birth to the phytophthora amplification to the DNA band of long 616 bp from the winter to primer, and other 19 kinds of epidemic diseases are mould and other fungi all without amplified band.Its detection limit can reach the level that per 25 μ L reaction systems only need 10 fg genomic dnas, but the amplified band of the oranges and tangerines sample that carries disease germs is unobvious especially.Therefore a kind of detection method of more high sensitivity is provided, has become the study hotspot in this area.
Summary of the invention
The invention provides the method that a kind of high sensitivity detects the mould brown rot pathogenic bacteria of living epidemic disease of oranges and tangerines winter, the method can be quick, sensitive the oranges and tangerines winter that detects give birth to the mould brown rot pathogenic bacteria of epidemic disease, detection method provided by the invention is than traditional detection method of utilizing pcr amplification, and its susceptibility will exceed 10 * 4 at least
9Doubly.
Realize that the technical scheme that above-mentioned purpose of the present invention adopts is:
A kind of high sensitivity detects the method for the mould brown rot pathogenic bacteria of living epidemic disease of oranges and tangerines winter, may further comprise the steps:
(1), in aseptic centrifuge tube, add successively sterilization deionized water, phi29 DNA polymerase buffer, sense primer PHIB1, antisense primer PHIB2 and target compound DNA, then it is mixed rear centrifugal, reaction solution is centrifugal to managing the end;
(2), centrifuge tube is used the PCR instrument at 95 ℃ of lower heating 3-5min, then rapid ice bath 10-20min;
(3), in centrifuge tube, add successively dNTPs mixture, BSA and phi29 DNA polysaccharase, mix rear centrifugal, reaction solution is centrifugal to managing the end;
(4), centrifuge tube is placed the PCR instrument, under 30 ℃ condition, react 10-15h, after reaction finishes, with centrifuge tube heating bath 10min under 65 ℃ of conditions, phi29 DNA polysaccharase is lost activity, can make the RCA reaction product, prepared RCA reaction product should in time be used or-20 ℃ of lower cryopreservation;
(5), get in addition an aseptic centrifuge tube, in centrifuge tube, add successively sterilization deionized water, PCR damping fluid, dNTPs mixture, sense primer PHIB1, antisense primer PHIB2, RCA reaction product and TaqDNA polysaccharase, then that reaction solution is centrifugal to managing the end;
(6), centrifuge tube placed on the PCR instrument increases, obtain the PCR reaction product after the amplification;
(7), the PCR product is with the separation of 2%-3% agarose gel electrophoresis, behind ethidium bromide solution dyeing 12-20min, at ultraviolet transilluminator observation PCR reaction result.
Phi29 DNA polymerase buffer described in the step (1) is 10 * phi29 DNA polymerase buffer, volume ratio between the deionized water that adds, phi29 DNA polymerase buffer, sense primer PHIB1, antisense primer PHIB2 and the target compound DNA is 5.9:1:0.5:.05:1, wherein the concentration of sense primer PHIB1 and antisense primer PHIB2 is 10 μ M, and the concentration of target compound DNA is 100-200 ng/ μ l; The volume ratio of dNTPs mixture, BSA, phi 29 DNA polysaccharases and the target compound DNA that adds in the step (3) is 0.5:0.1:0.5:1, wherein the concentration of dNTPs mixture is 2.5mM each, the concentration of BSA is that the concentration of 1%, phi, 29 DNA polysaccharases is 10U/ μ l.
In the step (2) centrifuge tube is heated 3min with the PCR instrument under 95 ℃, then rapid ice bath 15min.
The component that adds in the step (5) is that dNTPs mixture, the concentration of 2.5mM each is that sense primer PHIB1, the concentration of 10 μ M is that antisense primer PHIB2, RCA reaction product and the concentration of 10 μ M is the TaqDNA polysaccharase of 5U/ μ l for sterilization deionized water, PCR damping fluid, concentration, and the volume ratio between each material is: 18:2.5:1:1:1:1: 0.5.
The program of amplification is in the step (6): 94 ℃ of 3min, 94 ℃ of 30S, 62 ℃ of 30S, 72 ℃ of 30S, circulation 32-36 time, last 72 ℃ of added time 5min.
Detection method provided by the invention is at first carried out the RCA amplification to total DNA, and RCA has superior signal amplifying function, is a kind of additional development of round pcr.Rolling circle amplification (RCA) the specific DNA that can not only directly increase realizes that the signal of these target nucleic acids amplifies, and amplification realizes that under constant temperature condition is relatively gentleer.Utilize rolling circle amplification technology DNA amplification, carry out rolling circle amplification by the micro-template to the pg order of magnitude, can obtain the high quality amplified production of the mg order of magnitude.
In independent pcr amplification, total the mycelia DNA after repeatedly diluting is made template, even carry out continuously pcr amplification with previous round PCR product as masterplate, the purpose that can not increase fragment, because the template DNA molecule is very little, PCR can not obtain any result for the first time.And among the present invention, the RCA amplification of at first carrying out guarantees for follow-up pcr amplification provides the DNA masterplate of sufficient amount, so that RCA-PCR is sensitiveer than independent PCR.
In the present invention simultaneously, because the linear dsdna molecule has replaced the circular double stranded DNA molecule of RCA amplification on the traditional sense, therefore this research is not proper DNA rolling-circle replication, and only be to have utilized the superpower DNA chain of phi29 DNA polysaccharase to replace activity and polymerization activity, at room temperature finish synthesis reaction of DNA.Phi29 DNA polysaccharase also has the function that self-healing is corrected, and therefore can not affect the fidelity of DNA order type.Easy and simple to handle, quick, highly sensitive, the good reproducibility of RCA-PCR detection method provided by the invention can be as the desirable desirable detection of pathogens means of a kind of pathogenic bacteria.
Description of drawings
Fig. 1 is the gel electrophoresis figure as a result in the embodiments of the invention 1.
Embodiment
Below in conjunction with specific embodiment the present invention is done detailed specific description.
The bacterial classification oranges and tangerines winter gives birth to the mould brown rot pathogenic bacteria of epidemic disease (Phytophthora hibernalis Carne) and available from U.S. ATCC DSMZ, is numbered 32995 in the present embodiment
TM, and in strict accordance with the quarantine dangerous bacterial classification carry out Operation ﹠ use.The pcr amplification of process DNA, the checking of Cloning and sequencing before using.
The extracting method of target compound DNA is as follows in the present embodiment: be 10 with 100 μ l concentration
7Spore suspension is coated on the PDA and V8 flat board that is covered with the sterilization glassine paper, 18 ℃ of lower cultivations 7-10 days, and the scraping mycelia is freezing for subsequent use.Get the 0.5g mycelia, place centrifuge tube, add in the CTAB Extraction buffer of the own preheating of 2ml (65 ℃), centrifuge tube is put upside down mixing, 65 ℃ of water-bath 5min, gently mixings; Add isopyknic phenol: chloroform: primary isoamyl alcohol (25:24:l), mixing, the centrifugal 15min of 12000r/min gets supernatant liquor in centrifuge tube, adds the equal-volume chloroform again: primary isoamyl alcohol (24:l) mixing, the centrifugal 15min of 12000r/min, get supernatant liquor in centrifuge tube, 100% ethanol that adds 2 times of volumes is put upside down mixing, the centrifugal 10min of 12000r/min, the supernatant liquor that inclines, with 70% ethanol with twice of washing of precipitate.The centrifuge tube that will contain DNA after washing finishes places 37 ℃ of incubators dry.Add 30 μ l TE dissolution precipitations after the drying, save backup in-20 ℃.
The nucleotide sequence of sense primer PHIB1 is shown in SEQ ID NO:1 in following examples: 5 '-TCGGGTCTGAGCTAGTAGTCTT-3 '; The nucleotide sequence of antisense primer PHIB2 is shown in SEQ ID NO:2: 5 '-CTTCCACAACCAATTCCATTATGC-3
The cumulative volume of RCA reaction system is 10 μ l in the present embodiment.Concrete detection method is as follows: (1) adds the target compound DNA volume 1 μ l that sense primer PHIB1 volume 0.5 μ l that sterilization deionized water 5.9 μ l, 10 * phi29 DNA polymerase buffer, 1 μ l, concentration are 10 μ M, antisense primer PHIB2 volume 0.5 μ l that concentration is 10 μ M and concentration are 200 ng/ μ l successively in aseptic centrifuge tube, then it is mixed rear centrifugal, reaction solution is centrifugal to managing the end;
(2), centrifuge tube is used the PCR instrument at 95 ℃ of lower heating 3min, then rapid ice bath 15min;
(3), to add successively concentration in centrifuge tube be that dNTPs mixture 0.5 μ l, the concentration of 2.5mM each is the phi29 DNA polysaccharase 0.5 μ l that 1% BSA 0.1 μ l and concentration are 10 U/ μ l, mix rear centrifugal, reaction solution is centrifugal to managing the end;
(4), centrifuge tube is placed the PCR instrument, under 30 ℃ condition, react 11h, after reaction finishes, with centrifuge tube heating bath 10min under 65 ℃ of conditions, phi29 DNA polysaccharase is lost activity, can make the RCA reaction product, prepared RCA reaction product should in time be used or-20 ℃ of lower cryopreservation;
(5), get in addition an aseptic centrifuge tube, in centrifuge tube, add successively sterilization deionized water 18 μ l, 10 * PCR damping fluid, 2.5 μ l, concentration and be the TaqDNA polysaccharase 0.5 μ l that the dNTPs mixture 1 μ l of 2.5mM each, sense primer PHIB1 volume 1 μ l that concentration is 10 μ M, antisense primer PHIB2 volume 1 μ l, RCA reaction product 1 μ l that concentration is 10 μ M and concentration are 5U/ μ l, then that reaction solution is centrifugal to managing the end;
(6), centrifuge tube placed on the PCR instrument increases, amplification program is 94 ℃ of lower 3min, 94 ℃ of lower 30S, 62 ℃ of lower 30S, 72 ℃ of lower 30S circulate 36 times, last 72 ℃ of 5min of lower added time obtain the PCR reaction product after the amplification;
(7), the PCR product separates with 3% agarose gel electrophoresis, behind ethidium bromide solution dyeing 20min, observes the PCR reaction result at ultraviolet transilluminator, can observe on sepharose only has a specific dna fragmentation (407 bp).1 μ l RCA reaction solution is diluted, through test, be diluted to 4
16The obvious DNA band that can also increase doubly has high susceptibility, and as shown in Figure 1, among Fig. 1, swimming lane 1: the winter is given birth to phytophthora+water; Swimming lane 2-11 is the RCA reaction solution+PHIB after the gradually dilution, and the RCA reaction solution is diluted to 4 in the swimming lane 11
16Obvious DNA band can also increase doubly.
The cumulative volume of RCA reaction system is 10 μ l in the present embodiment.Concrete detection method is as follows: (1) adds the target compound DNA volume 1 μ l that sense primer PHIB1 volume 0.5 μ l that sterilization deionized water 5.9 μ l, 10 * phi29 DNA polymerase buffer, 1 μ l, concentration are 10 μ M, antisense primer PHIB2 volume 0.5 μ l that concentration is 10 μ M and concentration are 110 ng/ μ l successively in aseptic centrifuge tube, then it is mixed rear centrifugal, reaction solution is centrifugal to managing the end;
(2), centrifuge tube is used the PCR instrument at 95 ℃ of lower heating 5min, then rapid ice bath 18min;
(3), to add successively concentration in centrifuge tube be that dNTPs mixture 0.5 μ l, the concentration of 2.5mM each is the phi29 DNA polysaccharase 0.5 μ l that 1% BSA 0.1 μ l and concentration are 10 U/ μ l, mix rear centrifugal, reaction solution is centrifugal to managing the end;
(4), centrifuge tube is placed the PCR instrument, under 30 ℃ condition, react 15h, after reaction finishes, with centrifuge tube heating bath 10min under 65 ℃ of conditions, phi29 DNA polysaccharase is lost activity, can make the RCA reaction product, prepared RCA reaction product should in time be used or-20 ℃ of lower cryopreservation;
(5), get in addition an aseptic centrifuge tube, in centrifuge tube, add successively sterilization deionized water 18 μ l, 10 * PCR damping fluid, 2.5 μ l, concentration and be the TaqDNA polysaccharase 0.5 μ l that the dNTPs mixture 1 μ l of 2.5 mM each, sense primer PHIB1 volume 1 μ l that concentration is 10 μ M, antisense primer PHIB2 volume 1 μ l, RCA reaction product 1 μ l that concentration is 10 μ M and concentration are 5U/ μ l, then that reaction solution is centrifugal to managing the end;
(6), centrifuge tube placed on the PCR instrument increases, amplification program is 94 ℃ of lower 3min, 94 ℃ of lower 30S, 62 ℃ of lower 30S, 72 ℃ of lower 30S circulate 36 times, last 72 ℃ of 5min of lower added time obtain the PCR reaction product after the amplification;
(7), the PCR product separates with 2% agarose gel electrophoresis, behind ethidium bromide solution dyeing 13min, observes the PCR reaction result at ultraviolet transilluminator, can observe on sepharose only has a specific dna fragmentation (407 bp).1 μ l RCA reaction solution is diluted, through test, be diluted to 4
16The obvious DNA band that can also increase doubly has high susceptibility.
Claims (5)
1. a high sensitivity detects the method that the oranges and tangerines winter gives birth to the mould brown rot pathogenic bacteria of epidemic disease, it is characterized in that may further comprise the steps:
(1), in aseptic centrifuge tube, add successively sterilization deionized water, phi29 DNA polymerase buffer, sense primer PHIB1, antisense primer PHIB2 and target compound DNA, then it is mixed rear centrifugal, reaction solution is centrifugal to managing the end;
(2), centrifuge tube is used the PCR instrument at 95 ℃ of lower heating 3-5min, then rapid ice bath 10-20min;
(3), in centrifuge tube, add successively dNTPs mixture, BSA and phi29 DNA polysaccharase, mix rear centrifugal, reaction solution is centrifugal to managing the end;
(4), centrifuge tube is placed the PCR instrument, under 30 ℃ condition, react 10-15h, after reaction finishes, with centrifuge tube heating bath 10min under 65 ℃ of conditions, phi29 DNA polysaccharase is lost activity, can make the RCA reaction product, prepared RCA reaction product should in time be used or-20 ℃ of lower cryopreservation;
(5), get in addition an aseptic centrifuge tube, in centrifuge tube, add successively sterilization deionized water, PCR damping fluid, dNTPs mixture, sense primer PHIB1, antisense primer PHIB2, RCA reaction product and TaqDNA polysaccharase, then that reaction solution is centrifugal to managing the end;
(6), centrifuge tube placed on the PCR instrument increases, obtain the PCR reaction product after the amplification;
(7), the PCR product is with the separation of 2%-3% agarose gel electrophoresis, behind ethidium bromide solution dyeing 12-20min, at ultraviolet transilluminator observation PCR reaction result.
2. the detection oranges and tangerines winter according to claim 1 is given birth to the method for the mould brown rot pathogenic bacteria of epidemic disease, it is characterized in that: the phi29 DNA polymerase buffer described in the step (1) is 10 * phi29 DNA polymerase buffer, volume ratio between the deionized water that adds, phi29 DNA polymerase buffer, sense primer PHIB1, antisense primer PHIB2 and the target compound DNA is 5.9:1:0.5:.05:1, wherein the concentration of sense primer PHIB1 and antisense primer PHIB2 is 10 μ M, and the concentration of target compound DNA is 100-200 ng/ μ l; The volume ratio of dNTPs mixture, BSA, phi 29 DNA polysaccharases and the target compound DNA that adds in the step (3) is 0.5:0.1:0.5:1, wherein the concentration of dNTPs mixture is 2.5mM each, the concentration of BSA is that the concentration of 1%, phi, 29 DNA polysaccharases is 10U/ μ l.
3. the detection oranges and tangerines winter according to claim 1 is given birth to the method for the mould brown rot pathogenic bacteria of epidemic disease, it is characterized in that: in the step (2) centrifuge tube is heated 3min with the PCR instrument under 95 ℃, then rapid ice bath 15min.
4. the detection oranges and tangerines winter according to claim 1 is given birth to the method for the mould brown rot pathogenic bacteria of epidemic disease, it is characterized in that: the component that adds in the step (5) is that dNTPs mixture, the concentration of 2.5mM each is that sense primer PHIB1, the concentration of 10 μ M is that antisense primer PHIB2, RCA reaction product and the concentration of 10 μ M is the TaqDNA polysaccharase of 5U/ μ l for sterilization deionized water, PCR damping fluid, concentration, and the volume ratio between each material is: 18:2.5:1:1:1:1: 0.5.
5. the detection oranges and tangerines winter according to claim 1 is given birth to the method for the mould brown rot pathogenic bacteria of epidemic disease, it is characterized in that: the program of amplification is in the step (6): 94 ℃ of 3min, 94 ℃ of 30S, 62 ℃ of 30S, 72 ℃ of 30S, circulation 32-36 time, last 72 ℃ of added time 5min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210583427.XA CN103060448B (en) | 2012-12-28 | 2012-12-28 | Method for high sensitivity detection of Phytophthora hibernalis Carne |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210583427.XA CN103060448B (en) | 2012-12-28 | 2012-12-28 | Method for high sensitivity detection of Phytophthora hibernalis Carne |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103060448A true CN103060448A (en) | 2013-04-24 |
| CN103060448B CN103060448B (en) | 2015-06-10 |
Family
ID=48103340
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210583427.XA Expired - Fee Related CN103060448B (en) | 2012-12-28 | 2012-12-28 | Method for high sensitivity detection of Phytophthora hibernalis Carne |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103060448B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101619364A (en) * | 2008-07-04 | 2010-01-06 | 上海市免疫学研究所 | Method for detecting HPV and application of Phi29DNA polymerase in detecting HPV |
| CN102660651A (en) * | 2012-06-05 | 2012-09-12 | 湖南大学 | Degenerate primer for detecting streptomyces two-domain laccase gene and detection method of degenerate primer |
-
2012
- 2012-12-28 CN CN201210583427.XA patent/CN103060448B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101619364A (en) * | 2008-07-04 | 2010-01-06 | 上海市免疫学研究所 | Method for detecting HPV and application of Phi29DNA polymerase in detecting HPV |
| CN102660651A (en) * | 2012-06-05 | 2012-09-12 | 湖南大学 | Degenerate primer for detecting streptomyces two-domain laccase gene and detection method of degenerate primer |
Non-Patent Citations (3)
| Title |
|---|
| 《Biochemical and Biophysical Research Communications》 20081231 Yun Xu Rapid detection and identification of a pathogen's DNA using Phi29 DNA polymerase 522-525 1-5 第375卷, * |
| D. MONTENEGRO: "A selective PCR-based method for the identification of Phytophthora hibernalis Carne", 《SPANISH JOURNAL OF AGRICULTURAL RESEARCH》 * |
| YUN XU: "Rapid detection and identification of a pathogen’s DNA using Phi29 DNA polymerase", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103060448B (en) | 2015-06-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11306365B1 (en) | Molecular marker C42257 for rapidly identifying genetic sex of Marsupenaeus japonicus and applications thereof | |
| CN103740815B (en) | Primer for multi-PCR (Polymerase Chain Reaction) detection aiming at fusaria and application of primer | |
| CN108893557A (en) | A kind of method of three kinds of wheat rhizome portion diseases of quick detection | |
| CN105368948A (en) | Primer for sex identification of Nile tilapia and PCR (polymerase chain reaction) identification method | |
| CN106939346A (en) | Paddy rice bacterial leaf spot resistant gene xa5 molecular labeling and its application | |
| CN117344054A (en) | Molecular marker primer for amplifying wheat powdery mildew resistance gene pmXQ-0508 and application thereof | |
| CN103397099A (en) | Method for detecting quantity of pseudomonas fluorescens in rhizospheric soil during growth period of transgenic wheat by virtue of fluorescent quantitative PCR (Polymerase Chain Reaction) | |
| KR101615433B1 (en) | SNP marker for identifying the antlered form Ganoderma lucidum, and identifying method using the same | |
| CN1978664A (en) | Method for identifying goat early embryo sex via PCR | |
| CN111926104A (en) | SSR molecular marker and method for identifying authenticity of filial generation of sugarcane and saccharum arundinaceum | |
| CN103031383B (en) | Method for detecting phytophthora hibernalis carne and sphaeropsis tumefaciens hedges | |
| CN109680087B (en) | A kind of dual PCR detection method and its primer sets of Sugarcane white leaf phytoplasma and informal voucher Xanthomonas campestris | |
| CN103060448B (en) | Method for high sensitivity detection of Phytophthora hibernalis Carne | |
| CN107849566A (en) | For detecting the method and kit of powdery mildew | |
| CN103468677B (en) | Specific SS-COI (Species-specific Cytochrome Oxidase I) primers of aphis craccivora, kit containing primers and detecting method of kit | |
| CN106868147B (en) | Molecular detection primer for sigatoka bacteria and rapid detection method thereof | |
| CN106868198B (en) | Multiplex PCR primer group for simultaneously detecting four pathogenic bacteria of catfishes and monitoring method | |
| Prosdocimi et al. | Errors in ribosomal sequence datasets generated using PCR-coupled ‘panbacterial’pyrosequencing, and the establishment of an improved approach | |
| Selvi et al. | Identification of DNA Polymorphism induced by Gamma Ray Irradiation in Amla | |
| CN104328205A (en) | Establishment of rapid detection method for grain sclerospora graminicola by LAMP | |
| CN105506174A (en) | Detection method for sugarcane mosaic virus in corn | |
| CN103882108B (en) | Method for detecting temperature sensitive microspecies gx2 of F.moniliforme | |
| CN111621549B (en) | LAMP (loop-mediated isothermal amplification) rapid detection method for bacterial blight in rice seeds and application | |
| CN102605085B (en) | The authentication method of Americal rice leaf miner, south american leaf miner, Liriomyza trifolii | |
| CN103205489A (en) | Detection method for expression of MicroRNA and quantitative detection kit for MicroRNA |
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 |
Granted publication date: 20150610 Termination date: 20161228 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |