CN102086475A - Method for detecting drift treatment of target gene - Google Patents
Method for detecting drift treatment of target gene Download PDFInfo
- Publication number
- CN102086475A CN102086475A CN2010105927519A CN201010592751A CN102086475A CN 102086475 A CN102086475 A CN 102086475A CN 2010105927519 A CN2010105927519 A CN 2010105927519A CN 201010592751 A CN201010592751 A CN 201010592751A CN 102086475 A CN102086475 A CN 102086475A
- Authority
- CN
- China
- Prior art keywords
- drift
- goal gene
- detection method
- gene
- pcr reaction
- 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
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for detecting the drift treatment of a target gene. The method comprises the steps of: collecting liquids which possibly contain an exogenous gene such as transgenic cell culture solution, nuclear transplantation operation fluid, embryo culture solution and the like, performing high-pressure treatment, and then concentrating; and designing a primer for a gene sequence of the exogenous gene, specifically amplifying DNA fragments of the exogenous gene through a polymerase chain reaction (PCR) technology, and judging whether the exogenous gene still exists in the liquid produced during the screening of transgenic cells and the production and culturing of embryos and subjected to the treatment. The method for detecting the drift treatment of the target gene can completely avoid the drift of exogenous genes, and ensure the environmental safety during the production of transgenic animals. The method is applicable to the detection of genetic drift during the production of transgenic cows and goats derived from human defensin-3 genes.
Description
Technical field
Detection method is handled in the drift that the present invention relates to a kind of goal gene, and detection method is handled in the drift that is specifically related to goal gene in a kind of hBD-3 of commentaries on classics gene clone ox or the sheep production process.
Background technology
Transgenic cell and embryo contain the external source goal gene, and the drift of goal gene is an important step of transgenosis safety evaluation in operating process such as cell cultures and nuclear transplantation.It can not have the drift of other foreign genes, otherwise the drift of goal gene is unsafe.The drift of goal gene is meant the process of goal gene through the polymerase chain reaction.Wherein, goal gene-hBD3 assignment of genes gene mapping comprises 2 exons and 1 intron, total length 1156bp in No. 8 chromosomal p22～23 zones of people; Polymerase chain polymerase chain reaction (PCR) is meant that the templet gene sequence is earlier through the high-temperature denatured strand that becomes, under archaeal dna polymerase effect and suitable reaction conditions, according to two primers of template sequence design respectively with two chains of template DNA on corresponding one section complementary sequence take place to return goods and mutually combine, under the effect of archaeal dna polymerase, be substrate then with four kinds of thymus nucleic acids (dNTP), primer is extended, constantly repeat this circulation of sex change, annealing and extension then, the gene fragment that makes the desire amplification is with how much multiple amplifications.
Drift for the testing goal gene, collect the liquid that transgenic cell nutrient solution, nuclear transplantation operation liquid and embryo medium etc. may contain foreign gene, carry out a series of detection by the liquid that may contain foreign gene to these, judge whether the drift of goal gene has the drift of foreign gene to participate in.Present stage is difficult to accomplish not have the drift of going back foreign gene and participates in the bleach-out process of this goal gene, this constitutes a serious threat to the environmental safety in the whole transgenic animal production process.
Summary of the invention
The object of the present invention is to provide a kind of drift of goal gene to handle detection method, it has solved the technical problem that is difficult to accomplish not have the drift participation of going back foreign gene in the background technology.
Technical solution of the present invention is:
Detection method is handled in a kind of drift of goal gene, and its special character is that this method comprises:
1) collection of sample and processing
1.1) frozen storing liquid after transgenic cell waste liquid, the cell freezing of cultivating thawed, and the operation liquid in the nuclear transplantation process and the nutrient solution in the embryo culture process collect;
1.2) waste liquid of collecting is carried out autoclaving 30min under 121.5 ℃ temperature, pressure is at 103.4kPa;
1.3) collection liquid after autoclaving, get the centrifuge tube of 4 1.5ml, every centrifuge tube dress 1ml;
1.4) the centrifugal 1min of 3000rpm on the supercentrifuge.
1.5) centrifugate adopts minim DNA to extract that test kit extracts and concentrate;
2) PCR reaction
With the transgenosis plasmid and without the collection liquid of autoclaving as positive control, the non-transgenic cell culture fluid as negative control, replaces dna profiling as blank with water through the same back of handling; The PCR reaction system sees Table 1, and each reaction system is provided with two parallel reactors;
Table 1 PCR reaction system
3) PCR reaction cycle parameter
Behind the centrifugal 10s, the PCR pipe is inserted in the PCR instrument;
Response procedures is: 95 ℃ of sex change 5min; Carry out 94 ℃ of sex change 1min 35 times, 56 ℃ of annealing 30s, 72 ℃ of cyclic amplification reactions that extend 1min; 72 ℃ are extended 7min;
Reaction finishes the back and takes out the PCR reaction tubes, preserves stand-by to the PCR reaction product down at 4 ℃.
Detection method is handled in the drift of above-mentioned purpose gene, and its special character is that this method also comprises: PCR product electrophoresis detection step; Specifically:
1) an amount of agarose is added in 1 * TAE damping fluid, heating for dissolving is mixed with w/v concentration and is 1.0% agarose solution;
2) add EB solution in the ratio that adds 5 μ L EB solution in every 100mL agarose solution, mixing,
3) be poured on the electrophoresis plate, plug pecten, be frozen into gel under the room temperature after, put into 1 * TAE damping fluid, take out pecten vertically upward;
4) the PCR product of drawing 5 μ L mixes the back with an amount of sample loading buffer and adds in the point sample hole, adding dna molecular amount standard in point sample hole therein, connection power supply electrophoresis under the 2V/cm condition.
Detection method is handled in the drift of above-mentioned purpose gene, and its special character is that this method also comprises: the gel imaging analysis step; Specifically:
1) after electrophoresis finishes, takes out sepharose, place on the gel imaging instrument or imaging on the ultraviolet transilluminator;
2) according to the size of dna molecular amount standard estimation amplified band, electrophoresis result is formed electronic file file or take pictures with photographic system.
Detection method is handled in the drift of above-mentioned purpose gene, and its special character is that this method also comprises: the extract concentrations determination step; Specifically: ultraviolet spectrophotometry detects,
Sample DNA calculates the purity and the concentration of nucleic acid respectively with nucleic acid-protein analysis-e/or determining 260nm and 280nm place absorption value, and calculation formula is suc as formula (1), formula (2):
1) DNA purity=OD
260/ OD
280, ratio is 1.8-2.0;
2) double-stranded DNA concentration (μ g/mL)=50 * OD
280
Above-mentioned PCR reaction reagent dNTPs is: concentration is dATP, dTTP, the dGTP of 10mmol/L, the equal-volume mixing solutions of four kinds of deoxyribonucleotides of dCTP.
Above-mentioned 50 * TAE damping fluid is: take by weighing 242.2g Tris, with the dissolving of 500mL water heated and stirred, add 500mmol/L EDTA solution 100mL, transfer pH to 8.0 with glacial acetic acid, add water then and be settled to 1000mL; Described 50 * TAE damping fluid is diluted with water to 1 * TAE when using.
Above-mentioned agarose; Ethidium bromide (EB) solution: 10mg/mL; Described sample loading buffer: 6 * loading buffer.
Above-mentioned TE damping fluid: add the Tris-HCl 10mL of 1mol/L pH 8.0 and the EDTA solution 2mL of 500mmol/LpH 8.0 respectively, add water and be settled to 1000mL.
The primer of above-mentioned PCR reaction:
hBD3-F：5’-AGCAGCTATGAGGATCCATTATCTT-3’
hBD3-R：5’-TCTAGATTTTATTTCTTTCTTCGGCATT-3’。
The primer solution of above-mentioned PCR reaction: the TE damping fluid with pH8.0 is diluted to 10umol/L with above-mentioned primer.
This processing detection method of the present invention can be avoided the drift of foreign gene fully, has guaranteed the environmental safety in the transgenic animal production process.Be applicable to the detection of the genetic drift in the production process of the transgenosis milk cow of alexin-3 gene that derives from the people and goat.
Embodiment
1 scope
Comprise commentaries on classics human beta-defensin-3 gene ox and sheep cell culture fluid, nuclear transplantation operation liquid, the processing of all liquid and the qualitative PCR of goal gene drift detect in the operating process such as reconstruct embryo medium and commentaries on classics beta-defensin-3 gene ox, sheep offspring's cell cultures and nuclear transplantation.
Be applicable to the detection of the genetic drift in the production process of the transgenosis milk cow of alexin-3 gene that derives from the people and goat.
2 terms, definition and initialism
2.1 goal gene
The hBD3 assignment of genes gene mapping comprises 2 exons and 1 intron, total length 1156bp in No. 8 chromosomal p22～23 zones of people.
2.2 polymerase chain reaction polymerase chain reaction (PCR)
The templet gene sequence is earlier through the high-temperature denatured strand that becomes, under archaeal dna polymerase effect and suitable reaction conditions, according to two primers of template sequence design respectively with two chains of template DNA on corresponding one section complementary sequence take place to return goods and mutually combine, under the effect of archaeal dna polymerase, be substrate then with four kinds of thymus nucleic acids (dNTP), primer is extended, constantly repeat this circulation of sex change, annealing and extension then, the gene fragment that makes the desire amplification is with how much multiple amplifications.
2.3hBD3
The human alpha-defensin gene derives from the people
3 principles
Transgenic cell and embryo contain the external source goal gene, and the drift of goal gene is an important step of transgenosis safety evaluation in operating process such as cell cultures and nuclear transplantation.Collect the liquid that transgenic cell nutrient solution, nuclear transplantation operation liquid and embryo medium etc. may contain foreign gene, it is carried out autoclaving, then liquid is concentrated.Gene order design primer at foreign gene, pass through round pcr, the dna fragmentation of specific amplification foreign gene, according to the pcr amplification result, judge in the transgenic cell screening process and whether the liquid that is produced in Embryo Production and the culturing process also has the existence of foreign gene after treatment.
4 reagent
Except as otherwise herein provided, employed reagent is analytical pure or biochemical reagents.
4.1PCR reaction reagent dNTPs: concentration is dATP, dTTP, the dGTP of 10mmol/L, the equal-volume mixing solutions of four kinds of deoxyribonucleotides of dCTP.
50 * TAE damping fluid: take by weighing 242.2g Tris,, add 500mmol/L EDTA solution (pH 8.0) 100mL, transfer pH to 8.0, add water then and be settled to 1000mL with glacial acetic acid with the dissolving of 500mL water heated and stirred.Be diluted with water to 1 * TAE during use.
Agarose; Ethidium bromide (EB) solution: 10mg/mL; Sample loading buffer: 6 * loading buffer;
TE damping fluid (pH 8.0): add 1mol/L Tris-HCl (pH 8.0) 10mL and 500mmol/L EDTA (pH8.0) solution 2mL respectively, add water and be settled to 1000mL.20min sterilizes under 103.4kPa (121 ℃) condition; Dna molecular amount mark TRANS 5k (DIGI-KEY company); Be applicable to common PCR reaction Taq archaeal dna polymerase.
4.2 primer
hBD3-F：5’-AGCAGCTATGAGGATCCATTATCTT-3’
hBD3-R：5’-TCTAGATTTTATTTCTTTCTTCGGCATT-3’
4.3 primer solution: (pH8.0) is diluted to 10umol/L with above-mentioned primer with the TE damping fluid.
5 instruments
5.1PCR instrument.
5.2 electrophoresis apparatus.
5.3 gel analysis imaging system.
5.4 high speed tabletop centrifuge.
5.5 water-bath.
5.6 magnetic stirring apparatus.
5.7 high-pressure sterilizing pot.
5.8 microwave oven.
5.9 micro sample adding appliance: 0.1 μ L～2.5 μ L, 0.5 μ L～10 μ L, 10 μ L～100 μ L, 20 μ L～200 μ L, 200 μ L～1000 μ L.
5.10 electronic balance: sensitivity 0.1mg.
5.11 PCR reaction tubes: 200 μ L, two kinds of specifications of 500 μ L.
5.12 centrifuge tube: 1.5ml.
6 operation stepss
6.1 the collection of sample and processing
Frozen storing liquid after 6.1.1 transgenic cell waste liquid, the cell freezing of cultivating thawed, and the operation liquid in the nuclear transplantation process and the nutrient solution in the embryo culture process collect.
6.1.2 the waste liquid of collecting is carried out autoclaving, 121.5 ℃, 103.4kPa 30min.
6.1.3 the collection liquid after autoclaving is got 4 centrifuge tubes (1.5ml), every pipe dress 1ml.
6.1.4 the centrifugal 1min of 3000rpm on the supercentrifuge.
6.1.5 adopting minim DNA to extract test kit (MicroElute Genomic DNA Kit) extracts and concentrates.
6.2 extract concentrations is measured
6.2.1 ultraviolet spectrophotometry detects
Sample DNA calculates the purity and the concentration of nucleic acid respectively with nucleic acid-protein analysis-e/or determining 260nm and 280nm place absorption value, and calculation formula is suc as formula (1), formula (2):
(1) DNA purity=OD
260/ OD
280, ratio is that 1.8-2.0 is comparatively desirable.
(2) double-stranded DNA concentration (μ g/mL)=50 * OD
280
6.3PCR reaction
6.3.1 the PCR of sample reaction
With the transgenosis plasmid and without the collection liquid of autoclaving as positive control, the non-transgenic cell culture fluid as negative control, replaces dna profiling as blank with water through the same back of handling.The PCR reaction system sees Table 1, and each reaction system is provided with two parallel reactors.
Table 1 PCR reaction system
6.3.2PCR reaction cycle parameter
Behind the centrifugal 10s, the PCR pipe is inserted in the PCR instrument.Response procedures is: 95 ℃ of sex change 5min; Carry out 35 cyclic amplification reactions (72 ℃ are extended 1min for 94 ℃ of sex change 1min, 56 ℃ of annealing 30s); 72 ℃ are extended 7min.Reaction finishes the back and takes out the PCR reaction tubes, and the PCR reaction product is carried out electrophoresis detection or preserved stand-by down at 4 ℃.
6.4PCR product electrophoresis detection
An amount of agarose is added in 1 * TAE damping fluid, heating for dissolving, be mixed with the agarose solution that concentration is 1.0% (w/v), add EB solution, mixing in the ratio that adds 5 μ L EB solution in every 100mL agarose solution then, after the suitable slightly cooling, be poured on the electrophoresis plate, plug pecten, be frozen into gel under the room temperature after, put into 1 * TAE damping fluid, take out pecten gently vertically upward.The PCR product of drawing 5 μ L mixes the back and adds in the point sample hole with an amount of sample loading buffer, add dna molecular amount standard in a point sample hole therein, connects power supply electrophoresis under the 2V/cm condition.
6.5 gel imaging analysis
After electrophoresis finishes, take out sepharose, place on the gel imaging instrument lightly or imaging on the ultraviolet transilluminator.According to the size of dna molecular amount standard estimation amplified band, electrophoresis result is formed electronic file file or take pictures with photographic system
7 results
After PCR detected, band should not appear in negative control, blank and testing sample, and the 1156bp amplified band of expection size appears in positive plasmid contrast and undressed control group.This treatment process can be avoided the drift of foreign gene fully, has guaranteed the environmental safety in the transgenic animal production process.
Claims (10)
1. detection method is handled in the drift of a goal gene, it is characterized in that this method comprises:
1) collection of sample and processing
1.1) frozen storing liquid after transgenic cell waste liquid, the cell freezing of cultivating thawed, and the operation liquid in the nuclear transplantation process and the nutrient solution in the embryo culture process collect;
1.2) waste liquid of collecting is carried out autoclaving 30min under 121.5 ℃ temperature, pressure is at 103.4kPa;
1.3) collection liquid after autoclaving, get the centrifuge tube of 4 1.5ml, every centrifuge tube dress 1ml;
1.4) the centrifugal 1min of 3000rpm on the supercentrifuge.
1.5) centrifugate adopts minim DNA to extract that test kit extracts and concentrate;
2) PCR reaction
With the transgenosis plasmid and without the collection liquid of autoclaving as positive control, the non-transgenic cell culture fluid as negative control, replaces dna profiling as blank with water through the same back of handling; The PCR reaction system sees Table 1, and each reaction system is provided with two parallel reactors;
Table 1 PCR reaction system
3) PCR reaction cycle parameter
Behind the centrifugal 10s, the PCR pipe is inserted in the PCR instrument;
Response procedures is: 95 ℃ of sex change 5min; Carry out 94 ℃ of sex change 1min 35 times, 56 ℃ of annealing 30s, 72 ℃ of cyclic amplification reactions that extend 1min; 72 ℃ are extended 7min;
Reaction finishes the back and takes out the PCR reaction tubes, preserves stand-by to the PCR reaction product down at 4 ℃.
2. handle detection method according to the drift of the described goal gene of claim 1, it is characterized in that this method also comprises: PCR product electrophoresis detection step; Specifically:
1) an amount of agarose is added in 1 * TAE damping fluid, heating for dissolving is mixed with w/v concentration and is 1.0% agarose solution;
2) add EB solution in the ratio that adds 5 μ L EB solution in every 100mL agarose solution, mixing,
3) be poured on the electrophoresis plate, plug pecten, be frozen into gel under the room temperature after, put into 1 * TAE damping fluid, take out pecten vertically upward;
4) the PCR product of drawing 5 μ L mixes the back with an amount of sample loading buffer and adds in the point sample hole, adding dna molecular amount standard in point sample hole therein, connection power supply electrophoresis under the 2V/cm condition.
3. handle detection method according to the drift of the described goal gene of claim 2, it is characterized in that this method also comprises: the gel imaging analysis step; Specifically:
1) after electrophoresis finishes, takes out sepharose, place on the gel imaging instrument or imaging on the ultraviolet transilluminator;
2) according to the size of dna molecular amount standard estimation amplified band, electrophoresis result is formed electronic file file or take pictures with photographic system.
4. handle detection method according to the drift of the arbitrary described goal gene of claim 1～3, it is characterized in that this method also comprises: the extract concentrations determination step; Specifically: ultraviolet spectrophotometry detects,
Sample DNA calculates the purity and the concentration of nucleic acid respectively with nucleic acid-protein analysis-e/or determining 260nm and 280nm place absorption value, and calculation formula is suc as formula (1), formula (2):
1) DNA purity=OD
260/ OD
280, ratio is 1.8-2.0;
2) double-stranded DNA concentration (μ g/mL)=50 * OD
280
5. according to handling detection method according to the drift of the described goal gene of claim 4, it is characterized in that described PCR reaction reagent dNTPs is: concentration is dATP, dTTP, the dGTP of 10mmol/L, the equal-volume mixing solutions of four kinds of deoxyribonucleotides of dCTP.
6. handle detection method according to drift according to the described goal gene of claim 5, it is characterized in that, described 50 * TAE damping fluid is: take by weighing 242.2g Tris, dissolve with 500mL water heated and stirred, add 500mmol/L EDTA solution 100mL, transfer pH to 8.0 with glacial acetic acid, add water then and be settled to 1000mL; Described 50 * TAE damping fluid is diluted with water to 1 * TAE when using.
7. handle detection method according to drift, it is characterized in that described agarose according to the described goal gene of claim 6; Ethidium bromide (EB) solution: 10mg/mL; Described sample loading buffer: 6 * loadingbuffer.
8. handle detection method according to drift according to the described goal gene of claim 7, it is characterized in that, described TE damping fluid: add the Tris-HCl 10mL of 1mol/L pH 8.0 and the EDTA solution 2mL of 500mmol/L pH8.0 respectively, add water and be settled to 1000mL.
9. handle detection method according to the drift of described goal gene according to Claim 8, it is characterized in that, the primer of described PCR reaction:
hBD3-F：5’-AGCAGCTATGAGGATCCATTATCTT-3’
hBD3-R：5’-TCTAGATTTTATTTCTTTCTTCGGCATT-3’。
10. handle detection method according to the drift according to the described goal gene of claim 9, it is characterized in that, the primer solution of described PCR reaction: the TE damping fluid with pH8.0 is diluted to 10umol/L with above-mentioned primer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105927519A CN102086475A (en) | 2010-12-13 | 2010-12-13 | Method for detecting drift treatment of target gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105927519A CN102086475A (en) | 2010-12-13 | 2010-12-13 | Method for detecting drift treatment of target gene |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102086475A true CN102086475A (en) | 2011-06-08 |
Family
ID=44098480
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010105927519A Pending CN102086475A (en) | 2010-12-13 | 2010-12-13 | Method for detecting drift treatment of target gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102086475A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103858629A (en) * | 2014-02-14 | 2014-06-18 | 中国农业大学 | Method for evaluating influence of entomophilous insects on gene drift of transgenic crops |
| CN110244005A (en) * | 2019-06-15 | 2019-09-17 | 吉林省农业科学院 | A kind of rapid detection method that soybean plant strain is drifted about by external source Bar gene |
| CN111139311A (en) * | 2020-01-17 | 2020-05-12 | 南京农业大学 | Evaluation method and application of transgenic rice weediness gene drift risk |
-
2010
- 2010-12-13 CN CN2010105927519A patent/CN102086475A/en active Pending
Non-Patent Citations (6)
| Title |
|---|
| 《中华检验医学杂志》 20040930 刘军权等 清除聚合酶链反应扩增产物污染的方法探讨 第707－712页 9-10 第27卷, 第9期 * |
| 《农业生物技术学报》 20100828 马晶晶等 牛胎儿成纤维细胞beta－防御素（hBD3）基因转染及转基因克隆胚制备 第707－712页 1-3,5-10 第18卷, 第4期 * |
| 《环境科学学报》 20080531 李梦南等 重组质粒DNA在热处理过程中的降解/变性规律研究 第945－950页 1-3,5-8 第28卷, 第5期 * |
| 《环境科学学报》 20080531 李梦南等 重组质粒DNA在热处理过程中的降解/变性规律研究 第945－950页 4 第28卷, 第5期 * |
| 刘军权等: "清除聚合酶链反应扩增产物污染的方法探讨", 《中华检验医学杂志》 * |
| 马晶晶等: "牛胎儿成纤维细胞β－防御素（hBD3）基因转染及转基因克隆胚制备", 《农业生物技术学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103858629A (en) * | 2014-02-14 | 2014-06-18 | 中国农业大学 | Method for evaluating influence of entomophilous insects on gene drift of transgenic crops |
| CN110244005A (en) * | 2019-06-15 | 2019-09-17 | 吉林省农业科学院 | A kind of rapid detection method that soybean plant strain is drifted about by external source Bar gene |
| CN111139311A (en) * | 2020-01-17 | 2020-05-12 | 南京农业大学 | Evaluation method and application of transgenic rice weediness gene drift risk |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10253376B2 (en) | DNA-based detection and identification of eight mastitis pathogens | |
| CN108754000A (en) | Drug resistant gene mcr-4/5/8 fluorescent quantitative PCR detection methods | |
| CN103484571B (en) | LAMP (loop-mediated isothermal amplification) detection primer group, detection kit and detection method for infectious hypodermal and hematopoietic necrosis virus | |
| CN102154497B (en) | M-PCR (Multiplex Polymerase Chain Reaction) primers, probes and detection methods for vibrio cholerae, vibrio parahaemolyticus and salmonella | |
| JP5835702B2 (en) | Detection method and detection kit for cryptococcosis-causing bacteria | |
| CN102453762A (en) | Kit and method for detecting porcine proliferative enteropathy (PPE) pathogenic bacteria Lawsonia Intracellularis | |
| CN106086208A (en) | For detecting vibrio parahaemolyticus and the test kit of integron and method in food | |
| Gou et al. | The colorimetric isothermal multiple-self-matching-initiated amplification using cresol red for rapid and sensitive detection of porcine circovirus 3 | |
| CN104988246A (en) | Eriocheir sinensis white spot syndrome virus specificity PCR detection kit and method | |
| CN102086475A (en) | Method for detecting drift treatment of target gene | |
| CN1978664A (en) | Method for identifying goat early embryo sex via PCR | |
| CN105063187A (en) | Method and primer composition for rapid detection of resistance of botrytis cinerea to SDH1 fungicides | |
| CN106148483B (en) | Primer and method for detecting escherichia coli cell DNA | |
| CN110257544B (en) | Ergota germ fluorescent quantitative PCR detection reagent, detection kit and application | |
| CN105695579A (en) | Kit for rapidly detecting Avibacterium paragallinarum | |
| CN102827931B (en) | Detection primer for detecting listeria monocytogenes by PCR-pyrophosphate method, kit and detection method | |
| CN103122383B (en) | Horizontal fluorescent quantitation polymerase chain reaction (PCR) detection method of streptococcus suis strain | |
| CN112359098A (en) | Method for detecting content of herbicide-tolerant transgenic soybean J12 by real-time fluorescence quantitative PCR | |
| CN102605069B (en) | Enterohemorrhagic Escherichia coli O104: H4 detection kit and use method thereof | |
| CN107557456B (en) | LAMP (loop-mediated isothermal amplification) detection primer group and kit for ureaplasma urealyticum | |
| CN104313170A (en) | PCR (Polymerase chain reaction) rapid detection kit for fish myxosporea and detection method | |
| CN107435064B (en) | qPCR (quantitative polymerase chain reaction) method for rapidly and quantitatively detecting harmful golden algae in chlorella culture | |
| CN110923341B (en) | LAMP (loop-mediated isothermal amplification) detection method for Shewanella alga pathogenic bacteria of cultured fishes and application | |
| CN108660195A (en) | Functional nucleic acid rapid PCR methods and kit for the identification of MSTN gene knock-out pigs | |
| CN104178575B (en) | Method and the kit of a kind of quick discriminating campylobacter fetus and venereal disease subspecies |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20110608 |