CN103014173B - Method for identifying helix pomatia by PCR (polymerase chain reaction) detection - Google Patents
Method for identifying helix pomatia by PCR (polymerase chain reaction) detection Download PDFInfo
- Publication number
- CN103014173B CN103014173B CN201310016335.8A CN201310016335A CN103014173B CN 103014173 B CN103014173 B CN 103014173B CN 201310016335 A CN201310016335 A CN 201310016335A CN 103014173 B CN103014173 B CN 103014173B
- Authority
- CN
- China
- Prior art keywords
- pcr
- helix
- helix pomatia
- identifying
- primer
- 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.)
- Active
Links
- 238000000034 method Methods 0.000 title claims abstract description 26
- 241000237369 Helix pomatia Species 0.000 title abstract description 11
- 238000001514 detection method Methods 0.000 title abstract description 11
- 238000003752 polymerase chain reaction Methods 0.000 title abstract 7
- 238000006243 chemical reaction Methods 0.000 claims abstract description 15
- 238000011144 upstream manufacturing Methods 0.000 claims description 7
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 230000004087 circulation Effects 0.000 claims description 4
- 230000001186 cumulative effect Effects 0.000 claims description 4
- 238000004925 denaturation Methods 0.000 claims description 4
- 230000036425 denaturation Effects 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 238000012512 characterization method Methods 0.000 claims description 3
- 230000008676 import Effects 0.000 abstract description 2
- 102000053602 DNA Human genes 0.000 abstract 2
- 108020004414 DNA Proteins 0.000 abstract 2
- 241000607479 Yersinia pestis Species 0.000 abstract 1
- 230000003297 denaturating effect Effects 0.000 abstract 1
- 238000009792 diffusion process Methods 0.000 abstract 1
- 239000012154 double-distilled water Substances 0.000 abstract 1
- 230000009182 swimming Effects 0.000 description 22
- 241000237858 Gastropoda Species 0.000 description 13
- 239000000523 sample Substances 0.000 description 9
- 241001300044 Bradybaena Species 0.000 description 8
- 241000234282 Allium Species 0.000 description 7
- 235000002732 Allium cepa var. cepa Nutrition 0.000 description 7
- 238000012408 PCR amplification Methods 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 5
- 241000997379 Cathaica Species 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- 238000007689 inspection Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241000237367 Helix aspersa Species 0.000 description 3
- 241001441828 Helix lucorum Species 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 241000515128 Acusta Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000393427 Cepaea hortensis Species 0.000 description 2
- 241001137881 Cepaea nemoralis Species 0.000 description 2
- 241001299994 Cernuella virgata Species 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 241000114692 Nesiohelix swinhoei Species 0.000 description 2
- 241001609882 Plectotropis yonganensis Species 0.000 description 2
- 241001122315 Polites Species 0.000 description 2
- 241000020719 Satsuma Species 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 241000242541 Trematoda Species 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 2
- 229960005542 ethidium bromide Drugs 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000002002 slurry Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000009897 systematic effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- ZHPDOBDGGAXVTC-UHFFFAOYSA-N 2-cyanoguanidine;sulfuric acid Chemical compound OS(O)(=O)=O.NC(N)=NC#N ZHPDOBDGGAXVTC-UHFFFAOYSA-N 0.000 description 1
- 241001610131 Bradybaena sequiniana Species 0.000 description 1
- 101150087323 COI gene Proteins 0.000 description 1
- 108050004212 Cytochrome c oxidase subunit I Proteins 0.000 description 1
- 102000015884 Cytochrome c oxidase subunit I Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- 241000237852 Mollusca Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for identifying helix pomatia by PCR (polymerase chain reaction) detection, which uses specific primers to identify molecular characteristics of helix pomatia. The forward primer for the PCR reaction is HP-(P1): 5'-TTCGTTGAGAGTTAGG-3', and the reverse primer is HP-(P2): 5'-ATAGTAATAGCACCAGC-3'. The total volume of the PCR reaction system is 25 mu L, wherein the 2*TaqPCRMasterMix is 12.5 mu L, the forward and reverse primers are respectively 0.5 mu L, the DNA (deoxyribonucleic acid) template is 2 mu L, and the rest is sterilized ddH2O. The PCR reaction process comprises the following steps: pre-denaturating at 5 DEG C for 5 minutes; treating at 95 DEG C for 30 seconds, 45 DEG C for 30 seconds and 72 DEG C for 1 minute, and repeating 30 times; and extending at 72 DEG C for 10 minutes, and finishing the reaction. The method disclosed by the invention can quickly and accurately detect and identify helix pomatia. The invention provides a new technical means for detection and identification of helix pomatia in the Catalogue of Quarantine Pest for Import Plants to the People's Republic of China, and has important meanings for preventing helix pomatia from spreading and diffusion.
Description
Technical field
The present invention relates to molecular Biological Detection and identify field, be specifically related to a kind of PCR and detect the method for identifying housing helix.
Background technology
Housing helix
helix pomatia(Linnaeus, 1758) be the member that < < People's Republic of China (PRC) that China 2007 promulgates enters the territory in Plant Quarantine harmful organism register > >, its shell and the loose helix belonging to together
helix aspersa(M ü ller), bright helix
helix lucorum(Linnaeus, 1758) etc. are very similar, are often difficult to distinguish, and only have senior terrestrial mollush systematicalian to differentiate, the evaluation of ovum grain and young spiral shell is more difficult.Due to historical reason; the Plant Quarantine person overwhelming majority of frontier port is Plant Protection Specialty at present; classification to terrestrial mollush is known little about it, and it is passive that this situation has caused to the actual quarantine of China, is also the technical barrier of countries in the world inspection and quarantine department facing.Nearest two during the last ten years, and molecular systematics develop rapidly, by the taxonomy that relatively can differentiate species and the systematics status of gene order.Therefore form and characterization of molecules can be combined, the taxonomy of housing helix and allied species thereof is carried out to more deep and detailed research, quick and precisely identifying new technique means is provided for housing helix.And round pcr is quite universal at frontier port sanitary authority, if can design Auele Specific Primer, by pcr amplification method, carry out primary dcreening operation or evaluation, this difficult problem is just readily solved.The present invention has set up a kind of PCR method and has detected the technology of identifying housing helix, to adapt to the needs of current frontier port quarantine.
Summary of the invention
The present invention is directed to the existing deficiency of traditional form authenticate technology and conduct a research, aim to provide the detection method that a kind of accuracy is good, highly sensitive, with low cost, the rapid detection that is applied to housing helix is identified.
The invention provides a kind of PCR and detect the method for identifying housing helix, described method is that the characterization of molecules of application specific primer pair housing helix is identified, and described Auele Specific Primer is:
Upstream primer is HP-(P1): 5 '-TTCGTTGAGAGTTAGG-3 ',
Downstream primer is HP-(P2): 5 '-ATAGTAATAGCACCAGC-3 '.
PCR reaction system cumulative volume of the present invention is 25 μ L, 2 * Taq PCR MasterMix mixed solution, 12.5 μ L wherein, each 0.5 μ L of upstream and downstream primer (10 μ M), DNA profiling (100ng/ μ L) 2 μ L, the remaining sterilizing ddH that uses
2o supplies.
The program of described PCR reaction is: 95 ℃ of denaturation 5min; 95 ℃ of 30s, 45 ℃ of 30s, 72 ℃, 1min, 30 circulations; 72 ℃ are extended 10min, finish reaction.
Remarkable advantage of the present invention: the inventive method can detect quickly and accurately identifies housing helix.The present invention is in Plant Quarantine, the detection of quarantine harmful organisms housing helix being identified new technique means is provided, for prevent housing helix import into and spread significant.A kind of PCR provided by the present invention detects and identifies that the method for housing helix has following beneficial effect:
1, reliable results: the designed a kind of Auele Specific Primer of identifying housing helix that detects going out of the present invention, only for housing helix, detect, to the loose helix belonging to together, bright helix, equal garden green onion snail, forest green onion snail, the white snail in Mediterranean Sea, the representative classes that other section belongs to amounts to 35 kinds of snails and has carried out testing authentication (Fig. 1 shows 13 kinds), so result reliability has sufficient assurance.
2, high specificity: the primer adopting is the Auele Specific Primer of designing for the COI gene order of housing helix, and specificity is high.
3, highly sensitive: to the detection sensitivity of housing helix, on DNA level, can to reach 1 pg/ μ L.
4, practicality is good: when traditional taxonomy method is difficult to identify housing helix, the inventive method makes up with Protocols in Molecular Biology, thereby obtains the PCR method that housing helix is identified in quick, accurate, highly sensitive detection.Therefore present method is practical, can meet the needs that housing helix carried out to fast and reliable detection and evaluation.
Accompanying drawing explanation
Fig. 1 is housing helix PCR primer specificity test-results; Wherein: M is 100bp DNA ladder Marker; Swimming lane 1 is housing helix
helix pomatia(Dutch sample); Swimming lane 2 is housing helix
helix pomatia(German sample); Swimming lane 3 is garden green onion snail
cepaea hortensis; Swimming lane 4 is forest green onion snail
cepaea nemoralis; Swimming lane 5 is loose helix
helix aspersa; Swimming lane 6 is sky and water baby's spiral shells
pupinidius tianshuiensis; Swimming lane 7 is time bar snail
bradybaena(
bradybaena)
sequiniana; Swimming lane 8 is the white snail in Mediterranean Sea
cernuella virgata; Swimming lane 9 is ash point bar snail
bradybaena(
acusta)
ravida ravida; Swimming lane 10 is bright helix
helix lucorum; Swimming lane 11 is bar China snail
cathaica(
cathaica)
fasciola fasciola; Swimming lane 12 is Yongan ring rib spiral shell
plectotropis yonganensis; Swimming lane 13 is polite Hao Shi helix
nesiohelix swinhoei; Swimming lane 14 is flat slurry bar snail
bradybaena(
bradybaena)
uncopila; Swimming lane 15 is the arrowband Sa snail that rubs
satsuma stenozona.
Fig. 2 is housing helix (German sample) PCR detection sensitivity test-results; Wherein: M is 100bp DNA ladder Marker; Swimming lane 1 is 10ng/ μ L; Swimming lane 2 is 1ng/ μ L; Swimming lane 3 is 100pg/ μ L; Swimming lane 4 is: 10pg/ μ L; Swimming lane 5 is: 1pg/ μ L; Swimming lane 6 is: 100fg/ μ L; Swimming lane 7 is: 10fg/ μ L.
Embodiment
embodiment 1: the test of housing helix PCR primer specificity
1, the preparation of material
As follows for examination snail: housing helix
helix pomatia(Dutch sample), housing helix
helix pomatia(German sample), garden green onion snail
cepaea hortensis, forest green onion snail
cepaea nemoralis, loose helix
helix aspersa, sky and water baby's spiral shell
pupinidius tianshuiensis, inferior bar snail
bradybaena sequiniana, the white snail in Mediterranean Sea
cernuella virgata, ash point bar snail
bradybaena(
acusta)
ravida ravida, bright helix
helix lucorum, bar China snail
cathaica(
cathaica)
fasciola fasciola, Yongan ring rib spiral shell
plectotropis yonganensis, polite Hao Shi helix
nesiohelix swinhoei, flat slurry bar snail
bradybaena(
bradybaena)
uncopila, the arrowband Sa snail that rubs
satsuma stenozona.
For examination snail, be more than in national port quarantine, to intercept and capture or obtain by domestic investigation collection, after the mollusk quarantine of country of State Administration of Quality Supervision, Inspection and Quarantine identifies that key lab confirms, be placed in-20 ℃ and save backup.
2, the foundation of PCR method
2.1, the design of primer is synthetic: based on housing helix CO I(mitochondrial cytochrome C oxidase subunit I) gene order, with primer-design software Primer 5 and Oligo 6 design and analysis primers, after NCBIBlast checks its specificity, transfer to Shanghai Sheng Gong biotechnology Services Co., Ltd synthetic.Primer is as follows:
Upstream primer is HP-(P1): 5 '-TTCGTTGAGAGTTAGG-3 ',
Downstream primer is HP-(P2): 5 '-ATAGTAATAGCACCAGC-3 '.
Amplified fragments size is 511bp.
2.2, DNA extraction
2.2.1, different sulfuric acid cyanoguanidine method: by ready positive (housing helix, the place of production is respectively Holland and Germany) and negative control sample (13 kinds of snails such as helix, bright helix, garden green onion snail fall apart), shell and be placed in after shredding in mortar, add liquid nitrogen grind into powder and get 50mg, add again 200 μ L TE, mix; Add 400 μ L lysates, vortex mixes, and places 10min; Then add the 300 saturated phenol of μ L Tris-and 300 μ L chloroforms: primary isoamyl alcohol (24:1), concuss 15s, the centrifugal 10min of 13 000r/min; Get supernatant, add isopyknic chloroform: primary isoamyl alcohol (24:1), concuss 15s, the centrifugal 10min of 13 000r/min; Get supernatant, add isopyknic chloroform, concuss 15s, 13 000r/min, 10min; Get supernatant, add the Virahol of 0.8 times of volume, 12 000r/min, 10min, abandons supernatant; Get precipitation, add 70% dehydrated alcohol 1mL, centrifugal 13 000r/min, abandon supernatant, after in vacuum-drying instrument dry 2min, then add 100 μ L sterilizing ddH
2o dissolves.By the concentration of nucleic acid-protein analysis-e/or determining DNA, finally with TE solution, DNA is diluted to 100ng/ μ L, be placed in-20 ℃ and save backup.
2.2.2, isolation kit method: TIANGEN tissue gene group DNA extraction test kit extracts, and extracts DNA by working instructions operation.By the concentration of nucleic acid-protein analysis-e/or determining DNA, finally with TE solution, DNA is diluted to 100ng/ μ L, be placed in-20 ℃ and save backup.
2.3, pcr amplification reaction system: cumulative volume is 25 μ L, 2 * Taq PCR MasterMix mixed solution, 12.5 μ L wherein, each 0.5 μ L of upstream and downstream primer (10 μ M), DNA profiling (100ng/ μ L) 2 μ L, the remaining sterilizing ddH that uses
2o supplies.After mixing, putting into pcr amplification instrument increases.
2.4, pcr amplification reaction program is: 5 ℃ of denaturation 5min; 95 ℃ of 30s, 45 ℃ of 30s, 72 ℃ of 1min, 30 circulations; 72 ℃ are extended 10min, finish reaction.
2.5, PCR product detects and identifies: get PCR product 10 μ L upper with the about 30min of 130V electrophoresis at 1.5% sepharose (containing ethidium bromide), with gel imaging instrument, observe.If observed in the position of 511bp, occur band, the snail sample that explanation detects is housing helix.Detected result shows to only have 2 duplicate samples (Dutch sample and German sample) of housing helix in the position of 511bp, to amplify bright bright band, and amplified band (see figure 1) does not all appear in other samples, illustrates that this primer has very strong specificity.
embodiment 2: the test of housing helix PCR detection sensitivity
Adopt 10 times of concentration series dilution methods that the housing helix DNA stoste (100ng/ μ L) of extracting in embodiment 1 is diluted to 10ng/ μ L, 1ng/ μ L, 100 pg/ μ L, 10 pg/ μ L, 1 pg/ μ L, 100 fg/ μ L and 10 fg/ μ L are totally 7 different concns gradients.
Pcr amplification reaction system: cumulative volume is 25 μ L, 2 * Taq PCR MasterMix mixed solution, 12.5 μ L wherein, each 0.5 μ L of upstream and downstream primer (10 μ M), DNA profiling 2 μ L, the remaining sterilizing ddH that uses
2o supplies.After mixing, putting into pcr amplification instrument increases.
Pcr amplification reaction program is: 95 ℃ of denaturation 5min; 95 ℃ of 30s, 45 ℃ of 30s, 72 ℃ of 1min, 30 circulations; 72 ℃ are extended 10min, finish reaction.
PCR product detects and identifies: get PCR product 10 μ L upper with the about 30min of 130V electrophoresis at 1.5% sepharose (containing ethidium bromide), with gel imaging instrument, observe.If observed in the position of 511bp, there is band, illustrate that this concentration can be detected.Result (Fig. 2) shows, still has faint band to occur when DNA concentration is 1 pg/ μ L, shows that this method has higher sensitivity, can reach 1 pg/ μ L.
<110> Inspection & Quarantine Technology Center of Fujian Entry-Exit Inspection & Quar
<120> PCR detects the method for identifying housing helix
<160>?2
<210>?1
<211>16
<212>?DNA
<213> artificial sequence
<400>?1
ttcgttgaga?gttagg?16
<210>?2
<211>?17
<212>?DNA
<213> artificial sequence
<400>?2
atagtaatag?caccagc?17
Claims (3)
1. PCR detects a method of identifying housing helix, it is characterized in that: described method is that the characterization of molecules of application specific primer pair housing helix is identified, and described Auele Specific Primer is:
Upstream primer is HP-(P1): 5 '-TTCGTTGAGAGTTAGG-3 ',
Downstream primer is HP-(P2): 5 '-ATAGTAATAGCACCAGC-3 '.
2. a kind of PCR according to claim 1 detects the method for identifying housing helix, it is characterized in that: utilize the Auele Specific Primer described in claim 1 to carry out PCR reaction, PCR reaction system cumulative volume is 25 μ L, 2 * Taq PCR MasterMix mixed solution, 12.5 μ L wherein, each 0.5 μ L of upstream and downstream primer, DNA profiling 2 μ L, the remaining sterilizing ddH that uses
2o supplies.
3. a kind of PCR according to claim 2 detects the method for identifying housing helix, it is characterized in that: the program of described PCR reaction is: 95 ℃ of denaturation 5min; 95 ℃ of 30s, 45 ℃ of 30s, 72 ℃ of 1min, 30 circulations; 72 ℃ are extended 10min, finish reaction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310016335.8A CN103014173B (en) | 2013-01-17 | 2013-01-17 | Method for identifying helix pomatia by PCR (polymerase chain reaction) detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310016335.8A CN103014173B (en) | 2013-01-17 | 2013-01-17 | Method for identifying helix pomatia by PCR (polymerase chain reaction) detection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103014173A CN103014173A (en) | 2013-04-03 |
| CN103014173B true CN103014173B (en) | 2014-04-16 |
Family
ID=47963285
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310016335.8A Active CN103014173B (en) | 2013-01-17 | 2013-01-17 | Method for identifying helix pomatia by PCR (polymerase chain reaction) detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103014173B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103952491B (en) * | 2014-05-17 | 2016-04-27 | 福建出入境检验检疫局检验检疫技术中心 | The method of the white snail in a kind of PCR Testing and appraisal Mediterranean Sea |
| CN103993092B (en) * | 2014-06-24 | 2015-09-02 | 福建出入境检验检疫局检验检疫技术中心 | A kind of PCR Testing and appraisal falls apart the method for helix |
| CN106755524B (en) * | 2017-02-20 | 2019-08-23 | 福建出入境检验检疫局检验检疫技术中心 | A kind of method of fluorescence quantitative PCR detection identification Piza tea snail |
| CN107058592A (en) * | 2017-06-19 | 2017-08-18 | 福建出入境检验检疫局检验检疫技术中心 | A kind of method that fluorescence quantitative PCR detection identifies the white snail in Mediterranean |
| CN109486968B (en) * | 2019-01-11 | 2022-03-22 | 福建出入境检验检疫局检验检疫技术中心 | A method of fluorescence quantitative PCR detection and identification of upper spiral tooth snail |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1088217A (en) * | 1992-07-21 | 1994-06-22 | 皮特曼-穆尔公司 | vaccine |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012021830A (en) * | 2010-07-13 | 2012-02-02 | Tokai Univ | Diagnostic method of uterine cancer |
-
2013
- 2013-01-17 CN CN201310016335.8A patent/CN103014173B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1088217A (en) * | 1992-07-21 | 1994-06-22 | 皮特曼-穆尔公司 | vaccine |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103014173A (en) | 2013-04-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103014173B (en) | Method for identifying helix pomatia by PCR (polymerase chain reaction) detection | |
| CN102952885B (en) | Method for testing and identifying cepaea hortensis through polymerase chain reaction (PCR) | |
| CN103952491A (en) | Method for detecting and identifying Cernuella virgata Da Costa by PCR | |
| CN102409105A (en) | Biological type B and Q specific primers and rapid identification method of Bemisia tabaci | |
| CN101845508B (en) | PCR detection primer, kit and detection method for tiger-derived and leopard-derived DNA | |
| CN106755524B (en) | A kind of method of fluorescence quantitative PCR detection identification Piza tea snail | |
| CN104232755A (en) | Tobacco phytophthora LAMP detection primer and rapid detection method thereof | |
| CN107058592A (en) | A kind of method that fluorescence quantitative PCR detection identifies the white snail in Mediterranean | |
| CN104975083B (en) | A kind of primer and its authentication method of two hidden kind of Rapid identification Bemisia tabaci MEAM1, MED | |
| CN101792816B (en) | Primer for amplifying marten species cytochrome b gene and method for identifying sable and pine marten | |
| CN118147314B (en) | Method for identifying biogas digester by PCR (polymerase chain reaction) detection | |
| CN109136396A (en) | A kind of specific detection primer and detection kit of clostridium welchii disease | |
| CN101805795A (en) | Detection reagent kit and detection method of soybean phytophthora | |
| CN103834735A (en) | Nucleotide sequence and molecular probe for identifying dendrobium officinale and applications of molecular probe | |
| CN103993092B (en) | A kind of PCR Testing and appraisal falls apart the method for helix | |
| CN106636411A (en) | Xyleborus sp insect gene barcode detection kit and detection method thereof | |
| CN112322768A (en) | A rapid RPA detection method for the diagnosis of sea buckthorn branch dry wilt and pathogenic bacteria | |
| Endo et al. | Molecular based method for the detection and quantification of larvae of the golden mussel Limnoperna fortunei using real-time PCR | |
| CN111876498B (en) | Molecular identification method for Spanish mackerel and Spanish mackerel | |
| CN105385699A (en) | cytB specific sequences of four rats in river ports of three gorges area and molecular identification method | |
| CN101597645B (en) | A kind of sex-specific detection primer, detection kit and detection method of Schistosoma japonicum | |
| CN101608238B (en) | Primer pair for identifying booklice in stored booklices and application thereof | |
| CN105132540B (en) | The double PCR identification method of suspension culture of Aquilaria sinensis, primer and probe | |
| CN108588260B (en) | Osmanthus Radix Curcumae PCR identification kit and identification method | |
| CN105274241A (en) | Molecular detection primer and quick detection method for thielaviopsis basicola |
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 |