CN101985658B - Loop-mediated isothermal amplification method for detecting anaplasma ovis pathogeny - Google Patents
Loop-mediated isothermal amplification method for detecting anaplasma ovis pathogeny Download PDFInfo
- Publication number
- CN101985658B CN101985658B CN 201010542863 CN201010542863A CN101985658B CN 101985658 B CN101985658 B CN 101985658B CN 201010542863 CN201010542863 CN 201010542863 CN 201010542863 A CN201010542863 A CN 201010542863A CN 101985658 B CN101985658 B CN 101985658B
- Authority
- CN
- China
- Prior art keywords
- sheep
- primer
- slurry
- loop
- disease
- 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
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an application of a loop-mediated isothermal amplification (LAMP) in the preparation of a reagent for detecting anaplasma ovis pathogeny. In the method, three pairs of specific primers are designed by taking a main surface protein 4 gene of anaplasma ovis as a target gene, and a specific primer reaction mixture is prepared to perform deoxyribonucleic acid (DNA) amplification under the condition of constant temperature. The anaplasma ovis pathogeny is detected only by the conventional water bath kettle without complex instrument and polymerase chain reaction (PCR) amplification instruments, so the LAMP method has the characteristics of quickness, sensibility, high specificity and simplicity of operation, and can detect the pathogeny in the conventional laboratory. Therefore, the LAMP method is suitable for the detection of the anaplasma ovis pathogeny and large-scale epidemiological survey, and can obtain detection results with high sensibility.
Description
Technical field
The present invention relates to does not a kind ofly have main surface protein 4 (MSP4) gene order of slurry (Anaplasma ovis) according to sheep, detects the isothermal amplification technology (LAMP) that sheep does not have the ring mediation of slurry cause of disease.
Background technology
Sheep does not have the arbo Li Keshi body disease that the slurry disease is an obligatory parasitism in the sheep and goat red corpuscle.The a large amount of distribution in the Northwest of China of this disease exists certain potential threat to the development of sheep husbandry.Schellhase in 1912 and Bevan are reported in East Africa and have found that the sheep that parasitizes sheep, goat does not have the slurry disease.The clinical symptom that sheep does not have a slurry is comparatively gentle, and the morbidity animal often shows as slight fever, but pathogenic stronger to goat.In China; Since Ma Liangyu (1982) and fourth intelligence refined (1985) are found respectively and have been reported that parasitizing the endoerythrocytic sheep of China's sheep and goat does not have slurry; Have only suitable grade of Lv Wen in the period of 1989-1990, this disease to be carried out comparatively careful research; Confirmed that this disease is distributed widely in the great Northwest of China, and at these province sheep husbandrys in occupation of crucial status.Song goes on foot firm grade (1989) report, and the Ejina Banner popular sheep of Inner Mongol does not have the sick sickness rate of slurry up to 40%-50%, and mortality ratio reaches 17%, endangers quite serious.Do not have the sick M & M of slurry for other geographic sheep owing to lack data, so be difficult to the direct economic loss of estimating that it causes sheep husbandry, but the existence that can affirm this disease to the sheep husbandry of China potential certain threat.The cause of disease vitro culture of no slurry exists big difficulty always, although the edge vitro culture of not having a slurry has obtained certain progress in recent years, sheep does not have the vitro culture of slurry and do not appear in the newspapers as yet so far.Owing to lack vitro culture system and experimental animal model.Tradition dyes with Ji's nurse Sa, microscopy then, and most of endoerythrocytic edges do not have slurry and sheep does not have the edge that slurry is positioned at cell, and the no slurry of central authorities then is positioned at central authorities.Difference can provide strong evidence for microscopy although Different Kinds of Pathogens is in endoerythrocytic position, and these detections need the professional, and dyes the worm rate and omission takes place easily when low, and this all becomes the obstacle of this cause of disease of tradition diagnosis.
Summary of the invention
The present invention will solve the defective that prior art exists, and main surface protein 4 (MSP4) gene is incorporated into sheep not to be had in the sick diagnostic techniques of slurry, provides a kind of loop-mediated isothermal amplification primer to detect sheep and do not have the purposes in the reagent of slurry cause of disease in preparation.
Technical problem of the present invention realizes through following technical proposals:
A kind of loop-mediated isothermal amplification primer detects sheep and does not have the purposes in the reagent of slurry cause of disease in preparation; Not having main surface protein 4 genes of slurry with sheep is target gene; Designed 3 pairs of Auele Specific Primers, and be mixed with the Auele Specific Primer reaction mixture by a certain percentage, employed primer sequence is following:
The outside upstream primer F3:5 ' of forward-GTGTTGCACACAGATTTGCC-3 '
Adverse external downstream primer B3:5 '-AGGCTTTTGCTTCTCCGG-3 '
The inner primers F IP of forward:
5’-GCCCCTGTAGGCTAGCTTTGTGgaattcCCCATATGTGTGTGCCGG-3’
Reverse inner primer BIP:
5’-TGGTGGTAGGTGGGTTCTACCAgaattcATGTGCGGGTATGTCCTTG-3’
Ring upstream primer LF:5 '-TGTCGACAAAGCTAGCACC-3 '
Ring downstream primer LB:5 '-CGGACTCTTTGACGAGTCTT-3 '
Said Auele Specific Primer reaction mixture comprises: final concentration is FIP and the BIP of 40pmol; Final concentration is LF and the LB of 20pmol; Final concentration is F3 and the B3 of 5pmol.
The method of loop-mediated isothermal amplification that said detection sheep does not have the slurry cause of disease adopts the following step to accomplish:
A, constant-temperature amplification: the loop-mediated isothermal amplification that sheep does not have the slurry cause of disease is Auele Specific Primer reaction mixture 1.0uL; 2 * LAMP reaction buffer 12.5uL; Sterile purified water 9.5uL; Genomic dna-positive or blank sample 1.0uL; Bst archaeal dna polymerase 1.0uL; Amount to 25uL, 65 ℃ of amplifications 30 minutes, 80 ℃ of deactivations 2 minutes;
B, electrophoresis detection: with the sepharose of 1 * TAE damping fluid preparation 2%, every hole adds 6 * sample-loading buffer of above-mentioned LAMP amplified production of 3uL and 1.5uL, and 100 volts, 75 peaces, electrophoresis 80 minutes, observations under ultraviolet gel imaging appearance;
C, judge to detect sheep by judgement criteria and do not have the slurry cause of disease.
Each component volume ratio is following in the said amplification reaction system:
Auele Specific Primer reaction mixture: 2 * LAMP reaction buffer: sterile purified water: genomic dna-positive or blank sample: Bst archaeal dna polymerase=1: 12.5: 9.5: 1: 1.
Final concentration at the said Auele Specific Primer reaction mixture of this reaction system is FIP and BIP, the LF of 20pmol and F3 and the B3 of LB and 5pmol of 40pmol.
The present invention adopts the detection method of the isothermal amplification technology of ring mediation; Detect and do not need complicated plant and instrument when sheep does not have the slurry cause of disease; Only need conventional water-bath just can accomplish detection; Can carry out the detection and large-scale epidemiology survey that sheep does not have the slurry cause of disease in ordinary laboratory, and can obtain having the detected result of hypersensitivity.
Description of drawings
Fig. 1 is a different time LAMP amplification electrophorogram;
The specificity that Fig. 2 does not have a slurry LAMP method for sheep is electrophorogram as a result;
The susceptibility that Fig. 3 does not have a slurry LAMP method for sheep is electrophorogram as a result.
Embodiment
Detection method of the present invention is carried out in the centrifuge tube of 1.5mL, and employed primer and reagent are following:
1) special primer
The outside upstream primer F3:5 ' of forward-GTGTTGCACACAGATTTGCC-3 '
Adverse external downstream primer B3:5 '-AGGCTTTTGCTTCTCCGG-3 '
The inner primers F IP of forward:
5’-GCCCCTGTAGGCTAGCTTTGTGgaattcCCCATATGTGTGTGCCGG-3’
Reverse inner primer BIP:
5’-TGGTGGTAGGTGGGTTCTACCAgaattcATGTGCGGGTATGTCCTTG-3’
Ring upstream primer LF:5 '-TGTCGACAAAGCTAGCACC-3 '
Ring downstream primer LB:5 '-CGGACTCTTTGACGAGTCTT-3 '
2) FIP of Auele Specific Primer reaction mixture: 40pmol and BIP; The LF of 20pmol and LB; The F3 of 5pmol and B3.
3) 2 * LAMP reaction buffer: 40mM Tris-HCl, pH 8.8; 20mM KCl, 16mMMgSO4; 20mM (NH4) 2SO4; 0.2%Tween 20,1.6M betaine and 2.5mM dNTP.
4) Bst archaeal dna polymerase: M0275L, BioLabs.
5) the standard sheep does not have slurry positive gene group DNA.
6) standard sheep genomic dna.
7) 6 * sample-loading buffer: 0.25% tetrabromophenol sulfonphthalein, 0.25% YLENE is blue or green, 40% aqueous sucrose solution.
8) 1 * TAE damping fluid: 0.04M Tris-acetate, 0.001M EDTA.
9) 2% agarose: prepare with 1 * TAE damping fluid.
One, the detection of standard test specimen
(1) the standard sheep does not have the preparation process of slurry positive gene group DNA:
Do not have the slurry suspension with the sheep that preserves in the liquid nitrogen, shake thaws fast in 38 ℃ of water-baths, removes spleen sheep with 2mL red corpuscle blood cell mud (the former worm rate 5% of staining with blood) through neck subcutaneous vaccination sheep.After one week day by day have sharp ears blood sampling make blood smear, after the smear dry air, fix with methyl alcohol; With 10% Ji's nurse Sa solution-dyed, opticmicroscope is observed down in PBS (pH7.2), when the worm rate of dying of not having a slurry when sheep peaks; Gather anti-freezing by jugular vein and contain worm blood,, abandon the upper plasma part with the centrifugal 10min of 2000r/min; Again with equivalent or more 10mmol/L Tris-HCl damping fluids mixing gently, the centrifugal 10min of 2000r/min.With 10mM Tris-HCl damping fluid repeated washing two, during washing, with suction pipe the throw out in centrifuge tube piping and druming evenly after; Under 4 ℃, with 2000 rev/mins centrifugal 1 minute, take out supernatant with syringe then; Remove yellow blood layer (leukocytic cream) as far as possible, add washings then again, piping and druming; Centrifugal, so repeatedly.Till supernatant is clear and bright.The last centrifugal pcv of measuring afterwards.0.8% Sodium Citrate with 10 times of amounts of pcv comes cracking, till the erythrocyte splitting of microscopically 80.0%, after cracking well, recall to etc. and to ooze.Under 4 ℃ of the good blood of cracking, with 2000 rev/mins centrifugal 15 minutes, get supernatant, abandon deposition.The supernatant behind the differential centrifugation, under 4 ℃, with 6000 rev/mins centrifugal 20 minutes, abandon supernatant, stay deposition.Deposition suspends with the Tris-EDTA of pH 7.4, with suction pipe piping and druming evenly, and the same again high speed centrifugation; So repeatedly twice, in the mini desktop supercentrifuge, centrifugal 2 minutes for the last time with 9000 rev/mins; After the centrifugal end, abandon supernatant, stay deposition; Tris-EDTA damping fluid with pH 7.4 washs 2 times, and collecting precipitation is exactly a thalline, and-20 ℃ of preservations are subsequent use.
The extraction of genomic dna: the applying gene group is extracted test kit (Gentra), according to specification sheets the thalline of above-mentioned preservation is carried out the extraction that sheep does not have the slurry genomic dna.Measure the concentration of prepared genomic dna, and use diluted and become 100ng/uL ,-20 ℃ of preservations are subsequent use.
(2) preparation of standard sheep genomic dna
The screening sheep does not have the negative sheep of slurry, and the applying gene group is extracted test kit (Gentra) and carried out the extraction of genomic dna according to specification sheets.Measure the concentration of prepared genomic dna, and use diluted and become 100ng/uL ,-20 ℃ of preservations are subsequent use.
The preparation of (three) 2 * LAMP reaction buffers:
At first prepare 2 * reaction buffer storage liquid of no dNTP, can preserve-20 ℃ of prolonged preservation 3 months at 4 ℃ with the ultrapure water according to the form below ratio of sterilization.
Add 100uL 25mM dNTP before the use in 2 * reaction buffer storage liquid of every 900uL, mixing is prepared into 2 * reaction buffer working fluid, and-20 ℃ of preservations are subsequent use.
(3) testing process
In the centrifuge tube of 1.5mL by adding sample (2 * reaction buffer working fluid 12.5uL respectively; Primer reaction mixture 1uL; The ultrapure water 9.5uL of sterilization; DNA sample 1.0uL; BstDNA polysaccharase 1.0uL), TV amounts to 25uL., the barren contrast of setting up standard positive, negative genomic dna and ultrapure water simultaneously: mixing gently, moment is centrifugal.Amplification is 30 minutes in 65 degree water-baths, changes deactivation 2 minutes in the 80 degree water-baths at once over to.Getting amplified production 3uL, is damping fluid with 1 * TAE, and in the sepharose 2% (containing the 0.5ug/mL ethidium bromide), at 100 volts, 75 peaces detect in 80 minutes the nucleic acid electrophoresis of electrophoresis.
In the centrifuge tube of 1.5mL, add sample respectively, comprising: 2 * reaction buffer working fluid 12.5uL; Primer reaction mixture 1uL; The ultrapure water 9.5uL of sterilization; DNA sample 1.0uL; Bst archaeal dna polymerase 1.0uL), TV amounts to 25uL.The DNA sample is used sheep does not respectively have slurry genomic dna, standard sheep genomic dna and sterilization ultrapure water.Mixing gently, moment is centrifugal.65 degree amplifications are 15 minutes, 30 minutes and 45 minutes in water-bath, change in the 80 degree water-baths deactivation at once over to 2 minutes.Getting amplified production 3uL, is damping fluid with 1 * TAE, and in the sepharose 2% (containing the 0.5ug/mL ethidium bromide), at 100 volts, 75 peaces detect in 80 minutes the nucleic acid electrophoresis of electrophoresis.Detected result shows (as shown in Figure 1): 30 minutes 65 degree just can amplify sheep is not had the slurry genomic dna.
In the centrifuge tube of 1.5mL,, comprising: 2 * reaction buffer working fluid 12.5uL by adding sample respectively; Primer reaction mixture 1uL; The ultrapure water 9.5uL of sterilization; DNA sample 1.0uL; Bst archaeal dna polymerase 1.0uL, TV amounts to 25uL.The DNA sample use respectively Yuzhong A.ovis genome, Yongjing A.ovis genome, Yuzhong A.ovis MSP4 plasmid, chlamydia trachomatis gene group, mycoplasma genome, sheep Taylor, Taylor You Shi, Taylor Lv Shi, edge do not have slurry, Xinjiang BABEI this, sheep genome and sterilization ultrapure water.Mixing gently, moment is centrifugal.65 degree amplifications are 30 minutes in water-bath, change in the 80 degree water-baths deactivation at once over to 2 minutes.Getting amplified production 3uL, is damping fluid with 1 * TAE, and in the sepharose 2% (containing the 0.5ug/mL ethidium bromide), at 100 volts, 75 peaces detect in 80 minutes the nucleic acid electrophoresis of electrophoresis.Detected result is seen Fig. 2, and the result is illustrated in 65 degree Yuzhong A.ovis genomes, Yongjing A.ovis genome, Yuzhong A.ovis MSP4 plasmid and all can amplifies; And chlamydia trachomatis gene group, mycoplasma genome, sheep Taylor, Taylor You Shi, Taylor Lv Shi, edge do not have slurry, Xinjiang BABEI this, sheep genome and sterilization ultrapure water all do not increased; This LAMP of experimental result proof method specificity is good.
In the centrifuge tube of 1.5mL,, comprising: 2 * reaction buffer working fluid 12.5uL by adding sample respectively; Primer reaction mixture 1uL; The ultrapure water 9.5uL of sterilization; DNA sample 1.0uL; Bst archaeal dna polymerase 1.0uL, TV amounts to 25uL.The DNA sample is used Yuzhong A.ovis MSP4 plasmid 1000 copies, 100 copies, 10 copies, 1 copy, 0.1 copy, 0.01 copy, 0.001 copy respectively, and detected result is seen Fig. 3, and the result shows that this LAMP method can detect 1 copy number.
Claims (5)
1. a loop-mediated isothermal amplification primer detects sheep and does not have the purposes in the reagent of slurry cause of disease in preparation; It is characterized in that not having main surface protein 4 genes of slurry with sheep is target gene; 3 pairs of Auele Specific Primers have been designed; And be mixed with the Auele Specific Primer reaction mixture by a certain percentage, employed primer sequence is following:
The outside upstream primer F3:5 ' of forward-GTGTTGCACACAGATTTGCC-3 '
Adverse external downstream primer B3:5 '-AGGCTTTTGCTTCTCCGG-3 '
The inner primers F IP of forward:
5’-GCCCCTGTAGGCTAGCTTTGTGgaattcCCCATATGTGTGTGCCGG-3’
Reverse inner primer BIP:
5’-TGGTGGTAGGTGGGTTCTACCAgaattcATGTGCGGGTATGTCCTTG-3’
Ring upstream primer LF:5 '-TGTCGACAAAGCTAGCACC-3 '
Ring downstream primer LB:5 '-CGGACTCTTTGACGAGTCTT-3 '
2. a kind of loop-mediated isothermal amplification primer according to claim 1 detects sheep and does not have the purposes in the reagent of slurry cause of disease in preparation, and it is characterized in that said Auele Specific Primer reaction mixture comprises: final concentration is FIP and the BIP of 40pmol; Final concentration is LF and the LB of 20pmol; Final concentration is F3 and the B3 of 5pmol.
3. detect sheep and do not have the purposes in the reagent of slurry cause of disease according to claim 1 and 2 described a kind of loop-mediated isothermal amplification primers in preparation, it is characterized in that adopting the following step to accomplish:
A, constant-temperature amplification: the loop-mediated isothermal amplification that sheep does not have the slurry cause of disease is Auele Specific Primer reaction mixture 1.0uL; 2 * LAMP reaction buffer 12.5uL; Sterile purified water 9.5uL; Genomic dna-positive or blank sample 1.0uL; Bst archaeal dna polymerase 1.0uL; Amount to 25uL, 65 ℃ of amplifications 30 minutes, 80 ℃ of deactivations 2 minutes;
B, electrophoresis detection: with the sepharose of 1 * TAE damping fluid preparation 2%, every hole adds 6 * sample-loading buffer of above-mentioned LAMP amplified production of 3uL and 1.5uL, and 100 volts, 75 peaces, electrophoresis 80 minutes, observations under ultraviolet gel imaging appearance;
C, judge to detect sheep by judgement criteria and do not have the slurry cause of disease.
4. a kind of loop-mediated isothermal amplification primer according to claim 1 detects sheep and does not have the purposes in the reagent of slurry cause of disease in preparation, it is characterized in that each component volume ratio is following in the said amplification reaction system:
Auele Specific Primer reaction mixture: 2 * LAMP reaction buffer: sterile purified water: genomic dna-positive or blank sample: Bst archaeal dna polymerase=1: 12.5: 9.5: 1: 1.
5. detect sheep and do not have the purposes in the reagent of slurry cause of disease according to claim 3 and 4 described a kind of loop-mediated isothermal amplification primers in preparation, it is characterized in that the final concentration at the said Auele Specific Primer reaction mixture of this reaction system is FIP and BIP, the LF of 20pmol and F3 and the B3 of LB and 5pmol of 40pmol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010542863 CN101985658B (en) | 2010-11-12 | 2010-11-12 | Loop-mediated isothermal amplification method for detecting anaplasma ovis pathogeny |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010542863 CN101985658B (en) | 2010-11-12 | 2010-11-12 | Loop-mediated isothermal amplification method for detecting anaplasma ovis pathogeny |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101985658A CN101985658A (en) | 2011-03-16 |
| CN101985658B true CN101985658B (en) | 2012-10-17 |
Family
ID=43710058
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010542863 Active CN101985658B (en) | 2010-11-12 | 2010-11-12 | Loop-mediated isothermal amplification method for detecting anaplasma ovis pathogeny |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101985658B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102634597B (en) * | 2012-05-04 | 2014-06-04 | 中国农业科学院兰州兽医研究所 | Kit for detecting anaplasma ovis |
| CN104293936A (en) * | 2014-09-18 | 2015-01-21 | 黑龙江八一农垦大学 | Use of loop-mediated isothermal amplification primer in preparing reagent for detecting bovine anaplasma marginale antigen |
| CN106771190B (en) * | 2016-12-26 | 2018-11-13 | 中国农业科学院兰州兽医研究所 | A kind of sheep anaplasmosis indirect ELISA diagnostic reagent kit and preparation method thereof |
-
2010
- 2010-11-12 CN CN 201010542863 patent/CN101985658B/en active Active
Non-Patent Citations (6)
| Title |
|---|
| 实时荧光PCR检测牛边缘无浆体方法的建立及应用;王贵强等;《中国预防兽医学报》;20070815(第08期);全文 * |
| 牛、羊无浆体病的诊断与治疗;王伟东等;《今日畜牧兽医》;20091008(第10期);全文 * |
| 王伟东等.牛、羊无浆体病的诊断与治疗.《今日畜牧兽医》.2009,(第10期), |
| 王贵强等.实时荧光PCR检测牛边缘无浆体方法的建立及应用.《中国预防兽医学报》.2007,(第08期), |
| 边缘无浆体MSP5重组抗原间接ELISA检测方法的建立;马米玲等;《中国兽医科学》;20080820(第08期);全文 * |
| 马米玲等.边缘无浆体MSP5重组抗原间接ELISA检测方法的建立.《中国兽医科学》.2008,(第08期), |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101985658A (en) | 2011-03-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Fotedar et al. | Laboratory diagnostic techniques for Entamoeba species | |
| CN102277455B (en) | Primer, probe and assay kit used for detecting porcine circovirus, porcine pseudorabies virus and porcine parvovirus | |
| Nagdev et al. | Determination of polymerase chain reaction efficiency for diagnosis of tuberculous meningitis in Chelex-100® extracted DNA samples | |
| CN103074451A (en) | Kit for synchronously detecting twenty-two respiratory tract pathogens and detection method of kit | |
| CN101985658B (en) | Loop-mediated isothermal amplification method for detecting anaplasma ovis pathogeny | |
| CN113025726A (en) | Primer, probe, kit and method for visual rapid detection of schistosoma japonicum nucleic acid by LFD-RPA | |
| CN101831507A (en) | Real-time fluorescence quantitative PCR (polymerase chain reaction) detection method of grass carp reovirus SYBR Green I | |
| CN104388592B (en) | A kind of Porcine epidemic diarrhea virus S gene RT LAMP detection kits and detection method | |
| CN114540526A (en) | Primer, probe and method for typing detection of five input plasmodium | |
| CN109439800B (en) | Kit and method for detecting gene mutation of PR region and RT region of HIV-1 gene | |
| CN101724714B (en) | Loop mediated isothermal amplification kit for detecting encephalitis B virus | |
| CN104911275B (en) | A kind of bacterial vaginitis detection kit | |
| CN101886113B (en) | Method for detecting Lyme disease pathogen in tick bodies and kit | |
| CN101864492A (en) | Nucleic acid detection kit for aided diagnosis of ankylosing spondylitis | |
| CN103276091A (en) | Kit for detecting proviral DNA (Deoxyribonucleic Acid) of HIV (Human Immunodeficiency Virus) | |
| CN108130385A (en) | A kind of human cytomegalovirus kit for detecting nucleic acid | |
| CN102703604B (en) | Kit for rapidly detecting banana bunchy top virus by isothermal gene amplification and use method of kit | |
| CN102277439B (en) | Rapid detection kit for goat contagious pleuropneumonia and preparation method thereof | |
| CN109680101B (en) | Rapid detection method for distinguishing strong and weak viruses of H7N9 subtype avian influenza virus | |
| CN102154270B (en) | Cronobacter sakazakii O antigen specific nucleotides and use thereof | |
| CN103757109B (en) | Primer composition and multiple-gene detection kit for guiding administration of thiazine diuresis drugs and application method thereof | |
| CN103290141A (en) | Kit for detection of 16 high-risk and 5 low-risk HPV | |
| CN111235261A (en) | Kit for detecting human platelet-specific antigen HPA 1-29 genotyping | |
| CN102827944B (en) | Mycobacterium detection kit and using method thereof | |
| CN110205390A (en) | A kind of method and Primer composition detecting American hookworm |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C53 | Correction of patent for invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Yin Hong Inventor after: Liu Junlong Inventor after: Niu Qingli Inventor after: Niu Qingli Inventor after: Luo Jianxun Inventor after: Ma Miling Inventor after: Liu Zhijie Inventor after: Yang Jifei Inventor after: Guan Guiquan Inventor after: Li Youquan Inventor after: Liu Aihong Inventor after: Ren Qiaoyun Inventor before: Yin Hong Inventor before: Liu Junlong Inventor before: Niu Qingli Inventor before: Luo Jianxun Inventor before: Ma Zhuling Inventor before: Liu Zhijie Inventor before: Yang Zhifei Inventor before: Guan Guiquan Inventor before: Li Youai Inventor before: Liu Aihong Inventor before: Ren Qiaoyun |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: YIN HONG LUO JIANXUN MA ZHULING LIU ZHIJIE YANG ZHIFEI GUAN GUIQUAN LI YOUAI LIU AIHONG REN QIAOYUN LIU JUNLONG NIU QINGLI TO: YIN HONG LUO JIANXUN MA MILING LIU ZHIJIE YANG JIFEI GUAN GUIQUAN LI YOUQUAN LIU AIHONG REN QIAOYUN LIU JUNLONG NIU QINGLI NIU QINGLI |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |