CN102533961B - For detecting target sequence and the test kit of New Delhi metal beta lactamase-1 gene - Google Patents
For detecting target sequence and the test kit of New Delhi metal beta lactamase-1 gene Download PDFInfo
- Publication number
- CN102533961B CN102533961B CN201010621899.0A CN201010621899A CN102533961B CN 102533961 B CN102533961 B CN 102533961B CN 201010621899 A CN201010621899 A CN 201010621899A CN 102533961 B CN102533961 B CN 102533961B
- Authority
- CN
- China
- Prior art keywords
- gene
- test kit
- ndm
- seqidno
- fluorescent probe
- 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
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention is target sequence and the test kit that a kind of application Fluorescence PCR assay detects New Delhi metal beta lactamase-1 gene (NDM-1), it adopts region in pair of primers amplification NDM-1 gene target sequence SEQIDNo:1, and detect amplified production by fluorescent probe, the existence of NDM-1 gene is judged with template fluorescent signal and cycle threshold.Reagent constituents comprises PCR damping fluid, fluorescent probe, Taq enzyme, negative control, positive control and nucleic acid extraction liquid.Test kit uses and comprises sample process and augmentation detection two step, easy and simple to handle, highly sensitive, high specificity, can be widely used in clinical in the rapid detection of Multiple Classes of Antibiotics multidrug resistant disease indigenous bacteria that caused by NDM-1 gene.
Description
Technical field
The invention belongs to technological field of biochemistry, be specifically related to target sequence and test kit that a kind of application Fluorescence PCR assay detects New Delhi metal beta lactamase-1 (NDM-1) gene.
Background technology
2010, in India, a kind of novel bacteria mutated genes that the South Asian nations such as Pakistan occur is likely at global spread, have the bacterium of this gene to comprising cephalosporin, carbapenems, most microbiotic such as aminoglycoside has resistance (at present only to replacing family's ring element and polymyxin two kinds of antibiotic sensitive), therefore this unnamed gene is New Delhi metal beta lactamase-1 (NewDelhimetallo-β-lactamase1 by medical expert, be called for short NDM-1) gene, originally it be present on the plasmid of the bacteriums such as intestinal bacteria, express the enzyme produced and can decompose above-mentioned microbiotic, NDM-1 gene can be propagated along with the transfer of plasmid between bacterium, cause the bacterium obtaining this gene to the antibiotic resistance (YongD of major part, etal.AntimicrobialAgentsandChemotherapy, 2009, 53 (12): 5046-5054.RolainJM, etal, ClinicalMicrobiologyandInfection, 2010, 16 (12): 1699-1701.).So far, microbiotic multi-drug resistant bacteria containing this super drug resistant gene of NDM-1 has spread from South Asia region to be propagated to the whole world, successively and domestic discovery national in Britain, the U.S., Canada, Australia, Holland etc., to the health of the mankind define new threat (
www.theLancet.com/Infection, publishedonlineAugust11,2010DOI:10.1016/S1473-3099 (10) 70143-2).
Clinically to the traditional detection of this microbiotic multi-drug resistant bacteria be separation and Culture in conjunction with susceptibility authentication method, need the soonest to go out detected result week more than one, far can not meet the needs clinically to Pathogenic Microorganisms On Tropical and medication guide.Therefore adopt molecular biology method qualification to be inevitable choice clinically containing the multi-drug resistant bacteria of NDM-1 gene, wherein fluorescence PCR method is especially due to sensitive, feature and have advantage fast.
Summary of the invention
The object of this invention is to provide one utilizes Fluorescence PCR assay to detect target sequence and the test kit of New Delhi metal beta lactamase-1 (NDM-1) gene, test kit contains primer and the probe of with good grounds target sequence SEQIDNO:1 design, thus the accuracy of real-time PCR detection NDM-1 gene can be improved, be suitable for the microbiotic multidrug resistant pathogenetic bacteria of clinical Rapid identification containing NDM-1 gene.
1. the Design and synthesis of real-time fluorescence PCR primer probe
By inquiring about New Delhi metal beta lactamase-1 gene order in GenBank, compare by the gene order of molecular biology professional software to various source, select high conservative and the sequence SEQIDNo:1 with good homology as detection target sequence, according to the principle of TaqMan fluorescent PCR design, devise according to this target sequence the conservative region that a pair NDM-1 gene-specific primer increase on this gene, and detect in real time with the specificity T aqMan probe of 5 ' end FAM mark.Primer and probe base sequence as follows:
Upstream primer is 5 '-ggCggggATTgCgACTTATg-3 ', as shown in SEQIDNO:2;
Downstream primer is 5 '-AATACCTTgAgCgggCCAAAg-3 ', as shown in SEQIDNO:3;
Fluorescent probe is 5 '-AATgCgTTgTCgAACCAgCTTgCC-3 ' (5 ' flag F AM, 3 ' mark TAMRA or BHQ-1), as shown in SEQIDNO:4.
By above-mentioned design, obtain pair of primers and probe sequence by NDM-1 gene special, it is 25 μMs that above-mentioned primed probe is dissolved to concentration with aseptic double-distilled water after being synthesized by business sequent synthesis company (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd etc.) ,-20 DEG C of preservations.
2. the preparation of test kit
NDM-1 gene by fluorescence PCR detection kit (32 person-portion) main ingredient designed for target sequence SEQIDNO:1 is as follows:
(1) PCR damping fluid 672 μ l, main component has Tris-HCl (pH8.3), KCl, gelatin, dATP, dGTP, dCTP, dUTP, MgCl
2, upstream and downstream primer (SEQIDNo:2 and 3).
(2) fluorescent probe 96 μ l, containing fluorescent probe (SEQIDNo:4).
(3) Taq enzyme 64 μ l, containing Taq DNA polymerase 40U and uracil-DNA glycosylase (UNG enzyme) 16U.
(4) negative control 200 μ l is culture of Escherichia coli, 10
4individual bacterium/ml.
(5) positive control 200 μ l is the artificial constructed culture of Escherichia coli containing SEQIDNo:1 sequence plasmid, 10
3individual bacterium/ml.
(6) DNA extraction liquid 1.5ml, main component is 50mmol/LNaOH, 5mMEDTA, 1%TritonX-100,1%NP-40.
The 30 μ l reaction systems be made up of after adding template 4 μ l after mixing mentioned reagent box component PCR damping fluid 21 μ l, fluorescent probe 3 μ l, Taq enzyme 2 μ l, containing Tris-HCl (pH8.3) 10mM, KCl50mM, gelatin 0.1mg/ml, dATP, dGTP, dCTP, dUTP each 0.2-0.4mM, MgCl
2the each 0.2-0.5 of 1.5-5mM, primer μM, fluorescent probe 0.1-0.5 μM, Taq DNA polymerase 1-5U, UNG enzyme 0.1-1U, fluorescent PCR instrument carries out amplified reaction.
More specifically, Tris-HCl (pH8.3) 10mM, KCl50mM, gelatin 0.1mg/ml, dATP, dGTP, dCTP, dUTP each 0.2mM, MgCl is contained in the reaction system that said components is mixed with
2each 0.35 μM of 3.5mM, primer, fluorescent probe 0.2 μM, Taq DNA polymerase 1.25U, UNG enzyme 0.5U, fluorescent PCR instrument carries out amplified reaction.
The test kit that said components is assembled into is at-20 DEG C of storage and transport.
3. test kit using method
Test kit of the present invention containing one of use step in NDM-1 gene microbiotic multidrug resistant pathogenetic bacteria testing process is being:
(1) sample preparation
Get clinical sample or the enrichment liquid of collection, be placed in centrifuge tube 13,000rpm centrifugal 6 minutes, carefully inhale with pipettor and abandon supernatant (for preventing siphoning away precipitation, 5 μ l raffinates can be retained), in precipitation, add 1ml physiological saline, after vortex oscillation mixing, repeated centrifugation is inhaled and is abandoned supernatant.In precipitation, add 50 μ lDNA extracting solutions, latter 100 DEG C of vortex vibration mixing keeps 10 minutes, and 13,000rpm gets supernatant in centrifugal 2 minutes and carries out fluorescent PCR augmentation detection.Test kit is negative identical with sample liquid with positive control treatment process.
(2) augmentation detection
According to detected sample number n+2, get test kit PCR damping fluid 21 μ l × (n+2), fluorescent probe 3 μ l × (n+2), Taq enzyme 2 μ l × (n+2) component, each amplification pipe packing 26 μ l after mixing, adds the test kit negative control, positive control and each 4 μ l of the above-mentioned n handled well a sample form that handle well respectively.Each reaction tubes volume is 30 μ l, is put into by above-mentioned amplification pipe on two channels real-time fluorescence PCR instrument and runs according to the circulate program of 40 times of 94 DEG C of 10sec after 50 DEG C of 2min, 94 DEG C of 2min, 60 DEG C of 45sec.
(3) result judges
Use after fluorescent PCR instrument end of run and carry software analysis result, test kit negative control should be that feminine gender, positive control Ct value < 30 are for positive without Ct value.Sample amplification Ct value < is for containing NDM-1 gene masculine, and sample infects NDM-1 gene pathogenic bacteria of drug-resistant; Detected result is NDM-1 gene negative without Ct value person; If 38 < Ct value <, for detecting gray area, need duplicate detection 2 times, as detected result at least 1 Ct value < 40 judges that this pipe is NDM-1 gene masculine, otherwise, judge that this pipe is as negative.
4. beneficial effect
The present invention is according to the primed probe of above-mentioned design and technical scheme, and be developed into NDM-1 gene by fluorescence PCR detection kit by optimizing reaction system and amplification condition, its principal feature is as follows:
(1) test kit adopts the NDM-1 gene target sequence of Auele Specific Primer probe in detecting high conservative, ensure that detection specificity and sensitivity;
(2) test kit stopped pipe detects, fast easy and simple to handle, can report detected result in 2 hours;
(3) the dUTP-UNG enzyme system in amplification system further avoids the result false positive that gene-amplification product pollution causes.
The These characteristics of test kit, is the primed probe combined with fluorescent round pcr Combination application that adopt and design according to preferred target sequence and directly causes.From the principle, test kit of the present invention passes through the analysis to real-time fluorescent PCR amplification signal in NDM-1 genopathy substance clinical sample detects, judge the existence of NDM-1 gene nucleic acid specific fragment, as a kind of means of NDM-1 gene nucleic acid rapid detection, Technical Design of the present invention is reasonable, can be widely used in clinical in Rapid identification containing the super drug resistant gene pathogenetic bacteria of NDM-1.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can do various technical change or amendment by technology general knowledge to the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
Main agents involved in the present invention illustrates:
Taq DNA polymerase, uracil-DNA glycosylase (UNG enzyme), dNTPs (dATP, dUTP, dCTP, dGTP) are Shanghai Hong Yi company and produce, and primed probe and other chemical reagent are purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
The preparation of embodiment 1:NDM-1 gene by fluorescence PCR detection kit
(1) primed probe synthesis
Business sequent synthesis company (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) is entrusted to synthesize following sequence:
Upstream primer is 5 '-ggCggggATTgCgACTTATg-3 ', as shown in SEQIDNo:2;
Downstream primer is 5 '-AATACCTTgAgCgggCCAAAg-3 ', as shown in SEQIDNO:3;
Fluorescent probe is 5 '-AATgCgTTgTCgAACCAgCTTgCC-3 ' (5 ' flag F AM, 3 ' mark TAMRA or BHQ-1), as shown in SEQIDNo:4.
Being dissolved to concentration with aseptic double-distilled water is respectively 25 μMs ,-20 DEG C of preservations.
(2) NDM-1 gene PCR damping fluid is prepared
Containing Tris-HCl (pH8.3) 14.2mM, KCl71.4mM, gelatin 0.14mg/ml, dATP, dGTP, dCTP, dUTP each 0.29mM, MgCl
25mM, each 5 μMs of upstream and downstream primer (SEQIDNo:2 and 3), according to 672 μ l/ pipe packing.
(3) fluorescent probe is prepared
Containing NDM-1 gene by fluorescence probe (SEQIDNo:4) 2 μMs, according to 96 μ l/ pipe packing.
(4) Taq enzyme is prepared
Containing Taq DNA polymerase 40U and uracil-DNA glycosylase (UNG enzyme) 16U, according to 64 μ l/ pipe packing.
(5) DNA extraction liquid is prepared
Containing 50mmol/LNaOH, 5mMEDTA, 1%TritonX-100,1%NP-40.According to the packing of 1.5ml/ pipe.
(6) test kit feminine gender, positive reference substance is prepared
Cultivate intestinal bacteria, intestinal bacteria containing SEQIDNO:1 artificial sequence plasmid respectively, and measure nutrient solution concentration by viable plate count method, be diluted to 1 × 10
4individual bacterium/ml and 1 × 10
3individual bacterium/ml, respectively as test kit negative control and positive control.According to 200 μ l/ pipe packing.
(7) kind of the component of 5 after above-mentioned packing is placed in same container, is assembled into test kit, encapsulation.
Preparation technology is summarized as follows:
(1) primed probe synthesized quantitatively is prepared, Concentration Testing, sampling quality inspection.
(2) PCR damping fluid, fluorescent probe, Taq enzyme, DNA extraction liquid is aseptic subpackaged, sampling quality inspection.
(3) feminine gender, positive reference substance are carried out concentration determination, packing, sampling quality inspection.
(4) assemble finished product test kit, be stored in-20 DEG C.
The application of embodiment 2:NDM-1 gene by fluorescence PCR detection kit
(1) sample preparation
Get clinical sample or enrichment liquid 20 example of collection, be placed in centrifuge tube 13,000rpm centrifugal 6 minutes, carefully inhale with pipettor and abandon supernatant (for preventing siphoning away precipitation, 5 μ l raffinates can be retained), in precipitation, add 1ml physiological saline, after vortex oscillation mixing, repeated centrifugation is inhaled and is abandoned supernatant.In precipitation, add 50 μ lDNA extracting solutions, latter 100 DEG C of vortex vibration mixing keeps 10 minutes, and 13,000rpm gets supernatant in centrifugal 2 minutes and carries out fluorescent PCR augmentation detection.Test kit is negative identical with sample liquid with positive control treatment process.
(2) augmentation detection
According to detected sample number n+2 (n=20), get test kit PCR damping fluid 21 μ l × (n+2), fluorescent probe 3 μ l × (n+2), Taq enzyme 2 μ l × (n+2) component, each amplification pipe packing 26 μ l after mixing, adds respectively according to the test kit negative control of sample processing method process, positive control and each 4 μ l of the above-mentioned n handled well a sample form.Each reaction tubes volume is 30 μ l, is put into by above-mentioned amplification pipe on ABI7500 fluorescent PCR instrument and runs according to the circulate program of 40 times of 94 DEG C of 10sec after 50 DEG C of 2min, 94 DEG C of 2min, 60 DEG C of 45sec.
(3) result judges
Use after fluorescent PCR instrument end of run and carry software analysis result, test kit negative control should be judged as feminine gender without Ct value, positive control Ct value < 30 is judged as the positive.Sample amplification Ct value < is NDM-1 gene masculine, containing NDM-1 gene pathogenic bacteria of drug-resistant in sample; Sample amplification is that NDM-1 gene resistant organism is negative without Ct value person; If 38 < Ct value < 40, be judged as detecting gray area, need duplicate detection 2 times, as detected result at least 1 Ct value < 40 judges that this pipe is NDM-1 gene masculine, otherwise, judge that this pipe is as negative.
According to the above results determination methods, have 1 example to detect Ct value < 38, be judged as that NDM-1 gene pathogenic bacteria of drug-resistant is positive in the 20 routine samples that experiment detects, all the other 19 examples are that NDM-1 gene pathogenic bacteria of drug-resistant is negative.
Claims (5)
1. one kind is detected the fluorescent PCR kit of New Delhi metal beta lactamase-1 (NDM-1) gene, it is characterized in that, test kit designs primed probe according to target sequence SEQIDNO:1, article two, primer has SEQIDNo:2 and SEQIDNo:3 sequence, and a fluorescent probe has SEQIDNo:4 sequence
.
2. test kit as claimed in claim 1, fluorescent probe SEQIDNo:4 sequence 5 ' end flag F AM fluorophor, the fluorescent quenching group of 3 ' end mark is TAMARA or BHQ-1.
3. test kit as claimed in claim 1, NDM-1 gene detecting kit component comprises PCR damping fluid, fluorescent probe, Taq enzyme, negative control, positive control and DNA extraction liquid.
4. test kit as claimed in claim 1, the reaction solution that reagent constituents is mixed with according to following concentration carries out amplified reaction: Tris-HCl10mM, the KCl50mM of pH8.3, each 0.2 ~ 0.4mM of gelatin 0.1mg/ml, dATP, dGTP, dCTP, dUTP, MgCl
2each 0.2 ~ 0.5 μM of 1.5 ~ 5mM, primer, fluorescent probe 0.1 ~ 0.5 μM, Taq DNA polymerase 1 ~ 5U, UNG enzyme 0.1 ~ 1U.
5. test kit as claimed in claim 1, more specifically, its amplification reaction solution each composition final concentration is: Tris-HCl10mM, the KCl50mM of pH8.3, gelatin 0.1mg/ml, dATP, dGTP, dCTP, dUTP each 0.2mM, MgCl
2each 0.35 μM of 3.5mM, primer, fluorescent probe 0.2 μM, Taq DNA polymerase 1.25U, UNG enzyme 0.5U.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010621899.0A CN102533961B (en) | 2010-12-30 | 2010-12-30 | For detecting target sequence and the test kit of New Delhi metal beta lactamase-1 gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010621899.0A CN102533961B (en) | 2010-12-30 | 2010-12-30 | For detecting target sequence and the test kit of New Delhi metal beta lactamase-1 gene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102533961A CN102533961A (en) | 2012-07-04 |
| CN102533961B true CN102533961B (en) | 2016-03-02 |
Family
ID=46341987
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010621899.0A Active CN102533961B (en) | 2010-12-30 | 2010-12-30 | For detecting target sequence and the test kit of New Delhi metal beta lactamase-1 gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102533961B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102965377B (en) * | 2012-08-30 | 2014-04-16 | 广东医学院 | New Delhi metallo-beta-lactamase-1 aptamer, its screening method and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5538862A (en) * | 1994-02-04 | 1996-07-23 | California Institute Of Technology | Heat-inducible N-degron module |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6777014B2 (en) * | 2001-07-03 | 2004-08-17 | Harjit Singh | Agglomerated milk in coffee and tea |
| FI20095544A0 (en) * | 2009-05-15 | 2009-05-15 | Juha Kirveskari | Method and kit for identifying antibiotic resistant bacteria |
-
2010
- 2010-12-30 CN CN201010621899.0A patent/CN102533961B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5538862A (en) * | 1994-02-04 | 1996-07-23 | California Institute Of Technology | Heat-inducible N-degron module |
Non-Patent Citations (1)
| Title |
|---|
| 携带blaNDM-1基因泛耐药肠杆菌科细菌的检测方案;郑波等;《Chin J Clin Pharmacol》;20101130;第26卷(第11期);第847页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102533961A (en) | 2012-07-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106222248A (en) | A kind of detect the primer of methicillin-resistant staphylococcus aureus drug resistance gene, probe, method and test kit | |
| CN103397105B (en) | Kit for detecting GII type norovirus and applications thereof | |
| CN106520984A (en) | Pseudomonas aeruginosa nucleic acid fluorescent PCR (polymerase chain reaction) detection kit and detection method | |
| CN101429539B (en) | Reagent kit for real time fluorescence quantitative PCR detection of bacillus pyocyaneus | |
| CN102719540A (en) | Detection kit for detecting relative expression level of PML-RAR (alpha) fusion gene by use of fluorescent quantitative PCR (polymerase chain reaction) technology | |
| CN109735639A (en) | It is a kind of for detecting the primer and probe composition and kit of Klebsiella Pneumoniae and three kinds of main carbapenems | |
| CN102676664B (en) | Fluorescent quantitative polymerase chain reaction (PCR) primers and probes for detecting pathogenic bacteria of multiple aquatic products simultaneously and detection method | |
| CN101736078A (en) | Method for detecting viable bacteria of Mycobacterium tuberculosis through isothermal amplification of nucleic acid and kit | |
| CN102071247B (en) | Detection method for distinguishing non-pneumophilia legionella and legionella pneumophilia and kit | |
| CN102154487A (en) | Reagent for detecting francisella tularensis and complex probe and fluorescent quantitative polymerase chain reaction (PCR) method for detecting francisella tularensis | |
| CN102978282B (en) | Typhoid fever salmonella and salmonella paratyphi fluorescent quantitative polymerase chain reaction (PCR) detection kit and application thereof | |
| CN102533961B (en) | For detecting target sequence and the test kit of New Delhi metal beta lactamase-1 gene | |
| CN101781677A (en) | Kit for detecting leukemia broad-spectrum marker WT1 gene mRNA expression | |
| CN101705301A (en) | Special probe and gene chip used for identifying pathogenic aspergillus | |
| CN116287356A (en) | Primer probe composition and kit for detecting multiple respiratory tract pathogenic bacteria and drug resistance genes | |
| CN102417931B (en) | Polymerase chain reaction (PCR) fluorescence detection kit and detection method for candida albicans | |
| CN102146467A (en) | Reagent for detecting Yersinia pestis and method for carrying out fluorescence quantitative PCR (Polymerase Chain Reaction) detection on Yersinia pestis | |
| CN103388032A (en) | Coxsackie virus type A16 (CA16) real-time fluorescent nucleic acid isothermal amplification detection kit | |
| CN106048051B (en) | A kind of candida krusei fluorescence PCR detection reagent kit | |
| CN102134588B (en) | Legionella pneumophilia test kit and application thereof | |
| CN102344970B (en) | Primer sequences and quantitative determination kit used for simultaneously detecting human CMV and BK virus DNA | |
| CN108034736B (en) | Detection kit for drug resistance of Escherichia coli fluoroquinolone antibiotics, detection method and application | |
| CN102399899A (en) | Liquid phase chip method and kit for detecting polymorphism of cytochrome P450 1A2 (CYP1A2) genes | |
| CN101781678A (en) | Kit for detecting fusion gene Bcl2-IgH rearrangement | |
| CN101818212A (en) | Loop-mediated isothermal amplification kit for rapidly detecting porcine parvovirus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| DD01 | Delivery of document by public notice |
Addressee: Shanghai Fosun medical science and Technology Development Co., Ltd. Wu Dazhi Document name: Notification of Publication of the Application for Invention |
|
| DD01 | Delivery of document by public notice |
Addressee: Wu Dazhi Document name: Notification of before Expiration of Request of Examination as to Substance |
|
| CB02 | Change of applicant information |
Address after: 201315 Shanghai city Pudong New Area Kangshi Road No. 17 Applicant after: Shanghai Xingyao Medical Technology Development Co., Ltd. Applicant after: Shanghai Fosun Pharmaceutical (Group) Limited by Share Ltd. Address before: 200233 Yishan Road, Shanghai, No. 1289 Applicant before: Shanghai Fosun Pharmaceutical (Group) Corporation Applicant before: Shanghai Fosun Pharmaceutical (Group) Limited by Share Ltd. |
|
| CB02 | Change of applicant information | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: APPLICANT; FROM: FUXING MEDICAL SCIENCE -TECHNOLOGY DEVELOPMENT CO., LTD., SHANGHAI TO: SHANGHAI XINGYAO MEDICAL TECHNOLOGY DEVELOPMENT CO., LTD. |
|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |