CN105695625B - Detection kit for auxiliary diagnosis of primary hypertension and application thereof - Google Patents
Detection kit for auxiliary diagnosis of primary hypertension and application thereof Download PDFInfo
- Publication number
- CN105695625B CN105695625B CN201610277641.0A CN201610277641A CN105695625B CN 105695625 B CN105695625 B CN 105695625B CN 201610277641 A CN201610277641 A CN 201610277641A CN 105695625 B CN105695625 B CN 105695625B
- Authority
- CN
- China
- Prior art keywords
- methylation
- methylation specific
- primer
- promoter region
- nucleotide sequence
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Abstract
The invention discloses a detection kit for auxiliary diagnosis of essential hypertension, which comprises a pair of methylation specific amplification primers of a Toll-like receptor 2 gene promoter region and a methylation specific sequencing primer: nucleotide sequence of methylation specific forward primer: 5 '-Biotin-GGTAGTTGTAGGGGTAGGAT-3'; nucleotide sequence of methylation specific downstream primer: 5'-ACCCAAAAAAACTCTAAACCTC-3', respectively; nucleotide sequence of methylation specific sequencing primer: 5'-TTCCAAACAAATAACC-3' are provided. The method has the advantages of conveniently and quickly detecting the essential hypertension at a molecular level, high detection efficiency and strong pertinence.
Description
Technical Field
The invention relates to the technical field of auxiliary diagnosis of essential hypertension, in particular to a detection kit for auxiliary diagnosis of essential hypertension and application thereof, and particularly relates to a kit for detecting methylation degree of a Toll-like receptor 2 gene promoter region related to essential hypertension and application thereof.
Background
Essential Hypertension (EH) is a syndrome which is caused by various factors such as genetic factors, environmental factors and individual differences and is mainly manifested by the increase of systemic arterial blood pressure, and is also one of the worldwide public health problems seriously harming human health. The research on the causes of the primary hypertension increasingly becomes a hot point of domestic and foreign research. The search for EH-related genes to elucidate the genetic mechanism of elevated blood pressure has become a hot spot of current research. Although more and more medical research institutes pay attention to and develop the etiology research of EH and the research focuses on the correlation between the single nucleotide polymorphism of the related candidate gene and EH, the pathogenesis of EH is still not completely elucidated, which undoubtedly hinders the improvement of the prevention and treatment level of EH.
Toll-like receptors (TLRs), members of the interleukin receptor superfamily, are transmembrane receptors that are extracellular rich in leucine repeats and important signaling domains within cells. Toll-like receptor 2(TLR2) is a receptor with the widest expression range in a cloned Toll-like receptor family, has an important role in resisting the anti-inflammatory response of cytotoxin, and becomes a new target for treating various diseases. In a TLR2 signaling pathway, TLR2 can cooperate with TLR1 or TLR6 to activate the expression of downstream target genes through a Mal/MyD88 and NF-kB pathway mechanism, and finally, the release of inflammatory factors is caused. It has been shown that elevated levels of inflammatory factors associated with TLRs signaling pathways in the peripheral vascular circulation, such as TNF- α and IL-6, C-reactive protein, are considered important risk factors for hypertension. Toll-like receptors are thought to play an important role in the pathogenesis of EH and atherosclerotic diseases; however, the underlying molecular mechanism is still unclear.
At present, no relevant research report about a kit for detecting the methylation degree of a Toll-like receptor gene promoter region related to essential hypertension is published at home and abroad.
Disclosure of Invention
The invention aims to solve the technical problem of providing a detection kit for auxiliary diagnosis of essential hypertension and application thereof, which have high detection efficiency and strong pertinence, aiming at the current situation of the prior art, wherein the methylation level of a Toll-like receptor 2 gene promoter region is negatively related to the prevalence rate of the essential hypertension.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a detection kit for auxiliary diagnosis of essential hypertension comprises a pair of methylation specific amplification primers of a Toll-like receptor 2 gene promoter region and a methylation specific sequencing primer:
nucleotide sequence of methylation specific forward primer:
5'- Biotin-GGTAGTTGTAGGGGTAGGAT-3'；
nucleotide sequence of methylation specific downstream primer:
5'- ACCCAAAAAAACTCTAAACCTC-3'；
nucleotide sequence of methylation specific sequencing primer:
5'-TTCCAAACAAATAACC-3'。
the application of the detection kit for the auxiliary diagnosis of the essential hypertension can be used for the auxiliary diagnosis, detection or screening of the essential hypertension.
Compared with the prior art, the invention has the advantages that: the invention discloses a detection kit for auxiliary diagnosis of essential hypertension and application thereof, wherein the detection kit is used for detecting methylation degree of a Toll-like receptor 2 gene promoter region related to the essential hypertension, and the low methylation level of the Toll-like receptor 2 gene promoter region leads to high expression of a TLR2 gene, so that the expression of an inflammatory factor gene is activated to lead to the release of an inflammatory factor, and further the generation and development of the essential hypertension are influenced. Therefore, the methylation level of the Toll-like receptor 2 gene promoter region is in negative correlation with the prevalence rate of essential hypertension, and the detection kit based on the detection of the methylation level of the Toll-like receptor 2 gene promoter region can conveniently and quickly realize the detection of the essential hypertension on a molecular level, and has high detection efficiency and strong pertinence; meanwhile, the medicine taking Toll-like receptor 2 gene promoter region methylation as a target is expected to become a new technical method for auxiliary diagnosis, detection and screening of essential hypertension.
Drawings
FIG. 1 shows the region where the detected sequence is located (the specific positions are Chr4:154605441 and 154627242), and the correlation analysis results between the detected 8 CpG points (for example, the correlation coefficient between CpG1 and CpG2 is 0.588, and the correlation coefficient between CpG2 and CpG3 is 0.522);
FIG. 2 is an example of the results of the methylation level measurements (the percentages indicated in the figure are the degree of methylation at the corresponding CpG sites, as indicated by the 6%, 5%, 3%, 5%, 6%, 10%, 9%, 18% methylation for CpG1 through CpG8, respectively);
FIG. 3 shows the results of ROC curve analysis at position 6(CpG6) (area under the curve AUC: 0.834; 95% confidence interval CI 0.766-0.892).
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
A detection kit for auxiliary diagnosis of essential hypertension comprises a pair of methylation specific amplification primers of a Toll-like receptor 2 gene promoter region and a methylation specific sequencing primer:
nucleotide sequence of methylation specific forward primer:
5'- Biotin-GGTAGTTGTAGGGGTAGGAT-3'；
nucleotide sequence of methylation specific downstream primer:
5'- ACCCAAAAAAACTCTAAACCTC-3'；
nucleotide sequence of methylation specific sequencing primer:
5'-TTCCAAACAAATAACC-3'。
the application of the detection kit for the auxiliary diagnosis of the essential hypertension can be used for the auxiliary diagnosis, detection or screening of the essential hypertension.
1. Collection of study subjects
The multi-stage sampling based on community population is adopted to carry out case contrast research, 3000 Han population of 3 generations and over 35 to 70 years old in Ningbo city are selected in the first stage by a layered random sampling method to carry out preliminary questionnaire survey of demographic characteristics, environmental factors and the like and corresponding physical examination. On the basis of the analysis of the investigation result, 3000 people are divided into two groups of newly-occurring hypertension patients and normal subjects without hypertension and other concerned vascular diseases, the hypertension patients are required to meet the hypertension diagnosis standard established by the WHO in 1999, and patients with secondary hypertension and renal artery stenosis, and liver, kidney, heart disease, diabetes, thyroid disease and cerebrovascular disease are excluded. On the basis, according to the principle of sex and age 1:1 matching, study objects are randomly extracted from three groups respectively and divided into 96 new cases and 96 control groups. Performing questionnaire survey on the demographic characteristics and the environmental factors by adopting a unified questionnaire, and performing physical examination, blood sample collection and laboratory examination; laboratory tests mainly include blood routine and biochemical indicators.
2. Extraction of genomic DNA
The whole blood genome DNA of the sample obtained in the above step is extracted by using a Lab-Aid 820 full-automatic nucleic acid extractor (Shanghai, China Xiamen, China Biotech), and the concentration of the obtained DNA is detected by a NanoDrop2000 ultramicro spectrophotometer (ThermoFisher Scientific, USA) so as to detect the DNA methylation level of the TLR2 gene promoter region.
3. DNA methylation level determination
DNA methylation level analysis was performed on 8 CpG sites (shown in FIG. 1) in the promoter region of TLR2 gene by pyrosequencing. Pyrosequencing technology adopts the basic principle that pyrosequencing adopts biotin-labeled primers to perform PCR amplification, and after a PCR product is purified and denatured into a single strand, a mixture of four enzymes is added: DNA polymerase (synthesizes a DNA double strand, releases a pyrophosphate group of dNTP, and the amount of released pyrophosphate group is proportional to the amount of dNTP bound to the template), ATP sulfurylase (catalyzes the formation of ATP from a pyrophosphate group in the presence of adenylosine 5' phosphate), luciferase (converts luciferin into oxyluciferin under the mediation of ATP, and oxyluciferin releases a visible light signal proportional to the amount of ATP), and apyrase (degrades dNTP and ATP that are not incorporated into a new strand, and quenches fluorescence). In the sequencing process, one type of dNTP is added each time, if the dNTP can be complementarily paired with a template strand, a series of reactions occur under the action of four enzymes, and finally, a fluorescence signal is converted into an electric signal to be reflected and displayed as peaks with different heights, wherein the heights of the peaks are in direct proportion to the number of bases. Conversely, when a dNTP cannot bind to the template strand, it will be directly degraded by apyrase and, correspondingly, will not show a peak. In the research, PyroMarkAssay Design software is adopted for primer Design, and PCR amplification primers and sequencing primers used for experiments are as follows:
(1) methylation specific Forward primer (Forward primer):
5'- Biotin-GGTAGTTGTAGGGGTAGGAT-3' (SEQ ID NO.1)；
(2) methylation specific Reverse primer (Reverse primer):
5'- ACCCAAAAAAACTCTAAACCTC-3' (SEQ ID NO.2)；
(3) methylation specific Sequencing primer (Sequencing primer):
5'-TTCCAAACAAATAACC-3'(SEQ ID NO.3)。
the specific experimental steps are as follows:
A. bisulfite conversion of sample DNA was performed using EZ DNA Methylation Kit-Gold (EZ DNA Methylation-Gold TM Kit; ZYMO RESEARCH).
B. And (2) adding 20ng of the DNA sample converted in the step (A) into Zymo Taq TM PreMix enzyme (Zymo Taq TM PreMix, ZYMO RESEARCH), adding the pair of TLR2 gene promoter region methylation specific amplification primers, and performing PCR amplification under the amplification conditions: firstly, denaturation at 95 ℃ for 10 min; then carrying out 45 cycles of annealing reaction at 95 ℃ for 30s, Tm for 40s and 72 ℃ for 50 s; then the extension reaction was carried out at 72 ℃ for 7 min. (Note: Tm is determined in the experiment based on running PCR gradient temperature)
C. Early preparation of pyrophosphate sequencing: mu.l of an annealing Buffer (PyroMark annealing Buffer; Qiagen) containing 0.3. mu.M of the above methylation specific sequencing primer was added to the PSQ96 plate in advance; transfer the total amount of mixed agarose beads (3. mu.l per sample) needed to be used into an Eppendorf tube; adding a Binding Buffer (Qiagen) to the agarose beads so that on average there is a volume of about 50. mu.l per sample, and mixing the mixture; the above mixture was added to the PCR product (50. mu.l reaction volume) in 50. mu.l per sample; mixing the PCR product at normal temperature for 10min to combine the magnetic beads and biotin; in the vacuum preparation workstation, 180ml of high purity water, 70% ethanol, washing Buffer (Pyromark Wash Buffer; Qiagen;) and 120ml of Denaturation Buffer (Pyromark delivery Solution; Qiagen) were added to the four sample plates in sequence; starting a pump of the vacuum preparation workstation, and cleaning the vacuum preparation tool in high-purity water for 30 seconds; then the Vacuum preparation tool (Pyromark Vacuum Prep Filter Probes; Qiagen) is moved to the PCR plate, grabbing agarose beads (this is done within three minutes after the beads have bound to the PCR product); pick up the PCR plate and check if most of the magnetic beads are attached to the vacuum preparation tool; placing the vacuum preparation tool in 70% ethanol for 5 seconds; then transferred to denaturation buffer for 5 seconds; then moving the mixture into a washing buffer solution for washing for 5-10 seconds; the pump is turned off; placing the vacuum preparation tool in a plate containing the sequencing primers, shaking, and releasing agarose beads (sequencing primers can also be added last); cleaning the vacuum preparation tool with high purity water; the PSQ96 plate with the sample placed thereon was heated to 80 ℃ for 2 minutes on a hot plate and then cooled to room temperature, whereby the pyrosequencing reaction was carried out.
D. Pyrosequencing: the samples from the PSQ96 plates from step C were sequenced on a Pyromark Q96 pyrophosphate sequencer using the Pyromark Gold Q96 kit (Pyromark Gold Q96 Reagents; Qiagen) and the results were subjected to methylation analysis using Pyromark CpG software (see FIG. 2 for an example of methylation level detection results).
4. Data analysis
In this study, the data are sorted and analyzed by using SPSS 19.0, and we find that: as shown in FIG. 1, 8 CpG sites are correlated (0.131< correlation coefficient <0.708, P <0.05, statistically significant, note that P <0.05 is statistically significant, and the same applies hereinafter), and significant differences in methylation levels at site 1(CpG1), site 6(CpG6) and site 8(CpG8) between the case group and the control group are found in subsequent analysis site by site due to the different correlation coefficients (P <0.001, see Table 1). A receiver operating characteristic curve (ROC curve for short) analysis is carried out on the CpG6 to find that the CpG6 has a large diagnostic value on essential hypertension (the area AUC under the curve is 0.834, p is less than 0.001, and the area under the curve is shown in figure 3).
The kit for detecting the methylation degree of the TLR2 gene promoter region related to essential hypertension has the advantages of accuracy, reliability, flexibility, rapidness, economy and economy. The kit is used for detecting the methylation level of the TLR2 gene promoter region, and can quickly and reliably provide reference for auxiliary diagnosis, detection or screening of primary hypertension.
Table 1 comparison between case and control groups (n =190)
Note: in table 1, n is the number of samples; in the data of the table,pthe value is less than 0.05, which has statistical significance.
While the preferred embodiments of the present invention have been illustrated, various changes and modifications may be made by one skilled in the art without departing from the scope of the present invention.
SEQUENCE LISTING
<110> Ningbo university
<120> detection kit for auxiliary diagnosis of primary hypertension and application thereof
<130> 2016
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<400> 1
biotin-ggtagttgta ggggtaggat
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence
<400> 2
acccaaaaaa actctaaacc tc
<210> 3
<211> 16
<212> DNA
<213> Artificial sequence
<400> 3
ttccaaacaa ataacc
Claims (1)
1. The application of a primer group for detecting the methylation of a TLR2 gene promoter region in preparing a detection kit for the auxiliary diagnosis of essential hypertension is characterized in that: the primer group comprises a pair of TLR2 gene promoter region methylation specific amplification primers and a methylation specific sequencing primer:
nucleotide sequence of methylation specific forward primer:
5'- Biotin-GGTAGTTGTAGGGGTAGGAT-3'；
nucleotide sequence of methylation specific downstream primer:
5'- ACCCAAAAAAACTCTAAACCTC-3'；
nucleotide sequence of methylation specific sequencing primer:
5'-TTCCAAACAAATAACC-3'；
wherein the methylation level of the TLR2 gene promoter region is negatively correlated with the prevalence rate of essential hypertension, and the low methylation level of the TLR2 gene promoter region leads to high expression of the TLR2 gene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610277641.0A CN105695625B (en) | 2016-04-29 | 2016-04-29 | Detection kit for auxiliary diagnosis of primary hypertension and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610277641.0A CN105695625B (en) | 2016-04-29 | 2016-04-29 | Detection kit for auxiliary diagnosis of primary hypertension and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105695625A CN105695625A (en) | 2016-06-22 |
| CN105695625B true CN105695625B (en) | 2021-12-17 |
Family
ID=56217731
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610277641.0A Active CN105695625B (en) | 2016-04-29 | 2016-04-29 | Detection kit for auxiliary diagnosis of primary hypertension and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105695625B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101688239A (en) * | 2007-02-12 | 2010-03-31 | 约翰·霍普金斯大学 | The early detection of colorectal carcinoma and prognosis |
| CN102281880A (en) * | 2008-10-06 | 2011-12-14 | 艾德拉药物股份有限公司 | Use of inhibitors of toll-like receptors in the prevention and treatment of hypercholesterolemia and hyperlipidemia and diseases related thereto |
| CN103122378A (en) * | 2012-12-20 | 2013-05-29 | 宁波大学 | Kit capable of being used for detecting methylation degree of alpha-adduction protein gene promoter region related to primary hypertension and application thereof |
-
2016
- 2016-04-29 CN CN201610277641.0A patent/CN105695625B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101688239A (en) * | 2007-02-12 | 2010-03-31 | 约翰·霍普金斯大学 | The early detection of colorectal carcinoma and prognosis |
| CN102281880A (en) * | 2008-10-06 | 2011-12-14 | 艾德拉药物股份有限公司 | Use of inhibitors of toll-like receptors in the prevention and treatment of hypercholesterolemia and hyperlipidemia and diseases related thereto |
| CN103122378A (en) * | 2012-12-20 | 2013-05-29 | 宁波大学 | Kit capable of being used for detecting methylation degree of alpha-adduction protein gene promoter region related to primary hypertension and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| " Association between blood pressure and DNA methylation of retrotransposons and pro-inflammatory genes";Alexeeff S E 等;《International Journal of Epidemiology》;20130228;第42卷(第1期);摘要，第272页左栏第4段至右栏第2段，第278页右栏第4段，supplementary Table 2 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105695625A (en) | 2016-06-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Correia et al. | Circulating microRNAs as potential biomarkers of infectious disease | |
| JP2017184642A (en) | Dementia marker, evaluation method of dementia using same, evaluation reagent, and evaluation kit | |
| CN113249486B (en) | Kit for detecting DNA methylation degree of VGLL3 gene and application thereof | |
| KR101220806B1 (en) | Identification of group of hypertension-susceptibility genes | |
| CN105177115A (en) | UGT1A1 combined gene locus fluorescence detection kit for guiding irinotecan chemotherapeutic drug individualized treatment | |
| JP2011519275A5 (en) | ||
| CN104328200B (en) | The detection kit of auxiliary diagnosis alzheimer's disease and detection method thereof | |
| CN103160582B (en) | Kit for detecting methylation degree for GCK (glucokinase) gene associated with type 2 diabetes and application thereof | |
| CN105695625B (en) | Detection kit for auxiliary diagnosis of primary hypertension and application thereof | |
| CN110499368A (en) | One kind SNP marker relevant to carcinoma of mouth prognosis prediction and its application | |
| CN104328201B (en) | A kind of detection kit of auxiliary diagnosis alzheimer's disease and detection method thereof | |
| CN109988855B (en) | LAMP primer combination for detecting six kinds of aspergillus and application thereof | |
| CN103122378B (en) | Kit capable of being used for detecting methylation degree of alpha-adduction protein gene promoter region related to primary hypertension and application thereof | |
| CN113999901B (en) | Myocardial specific methylation markers | |
| Yang et al. | Recent advances of human leukocyte antigen (HLA) typing technology based on high-throughput sequencing | |
| CN108473955B (en) | Method for separating target cells from blood sample and application thereof | |
| CN111549137B (en) | Genetic molecular marker related to gastric cancer auxiliary diagnosis and application thereof | |
| CN103014165A (en) | Kit capable of being used for detecting methylation degree of PLA2G7 gene promoter region relevant to coronary heart disease and application of kit | |
| CN109880903B (en) | SNP marker for auxiliary diagnosis of non-small cell lung cancer and application thereof | |
| JP4889258B2 (en) | Method for determining resistance to the onset of bovine leukemia | |
| CN113981072A (en) | Primers, probes, kit and method for detecting HLA-A29 gene | |
| CN105886641B (en) | Detection kit and its application for auxiliary diagnosis essential hypertension | |
| RU2805860C1 (en) | Method of genotyping tlr2 gene using rs3804100 polymorphism and a set of oligonucleotide primers and probes for its implementation | |
| CN103966340B (en) | A kind of detection kit for auxiliary diagnosis alzheimer's disease and application thereof | |
| RU2805863C1 (en) | Method of genotyping tlr6 gene using rs5743810 polymorphism and a set of oligonucleotide primers and probes for its implementation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |