CN108060232A - For expanding tortoise androgen receptor（AR）The RT-qPCR primers and method of gene - Google Patents
For expanding tortoise androgen receptor（AR）The RT-qPCR primers and method of gene Download PDFInfo
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- CN108060232A CN108060232A CN201711325928.7A CN201711325928A CN108060232A CN 108060232 A CN108060232 A CN 108060232A CN 201711325928 A CN201711325928 A CN 201711325928A CN 108060232 A CN108060232 A CN 108060232A
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- 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
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- 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
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
Abstract
The invention discloses a kind of for expanding the RT qPCR primers and method of tortoise androgen receptor (AR) gene, RT qPCR primers include SEQ ID No：1 and SEQ ID No：Primer pair shown in 2.Method includes：1) total serum IgE of sample to be amplified is extracted；2) by total serum IgE reverse transcription into cDNA；3) expanded using RT qPCR primer pairs cDNA；RT qPCR primers are RT qPCR primers described above.The present invention is by designing one group of RT qPCR primer, first the total serum IgE of sample to be amplified is extracted, again by above-mentioned total serum IgE reverse transcription, cDNA is obtained, and the cDNA is expanded using above-mentioned RT qPCR primers, simultaneously, amplification template used can be extracted directly from female tortoise fallopian tubal, the difficulty of sampling is greatly reduced, and this method is simple and efficient, it is economical and practical.
Description
Technical field
The present invention relates to molecular biology research fields, and in particular, to for expanding tortoise androgen receptor (AR) base
The RT-qPCR primers and method of cause.
Background technology
Tortoise (Chinemys reevesii) is under the jurisdiction of Reptilia, Chelonia, fresh water Testudinidae, tortoise category.Tortoise has higher
Medicinal and nutritional values, it is very popular.In recent decades, artificial economic activity constantly aggravates, and Chelonian is depended on for existence
Ecological environment seriously destroyed, in addition to the overfishing of wild tortoise, habitat destruction, chemical contamination and river Jian Ba
Cause the fragmentation, islanding in its waters of living, the quantity of wild tortoise has been made drastically to decline, wildlife species, which have, faces extinction
Danger.As it can be seen that extremely urgent to the wild stocks protection of tortoise.As wild stocks is increasingly rare, tortoise it is artificial
Cultivation scale is but constantly expanding；Since the eighties in last century, with scientific research and the continuous development of cultural technique, China
Tortoise breeding has obtained unprecedented development, and cultured output is occupied first of the world.And during tortoise breeding, the female defeated ovum of tortoise
The variation of pipe hormone affects the sperm of male tortoise, can be stored for sperm in female fallopian tubal and provide advantage.
Therefore, the variation of female tortoise fallopian tubal androgen levels is studied, tortoise can be best understood from breeding process
In the breeding run into and the problem of mating.
The content of the invention
For the above-mentioned prior art, it is an object of the invention to provide one kind can identify tortoise androgen receptor (i.e.
Androgen receptor or AR) gene primer, and can apply to gene RT-qPCR experiment, cost-effective
Under conditions of, quickly, tortoise androgen receptor (AR) gene is stably and accurately expanded, it is male sharp can more intuitively to observe tortoise
Plain receptor (AR) gene is in the variation of its mRNA level in-site of different parts.
To achieve these goals, the present invention provides a kind of for expanding the RT- of tortoise androgen receptor (AR) gene
QPCR primers, wherein, the RT-qPCR primers include SEQ ID No：1 and SEQ ID No：Primer pair shown in 2.
The present invention also provides a kind of method using RT-qPCR primer amplification tortoise androgen receptor (AR) gene,
In, the described method includes：
1) total serum IgE of sample to be amplified is extracted；
2) by the total serum IgE reverse transcription of extraction in step 1) into cDNA；
3) expanded using the cDNA of reverse transcription in RT-qPCR primer pair steps 2)；Wherein,
The RT-qPCR primers are the RT-qPCR primers described in claim 1.
Through the above technical solutions, it is of the invention by designing one group of RT-qPCR primer, first by the total serum IgE of sample to be amplified
Extract, then by above-mentioned total serum IgE reverse transcription, obtain cDNA, further, to the cDNA using above-mentioned RT-qPCR primers into
Row amplification, meanwhile, amplification template used can be extracted directly from female tortoise fallopian tubal, greatly reduce the difficulty of sampling,
And this method is simple and efficient, and it is economical and practical.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Description of the drawings
Attached drawing is for providing a further understanding of the present invention, and a part for constitution instruction, with following tool
Body embodiment is together for explaining the present invention, but be not construed as limiting the invention.In the accompanying drawings：
Fig. 1 is the sugared gel electrophoresis figure of the amplified production of embodiment 1-5 and comparative example 1-5；Wherein, swimming lane 1 and swimming lane 6
The uterine part sample of portio vaginalis sample, swimming lane 2 and 7 from female tortoise fallopian tubal, swimming lane 3 and 8 from female tortoise fallopian tubal
The magnum sample of isthmus sample from female tortoise fallopian tubal, swimming lane 4 and 9 from female tortoise fallopian tubal, swimming lane 5
It is molecular weight marker with the 10 pars infundibularis samples from female tortoise fallopian tubal, swimming lane M.
Fig. 2 a are the amplification curves of embodiment 6；
Fig. 2 b are the solubility curves of embodiment 6.
Specific embodiment
The specific embodiment of the present invention is described in detail below.It is it should be appreciated that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to limit the invention.
The endpoint of disclosed scope and any value are not limited to the accurate scope or value herein, these scopes or
Value should be understood to comprising the value close to these scopes or value.For numberical range, between the endpoint value of each scope, respectively
It between the endpoint value of a scope and individual point value and can be individually combined with each other between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
The present invention provides a kind of for expanding the RT-qPCR primers of tortoise androgen receptor (AR) gene, wherein, it is described
RT-qPCR primers include SEQ ID No：1 and SEQ ID No：Primer pair shown in 2.
The present invention also provides a kind of method using RT-qPCR primer amplification tortoise androgen receptor (AR) gene,
In, the described method includes：
1) total serum IgE of sample to be amplified is extracted；
2) by the total serum IgE reverse transcription of extraction in step 1) into cDNA；
3) expanded using the cDNA of reverse transcription in RT-qPCR primer pair steps 2)；Wherein,
The RT-qPCR primers are the RT-qPCR primers described in claim 1.
In the present invention, in order to reach better PCR amplification effect so that obtained agarose gel electrophoresis figure is apparent
As it can be seen that convenient for preferably determining experimental result, the amplification procedure expands for regular-PCR, and with the PCR amplification system of 12.5 μ L
On the basis of, the concentration of every primer is respectively 0.01-0.03pmol/L, and the dosage of cDNA templates is 30-80ng.Wherein, it is used for
The polymerase and buffer solution of PCR amplification can be polymerase and buffer type commonly used in the art.
In another preferred embodiment, the amplification procedure expands for RT-qPCR, and is expanded with the RT-qPCR of 20 μ L
On the basis of system, the concentration of every primer is respectively 0.01-0.05pmol/L, and the dosage of cDNA templates is 50-150ng.Wherein,
RT-qPCR amplifications can be operated using such kit commonly used in the art, and dosage is specifically referred to produce
Detailed description in product specification.In this not go into detail by the present invention.
In a kind of embodiment being more highly preferred to, the amplification procedure is the regular-PCR amplification and RT-qPCR sequentially carried out
Amplification；Wherein,
In regular-PCR amplification procedure, on the basis of the PCR amplification system of 12.5 μ L, the concentration of every primer is respectively
The dosage of 0.01-0.03pmol/L, cDNA template is 30-80ng；
In RT-PCR amplification procedures, on the basis of the RT-qPCR amplification systems of 20 μ L, the concentration of every primer is respectively
The dosage of 0.01-0.05pmol/L, cDNA template is 50-150ng.
In order to carry out preferably judging and detecting to the product after amplification, the method, which further includes, takes regular-PCR to expand
The product of amplification afterwards does agarose gel electrophoresis detection.
In further preferred embodiment, contained DNA fragmentation in the band occurred in the swimming lane of the product of amplification
Length be 350bp.
Annealing process in amplification procedure for the conventional use of annealing way in this field and can return fiery parameter, in this hair
In bright, better expanding effect in order to obtain, the annealing temperature of the amplification procedure is 50-60 DEG C, annealing time 15-25s.
In addition, the setting for Denaturing, the extension parameters such as condition and period in regular-PCR amplification and RT-qPCR
It is operated with the setting according to this field routine, in this not go into detail by the present invention.
The present invention will be described in detail by way of examples below.In following embodiment, the primer is given birth to by raw work
Object engineering (Shanghai) Co., Ltd. synthesizes and primer concentration is that (primer in embodiment and comparative example is 10 times of dilution to 5pmol/L
After used, i.e., the concentration of the primer in embodiment and comparative example is 0.5pmol/L), the dNTPMix that uses is in the present invention
The commercially available product of Takara companies, using RT-PCR kit be Tiangeng biotech firm commercially available product, the model of the kit
SuperReal PreMix Plus (SYBR Green), remaining used chemical reagent are conventional commercial analytical reagents.
Wherein, female tortoise fallopian tubal is divided into five parts：Portio vaginalis, uterine part, isthmus, magnum and pars infundibularis.
Embodiment 1
1) extraction of total serum IgE：Using fast speed heat phenol extraction method, the tissue samples of 100mg fallopian tubal portio vaginalises are taken, is put into and grinds
After fully being pulverized in grinder, the EP pipes of sterilized 2.0mL are placed in；The Trizol solution of 1mL, incubation at room temperature are added in EP pipes
5min makes it fully crack；Centrifugal force is 12000g, centrifuges 10min under conditions of 4 DEG C, takes out upper strata aqueous phase to another
In 2.0mLEP pipes；0.2mL chloroforms (dichloromethane) and 1mL Trizol solution are added in EP pipes, jiggles 15s, room temperature
Place 2min；And centrifugal force is placed on as 12000g, 15min is centrifuged under conditions of 4 DEG C, takes out upper strata aqueous phase to another
In 2.0mLEP pipes；0.5ml isopropanols and 1mL Trizol are added in EP pipes, abundant mixing is incubated at room temperature 10min；Centrifugal force
For 12000g, 10min is centrifuged at 4 DEG C, abandons supernatant；Add in 75% alcohol washes (gently overturning EP pipes up and down) of 1mL；From
Mental and physical efforts are 7500g, and (25 DEG C or so) centrifugation 5min of room temperature abandon supernatant, and room temperature is dried；Add in 50 μ L RNase-Free ddH2O,
In 60 DEG C of metal bath 10min；OD values are surveyed, total serum IgE is obtained, total serum IgE is put in -80 DEG C of preservations.
2) total serum IgE reverse transcription is into cDNA：The total of the 5*gDNA buffer and 5 μ L of 2 μ L is added in into the sample cell of PCR instrument
RNA, and with RNase-Free ddH2O polishings are to 10 μ L；Thorough mixing, brief centrifugation, 42 DEG C of incubation 3min are subsequently placed in ice
On；The FQ-RT Primer Mix of RT the Enzyme Mix, 2 μ L of 10*Fast the RT Buffer, 1 μ L of 2 μ L are added in, are used in combination
RNase-Free ddH2O polishings are to 20 μ L, and abundant mixing, 42 DEG C of incubation 15min, 95 DEG C are incubated 3min and are put on ice afterwards, obtain
The total cDNA arrived.(the total cDNA wherein, obtained is diluted to the concentration of total cDNA to be used after 50ng/ μ L using distilled water)
3) PCR amplification：HP buffer 1.25 μ L, each 0.5 μ L of every primer are added in into the sample cell of PCR instrument, 1 μ L's
Total cDNA of dNTPMix, the Taq archaeal dna polymerases of 0.2 μ L and 1 μ L, and with distilled water polishing to 12.5 μ L.PCR reaction conditions
For：95 DEG C of pre-degeneration 5min；Then carry out 34 cycle, the cycling including：95 DEG C of denaturation 30s, 58 DEG C of 20s and 72 DEG C of annealing are prolonged
Stretch 20s；Finally remake 72 DEG C of extension 10min.PCR amplification is carried out under the above-described reaction conditions.Product after amplification is subjected to fine jade
Sepharose electrophoresis, electrophoresis result are as shown in Figure 1.(swimming lane number is 1)
Embodiment 2
It is prepared by the preparation method according to embodiment 1, unlike, by the fallopian tubal portio vaginalis in embodiment 1 by defeated
Oviduct uterine part replaces, and electrophoresis result is as shown in Figure 1.(swimming lane number is 2)
Embodiment 3
It is prepared by the preparation method according to embodiment 1, unlike, by the fallopian tubal portio vaginalis in embodiment 1 by defeated
Oviduct isthmus replaces, and electrophoresis result is as shown in Figure 1.(swimming lane number is 3)
Embodiment 4
It is prepared by the preparation method according to embodiment 1, unlike, by the fallopian tubal portio vaginalis in embodiment 1 by defeated
Oviduct magnum replaces, and electrophoresis result is as shown in Figure 1.(swimming lane number is 4)
Embodiment 5
It is prepared by the preparation method according to embodiment 1, unlike, by the fallopian tubal portio vaginalis in embodiment 1 by defeated
Oviduct pars infundibularis replaces, and electrophoresis result is as shown in Figure 1.(swimming lane number is 5)
Embodiment 6
It is prepared by the preparation method according to embodiment 1, unlike, step 3) expands for RT-PCR, and amplification procedure
For：After total cDNA is obtained, the 2*SuperReal Premix Plus of 10 μ L are added in into the sample cell of RT-PCR instrument,
Total cDNA of every primer each 0.6 μ L and 2 μ L, and with RNase-Free ddH2O polishings are to 20 μ L.RT-PCR reaction conditions are：
95 DEG C of pre-degeneration 15min；Then carry out 40 cycle, the cycling including：95 DEG C of denaturation 10s, 20s and 72 DEG C of extension of 58 DEG C of annealing
20s carries out RT-PCR amplifications under the above-described reaction conditions.Amplification can be observed using software Bio-Rad CFX Manager
Amplification curve and solubility curve, as a result as shown in Figure 2 a and 2 b.
Comparative example 1
It is prepared by the preparation method according to embodiment 1, unlike, it is that internal reference β-Actin genes draw that primer, which uses,
Object sequence (its sequence such as SEQ ID No：3 and SEQ ID No：Shown in 4), electrophoresis result is as shown in Figure 1.(swimming lane number is 6).
Comparative example 2
It is prepared by the preparation method according to embodiment 2, unlike, it is that internal reference β-Actin genes draw that primer, which uses,
Object sequence (its sequence such as SEQ ID No：3 and SEQ ID No：Shown in 4), electrophoresis result is as shown in Figure 1.(swimming lane number is 7).
Comparative example 3
It is prepared by the preparation method according to embodiment 3, unlike, it is that internal reference β-Actin genes draw that primer, which uses,
Object sequence (its sequence such as SEQ ID No：3 and SEQ ID No：Shown in 4), electrophoresis result is as shown in Figure 1.(swimming lane number is 8).
Comparative example 4
It is prepared by the preparation method according to embodiment 4, unlike, it is that internal reference β-Actin genes draw that primer, which uses,
Object sequence (its sequence such as SEQ ID No：3 and SEQ ID No：Shown in 4), electrophoresis result is as shown in Figure 1.(swimming lane number is 9).
Comparative example 5
It is prepared by the preparation method according to embodiment 5, unlike, it is that internal reference β-Actin genes draw that primer, which uses,
Object sequence (its sequence such as SEQ ID No：3 and SEQ ID No：Shown in 4), electrophoresis result is as shown in Figure 1.(swimming lane is numbered
10)。
It can be seen that DNA contained in the band occurred in embodiment 1- embodiments 5 by electrophoretogram shown in FIG. 1
The length of segment is 350bp, and the length of contained DNA fragmentation is 260bp in the band occurred in comparative example 1- comparative examples 5,
Pass through the comparison of reference gene, it can be seen that RT-PCR primer of the invention can to female tortoise estrogen receptorαgene into
The effective amplification of row, and with the length of the specifically DNA fragmentation for the 220bp that amplification is needed to us.Also, pass through figure
1 can with it is further seen that, the present invention by designing above-mentioned primer, sample to be amplified is only needed by once common PCR amplification,
And by agarose gel electrophoresis detection as can be seen that the band in figure there was only one and display is very bright, it was confirmed that should
Primer amplification effect is clearly and very reliable.Meanwhile (wherein, left side peak is the molten of reference gene by Fig. 2 a and Fig. 2 b
Solution curve, the right peak are the solubility curve after the primer pair amplifies in the present invention) result can be seen that the primer and apply
The solubility curve (another unimodal curve for reference gene amplification) of apparent simple spike can be amplified in RT-qPCR, is special
Property amplified production.This experimental result illustrates that the primer can simply and rapidly amplify tortoise androgen receptor by RT-qPCR
Body (AR) genetic fragment.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail, within the scope of the technical concept of the present invention, a variety of simple variants can be carried out to technical scheme, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that the specific technical features described in the above specific embodiments, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Sequence table
<110>Anhui Normal University
<120>For expanding tortoise androgen receptor（AR）The RT-qPCR primers and method of gene
<130> 2017003
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial
<220>
Claims (8)
1. one kind is used to expand the RT-qPCR primers of tortoise androgen receptor (AR) gene, which is characterized in that the RT-qPCR
Primer includes SEQ ID No：1 and SEQ ID No：Primer pair shown in 2.
A kind of 2. method using RT-qPCR primer amplification tortoise androgen receptor (AR) gene, which is characterized in that the method
Including：
1) total serum IgE of sample to be amplified is extracted；
2) by the total serum IgE reverse transcription of extraction in step 1) into cDNA；
3) expanded using the cDNA of reverse transcription in RT-qPCR primer pair steps 2)；Wherein,
The RT-qPCR primers are the RT-qPCR primers described in claim 1.
3. according to the method described in claim 2, wherein, the amplification procedure is regular-PCR amplification, and with the PCR of 12.5 μ L
On the basis of amplification system, the concentration of every primer is respectively 0.01-0.03pmol/L, and the dosage of cDNA templates is 30-80ng.
4. according to the method described in claim 2, wherein, the amplification procedure is RT-qPCR amplifications, and with the RT- of 20 μ L
On the basis of qPCR amplification systems, the concentration of every primer is respectively 0.01-0.05pmol/L, and the dosage of cDNA templates is 50-
150ng。
5. according to the method described in claim 2, wherein, the amplification procedure is the regular-PCR amplification and RT- sequentially carried out
QPCR is expanded；Wherein,
In regular-PCR amplification procedure, on the basis of the PCR amplification system of 12.5 μ L, the concentration of every primer is respectively 0.01-
The dosage of 0.03pmol/L, cDNA template is 30-80ng；
In RT-PCR amplification procedures, on the basis of the RT-qPCR amplification systems of 20 μ L, the concentration of every primer is respectively
The dosage of 0.01-0.05pmol/L, cDNA template is 50-150ng.
6. according to the method described in claim 5, wherein, the method further includes the product of the amplification after taking regular-PCR amplification
Do agarose gel electrophoresis detection.
7. according to the method described in claim 6, wherein, contained DNA in the band occurred in the swimming lane of the product of amplification
The length of segment is 350bp.
8. according to the method described in claim 2, wherein, the annealing temperature of the amplification procedure is 50-60 DEG C, and annealing time is
15-25s。
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Citations (4)
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| US5650278A (en) * | 1992-12-22 | 1997-07-22 | Children's Hospital Of Philadelphia | Compositions and diagnostic kits for identifying alveolar rhabdomyosarcoma |
| AU3587599A (en) * | 1997-11-28 | 1999-06-16 | Invitrogen Corporation | Single chain monoclonal antibody fusion reagents that regulate transcript ion in vivo |
| CN103993084A (en) * | 2014-05-26 | 2014-08-20 | 温州医科大学附属第一医院 | Amplification and sequencing primers and kit for mutation detection of all coding regions of gene of androgen receptor |
| CN106170562A (en) * | 2015-07-01 | 2016-11-30 | 深圳市第二人民医院 | The test kit of the genetic marker that a kind of detection is relevant to idiopathic azoospermia |
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2017
- 2017-12-13 CN CN201711325928.7A patent/CN108060232A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5650278A (en) * | 1992-12-22 | 1997-07-22 | Children's Hospital Of Philadelphia | Compositions and diagnostic kits for identifying alveolar rhabdomyosarcoma |
| AU3587599A (en) * | 1997-11-28 | 1999-06-16 | Invitrogen Corporation | Single chain monoclonal antibody fusion reagents that regulate transcript ion in vivo |
| CN103993084A (en) * | 2014-05-26 | 2014-08-20 | 温州医科大学附属第一医院 | Amplification and sequencing primers and kit for mutation detection of all coding regions of gene of androgen receptor |
| CN106170562A (en) * | 2015-07-01 | 2016-11-30 | 深圳市第二人民医院 | The test kit of the genetic marker that a kind of detection is relevant to idiopathic azoospermia |
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Application publication date: 20180522 |