CN105886599A - ARMS-qPCR detection kit and detection method for ABCB1 genotyping - Google Patents
ARMS-qPCR detection kit and detection method for ABCB1 genotyping Download PDFInfo
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- CN105886599A CN105886599A CN201410587532.XA CN201410587532A CN105886599A CN 105886599 A CN105886599 A CN 105886599A CN 201410587532 A CN201410587532 A CN 201410587532A CN 105886599 A CN105886599 A CN 105886599A
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Abstract
The invention relates to the field of molecular biology and particularly relates to an ARMS-qPCR detection kit for ABCB1 genotyping and also relates to an ARMS-qPCR detection method for the ABCB1 genotyping on the basis of the kit. The kit includes a qPCR reaction system which comprises a qPCR mixture reaction solution, a reference primer, an ARMS primer and a positive pole reference sample. The qPCR mixture reaction solution comprises a PCR buffer liquid, dNTP, MgCl2, Tag enzyme, a PCR downstream primer and a TaqMan probe. Final concentrations of the components in the qPCR reaction system are described as follows: the PCR buffer liquid is 1X, the dNTP is 0.1-1.5 mM, the MgCl2 is 0.5-5 mM, the Tag enzyme is 0.05-0.1 U/[mu]l, the PCR downstream primer is 0.1-1 [mu]M, the TaqMan probe is 0.1-1 [mu]M, the reference primer is 0.5 [mu]M, the ARMS primer is 0.5 [mu]M, and the positive pole reference sample is 1-10 ng/[mu]l. The kit can quickly, sensitively and accurately detect different genotypes of the ABCB1 gene, and can detect the genome DNA of different sources, such as blood, mucous membrane of mouth, hairs and the like.
Description
Technical field
The present invention relates to biology field, be specifically related to a kind of for ABCB1 gene type
ARMS-qPCR detection kit, the invention still further relates to a kind of ABCB1 gene type ARMS-qPCR inspection
Survey method.
Background technology
ABCB1 gene is positioned at No. 7 chromosome (7q21) of human genome, encodes long 1280 ammonia
The albumen of base acid.It has the domain of 12 cross-films, at its intracellular binding site having ATP.This
Gene is a transport protein, and some medicines that can operate, to intracellular, play very important physiological function.
ABCB1 albumen high expressed in blood brain barrier, discharges many medicines and neurotoxin to protect brain.
Now there are some researches show, the potential morbidity of genotype different for ABCB1 and depression is closely related (Fuji et
al.2012)。
Depression, also known as melancholia, is with a depressed class mental illness as principal character, its clinical table
Existing light-duty patient's appearance is as usual, and heart has Affliction grade.Slightly heavy people can behave as depressed, knitted brows is bitter
Face, moaning and groaning, feel oneself inferior, some patient is frequently accompanied by neurosis symptom, such as: attention does not collects
In, the symptom such as hypomnesis, delay of response and insomnia and dreamful sleep.Major depression patient there will be pessimism and detests
Generation, despair, self-accusation fall sharply from crime, hallucination vain hope, inappetence, body weight, hypofunction with sternly
The suicidal attempt of weight, even has suicide, therefore constitutes a serious threat human health.
Along with quickening and the increase of pressure of modern life rhythm, increasing people suffers from melancholia, Beijing
In the city crowd of more than 15 years old, the lifelong attack rate of depressive disorder is 6.87%, and the attack rate in the whole nation is at 5-10%
Between, wherein the attack rate of women is the twice of male.According to World Health Organization (WHO) it is expected that to the year two thousand twenty, press down
Strongly fragrant disease will become the second largest Disease Spectrum source after coronary heart disease.
Scientific research finds that the different genotype of ABCB1 gene is closely related with the risk of depression,
The people of T3435 homozygous genotype and C3435 and C/T3435 heterozygous genotypes suffers from the risk of depression to be had
Difference (Fuji et al.2012) significantly.Therefore, the different genotype of ABCB1 gene 3435 position is pre-
Survey the fabulous biomarker of depression potential risk, the different genes of detection ABCB1 gene 3435 position
Type has great importance for prevention of depression in advance, save medical resources.
The technology that can be used for ABCB1 gene type is a lot, including such as DNA direct sequencing, hybridization hybrid chip
Method, Manganic pyrophosphate complex initiation method and denaturing high-performance liquid chromatography etc..Wherein, DNA sequencing method is gene type
Goldstandard, the method has a disadvantage in that the sensitivity of detection is the highest, and only the sudden change more than 15% is
Can detect；Time-consuming, operation complexity, operator are required height；Non-stopped pipe operates, and relates to PCR amplification
After operation, be the most easily contaminated, cause the undesirable of result；Flux is the least, the most at most can only do 24
Individual, it is difficult to carry out on a large scale.
Summary of the invention
First to be solved by this invention technical problem is that for state of the art offer a kind of highly sensitive, special
The ARMS-qPCR detection kit for ABCB1 gene type that the opposite sex is strong.
Second technical problem to be solved by this invention is to provide a kind of ABCB1 gene type
ARMS-qPCR detection method, this detection method is highly sensitive, high specificity, and simple to operate.
The present invention solves the technical scheme that above-mentioned first technical problem used: a kind of for ABCB1 base
Because of the ARMS-qPCR detection kit of typing, including qPCR reaction system, described qPCR reaction system
Including qPCR mixed reaction solution, with reference to primer, ARMS primer and positive controls sample；Described qPCR mixes
Close reactant liquor and include PCR buffer, dNTP, MgCl2, Tag enzyme, PCR downstream primer and TaqMan
Probe；
In described qPCR reaction system, it is 1X that the final concentration of each component is respectively as follows: PCR buffer；dNTP
It is 0.1～1.5mM；MgCl2It is 0.5～5mM；Tag enzyme is 0.05～0.1U/ μ l；PCR downstream primer is 0.1～1
μM；TaqMan probe is 0.1～1 μM；It it is 0.5 μM with reference to primer；ARMS primer is 0.5 μM；Sun
Property control sample is 1～10ng/ μ l.
Wherein, the sequence of described PCR downstream primer is as shown in ABCB1-R；
The sequence of described TaqMan probe is as shown in ABCB1-TaqMan；
The described sequence with reference to primer is as shown in ABCB1-C, it is possible to expand all different ABCB1 genotype；
Described ARMS primer is any one in two kinds of forward primer, difference corresponding A BCB1 CC3435,
TT3435 type genotype, the sequence of both forward primer is as shown in ABCB1-F1, ABCB1-F2；This
Two kinds of ARMS primers are respectively at 3 ' ends and different genotype Mismatchings, simultaneously reciprocal at its 3 ' end
2nd, 3 increase one or two base mispairings, to increase its specificity；For specific amplification
3435 genotype of ABCB1 are TT/CC/TC；
Described positive controls sample is respectively the plasmon DNA of ABCB1 different genotype base substitution.
Each primer sequence is listed below:
ABCB1-R:5 '-AAACAGGAAGTGTGGCCAGA-3 '
ABCB1-TaqMan:5 '-FAM-CTCTCTTCAAATAAACAGCC-3 '
ABCB1-C:5 '-CCTATGGAGACAACAGCCGG-3 '
ABCB1-F1:5 '-GTGGTGTCACAGGAAGAGGCT-3 '
ABCB1-F2:5 '-GTGGTGTCACAGGAAGAGGCC-3 '
Wherein, described Tag enzyme is Takara Tag enzyme or Goldstar Best Tag enzyme.
The present invention solves the technical scheme that above-mentioned second technical problem used: based on mentioned reagent box
ABCB1 gene type ARMS-qPCR detection method, the method is before this for ABCB1 gene design one
To with reference to primer, TaqMan probe, the different genotype for 3435 sites designs ARMS primer, instead
Answer in system addition qPCR mixed reaction solution, with reference to primer or ARMS primer, TaqMan primer with treat
Survey template DNA and carry out the detection of quantitative fluorescent PCR, specifically include following steps:
(1) design and screen containing 3435 different genotype of ABCB1 gene general with reference to primer,
TaqMan probe and two kinds of ARMS primers；
(2) from cell system or blood, genomic DNA is extracted as template DNA；
(3) carrying out real-time fluorescence quantitative PCR amplification, the detection of each sample divides 3 pipes to carry out, and each pipe adds
Identical qPCR mixed reaction solution, TaqMan probe and template DNA, often pipe be separately added into reference to primer and
One in two kinds of ARMS primers, carries out the qPCR detection of 3435 gene types of ABCB1 gene；
(4) result interpretation:
Positive with reference to primer pipe amplification curve, ABCB1-F1 pipe amplification curve is positive, and ABCB1-F2 pipe expands
Curve is negative or itself and the amplification curve Δ CT ＞ 10 with reference to primer pipe, and this sample results is judged to 3435TT
Genotype；
Positive with reference to primer pipe amplification curve, ABCB1-F1 pipe amplification curve is positive, and ABCB1-F2 pipe expands
Curve is positive, and this sample results is judged to 3435C/T genotype；
Positive with reference to primer pipe amplification curve, ABCB1-F1 pipe amplification curve negative or its with reference to primer pipe
Amplification curve Δ CT ＞ 10, ABCB1-F2 pipe amplification curve is positive, and this sample results is judged to 3435CC
Genotype；
Negative with reference to primer pipe amplification curve, point out this sample extraction failure, need to re-start extraction.
In described step (2), template DNA comes off carefully selected from blood of human body genomic DNA or oral mucosa
Born of the same parents or hair.
In described step (3), the condition carrying out pcr amplification reaction is, 95 DEG C of denaturations 2min, 95 DEG C of changes
Property 15s, 60 DEG C extend 1min, 46 circulations.
The Full Name in English of qPCR mentioned in technique scheme is Real-time Quantitative PCR
Detecting System.I.e. real time fluorescent quantitative nucleic acid amplification detecting system, is also named real-time quantitative gene amplification
Fluorescence detecting system, is called for short qPCR.
Compared with prior art, it is an advantage of the current invention that:
The invention provides the ARMS-qPCR detection kit of a kind of ABCB1 gene type, existing to solve
Have time-consuming in gene type detection technique, program is loaded down with trivial details and the problem such as easy pollution.This test kit can quickly,
Accurately, cheaply, high flux carry out typing to ABCB1 gene, its highly sensitive, high specificity, it is adaptable to
Common sample such as blood, oral mucosa and hair etc..
This test kit can detect ABCB1 different genotype quick, sensitive, accurately, can detect different next
The genomic DNA in source, such as blood, Oral Mucosal Cells, hair etc..With gene types such as direct Sequencings
Technology is compared, and the present invention has a high specificity, highly sensitive for ABCB1 gene type, simple to operate quickly,
The advantages such as high flux, sentence read result are reliable, easy.
The test kit of the present invention can be quick, cheap, accurate, with high throughput detection blood of human body or other
The type that in histiocyte, ABCB1 gene is different, for typing method, is that current cost performance is best
Method, it is simple to clinical or health detection is carried out on a large scale.
ARMS (amplification refractory mutation system) used by the present invention i.e. expands obstruction
Abruptly-changing system, for detecting known mutations gene.This method holds primers by designing two 5 ', one
Complementary with normal DNA, one complementary with mutant DNA, for homozygous mutant, is separately added into both
Primer and 3 ' end primers carry out two parallel PCR, only the most extensible with the primer of mutant DNA complete complementary
And obtain pcr amplification product.If mispairing is positioned at 3 ' ends of primer, causes PCR not extend, be then referred to as
ARMS。
The sensitivity of ARMS detection depends in specificity and the reaction condition such as reactant liquor of ARMS primer
The concentration of MgCl2, adds the optimization being not added with DMSO etc..In order to increase the specificity of primer, reduce primer with
The mispairing of the wrong timing of targeting DNA extends, and can be introduced additionally by the 2nd, 3 bases held at primer 3 '
One or two base mismatch, are allowed to form multiple mispairing between template and extend with IMPEDANCE WRONG.
Probe used by the present invention is the TaqMan probe of 5 ' end FAM labellings, and probe two ends labelling respectively is glimmering
Light reporter group (R) and fluorescent quenching group (Q).When probe is complete, i.e. in random manner with without PCR
During products thereof state, the fluorescence that reporter group sends is quenched group absorptions, can't detect the existence of fluorescence.
In ARMS-qPCR amplification procedure, when special PCR primer and TaqMan probe generation hybridization
Time, Goldstar Best Tag enzyme 5 ' end 5 prime excision enzyme activities also the base of TaqMan probe is sheared one by one,
The exometer that the fluorescence that reporter group is discharged just can be built in PCR instrument detects.PCR passes through
One circulation, fluorescence signal is also the same with purpose fragment, has the process of a sync index amplification, and fluorescence is believed
Number power just represent the number of template DNA copy number.Therefore the present invention is a work for good gene type
Tool.
The advantage combining ARMS primer and real-time fluorescence quantitative PCR, with gene types such as direct Sequencings
Technology compare, the present invention for ABCB1 genotyping gene detection have the advantage that
1, high specificity: the ARMS primer of design is respectively directed to ABCB1 TT 3435, the spy of CC3435
Different series of variation, can expand corresponding genotype DNA specifically；The inverse the 2nd held at ARMS primer 3 ',
3 bases introduce another one or two base mismatch, increase the specificity of ARMS primer.
2, the sensitivity of the technology of the present invention is up to 1%, far above the sensitivity of DNA direct Sequencing 15%；
3, detection process is stopped pipe reaction, greatly reduces pollution and the probability of result error；
4, operation quickly and easily, can complete to obtaining result from sample censorship in 3 hours.And directly survey
Sequence method detecting step is loaded down with trivial details: censorship specimen → extraction DNA → PCR expands → verify PCR primer (electrophoresis)
→ purified pcr product → direct Sequencing → interpretation of result, it is dirty through the electrophoresis process of two PCR primer
Dye probability is very big, is not suitable for carrying out on a large scale in hospital；
5, sentence read result is the most objective, result can be carried out quantitative analysis time if desired；
6, high flux, once can detect 96 samples；
7, safety, does not comprise poisonous and harmful substance, it is not necessary to the post processing of PCR primer, to behaviour in whole system
Make personnel and environment is the most harmless.
In a word, compared with the technology of the gene type such as direct Sequencing, the present invention has for ABCB1 gene type
High specificity, highly sensitive, simple to operate quickly, high flux, sentence read result reliable, the advantage such as easy.
Accompanying drawing explanation
Fig. 1 is for detect a patients with depression ABCB1 with ABCB1 ARMS-qPCR genotyping kit
The amplification curve of 3435 position genotype, after testing, this sample genotype in 3435 positions is ABCB1
TT3435；
Fig. 2 is for detecting normal person ABCB1 3435 with ABCB1 ARMS-qPCR genotyping kit
The amplification curve of tps gene type, after testing, this sample genotype in 3435 positions is ABCB1 CC3435；
Fig. 3 is for detecting normal person ABCB1 3435 with ABCB1 ARMS-qPCR genotyping kit
The amplification curve of tps gene type, after testing, this sample genotype in 3435 positions is ABCB1 C/T3435.
Detailed description of the invention
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Collect 100 example patients with depression and the blood sample of 50 example normal persons, by the method for ARMS-qPCR to it
ABCB1 gene 3435 position carries out typing.
Design and screen ARMS primer each one and the reference primer one of the energy above-mentioned 3 kinds of genotype of specific detection
Bar, designs general downstream primer one, designs and screen TaqMan general probe one, each primer, probe
Sequence as follows:
ABCB1-R:5 '-AAACAGGAAGTGTGGCCAGA-3 '
ABCB1-TaqMan:5 '-FAM-CTCTCTTCAAATAAACAGCC-3 '
ABCB1-C:5 '-CCTATGGAGACAACAGCCGG-3 '
ABCB1-F1:5 '-GTGGTGTCACAGGAAGAGGCT-3 '
ABCB1-F2:5 '-GTGGTGTCACAGGAAGAGGCC-3 '
The optimization of reaction system:
(1) optimization of primer concentration, in the case of in reaction system, other conditions are identical, by primer concentration
Make multiple proportions serial dilution from 0.1 μM/L～1.5 μMs/L, determine that optium concentration is 0.5 by the analysis of experimental result
μM/L。
(2) optimization of concentration and probe concentration, in the case of in reaction system, other conditions are identical, by primer concentration
Make multiple proportions serial dilution from 0.05 μM/L～0.5 μM/L, determine that optium concentration is by the analysis of experimental result
0.1μM/L。
(3) optimization of annealing temperature, in the case of other conditions are identical in reaction system, carries out grads PCR
By the analysis of experimental result, (55 DEG C～65 DEG C), determine that optimum temperature is 60 DEG C.
(4) optimization of amplified reaction Tag enzyme, expands Tag enzyme, choosing by comparing the various PCR on market
Take Goldstar Best Tag enzyme.
(5) after optimizing, final reaction system is 25 μ l, including qPCR mixed reaction solution 23 μ l, with reference to drawing
Thing and each 0.5 μ l of ARMS primer (0.5 μM/L of final concentration), template DNA 1 μ l (final concentration 1-10ng/ μ l),
QPCR mix component and final concentration of:
ARMS-qPCR detects each sample on ABI 7900 detector and carries out 3 tube reactions, every tube reaction
Add identical qPCR mix (i.e. qPCR mixed reaction solution), general downstream primer, TaqMan probe,
Each pipe is except that with reference to primer or ARMS primer.QPCR reaction condition is: 95 DEG C of denaturations
10min, 95 DEG C of 15s, 60 DEG C of 1min, amplified reaction is 46 circulations.
Result is: have 79 people in 100 example patients with depression for ABCB1 gene TT3435 genotype, 11
Artificial T/C3435,10 people are CC3435 genotype；50 people normal persons have 37 people for CC3435 type,
9 people are T/C3435 genotype, and 4 people are TT3435 genotype, verify through DNA direct Sequencing, completely
Consistent with ARMS-qPCR result.
The above results shows, the ABCB1 Gene A RMS-qPCR classifying method method of the present invention is reliable, spirit
Sensitivity is high, and simple to operate, sentence read result is objective, and beneficially larger scale clinical is carried out.
Above content is only presently preferred embodiments of the present invention, for those of ordinary skill in the art, according to this
The thought of invention, the most all will change, and this specification content is not
It is interpreted as limitation of the present invention.
Claims (5)
1. the ARMS-qPCR detection kit for ABCB1 gene type, it is characterised in that:
Including qPCR reaction system, described qPCR reaction system include qPCR mixed reaction solution, with reference to primer,
ARMS primer and positive controls sample；Described qPCR mixed reaction solution include PCR buffer, dNTP,
MgCl2, Tag enzyme, PCR downstream primer and TaqMan probe；
In described qPCR reaction system, it is 1X that the final concentration of each component is respectively as follows: PCR buffer；dNTP
It is 0.1～1.5mM；MgCl2It is 0.5～5mM；Tag enzyme is 0.05～0.1U/ μ l；PCR downstream primer is 0.1～1
μM；TaqMan probe is 0.1～1 μM；It it is 0.5 μM with reference to primer；ARMS primer is 0.5 μM；Sun
Property control sample is 1～10ng/ μ l；
The sequence of described PCR downstream primer is as shown in ABCB1-R；
The sequence of described TaqMan probe is as shown in ABCB1-TaqMan；
The described sequence with reference to primer is as shown in ABCB1-C, it is possible to expand all different ABCB1 genotype；
Described ARMS primer is any one in two kinds of forward primer, difference corresponding A BCB1CC3435,
TT3435 type genotype, the sequence of both forward primer is as shown in ABCB1-F1, ABCB1-F2；This
Two kinds of ARMS primers, respectively at 3 ' ends and different genotype Mismatchings, fall at its 3 ' end simultaneously
Several 2nd, 3 increase one or two base mispairings, for the ABCB13435 position of specific amplification
Genotype is TT/CC/TC；
Described positive controls sample is respectively the plasmon DNA of ABCB1 different genotype base substitution.
ARMS-qPCR detectable for ABCB1 gene type the most according to claim 1
Box, it is characterised in that: described Tag enzyme is Takara Tag enzyme or Goldstar Best Tag enzyme.
3. ABCB1 gene type ARMS-qPCR based on test kit described in claim 1 or 2 detection
Method, it is characterised in that comprise the steps:
(1) design and screen containing 3435 different genotype of ABCB1 gene general with reference to primer,
TaqMan probe and two kinds of ARMS primers；
(2) from cell system or blood, genomic DNA is extracted as template DNA；
(3) carrying out real-time fluorescence quantitative PCR amplification, the detection of each sample divides 3 pipes to carry out, and each pipe adds
Identical qPCR mixed reaction solution, TaqMan probe and template DNA, often pipe is separately added into reference to primer
With the one in two kinds of ARMS primers, carry out the qPCR detection of 3435 gene types of ABCB1 gene；
(4) result interpretation:
Positive with reference to primer pipe amplification curve, ABCB1-F1 pipe amplification curve is positive, and ABCB1-F2 pipe expands
Increasing that curve is negative or itself and the amplification curve Δ CT ＞ 10 with reference to primer pipe, this sample results is judged to
3435TT genotype；
Positive with reference to primer pipe amplification curve, ABCB1-F1 pipe amplification curve is positive, and ABCB1-F2 pipe expands
Increasing curve positive, this sample results is judged to 3435C/T genotype；
Positive with reference to primer pipe amplification curve, ABCB1-F1 pipe amplification curve negative or its with reference to primer
The amplification curve Δ CT ＞ 10 of pipe, ABCB1-F2 pipe amplification curve is positive, and this sample results is judged to 3435CC
Genotype；
Negative with reference to primer pipe amplification curve, point out this sample extraction failure, need to re-start extraction.
Detection method the most according to claim 3, it is characterised in that: in described step (2), template
DNA is selected from blood of human body genomic DNA or oral mucosa exfoliative cyte or hair.
Detection method the most according to claim 3, it is characterised in that: in described step (3), carry out
The condition of pcr amplification reaction is, 95 DEG C of denaturations 2min, 95 DEG C of degeneration 15s, 60 DEG C of extension 1min, 46
Individual circulation.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106755530A (en) * | 2017-02-24 | 2017-05-31 | 陕西佰美基因股份有限公司 | One kind detection HLA A*31:The MGB probe for real-time fluorescence PCR method and its primer combination of probe of 01 allele |
| CN108048531A (en) * | 2017-11-16 | 2018-05-18 | 苏州吉玛基因股份有限公司 | A kind of super retardance fluorescence quantifying PCR method of highly sensitive detection rare mutation |
| CN109295229A (en) * | 2018-10-22 | 2019-02-01 | 北京华夏时代生物工程有限公司 | Detection method is sequenced in the SNP fluorescence in situ hybridization of ABCB1 and SLCO1B1 |
| CN116516012A (en) * | 2023-05-24 | 2023-08-01 | 北京阅微基因技术股份有限公司 | Composite amplification system and kit for POLE genotyping detection |
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| WO2007058896A3 (en) * | 2005-11-10 | 2007-10-04 | Us Gov Health & Human Serv | Materials and methods for abcb1 polymorphic variant screening, diagnosis, and treatment |
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| WO2007058896A3 (en) * | 2005-11-10 | 2007-10-04 | Us Gov Health & Human Serv | Materials and methods for abcb1 polymorphic variant screening, diagnosis, and treatment |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755530A (en) * | 2017-02-24 | 2017-05-31 | 陕西佰美基因股份有限公司 | One kind detection HLA A*31:The MGB probe for real-time fluorescence PCR method and its primer combination of probe of 01 allele |
| CN106755530B (en) * | 2017-02-24 | 2021-01-01 | 陕西佰美基因股份有限公司 | MGB probe real-time fluorescence PCR method for detecting HLA-A31: 01 allele and primer probe combination thereof |
| CN108048531A (en) * | 2017-11-16 | 2018-05-18 | 苏州吉玛基因股份有限公司 | A kind of super retardance fluorescence quantifying PCR method of highly sensitive detection rare mutation |
| CN108048531B (en) * | 2017-11-16 | 2021-06-18 | 苏州吉玛基因股份有限公司 | Ultra-blocking fluorescent quantitative PCR method for detecting rare mutation with high sensitivity |
| CN109295229A (en) * | 2018-10-22 | 2019-02-01 | 北京华夏时代生物工程有限公司 | Detection method is sequenced in the SNP fluorescence in situ hybridization of ABCB1 and SLCO1B1 |
| CN116516012A (en) * | 2023-05-24 | 2023-08-01 | 北京阅微基因技术股份有限公司 | Composite amplification system and kit for POLE genotyping detection |
| CN116516012B (en) * | 2023-05-24 | 2023-10-13 | 北京阅微基因技术股份有限公司 | Composite amplification system and kit for POLE genotyping detection |
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Application publication date: 20160824 |