CN104762382A - Primer pair for identifying radish seeds and application of primer pair - Google Patents
Primer pair for identifying radish seeds and application of primer pair Download PDFInfo
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Abstract
The invention discloses a primer pair for identifying radish seeds and an application of the primer pair. The primer pair for identifying or helping to identify the radish seeds is specifically composed of two single-chain DNA molecules as shown in a sequence 1 and a sequence 2 in the sequence table. Experiments prove that the primers are adopted to realize quick and accurate identification of radish seeds, Chinese dodder seeds, perilla fruits, white mustard seeds, feather cockscomb seeds, flastem milkvetch seeds, seeds of garden balsam, chingma abutilon seeds, rapeseeds and the like are identified by use of a fast PCR method and a fluorescent staining method; and a fluorescent dye method is introduced to detect the true and false identification results, and technical support is provided for the field application of molecular identification of medicinal materials.
Description
Technical field
The invention belongs to technical field of molecular biology, relate to Chinese medicine and Materia Medica Identification field, particularly a kind of primer pair for the identification of Semen Raphani and application thereof.
Background technology
According to version " Chinese Pharmacopoeia " record in 2010, Semen Raphani source was the dry mature seed of cress radish Raphanussativus L..Semen Raphani is clinical comparatively conventional Chinese medicine, has effect of the de-swelling that helps digestion, lowering the adverse-rising QI to resolve phlegm, and for retention of food and drink, abdominal distention, constipation, stagnates and rush down dysentery, and phlegm heap soil or fertilizer over and around the roots is breathed with cough.In recent years, Semen Raphani often obscures use with the seed of equal plant Europe sinapsis alba, rape and the plant such as convolvulus cuscuta plant or Cuscuta australis in commodity, for guaranteeing quality and the clinical efficacy thereof of Semen Raphani in medicinal material market, need set up more fast and accurately method to identify Semen Raphani adulterant mixed with it.
Different researcher adopts tlc, technique of polyacrylamide gel electrophoresis, microscopic method etc. to identify (Zhang Suqin to Semen Raphani respectively, Liu Juan, Ran Mei etc. the protein electrophoresis of Semen Raphani and adulterant thereof is differentiated, CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2006,31 (17): 1467; Zhu Ying. the Identification method of White Mustard Seed and Semen Raphani. practical medical technologies magazine, 2006,13 (5): 743.), but these Measures compare are loaded down with trivial details, are unfavorable for promoting.
DNA molecular marker technology is that recent two decades rises and the new detection technique of continuous development and improvement.And with regard to DNA molecular marker technology medicinal plant qualification in application with regard to, DNA molecular marker is then the difference reflected objectively from molecular level between detected materials gene fragment, identification result is not by the impact of environmental factors, sample morphology and material source, the general modes such as hybridization or electrophoresis of passing through just can embody intuitively, and result more accurately and reliably.
In sum, different researcher adopts micro-, physics and chemistry and protein electrophoresis method to carry out identification research to Semen Raphani medicinal material respectively, but micro-, these traditional method subjectivities of physics and chemistry strong, is unfavorable for stdn, standardization.The protein electrophoresis technical testing time is longer, and needs electrophoresis detection just can complete, and fluorescent staining method of therefore comparing is loaded down with trivial details, is unfavorable for application and the popularization of on-the-spot discriminating fast.
Summary of the invention
First object of the present invention be to provide a kind of for the identification of or the primer pair of assistant identification Semen Raphani.
Provided by the present invention for the identification of or the primer pair of assistant identification Semen Raphani, be specially the primer pair be made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2.
In described primer pair, two single strand dnas both can individually be packed, packaging together after the ratio that can be also 1:1 according to mol ratio mixes.
Second object of the present invention be to provide a kind of for the identification of or the test kit of assistant identification Semen Raphani.
Provided by the present invention for the identification of or the test kit of assistant identification Semen Raphani, specifically can contain described primer pair, dNTP and archaeal dna polymerase.
As required, also fluorescence dye SYBR Green I can be contained in described test kit.
The method preparing described primer pair also belongs to protection scope of the present invention.
Prepare the method for described primer pair, specifically can comprise the step of individually being packed by two single strand dnas of the described primer pair of composition.
The method preparing described test kit also belongs to protection scope of the present invention.
Prepare the method for described test kit, specifically can comprise following A) or step B):
A), after two single strand dnas of the described primer pair of composition individually being packed, be packaged in same test kit with the dNTP individually packed and archaeal dna polymerase:
B), after two single strand dnas of the described primer pair of composition individually being packed, be packaged in same test kit with the dNTP individually packed, archaeal dna polymerase and SYBR Green I.
Described primer pair, or described test kit, the application in qualification or assistant identification Semen Raphani also belongs to protection scope of the present invention.
3rd object of the present invention is to provide a kind of method utilized whether containing Semen Raphani in described primer pair or the qualification of described test kit or assistant identification testing sample.
The method utilized whether containing Semen Raphani in described primer pair or the qualification of described test kit or assistant identification testing sample provided by the present invention, specifically can comprise the steps:
A () extracts genomic dna as template from testing sample, adopt described primer pair (sequence 1 and sequence 2) to carry out pcr amplification;
B () is following (b1) or (b2):
(b1) according to the size of step (a) gained PCR primer, to determine in testing sample whether containing Semen Raphani as follows: if the DNA fragmentation containing 250-500bp (as 350-370bp) in PCR primer, then in described testing sample containing or candidate contain Semen Raphani; If not containing the DNA fragmentation of 250-500bp (as 350-370bp) in PCR primer, then in described testing sample not containing or candidate not containing Semen Raphani;
(b2) in the reaction system after step (a) amplification, fluorescence dye SYBR Green I is added, to determine in testing sample whether containing Semen Raphani as follows: if observe described reaction system to produce green fluorescence, then in described testing sample containing or candidate contain Semen Raphani; If do not observe described reaction system to produce green fluorescence, then described testing sample not containing or candidate not containing Semen Raphani.
In the step (b2) of described method, when adding fluorescence dye SYBR Green I in described reaction system, the add-on of described fluorescence dye SYBR Green I for: add 1 μ L 100 × SYBR Green I (Invitrogen company, its catalog number is 1135054) in reaction system described in every 20 μ L.
In the step (b2) of described method, determine whether described reaction system produces that green fluorescence specifically carries out detecting under 365nm ultraviolet wavelength.
In the process, described Semen Raphani is Chinese medicinal materials.Certain described method also may be used for the former plant of base---the radish (Raphanus sativus L.) detecting Semen Raphani.Accordingly, described testing sample both can be Chinese medicinal materials finished product that is single or mixing, also can be plant.
4th object of the present invention is to provide one and utilizes described primer pair or described test kit to identify or assistant identification testing sample is Semen Raphani, or the method for any one in South Dodder Seed Chinese Dodder Seed, perilla seed, White Mustard Seed, Semen Celosiae, Semen Astragali Complanati, the seed of garden balsam, Semen Abutili and coleseed.
Provided by the present inventionly described primer pair or described test kit is utilized to identify or assistant identification testing sample is Semen Raphani, or the method for any one in South Dodder Seed Chinese Dodder Seed, perilla seed, White Mustard Seed, Semen Celosiae, Semen Astragali Complanati, the seed of garden balsam, Semen Abutili and coleseed, specifically can comprise the steps:
A () extracts genomic dna as template from testing sample, adopt described primer pair to carry out pcr amplification;
B () is according to the size of step (a) gained PCR primer, determine that described testing sample is Semen Raphani as follows, or any one in South Dodder Seed Chinese Dodder Seed, perilla seed, White Mustard Seed, Semen Celosiae, Semen Astragali Complanati, the seed of garden balsam, Semen Abutili and coleseed: if the DNA fragmentation containing 250-500bp in PCR primer, then described testing sample is or candidate is Semen Raphani; If not containing the DNA fragmentation of 250-500bp in PCR primer, then described testing sample is or candidate is any one in South Dodder Seed Chinese Dodder Seed, perilla seed, White Mustard Seed, Semen Celosiae, Semen Astragali Complanati, the seed of garden balsam, Semen Abutili and coleseed;
Described testing sample is any one (namely can be Chinese medicinal materials and also can be the former plant of base) in Semen Raphani, South Dodder Seed Chinese Dodder Seed, perilla seed, White Mustard Seed, Semen Celosiae, Semen Astragali Complanati, the seed of garden balsam, Semen Abutili and coleseed.
In the step (a) of above-mentioned two methods, the annealing temperature adopted when carrying out described pcr amplification can be 70 DEG C.
In the present invention, the concrete reaction conditions adopted when carrying out described pcr amplification is as follows: 94 DEG C of 1min; 90 DEG C of 15s, 70 DEG C of 20s, 28 circulations.
In the step (a) of above-mentioned two methods, in the reaction system of carrying out described pcr amplification, in described primer pair, the final concentration of every bar single strand dna is 125nM.
In the present invention, the concrete reaction system adopted when carrying out described pcr amplification is as follows: 2.0 μ L 10 × buffer damping fluids, 1.6 μ L concentration are the dNTP of 2.5mM, 0.5 μ L concentration is the primers F 1 shown in the sequence 1 of 5 μMs, 0.5 μ L concentration is primers F 2,0.2 μ L SpeedStar HS Taq archaeal dna polymerase (Takara company, 5U/ μ L) shown in the sequence 2 of 5 μMs, 0.5 μ L template DNA, aseptic double-distilled water complements to 20 μ L.
In above-mentioned two methods, the DNA fragmentation of described 250-500bp (as 350-370bp) is specially the DNA fragmentation (band on agarose gel electrophoretogram between DNA molecular amount standard 250 and 500bp) that size is about 360bp.
In actual applications, judge the DNA fragmentation whether containing 250-500bp (as 350-370bp) in described PCR primer, detect by described PCR primer is carried out agarose gel electrophoresis, if electrophoresis result display is containing 250-500bp (as 350-370bp) object band, then the DNA fragmentation containing 250-500bp (as 350-370bp) in described PCR primer; Otherwise, then the DNA fragmentation not containing 250-500bp (as 350-370bp).Certainly also judge by the method for order-checking the DNA fragmentation whether containing 250-500bp (as 350-370bp) in described PCR primer.
Experiment proves, adopt primer provided by the present invention, by the method for rapid PCR methods and fluorescent dye achieve the mixed adulterant such as Semen Raphani and perilla seed, White Mustard Seed, South Dodder Seed Chinese Dodder Seed, Semen Celosiae, Semen Astragali Complanati, coleseed, the seed of garden balsam, Semen Abutili quick, accurately differentiate, and introduce fluorescent dye determination real and fake discrimination result is detected, provide technical support for the scene realizing medicinal material molecular identificalion uses.
Accompanying drawing explanation
Fig. 1 is Semen Raphani adulterant PCR identification result mixed with it.Wherein, swimming lane 1 is negative control; Swimming lane 2-4 is Semen Raphani (Hui nationality); Swimming lane 5-6 is perilla seed (Hui nationality); Swimming lane 7-8 is Semen Astragali Complanati (Hui nationality); 9-10 is impetuous (Hui nationality); Swimming lane 11-12 is South Dodder Seed Chinese Dodder Seed (Hui nationality); Swimming lane 13-14 is coleseed (Hubei); Swimming lane 15-16 is Semen Celosiae (Jinghong City, Yunnan Province); Swimming lane 17-18 is White Mustard Seed (Hui nationality); Swimming lane 19-20 is Semen Abutili (Hui nationality); Swimming lane M is DNA molecular amount standard: be followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom.
Fig. 2 is Semen Raphani and White Mustard Seed and Semen Abutili fluorescence identification result.Wherein, 1 is negative control; 2 is Semen Raphani (Hui nationality); 3 is White Mustard Seed (Hui nationality); 4 is Semen Abutili (Hui nationality).A:PCR product stoste; B-E:PCR product dilutes 2 times, 4 times, 8 times, 20 times respectively.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
In following embodiment, material therefor is as shown in table 1:
Table 1 traditional Chinese medicinal material samples source table
| Numbering | Sample ID | Source (criticizing) |
| 1 | Semen Raphani | Hui nationality (2) |
| 2 | South Dodder Seed Chinese Dodder Seed | Hui nationality (5) |
| 3 | Perilla seed | Hui nationality (3) |
| 4 | White Mustard Seed | Hui nationality (2); Guangxi Yulin (1); Chongqing (1) |
| 5 | Semen Celosiae | Jinghong City, Yunnan Province (4) |
| 6 | Semen Astragali Complanati | Hui nationality (2) |
| 7 | Impetuous | Hui nationality (1) |
| 8 | Semen Abutili | Hui nationality (1) |
| 9 | Coleseed | Hubei (1) |
In table, each sample all meets the relevant regulations under Chinese Pharmacopoeia (version in 2010) each medicinal material item of text.Be tested and appraised, ingredients material object conforms to title, quality conformance with standard.
Embodiment 1, method of preparation and use for the identification of the test kit of Semen Raphani
One, for the identification of the Design and synthesis of the primer pair of Semen Raphani
Selection universal primer ITS (1F:5 '-TCCGTAGGTGAACCTGCGG-3 '; 4R:5 '-TCCTCCGCTTATTGATATGC-3 ') Semen Raphani and perilla seed, White Mustard Seed, South Dodder Seed Chinese Dodder Seed, Semen Celosiae, Semen Astragali Complanati, coleseed, the seed of garden balsam, Semen Abutili sample are increased, order-checking, then Semen Raphani sequence (GenBank accession number: AF128105 is downloaded to gained sequence and GenBank, AF128104, AY746462, AY563100, AY662290, FJ980407) homology comparison is carried out, a pair special primer F1, F2 is designed, under sequence is according to its region of variability:
F1:5 '-ATGCCTTCCGATTCCGTGGTTATTT-3 ' (sequence 1);
F2:5 '-ATGGGGGGATGACGATTTGTGAC-3 ' (sequence 2).
Two, for the identification of the assembling of the test kit of Semen Raphani
After the primers F 1 of step one design and synthesis and F2 are individually packed, be packaged in same test kit with the dNTP individually packed, archaeal dna polymerase, 10 × buffer damping fluid, fluorescence dye SYBR Green I etc., namely obtain the test kit of the present invention for the identification of Semen Raphani.
Three, the method whether containing Semen Raphani in testing sample is identified
The test kit of step 2 is adopted whether to contain Semen Raphani according in the method qualification testing sample comprised the steps:
1, pcr amplification
From testing sample, extract genomic dna as template, the primers F 1 of employing step one design and synthesis and F2 (sequence 1 and sequence 2) are according to carrying out pcr amplification as follows:
PCR reaction is carried out in 200 μ L PCR reaction tubess, reaction cumulative volume 20 μ L, comprise following reagent: 2.0 μ L10 × buffer damping fluid (Takara companies, its catalog number is A1101E), 1.6 μ L concentration are the dNTP of 2.5mM, 0.5 μ L concentration is the primers F 1 of 5 μMs, 0.5 μ L concentration is the primers F 2 of 5 μMs, 0.2 μ L SpeedStar HSTaq archaeal dna polymerase (Takara company, Extaq 5U/ μ L), 0.5 μ L template DNA, 14.7 μ L aseptic double-distilled waters.
PCR reaction solution shakes mixing after having prepared gently, and PCR pipe is put into PCR instrument, adopts two-step approach to carry out pcr amplification, specifically reacts as follows: denaturation temperature 94 DEG C, time 1min; Denaturation temperature 90 DEG C, time 15s, annealing temperature 70 DEG C, time 20s, 28 circulations; 4 DEG C of preservations, obtain amplified production.
2, PCR primer detects
Two kinds of methods can be adopted to detect PCR primer:
(1) agarose gel electrophoresis method
Get 3 μ L amplified productions, adopt the sepharose of 1%, under voltage 100-150V, electrophoresis 15 minutes, observes under gel imaging system and takes pictures.According to the size of object band, determine whether contain Semen Raphani in testing sample as follows: if acquisition size is about the object band of 360bp, then contain Semen Raphani in described testing sample; The object band that size is 360bp if there is no, then do not contain Semen Raphani in described testing sample.
(2) fluorescence colour
1 μ L 100 × SYBR Green I (Invitrogen company is added in pcr amplification product (20 μ L), its catalog number is 1135054) under 365nm ultraviolet wavelength, detect fluorescence, determine whether contain Semen Raphani in testing sample as follows: if observe described reaction system to produce green fluorescence, then contain Semen Raphani in described testing sample; If do not observe described reaction system to produce green fluorescence, then described testing sample is not containing Semen Raphani.
Embodiment 2, the test kit qualification Semen Raphani adopting embodiment 1 to prepare
Testing sample: kind of the traditional Chinese medicinal material samples of 9 shown in table 1.
One, from testing sample, genomic dna is extracted
The dry medicinal material chosen without going mouldy is about 0.03g, is placed in pulverizer and grinds, and crosses 40 mesh sieves.By powder transfer in the Eppendorf tube of 2.0mL, add the sterilized CTAB extracting solution of 900 μ L (formula: 2% (2g/100ml) CTAB, 100mmol/L Tris-HCl pH=8.0,20mmol/L EDTA, 1.4mol/L NaCl), 0.02g PVP 40000,10 μ L beta-mercaptoethanol fully vibrates mixing, 65 DEG C of water-bath 1.5h-2h, period jog 2-3 time.Take out after terminating and be cooled to room temperature, add 900 μ L chloroform-isoamyl alcohol (volume ratio 24:1), mixing of fully vibrating, the centrifugal 10min of 12000g.Get supernatant, add equal-volume chloroform-isoamyl alcohol (volume ratio 24:1), mixing of fully vibrating, the centrifugal 10min of 12000g.Get supernatant, add the aqueous isopropanol of 2/3 volume precooling, place more than 0.5h for-20 DEG C.Take out, the centrifugal 10min of 12000g, abandons supernatant, and precipitate by 70% (volume fraction) washing with alcohol twice, 37 DEG C volatilize ethanol, dissolve with appropriate aqua sterilisa ,-20 DEG C of preservations.
Two, pcr amplification
The genomic dna extracted from testing sample with step one is template, and the primer pair (primers F 1 and primers F 2) adopting embodiment 1 step one to design carries out pcr amplification.Concrete reaction system and reaction conditions are with embodiment 1 step 31.It is that template is as negative control that experiment is arranged with distilled water simultaneously.Experiment in triplicate.
After reaction terminates, on the one hand, according to the method for step 3 in embodiment 12 (1), testing sample is identified.On the other hand, according to the method for step 3 in embodiment 12 (2), testing sample is identified.
Result as shown in Figure 1, as can be seen from the figure:
Primer pair of the present invention (primers F 1 and primers F 2) is utilized to detect 9 kinds of testing samples, the object band only having Semen Raphani can amplify clip size to be about 360bp.Other 8 kinds of Chinese medicinal materialss (perilla seed, White Mustard Seed, South Dodder Seed Chinese Dodder Seed, Semen Celosiae, Semen Astragali Complanati, coleseed, the seed of garden balsam, Semen Abutili) then all do not amplify object band.This shows that Semen Raphani and perilla seed, White Mustard Seed, South Dodder Seed Chinese Dodder Seed, Semen Celosiae, Semen Astragali Complanati, coleseed, the seed of garden balsam, Semen Abutili can identify by this reaction system accurately.
In addition, fluoroscopic examination result shows, Semen Raphani demonstrates brilliant green fluorescence, and perilla seed, Semen Celosiae, Semen Astragali Complanati, coleseed, the seed of garden balsam all do not send fluorescence, and White Mustard Seed, Semen Abutili also send fluorescence.Discovery is compared according to electrophoresis result, White Mustard Seed, primer dimer is all had to occur after Semen Abutili pcr amplification, therefore, what can judge to send fluorescence is primer dimer but not specific band, again respectively to Semen Raphani, White Mustard Seed, Semen Abutili PCR primer carries out different multiples dilution, be specially 2 times, 4 times, 8 times, 20 times, after dilution, volume is 20 μ L, add 1 μ L 100 × SYBR Green I again under 365nm ultraviolet wavelength, detect fluorescence and take pictures, after finding dilution 8 times, Semen Raphani still can show fluorescence, and White Mustard Seed and Semen Abutili have not shown fluorescence, therefore, its adulterant mixed with it can be identified out, see Fig. 2.
Claims (10)
1. for the identification of or the primer pair of assistant identification Semen Raphani, it is characterized in that: the primer pair of described primer pair for being made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2.
2. for the identification of or the test kit of assistant identification Semen Raphani, it is characterized in that: containing primer pair according to claim 1, dNTP and archaeal dna polymerase in described test kit.
3. test kit according to claim 2, is characterized in that: also containing fluorescence dye SYBR Green I in described test kit.
4. prepare the method for primer pair described in claim 1, comprise the step of individually being packed by two single strand dnas of the described primer pair of composition.
5. prepare the method for test kit described in Claims 2 or 3, comprise following A) or step B):
A), after two single strand dnas of the described primer pair of composition individually being packed, be packaged in same test kit with the dNTP individually packed and archaeal dna polymerase:
B), after two single strand dnas of the described primer pair of composition individually being packed, be packaged in same test kit with the dNTP individually packed, archaeal dna polymerase and SYBR Green I.
6. primer pair according to claim 1 or the test kit described in Claims 2 or 3, the application in qualification or assistant identification Semen Raphani.
7. utilize the primer pair described in claim 1 or the test kit described in Claims 2 or 3, identify or whether contain in assistant identification testing sample the method for Semen Raphani, comprise the steps:
A () extracts genomic dna as template from testing sample, adopt primer pair according to claim 1 to carry out pcr amplification;
B () is following (b1) or (b2):
(b1) according to the size of step (a) gained PCR primer, to determine in described testing sample whether containing Semen Raphani as follows: if the DNA fragmentation containing 250-500bp in PCR primer, then in described testing sample containing or candidate contain Semen Raphani; If not containing the DNA fragmentation of 250-500bp in PCR primer, then in described testing sample not containing or candidate not containing Semen Raphani;
(b2) in the reaction system after step (a) amplification, fluorescence dye SYBR Green I is added, to determine in testing sample whether containing Semen Raphani as follows: if observe described reaction system to produce green fluorescence, then in described testing sample containing or candidate contain Semen Raphani; If do not observe described reaction system to produce green fluorescence, then described testing sample not containing or candidate not containing Semen Raphani.
8. utilize the primer pair described in claim 1 or the test kit described in Claims 2 or 3, to identify or assistant identification testing sample is Semen Raphani, or the method for any one in South Dodder Seed Chinese Dodder Seed, perilla seed, White Mustard Seed, Semen Celosiae, Semen Astragali Complanati, the seed of garden balsam, Semen Abutili and coleseed, comprises the steps:
A () extracts genomic dna as template from testing sample, adopt primer pair according to claim 1 to carry out pcr amplification;
B () is according to the size of step (a) gained PCR primer, determine that described testing sample is Semen Raphani as follows, or any one in South Dodder Seed Chinese Dodder Seed, perilla seed, White Mustard Seed, Semen Celosiae, Semen Astragali Complanati, the seed of garden balsam, Semen Abutili and coleseed: if the DNA fragmentation containing 250-500bp in PCR primer, then described testing sample is or candidate is Semen Raphani; If not containing the DNA fragmentation of 250-500bp in PCR primer, then described testing sample is or candidate is any one in South Dodder Seed Chinese Dodder Seed, perilla seed, White Mustard Seed, Semen Celosiae, Semen Astragali Complanati, the seed of garden balsam, Semen Abutili and coleseed;
Described testing sample is any one in Semen Raphani, South Dodder Seed Chinese Dodder Seed, perilla seed, White Mustard Seed, Semen Celosiae, Semen Astragali Complanati, the seed of garden balsam, Semen Abutili and coleseed.
9. the method according to claim 7 or 8, is characterized in that: in step (a), and the annealing temperature adopted when carrying out described pcr amplification is 70 DEG C;
Concrete, the amplification program adopted when carrying out described pcr amplification is: 94 DEG C of 1min; 90 DEG C of 15s, 70 DEG C of 20s, 28 circulations.
10., according to described method arbitrary in claim 7-9, it is characterized in that: in step (b2), determine whether described reaction system produces green fluorescence and detect under 365nm ultraviolet wavelength.
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Cited By (1)
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Application publication date: 20150708 |