CN104962642B - The related a gene polymorphism sites of GP III of aspirin resistance and application - Google Patents

The related a gene polymorphism sites of GP III of aspirin resistance and application Download PDF

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Publication number
CN104962642B
CN104962642B CN201510423236.0A CN201510423236A CN104962642B CN 104962642 B CN104962642 B CN 104962642B CN 201510423236 A CN201510423236 A CN 201510423236A CN 104962642 B CN104962642 B CN 104962642B
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iii
patient
aspirin
aspirin resistance
resistance
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CN104962642A (en
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胡波
毛玲
夏远鹏
贺权威
靳慧娟
李亚男
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Wuhan Ourui Kangan Biotechnology Co Ltd
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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
    • 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
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    • C12Q2600/136—Screening for pharmacological compounds
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    • 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
    • C12Q2600/156—Polymorphic or mutational markers

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Abstract

The invention discloses the related a gene polymorphism sites of GP III of aspirin resistance and application, antiplatelet drug（Especially aspirin）Vital effect is played in the preventing and treating of Ischemic Stroke, even if but the survey showed that at present that many Ischemic Strokes have taken antiplatelet drug, still send out palsy again repeatedly, this phenomenon is referred to as antiplatelet drug resistance.Applicant specify that a GP III a gene polymorphism sites related to aspirin resistance：GPⅢa（A1166C）And the primer for detecting the loci polymorphism is devised for the site, the primer can be used for preparing screening aspirin resistance patient's kit, think that Ischemic Stroke selects antiplatelet drug, reduction aspirin resistance and Stroke Recurrence rate.

Description

The related a gene polymorphism sites of GP III of aspirin resistance and application
Technical field
Field is selected the present invention relates to medical conditions medicine, and in particular to the related a bases of GP III of aspirin resistance Because of pleomorphism site and application.
Technical background
With China human mortality aging, Cerebral Vascular Disease rate rises year by year, and latest survey result showed from 2010 Cerebrovascular disease has turned into the first big killer for surmounting tumour harm Chinese's health, annual China's new cases 2,000,000, ischemic Cerebrovascular disease accounts for 70~80%, and patients with recurrent accounts for 27%, and 70% patient leaves neurologic impairment, cerebrovascular disease it is high lethal Rate, disability rate and recurrence rate have had resulted in great burden on society and financial burden.Antiplatelet drug (especially Ah Si Woods) play vital effect in the preventing and treating of cerebrovascular disease, but the survey showed that at present many cerebrovascular sufferers Even if person has taken antiplatelet drug, palsy is still sent out again repeatedly, this phenomenon is referred to as antiplatelet drug resistance.
Antiplatelet drug resists, such as cerebrovascular disease Related Risk Factors (sex, diabetes, suction related to factors Cigarette, metabolic syndrome, acute coronary syndrome, the past cardiovascular event, hepatic and renal function exception, disease in the blood system etc.), patient Compliance or drug dose, drug interaction and gene pleiomorphism etc..Wherein gene pleiomorphism is current study hotspot.Previously Research is found, influences the factor of multiple links of aspirin effect to there is gene pleiomorphism in vivo, this polymorphism is direct Its function is influenceed, so that different to hematoblastic reaction after Different Individual Aspirin.According to incompletely statistics, it is clinical The patient of aspirin resistance is up to 40%, but because the target spot of influence aspirin effect is numerous, up to the present, A Si The related gene pleiomorphism of woods resistance is not known also.But aspirin is used as the weight of ICVD secondary prevention Medicine is wanted, clear and definite aspirin resistance related gene polymorphism is most important as early as possible.
Platelet glycoprotein GPⅢa is one of cell adhesion receptor integrin family member, blood platelet by a of GP III with The attachment proteinses such as fibrin, fibronectin are specifically bound, and participate in platelet adhesion reaction and aggregation.It is nearest based on applicant Research, applicant specify that a GP III a gene polymorphism sites related to aspirin resistance：The a of GP III (A1166C), The site can be used for selecting antiplatelet drug, reduction aspirin resistance and Stroke Recurrence rate for Ischemic Stroke.
The content of the invention
Object of the present invention is to provide the related a gene polymorphism sites of GP III of aspirin resistance：A1166C.Should Site is located at the 1166th of a full length genes of people GP III, and its base is A or C, carries a mutant 1166C allele of GP III Patient compared with wild type A1166 patient, be more easy to aspirin resistance occur.
It is another object of the present invention to provide a gene polymorphism sites of detection aspirin resistance correlation GP III A1166C primer.
Last purpose of the invention is the provision of the primer based on a gene polymorphism sites A1166C of GP III in preparation Screen the application in aspirin resistance patient's kit.The kit can be used for screening aspirin resistance patient, so as to more Good selects antiplatelet drug, reduction aspirin resistance and Stroke Recurrence rate for Ischemic Stroke.
To achieve these goals, the present invention uses following technical measures：
The GP III a gene polymorphism sites related to aspirin resistance, the site is at 1166 of a genes of people GP III A → C mutation is there occurs, the patient that a mutant 1166C of GP III are carried after testing is easier occur aspirin resistance.Base Primer is designed in the A1166C sites of a genes of GP III, then the site is detected.
The protection content of the present invention also includes a gene polymorphism sites A1166C's of detection aspirin resistance correlation GP III Primer, as long as the primer based on a genes of GP III A1166C sites design primer, and can Successful amplification go out including a of GP III The A1166C of gene DNA fragmentation, be the protection content of the present invention.
It is preferred that, it is according to the above-mentioned a gene polymorphism sites A1166C of GP III primers designed：
Positive-sense strand：5‘-TGTCAGACACTACACTCA-3’
Antisense strand：5‘-GGATAATCCTAGGAAAAT-3’；
Or
Positive-sense strand：5‘-TCCTGAACCTCACGGTCG-3’
Antisense strand：5‘-AAGGATGTAGAGGTTAAT-3’；
Or
Positive-sense strand：5‘-CTACGGTAGTACGTCCGA-3’；
Antisense strand：5‘-GTCATCAGCCCCAAAGAG-3’.
Primer based on a gene polymorphism sites A1166C of GP III is in screening aspirin resistance patient's kit is prepared Application, detected, taken if detecting using the DNA of GP III a (A1166C) site primer pair Ischemic Stroke Patient with a mutant 1166C of GP III, then the patient have the trend for being more also easy to produce aspirin resistance, detection method includes But it is not limited to the current existing detection technique for SNP site.
GP III a (A1166C) site primer provided using the present invention, related to other aspirin resistances is more State property site primer combines the kit of composition, and the DNA of patient is detected, aspirin resistance is more also easy to produce to screen Patient.The related pleomorphism site of described other aspirin resistances is preferably：People's P2Y1 gene polymorphism sites (A745G), primer：Positive-sense strand：5‘-CACGGGTGGCCATGTCGC-3’；Antisense strand：5‘-TACCAGGTAAATCGATTT-3’.
Compared with prior art, the invention has the characteristics that：
1) a gene polymorphism sites (A1166C) of GP III that the present invention is provided are related to aspirin resistance, it is intended that be scarce Courageous and upright apoplexy patient preferably selects antiplatelet drug, reduces Stroke Recurrence rate.
2) primer for detection GP III a gene polymorphism sites A1166C that the present invention is provided, it is intended that prepare screening Ah The kit of the patient of department woods resistance.
Embodiment
Technical scheme of the present invention, is the conventional scheme of this area if not otherwise specified.
Embodiment 1：
The excavation of the GP III a gene polymorphism sites (A1166C) related to aspirin resistance：
2010~2014 years apoplexy patient data are transferred from palsy database, according to Ischemic Stroke diagnostic criteria, sieve Choosing is diagnosed as Ischemic Stroke, and further chooses clear and definite Aspirin as secondary prevention (secondary prevention is For having had cerebral apoplexy symptom or having occurred the patient of Post stroke, these people need to prevent that cerebral apoplexy occurs again) Patient is that research object is used as total sample (totally 5033 people).
1. the number of Stroke Recurrence in clearly total sample, inquire into total sample a of GP III (A1166C) gene polynorphisms with The relation (table 2) of Stroke Recurrence；
2. in total sample, return institute further consultation patient (including recurred and the patient do not recurred, totally 3429 people), gather patient Peripheral blood, aspirin reaction member (ARU) is detected using VerifyNow instruments, determines the number of ARU >=550, inquires into GP III A (A1166C) gene polynorphisms and the relation (table 1) of ARU >=550.
In the embodiment of the present invention, following two situations any one be accordingly to be regarded as occur aspirin resistance：
If 1. during patient's Aspirin, still occurring palsy, being then defined as aspirin resistance；
2. using VerifyNow instruments detection aspirin reaction member (ARU), if ARU >=550, it is defined as blood Platelet high response, shows that this patient has aspirin resistance.
As a result show, Aspirin is carried out in the Ischemic Stroke of secondary prevention, compared with wild type patient, GP III a (A1166C) saltant type group patient aspirin reaction member ARU values are substantially high, and ARU >=550 patient numbers are substantially high In wild type group (table 1)；Stroke Recurrence rate is also substantially high (table 2) simultaneously.
Detection in the present embodiment for GP III a (A1166C) gene polynorphisms is realized by the following method：
It is as follows for a gene polymorphism sites (A1166C) of GP III design primer：
Positive-sense strand：5‘-TGTCAGACACTACACTCA-3’
Antisense strand：5‘-GGATAATCCTAGGAAAAT-3’.
Using patient's STb gene as template, expanded, expanding fragment length is 350bp, be sequenced using Standard PCR, detection is expanded Increase the 186th base of fragment.The DNA sequence dna of wild type patient is the DNA sequence dna of saltant type patient shown in SEQ ID NO.1 Shown in SEQ ID NO.2.The 186th base-pair answers in a genes of GP III in the sequence amplified using this pair of primer 1166 sites.
A (A1166C) the wild type patients of 1 GP of table III and saltant type patient's ARU values
A (A1166C) the wild type patients of 2 GP of table III and saltant type patient's Stroke Recurrence rate
As a result show, the screening of GP III a (A1166C) saltant type patient is can be used for based on the primer that the site is designed, so as to Preferably antiplatelet drug, reduction aspirin resistance and Stroke Recurrence rate are selected for Ischemic Stroke.
Embodiment 2：
A gene polymorphism sites (A1166C) primers of GP III related to aspirin resistance are supported in preparation screening aspirin Application in anti-patient's kit：
Ischemic Stroke occurs first to all, gives aspirin and carries out secondary prevention, patient is entered after 3 months Row follow-up, detects each patient ARU, and Stroke Recurrence situation.Peripheral blood in patients is taken to detect that patient is by gene sequencing simultaneously It is no to there is GP III a (A1166C) polymorphism, analysis saltant type and wild type patient's aspirin resistance incidence.
Begun one's study from August, 2013, case includes deadline in August, 2014, is diagnosed and marked according to Ischemic Stroke Standard, selection is diagnosed as Ischemic Stroke as research object, completes following work：
1. according to Ischemic Stroke and TIA secondary prevention guides, give all patient's aspirin and carry out secondary prevention；
2. all patients carry out follow-up when starting secondary prevention 90 days, detect that the ARU and palsy of follow-up investigation object are multiple Heat condition；
3. peripheral blood in patients is taken, the primer (positive-sense strand provided using the present invention：5‘-TGTCAGACACTACACTCA- 3’；Antisense strand：5 '-GGATAATCCTAGGAAAAT-3 '), enter performing PCR amplification, be sequenced, the fragment total length 350bp amplified, its In the 186th bit base correspondence GP III a genes the 1166th site.Therefore, carried out by the bit base of sequence the 1166th to amplification Judge, detection patient is a of GP III (A1166) wild types or GP III a (1166G) saltant type.
4. analyze saltant type and wild type patient ARU values and Stroke Recurrence rate.
As a result show, Aspirin is carried out in the Ischemic Stroke of secondary prevention, it is wild with a of GP III (A1166) Raw type patient compares, and GP III a (1166C) saltant type group patient's ARU values are higher, and the patient numbers of and ARU >=550 are apparently higher than wild Type group (table 3), Stroke Recurrence rate is also substantially high (table 4).
A (A1166C) the wild type patients of 3 GP of table III and saltant type patient's ARU values
A (A1166C) the wild type patients of 4 GP of table III and saltant type patient's Stroke Recurrence rate
Embodiment 3：
The excavation of the P2Y1 gene polymorphism sites (A745G) related to aspirin resistance：
With the same palsy database of embodiment 1, blood sample and method detect total sample (totally 5033 people, sample and reality Apply example 1 identical) in P2Y1 (A745G) gene polynorphisms and Stroke Recurrence relation (table 6)；And time institute further consultation patient (including Having recurred and patient that is not recurring, totally 3429 people, same as Example 1) in P2Y1 (A745G) gene polynorphisms and ARU >=550 relation (table 5).
As a result show, Aspirin is carried out in the Ischemic Stroke of secondary prevention, compared with wild type patient, P2Y1 (A745G) saltant type group patient aspirin reaction member ARU values are substantially high, the patient numbers of and ARU >=550 apparently higher than Wild type group (table 5)；Stroke Recurrence rate is also substantially high (table 6) simultaneously.
Detection in the present embodiment for P2Y1 (A745G) gene polynorphisms is realized by the following method：
It is as follows for P2Y1 gene polymorphism sites (A745G) design primer：
Positive-sense strand：5‘-CACGGGTGGCCATGTCGC-3’
Antisense strand：5‘-TACCAGGTAAATCGATTT-3’.
Using patient's STb gene as template, expanded, expanding fragment length is 350bp, be sequenced using Standard PCR, detection is expanded Increase the 185th base of fragment.The DNA sequence dna of wild type patient is the DNA sequence dna of saltant type patient shown in SEQ ID NO.9 Shown in SEQ ID NO.10.The 185th base-pair answers the 745th in P2Y1 genes in the sequence amplified using this pair of primer Site.
The P2Y1 of table 5 (A745G) wild type patients and saltant type patient's ARU values
The P2Y1 of table 6 (A745G) wild type patients and saltant type patient's Stroke Recurrence rate
As a result show, the screening of P2Y1 (A745G) saltant type patient is can be used for based on the primer that the site is designed, so as to more Good selects antiplatelet drug, reduction aspirin resistance and Stroke Recurrence rate for Ischemic Stroke.
Embodiment 4：
A gene polymorphism sites (A1166C) primers of GP III related to aspirin resistance are supported in preparation screening aspirin Application in anti-patient's kit：
The primer of kit described in the present embodiment includes：
GP III a gene polymorphism sites (A1166C) primer：Positive-sense strand：5 '-TGTCAGACACTACACTCA-3 ', antisense Chain：5 '-GGATAATCCTAGGAAAAT-3 ', and P2Y1 gene polymorphism sites (A745G) primer：Positive-sense strand：5‘- CACGGGTGGCCATGTCGC-3 ', antisense strand：5‘-TACCAGGTAAATCGATTT-3’.
Specific operation process is as follows：
With the same palsy database of embodiment 1 and blood sample and method, the detection a genes (A1166C) of GP III and P2Y1 Gene (A745G) at the same during for mutant and Stroke Recurrence relation (sample is 5033 people in embodiment 1)；And a bases of GP III Because of (A1166C) and P2Y1 genes (A745G) while the relation of during for mutant and ARU value >=500 is (during sample is embodiment 1 3429 people).As a result as shown in Table 7 and 8.
The detection method be the same as Example 2 of a genes (A1166C) of GP III, the detection method of P2Y1 (A745G) gene is with implementation Example 3.
The wild type patient of table 7 and the comparison of saltant type patient's ARU values
The wild type patient of table 8 and the comparison of saltant type patient's Stroke Recurrence rate
As known to table 7 and table 8, when a of GP III (A1166C) and P2Y1 (A745G) are mutant, patient is easier hair Raw aspirin resistance, it is therefore desirable to select suitable antiplatelet drug for the Ischemic Stroke, reduces aspirin Resistance and Stroke Recurrence rate.
SEQUENCE LISTING
<110>Wuhan Oerlikon peace bio tech ltd
<120>The related a gene polymorphism sites of GP III of aspirin resistance and application
<130>The related a gene polymorphism sites of GP III of aspirin resistance and application
<160> 12
<170> PatentIn version 3.1
<210> 1
<211> 350
<212> DNA
<213>Artificial sequence
<400> 1
acagtctgtg atgtgagttt ggaggacttg gagtgccagc tgtggctggc atagaatttg 60
tctcctctgc ctttgttttt tgttttcttt taacaggaaa agattggctg gaggaatgat 120
gcatcccact tgctggtgtt taccactgat gccaagactc atatagcatt ggacggaagg 180
ctggcaggca ttgtccagcc caatgacggg cagtgtcatg ttggtagtga caatcattac 240
tctgcctcca ctaccatggt gagatctctg gcacactgtg gtttctattc atgattgtga 300
tacatgagac gtcattaacc tctacatcct tcattttcct aggattatcc 350
<210> 2
<211> 350
<212> DNA
<213>Artificial sequence
<400> 2
acagtctgtg atgtgagttt ggaggacttg gagtgccagc tgtggctggc atagaatttg 60
tctcctctgc ctttgttttt tgttttcttt taacaggaaa agattggctg gaggaatgat 120
gcatcccact tgctggtgtt taccactgat gccaagactc atatagcatt ggacggaagg 180
ctggccggca ttgtccagcc caatgacggg cagtgtcatg ttggtagtga caatcattac 240
tctgcctcca ctaccatggt gagatctctg gcacactgtg gtttctattc atgattgtga 300
tacatgagac gtcattaacc tctacatcct tcattttcct aggattatcc 350
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence
<400> 3
tgtcagacac tacactca 18
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence
<400> 4
ggataatcct aggaaaat 18
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence
<400> 5
tcctgaacct cacggtcg 18
<210> 6
<211> 18
<212> DNA
<213>Artificial sequence
<400> 6
aaggatgtag aggttaat 18
<210> 7
<211> 18
<212> DNA
<213>Artificial sequence
<400> 7
ctacggtagt acgtccga 18
<210> 8
<211> 18
<212> DNA
<213>Artificial sequence
<400> 8
gtcatcagcc ccaaagag 18
<210> 9
<211> 350
<212> DNA
<213>Artificial sequence
<400> 9
gtgcccaccg gtacagcggt gtggtgtacc ccctcaagtc cctgggccgg ctcaaaaaga 60
agaatgcgat ctgtatcagc gtgctggtgt ggctcattgt ggtggtggcg atctccccca 120
tcctcttcta ctcaggtacc ggggtccgca aaaacaaaac catcacctgt tacgacacca 180
cctcagacga gtacctgcga agttatttca tctacagcat gtgcacgacc gtggccatgt 240
tctgtgtccc cttggtgctg attctgggct gttacggatt aattgtgaga gctttgattt 300
acaaagatct ggacaactct cctctgagga gaaaatcgat ttacctggta 350
<210> 10
<211> 350
<212> DNA
<213>Artificial sequence
<400> 10
gtgcccaccg gtacagcggt gtggtgtacc ccctcaagtc cctgggccgg ctcaaaaaga 60
agaatgcgat ctgtatcagc gtgctggtgt ggctcattgt ggtggtggcg atctccccca 120
tcctcttcta ctcaggtacc ggggtccgca aaaacaaaac catcacctgt tacgacacca 180
cctcggacga gtacctgcga agttatttca tctacagcat gtgcacgacc gtggccatgt 240
tctgtgtccc cttggtgctg attctgggct gttacggatt aattgtgaga gctttgattt 300
acaaagatct ggacaactct cctctgagga gaaaatcgat ttacctggta 350
<210> 11
<211> 18
<212> DNA
<213>Artificial sequence
<400> 11
cacgggtggc catgtcgc 18
<210> 12
<211> 18
<212> DNA
<213>Artificial sequence
<400> 12
taccaggtaa atcgattt 18

Claims (2)

186th base of the sequence shown in 1.SEQ ID NO.2 is in screening aspirin resistance patient's kit is prepared Using.
185th base of the sequence shown in the 186th base and SEQ ID NO.10 of the sequence shown in 2.SEQ ID NO.2 Application in screening aspirin resistance patient's kit is prepared.
CN201510423236.0A 2015-07-18 2015-07-18 The related a gene polymorphism sites of GP III of aspirin resistance and application Expired - Fee Related CN104962642B (en)

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CN102010900A (en) * 2010-06-08 2011-04-13 广州益善生物技术有限公司 Liquid chip and specific primer for detecting SNP of GPIIIa gene and liquid chip and specific primer for detecting SNP of GPIIIa and COX-1 genes

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Publication number Priority date Publication date Assignee Title
CN102010900A (en) * 2010-06-08 2011-04-13 广州益善生物技术有限公司 Liquid chip and specific primer for detecting SNP of GPIIIa gene and liquid chip and specific primer for detecting SNP of GPIIIa and COX-1 genes

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Genetic Variation of ITGB3 Is Associated with Asthma in Chinese Han Children;Yan Zhang等;《Plos One》;20130222;第8卷(第2期);附件TableS1 *
血小板膜GPⅢa 受体的基因多态性与阿司匹林在冠心病患者中疗效的关系;丁建平等;《中国现代医学杂志》;20110831;第21卷(第22期);第2722-2725页 *

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