CN114457148A - 一种单纯型甲基丙二酸血症基因突变检测引物对及试剂盒 - Google Patents
一种单纯型甲基丙二酸血症基因突变检测引物对及试剂盒 Download PDFInfo
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Abstract
本发明公开了一种单纯型甲基丙二酸血症基因突变检测引物对及试剂盒,所述引物对包括针对Mut基因的3个引物对,所述3个引物对的核苷酸序列分别如SEQ ID NO.1‑SEQ ID NO.6所示。所述试剂盒包括Mut基因检测液、扩增反应液、测序检测液、阴性对照品及阳性对照品;所述Mut基因检测液包括针对Mut基因的3个引物对。该试剂盒的检测液仅设计了针对三个热点突变区域的三组引物对,即可完成单纯型甲基丙二酸血症基因突变检测,并且还能够快速、准确且灵敏地检测出该基因三个热点突变区域突变位点,实验结果重复性好,精密度高。
Description
技术领域
本发明涉及生物检测技术领域,具体来说,涉及一种单纯型甲基丙二酸血症基因突变检测引物对及试剂盒。
背景技术
甲基丙二酸血症(methylmalonicacidemia,MMA)是一类遗传性代谢病,由甲基丙二酞辅酶A变位酶(methylmalonyl-CoA mutase,MCM)缺陷或钴胺素代谢障碍引起,患者体内甲基丙二酸和甲基枸椽酸(Me-citrate,MCA)等代谢物大量蓄积导致代谢紊乱和多器官损伤。根据是否合并同型半胱氨酸血症,MMA分为合并型和单纯型,两者均以常染色体隐性方式遗传。
合并型MMA属于钴胺素代谢障碍类疾病,包括多种亚型,由钴胺素代谢相关C基因(metabolism of cobalamin associated C gene,MMACHC)突变导致的钴胺素C(cobalaminC,cb1C)型是最常见者,占比超过80%。单纯型MMA多由MCM缺陷引起,由甲基丙二酞辅酶A变位酶基因(methylmalonyl-CoA mutase gene,MUT)基因突变引起的MCM缺陷(MMA due toMCM deficiency,mut)型是其中最常见者,比例超过一半,中国人群中mut型占比超过90%。mut型常见临床表现与ch1C型有相似性,包括代谢危象、嗜睡、呕吐、高氨血症性脑病、发育迟缓、肌张力减退等。通常mut型临床表现比cblC型更严重,尤其是对维生素B12(VitaminB12,VitB12)治疗无反应的酶活性完全缺失的mut0型。单纯型MMA的临床表现复杂多样,缺乏特异性。不同基因型的患者临床表现类似,多在出生后1周至1个月发病,主要临床表现为顽固性呕吐、喂养困难、嗜睡、发育落后、肌张力减低等,严重时出现呼吸困难、意识障碍、昏迷,早发型患者死亡。因此,新生儿筛查及高危筛查十分重要。
近年来,串联质谱技术、酶学检测及基因检测水平发展迅速,仅仅依靠GC/MS技术和血浆氨基酸检测已不能满足疾病分型的需要。基因突变分析是鉴定分型最可靠的依据,mut型因为对维生素不反应,预后较cblC型差,因此mut型和cblC型的鉴别在临床上非常重要,但目前,尚无专门针对MMA基因突变检测方案,MMA患者基因突变检测仅有二代测序,该方法通量大、覆盖度高,但是价格昂贵,技术要求高,临床推广具有一定困难。
发明内容
针对相关技术中的上述技术问题,本发明提出一种单纯型甲基丙二酸血症基因突变检测引物对及试剂盒,能够克服现有技术的上述不足。
为实现上述技术目的,本发明的技术方案是这样实现的:
一种单纯型甲基丙二酸血症基因突变检测引物对,包括针对Mut基因的3个引物对,所述3个引物对的核苷酸序列分别如SEQ ID NO.1-SEQ ID NO.6所示。
根据本发明的另一方面,提供了一种单纯型甲基丙二酸血症基因突变检测试剂盒,包括Mut基因检测液、扩增反应液、测序检测液、阴性对照品及阳性对照品;所述Mut基因检测液包括针对Mut基因的3个引物对,所述3个引物对的核苷酸序列分别如SEQ ID NO.1-SEQ ID NO.6所示。
进一步地,所述Mut基因检测液中各引物浓度为200-500nM。
进一步地,所述扩增反应液包括Taq DNA聚合酶、dNTPs、PCR扩增缓冲液、Mg2+、UNG酶和dUTP。
进一步地,所述测序检测液包括测序染料、测序反应液和甲酰胺。
进一步地,所述阳性对照品为含有Mut基因25个位点突变的质粒。
进一步地,所述阴性对照品为未突变的Mut基因质粒。
本发明的有益效果:本发明提供了一种快速简便的针对单纯型MMA基因检测的引物对及试剂盒,利用多重PCR-sanger测序技术,可同时检出mut基因上25种突变类型,操作简便,结果易于判读,临床推广性高;有效满足了临床单纯型MMA的基因检测需求,提供了mut基因热点突变区域检测的引物、试剂盒,能够判断出MMA缺失类型,对患者后续治疗及精准用药提供依据。该试剂盒的检测液仅设计了针对三个热点突变区域的三组引物对,即可完成上述检测,并且还能够快速、准确且灵敏地检测出该基因三个热点突变区域突变位点,实验结果重复性好,精密度高。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
根据本发明实施例所述的一种单纯型甲基丙二酸血症基因突变检测引物对,包括针对Mut基因的3个引物对,如表1所示,所述3个引物对包括第一引物对、第二引物对和第三引物对,第一引物对包括第一上游引物和第一下游引物,第二引物对包括第二上游引物和第二下游引物,第三引物对包括第三上游引物和第三下游引物,所述3个引物对的核苷酸序列分别如SEQ ID NO.1-SEQ ID NO.6所示。
Mut基因野生型序列如SEQ ID NO.7所示。
表1.Mut检测液序列
引物 | 序列(5′-3′) | SEQ ID NO. |
Mut–F1(第一引物) | GCTCCTATTCCACCCCTCTTCTA | 1 |
Mut-R1(第一引物) | GCAGTAACGACAGACATAAATTAGT | 2 |
Mut-F2(第二引物) | TGCTTAGTTATGGATTAATAGACCT | 3 |
Mut-R2(第二引物) | TAAATCTATAAATCTTGACTTGTAAGTT | 4 |
Mut-F3(第三引物) | CGCAGACCTCGTCTTCTTGTA | 5 |
Mut-R3(第三引物) | TGAAGACATAGCTTTACTCTCTTCT | 6 |
实施例2
根据本发明实施例所述的一种单纯型甲基丙二酸血症基因突变检测试剂盒,该试剂盒包括Mut基因检测液、扩增反应液、测序检测液、阴性对照品及阳性对照品,试剂盒保存于-20℃,试剂盒规格:50人份/盒,具体组分如表2所示。
其中,所述Mut基因检测液包括针对Mut基因的3个引物对,所述3个引物对的核苷酸序列分别如SEQ ID NO.1-SEQ ID NO.6所示。所述扩增反应液包括Taq DNA聚合酶、dNTPs、PCR扩增缓冲液、Mg2+、UNG酶和dUTP。所述测序检测液包括测序染料、测序反应液和去离子甲酰胺。所述阳性对照品为含有Mut基因25个常见位点突变的质粒,所述阴性对照品为未突变的Mut基因质粒。
所述Mut基因检测液中各引物浓度为200-500nM。
所述测序反应液包括三羟甲基氨基甲烷-盐酸缓冲液。
表2.试剂盒组分
实验例1
使用自建企业参考品对实施例2所述试剂盒进行性能验证,具体操作如下:
1、样本处理
选用企业参考品进行试剂盒性能验证。所有样本按照美基生物核酸提取试剂盒(产品编号:IVD4173)进行核酸提取,所得DNA样本保存于2-8℃;若样本长时间不使用可保存于-20℃。
2、PCR扩增
按表3配置PCR混合液(每个反应45μL)。将配置好的PCR混合液按45μL每个反应孔进行分装。将已提取好的样品DNA、阳性对照品、阴性对照品各5μL加入相应反应孔,上机进行PCR扩增。
表3.PCR混合液组分
组分 | 1个反应体积 |
Mut检测液 | 14μL |
核酸扩增反应液 | 31μL |
总体积 | 45μL |
3、扩增程序:
95℃5min(1个循环);95℃30sec,60℃90sec,72℃30sec(35个循环);72℃10min(1个循环);4℃∞。
4、产物纯化:
PCR扩增结束以后,取5μL PCR产物进行1-2%琼脂糖凝胶电泳,观察是否有目的条带扩增(目的条带大小为500bp),如观察到目的条带,则将上述PCR产物及时进行纯化,具体操作详见PCR产物纯化试剂盒说明书。按试剂盒步骤纯化获得的PCR产物进行电泳鉴定定值(PCR产物浓度范围为10-50ng),可立即进行测序PCR或置于-20±2℃保存备用(保存时间不超过2天)。
5、测序PCR:
经过电泳鉴定定值的PCR产物,按表4配制测序PCR体系。
表4.配制测序PCR体系
将各反应管放入定性PCR仪,按下列条件扩增:96℃1分钟→(96℃10秒→50℃5秒→60℃4分钟)×25个循环→4℃保温。
6、测序准备:
6.1取测序PCR反应结束后的反应管,加入2μL 125mmol/L EDTA和2μL 3mol/L醋酸钠(pH 5.2)到管底。
6.2加入50μL无水乙醇,盖紧管盖,震荡混匀,室温避光放置15min。
6.3 4℃,12 000rpm离心30min,立即小心弃去上清。
6.4每管加入150μL预冷70%乙醇,4℃下12 000rpm离心10min,立即小心去上清(尽量将管底的70%乙醇吸干净,若不能立即操作,请在操作前重新离心3分钟),此步骤可重复操作一次。
6.5室温避光放置15-30min(观察管底液体,使70%乙醇挥发干净),此步骤产物可密封避光置于-20±2℃保存5天。
6.6加入10μL Hi-Di Formamide,短时振荡溶解DNA,进行短时离心将管壁上的液体全部离心至管底。
6.7溶解后的样品在定性PCR仪上95℃变性5分钟,迅速置冰中冷却4分钟后,准备上样电泳。
7、基因分析仪测序:
7.1加样:变性后的测序产物加入与基因分析仪配套的96孔板,盖好,按加样顺序编辑样品列表。根据测序仪型号选用具有IVD标志的Seq_std_BDTV3.1_ASSYXL_POP7或Seq_std_BDTV3.1_ASSY_POP7进行测序。
7.2使用ABI基因分析仪时,应用ABI公司Data Collectio与Sequencing Analysis软件进行数据收集和分析。Data Collection与Sequencing Analysis分析软件的进一步信息请参阅Data Collection与Analysis Software用户手册。测序结果自动保存在预设位置,反应结束后打开测序结果进行分析,得到.ab1、.phd.1格式的文件。
8、结果分析:
8.1运行SeqScanner程序,导入测序结果。
8.2测序结果应用软件对比野生型序列逐个碱基比较,查找突变点,记录突变的碱基类型即可。
9、试剂盒性能:
9.1阳性符合率:对阳性企业参考品P1进行检测,如表5所示,检测结果应为阳性,阳性符合率为100%。
表5.mut突变位点阳性企业参考品
编号 | 阳性参考品 | 浓度(ng/μL) |
P1 | 含有25个突变位点的mut基因序列质粒 | 20 |
9.2阴性符合率:对阴性企业参考品N1进行检测,如表6所示,检测结果为阴性,符合率100%。
表6.mut突变位点阴性企业参考品
编号 | 阴性参考品 | 浓度(ng/μL) |
N1 | 不含mut基因突变的正常质粒 | 20 |
9.3重复性:对重复性企业参考品R1进行检测,重复检测10次,如表7所示,检测结果为阳性,CV≤5%。
表7.mut突变位点重复性企业参考品
编号 | 重复性参考品 | 浓度(ng/μL) |
R1 | 含有25个突变位点的mut基因序列质粒 | 20 |
9.4最低检出限:对稀释至不低于5ng/μL的最低检出限企业参考品L1进行检测,如表8所示,检测结果为阳性,符合率100%。
表8.mut突变位点最低检出限企业参考品
编号 | 最低检出限参考品 | 浓度(ng/μL) |
R1 | 含有25个突变位点的mut基因序列质粒 | 5 |
实验例2
使用实施例1所述试剂盒对50例单纯型MMA临床患者样本进行检测,所述50例样本均经高通量测序方法检测突变类型,其中,mut突变为41例。41例mut突变类型及利用不同方法检出率如表9所示。
由表9可知,利用高通量测序技术,共检测出25种mut突变,新发突变3种。在已知的突变类型中,c.729_730 insTT突变频次最高,占总突变频率的20.3%;除外显子6-外显子9上突变检测不出,本发明试剂盒突变检出频次与高通量测序检测率一致,可以达到替代高通量测序法的检出效果。
表9. 41例临床样本mut突变位点检出率比较
*表示新发突变
利用实施例2所述试剂盒对50例单纯型MMA患者基因突变检测(如表10所示),发现40例为mut基因突变,高通量测序检出mut突变为41例,本试剂盒在mut检出率上与高通量测序一致性为97.6%。本试剂盒总阳性检出率为80%,仅比高通量测序阳性检出率低10%,从成本及操作难易程度上,可以达到替代高通量测序的目的。
表10. 50例单纯性MMA临床样本检测结果
突变类型 | 高通量测序检出例数 | 本发明 |
MUT | 41人 | 40人 |
MMAA | 1人 | 0人 |
MMAB | 1人 | 0人 |
其他 | 2人 | 0人 |
未检出 | 5人 | 10人 |
检出率 | 90% | 80% |
综上所述,借助于本发明的上述技术方案,本发明设计的针对三个热点突变区域的三组引物对,即可完成mut基因上25种突变类型的检测,并且还能够快速、准确且灵敏地检测出该基因三个热点突变区域突变位点,实验结果重复性好,精密度高。
本发明不局限于上述可选的实施方式,任何人在本发明的启示下都可得出其他各种形式的产品,均属于本发明的保护范围。上述具体实施方式不应理解成对本发明的保护范围的限制,本领域的普通技术人员应当理解,在不背离本发明的范围下,可对前述各实施例所记载的技术方案进行修改,或对其中部分或者全部技术特征进行等同替换,与此同时这些修改或者替换,并不会使相应的技术方案的本质脱离本发明各实施例技术方案的范围。
序列表
<110> 北京华诺奥美基因医学检验实验室有限公司
<120> 一种单纯型甲基丙二酸血症基因突变检测引物对及试剂盒
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gctcctattc cacccctctt cta 23
<210> 2
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gcagtaacga cagacataaa ttagt 25
<210> 3
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tgcttagtta tggattaata gacct 25
<210> 4
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
taaatctata aatcttgact tgtaagtt 28
<210> 5
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cgcagacctc gtcttcttgt a 21
<210> 6
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tgaagacata gctttactct cttct 25
<210> 7
<211> 3963
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ttcttatagt agctcctatt ccacccctct tctaaatgtt ttactctatc tttctttttc 60
taggtcagtt cttatttcta ttgggtgttt ccatgctcca ccatgttaag agctaagaat 120
cagctttttt tactttcacc tcattacctg aggcaggtaa aagaatcatc aggctccagg 180
ctcatacagc aacgacttct acaccagcaa cagccccttc acccagaatg ggctgccctg 240
gctaaaaagc agctgaaagg caaaaaccca gaagacctaa tatggcacac cccggaaggg 300
atctctataa aacccttgta ttccaagaga gatactatgg acttacctga agaacttcca 360
ggagtgaagc cattcacacg tggaccatat cctaccatgt atacctttag gccctggacc 420
atccgccagt atgctggttt tagtactgtg gaagaaagca ataagttcta taaggacaac 480
attaaggtga gatttaatat agactaatta tgatagtgtc atatgctcta gttttatttt 540
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 600
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn tttgacatgt atgagcataa 660
aaatagttgt tataaaagtg tactgttatg agatgaatca acatttttag tagtgttaaa 720
agtaaatcat tttaccttga ttccagactc ttgaatctta cattattttt ctttatatat 780
ttaggctggt cagcagggat tatcagttgc ctttgatctg gcgacacatc gtggctatga 840
ttcagacaac cctcgacttc gtggtgatgt tggaatggct ggagttgcta ttgacactgt 900
ggaagatacc aaaattcttt ttgatggaat tcctttagaa aaaatgtcag tttccatgac 960
tatgaatgga gcagttattc cagttcttgc aaattttata gtaactggag aagaacaagg 1020
tgtacctaaa gagaaactta ctggtaccat ccaaaatgat atactaaagg aatttatggt 1080
tcgaaataca tacatttttc ctccagaccc atccatgaaa attattgctg acatatttga 1140
atatacagca aaggtatact gtggttatat aggtttttct atagttcctt gaatgtagga 1200
atttttacaa catgctatat actgaattta aaaatttagt ctgagtaata taatttagct 1260
ttgttatgtt actttgttta tgtactaatt tatgtctgtc gttactgctt taacatcatt 1320
ttaaaataat ggtttatgct tagttatgga ttaatagacc ttttaatatt tttacttatt 1380
caatgttaca attttttttt ttatgtagca catgccaaaa tttaattcaa tttcaattag 1440
tggataccat atgcaggaag caggggctga tgcattctgg agctgggcct atactttagc 1500
agatggattg gagtactcta gaactggact ccaggctggc ctgacaattg atgaatttgc 1560
accaaggtga gtaaattaaa cctagatatt gtttaacatt agaatgcata ggacattata 1620
tattgataat gattatnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1680
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnttta 1740
ctctctcaat caattagatt tattttctgc tctatattgt taactttaat taaattctgg 1800
acctaacgtt aacttatttt agggttgtct ttcttctggg gaattggaat gaatttccat 1860
atggaaatag caaagatgag agctggtaga agactctggg ctcacttaat agagaaaatg 1920
tttcagccta aaaactcaaa atctcttctt ctaagagcac actgtcagac atctggatgg 1980
tcacttactg agcaggtatg tatataattt aaaatgtaga attttaataa agttatatat 2040
atatattttt tctgagcaat gtggcacaag cagannnnnn nnnnnnnnnn nnnnnnnnnn 2100
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2160
nnnnnnnnnn nnnncagtgc gttatagtgc atatctatca ccgttcttga gttttgtttt 2220
ttatcaaata ttttaggatc cctacaataa tattgtccgt actgcaatag aagcaatggc 2280
agcagtattt ggagggactc agtctttgca cacaaattct tttgatgaag ctttgggttt 2340
gccaactgtg aaaagtgctc gaattgccag gaacacacaa atcatcattc aagaagaatc 2400
tgggattccc aaagtggctg atccttgggg aggttcttac atgatggaat gtctcacaaa 2460
tgatgtttat gatgctgctt taaaggtaag tttttccact ttgcaacttt gttttcagat 2520
ttatagattt gcaaagattt atagatttta aaacttacaa gtcaagattt atagatttaa 2580
atttataaat caatcactta cagattttat ataataattt aaacgatgtt ttgcggttca 2640
taaattcatg gaacgtgaag gtcgcagacc tcgtcttctt gtagcaaaaa tgggacaaga 2700
tggccatgac agaggagcaa aagttattgc tacaggattt gctgatcttg gttttgatgt 2760
ggacataggc cctcttttcc aggtactaaa aactgttggg gggaattcaa aagtatttac 2820
tagtcactta catttagaaa atgtagtaaa atgatgacag acattgagca aattaataca 2880
tctcctggta actggtatgt agcctctgac ttgcaaatga atttctctca ttccnnnnnn 2940
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3000
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnattgtc attgcttgtg tatgcttgca 3060
tctcatcatg agtcttgcca tagtatgtct gaatggtatt gcccagattg tcttccaggg 3120
tttttatagt cattatctag taataaacaa tgatttatct ttactttctg actgtttata 3180
ctcctcgtga agtggcccag caggctgtgg atgcggatgt gcatgctgtg ggcgtaagca 3240
ccctcgctgc tggtcataaa accctagttc ctgaactcat caaagaactt aactcccttg 3300
gacggccaga tattcttgtc atgtgtggag gggtgatacc acctcaggta ttttttatct 3360
ctatttttct agtactgtga tgggaatctt gagtaatgat agcttatttg aggttctaaa 3420
gataaacttg tagatttagg aacatattat ttctggtgat tcactaaata aattctacaa 3480
ggtctaaagc ggacagtgtt gtttgcattc aagttatatt gcaatttatt atgtataatt 3540
taacttaaca cttacaagct ggatccnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3600
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3660
nnnnnntgac aagagaagat agggttttat ttccggtaaa atggaaaata gtggctctta 3720
ttaattaaac acattcaaac aacaggatta tgaatttctg tttgaagttg gtgtttccaa 3780
tgtatttggt cctgggactc gaattccaaa ggctgccgtt caggtgcttg atgatattga 3840
gaagtgtttg gaaaagaagc agcaatctgt ataatatcct ctttttgttt tagcttttgt 3900
ctaaaatatt attttagtta tgatcaaaga agagagtaaa gctatgtctt caatttaatt 3960
tca 3963
Claims (7)
1. 一种单纯型甲基丙二酸血症基因突变检测引物对,其特征在于,包括针对Mut基因的3个引物对,所述3个引物对的核苷酸序列分别如SEQ ID NO.1-SEQ ID NO.6所示。
2.一种单纯型甲基丙二酸血症基因突变检测试剂盒,其特征在于,包括Mut基因检测液、扩增反应液、测序检测液、阴性对照品及阳性对照品;
所述Mut基因检测液包括针对Mut基因的3个引物对,所述3个引物对的核苷酸序列分别如SEQ ID NO.1-SEQ ID NO.6所示。
3. 根据权利要求2所述的单纯型甲基丙二酸血症基因突变检测试剂盒,其特征在于,所述Mut基因检测液中各引物浓度为200-500 nM。
4. 根据权利要求2所述的单纯型甲基丙二酸血症基因突变检测试剂盒,其特征在于,所述扩增反应液包括Taq DNA聚合酶、dNTPs、PCR扩增缓冲液、Mg2+、UNG酶和dUTP。
5.根据权利要求2所述的单纯型甲基丙二酸血症基因突变检测试剂盒,其特征在于,所述测序检测液包括测序染料、测序反应液和甲酰胺。
6.根据权利要求2所述的单纯型甲基丙二酸血症基因突变检测试剂盒,其特征在于,所述阳性对照品为含有Mut基因25个位点突变的质粒。
7.根据权利要求2所述的单纯型甲基丙二酸血症基因突变检测试剂盒,其特征在于,所述阴性对照品为未突变的Mut基因质粒。
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