CN108342468A - 用于AD筛查的circRNA标志物、试剂盒及基因芯片 - Google Patents
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Abstract
本发明公开了用于AD筛查的circRNA标志物、试剂盒及基因芯片,其中所述的标记物包括hsa_circRNA_0054102、hsa_circRNA_0062028、hsa_circRNA_0001929、hsa_circRNA_0023262、hsa_circRNA_0090857和hsa_circRNA_0092223六种circRNA的组合,提供的试剂盒能够检测外周血中上述标志物的含量;本发明提供的用于AD筛查的circRNA标志物、试剂盒及基因芯片具有高度稳定性、特异性和敏感性,灵敏度可达84.6%,特异度可达78.3%,有较高的临床参考价值。
Description
技术领域
本发明属于生物技术领域,涉及一种用于阿尔茨海默病诊断的circRNA标志物、试剂盒及基因芯片。
背景技术
阿尔茨海默病(Alzheimerdisease,AD),又叫老年性痴呆,是一种中枢神经系统变性病,起病隐袭,病程呈慢性进行性,是老年期痴呆最常见的一种类型。主要表现为渐进性记忆障碍、认知功能障碍、人格改变及语言障碍等神经精神症状,严重影响社交、职业与生活功能。AD的病因及发病机制尚未阐明,特征性病理改变为β淀粉样蛋白沉积形成的细胞外老年斑和tau蛋白过度磷酸化形成的神经细胞内神经原纤维缠结,以及神经元丢失伴胶质细胞增生等。AD的发病率逐年提高,美国约翰霍普金斯大学的研究报告显示,到2050年阿尔兹海默氏病患者人数将达到1亿7百万人,同时阿尔兹海默氏病又是致命的,从确诊到最终死亡大多数患者只用了7年的时间,早期发现早期预防可以延缓疾病进程。但是目前的诊断包括神经心理学测验、血液学检查、神经影像学检查、脑电图、脑脊液检测等方法,都难以在未发病和发病早期进行筛选,导致大量的患者错过了效果最佳的干预期。
经过几十年的探索,AD的发病机制虽然依旧不是很清楚,但已有大量研究表明,基因遗传是AD的主导因素,尤其与环状RNA表达水平联系紧密。上世纪70年代,研究发现了大量的编码蛋白质功能之外的非编码RNA。这类小的非编码RNA分布广泛,普遍存在于生物体内,能够调节多种生物学信号通路,具有负调控基因表达作用。最新研究发现,生命体中还存在一类闭合成环状的RNA分子(circRNA),这是不同于一般意义上为大家熟知的呈线性结构的RNA。这类circRNA由蛋白编码基因外显子的5’端与3’端首尾相接环化而成,因其结构上的特殊环状闭合使其能够避免受RNA核酸外切酶的影响,因此与传统的线性RNA相比性质更加稳定,可长期存在。由于circRNA的稳定性,如果能够确定circRNA与疾病的明确关系,其成为临床诊断疾病的生物标志物或疾病治疗的靶点潜力巨大,意义深远。
AD病因复杂,病理机制不明确,现有的诊断方法都难以在未发病和发病早期进行筛选,导致大量的患者错过效果最佳的干预期,因此,探讨AD的基因检测方法,对于早期发现早期预防阿尔兹海默症等均具有重要的意义和价值。
发明内容
针对现有的AD诊断方法都难以在未发病和发病早期进行筛查,导致大量的患者错过效果最佳的干预期的问题,本发明提供一种用于AD筛查的circRNA标志物、试剂盒及基因芯片,达到AD早期发现早期预防以可以延缓疾病进程的目的。
本发明通过以下技术方案实现的:
本发明提供一种用于AD筛查的circRNA标志物,所述标记物包括hsa_circRNA_0054102、hsa_circRNA_0062028、hsa_circRNA_0001929、hsa_circRNA_0023262、hsa_circRNA_0090857和hsa_circRNA_0092223六种circRNA的组合。所述hsa_circRNA_0054102、hsa_circRNA_0062028、hsa_circRNA_0001929、hsa_circRNA_0023262、hsa_circRNA_0090857和hsa_circRNA_0092223为相关数据库中的RNA代号,可以在数据库中检索得到。
本发明还提供了一种用于AD筛查的试剂盒,能够检测外周血中hsa_circRNA_0054102、hsa_circRNA_0062028、hsa_circRNA_0001929、hsa_circRNA_0023262、hsa_circRNA_0090857和hsa_circRNA_0092223的含量。
优选的,上述试剂盒含有hsa_circRNA_0054102、hsa_circRNA_0062028、hsa_circRNA_0001929、hsa_circRNA_0023262、hsa_circRNA_0090857和hsa_circRNA_0092223的引物和探针。
同时,本发明还提供了hsa_circRNA_0054102、hsa_circRNA_0062028、hsa_circRNA_0001929、hsa_circRNA_0023262、hsa_circRNA_0090857和hsa_circRNA_0092223在制备用于AD筛查的试剂盒中的应用。
优选的,上述hsa_circRNA_0054102的正向引物如SEQ ID NO.1所示,hsa_circRNA_0054102的反向引物如SEQ ID NO.2所示;hsa_circRNA_0062028的正向引物如SEQID NO.3所示,hsa_circRNA_0062028的反向引物如SEQ ID NO.4所示;hsa_circRNA_0001929的正向引物如SEQ ID NO.5所示,hsa_circRNA_0001929的反向引物如SEQ ID NO.6所示;hsa_circRNA_0023262的正向引物如SEQ ID NO.7所示,hsa_circRNA_0023262的反向引物如SEQ ID NO.8所示;hsa_circRNA_0090857的正向引物如SEQ ID NO.9所示,hsa_circRNA_0090857的反向引物如SEQ ID NO.10所示;hsa_circRNA_0092223的正向引物如SEQ ID NO.11所示,hsa_circRNA_0092223的反向引物如SEQ ID NO.12所示。
优选的,上述hsa_circRNA_0054102的探针序列如SEQ ID NO.13所示;hsa_circRNA_0062028的探针序列如SEQ ID NO.14所示;hsa_circRNA_0001929的探针序列如SEQ ID NO.15所示;hsa_circRNA_0023262的探针序列如SEQ ID NO.16所示;hsa_circRNA_0090857的探针序列如SEQ ID NO.17所示;hsa_circRNA_0092223的探针序列如SEQ IDNO.18所示。
进一步,本发明提供一种用于AD筛查的基因芯片,包括探针和固相支持物,所述的探针序列如SEQ ID NO.13~18所示。
所述AD筛查试剂盒中还包括PCR反应常用试剂和其它一些辅助试剂,这些试剂的特性以及它们的配制方法均是本领域技术人员所熟知的;在本发明的具体实施方案中,使用了定量PCR的方法。
通过实施本发明的技术方案,可以达到以下有益效果:
(1)本发明提供的用于AD筛查的circRNA标志物、试剂盒及基因芯片具有高度稳定性、特异性和敏感性,灵敏度可达84.6%,特异度可达78.3%,有较高的临床参考价值。
(2)本发明提供的用于AD筛查的circRNA标志物、试剂盒及基因芯片可以使AD的筛查、早期诊断不依靠主观经验判断,而是可以通过circRNA表达水平这种客观指标检查,与脑血管病等进行鉴别诊断,提高诊断准确率,简化诊断过程。
附图说明
图1显示为circRNA阿尔兹海默筛查价值的接受者操作特征曲线图。
具体实施方式
下面,举实施例说明本发明,但是,本发明并不限于下述的实施例。
主要试剂和耗材:
RNeasy mini kit(QIAGEN),Low Input Quick Amp WT Labeling Kit(Agilenttechnologies),Gene Expression Hybridization Kit(Agilent technologies),GeneExpression Wash Buffer Kit(Agilent technologies),反转录试剂盒:Rever Tra AceqPCR Kit(TOYOBO),PCR引物:Custom Plus TQMN RNA Assays(ABI);荧光定量PCR试剂:Power SYBR Green PCR Master Mix(ABI)。
主要仪器:
Agilent Bioanalyzer 2100电泳仪(Agilent technologies)HybridizationOven滚动杂交炉(Agilent technologies),staining dishes洗缸(Thermo Shandon),Agilent Microarray Scanner扫描仪(Agilent technologies),7900HT SequenceDetection System PCR仪(ABI);9600振荡器(Agilent);HB-100金属浴(博日);天美CT14RD型台式高速冷冻离心机(上海天美生化仪器设备工程有限公司);Nano Drop Lite超微量紫外分光光度计(Thermo fisher scientific),WH-2微型漩涡混合仪(上海沪西分析仪器厂有限公司),超低温冰箱(日本三洋公司)。
本发明所述的环状RNA为相关数据库中的RNA代号,选用的所有材料、试剂和仪器都为本领域熟知的,但不限制本发明的实施,其他本领域熟知的一些试剂和设备都可适用于本发明以下实施方式的实施。
实施例一:用于AD筛查的circRNA标志物
本发明提供一种用于AD筛查的circRNA标志物,标记物包括hsa_circRNA_0054102、hsa_circRNA_0062028、hsa_circRNA_0001929、hsa_circRNA_0023262、hsa_circRNA_0090857和hsa_circRNA_0092223六种circRNA的组合。
实施例二:用于AD筛查的试剂盒
本发明提供一种用于AD筛查的试剂盒,试剂盒中包括:Taq酶,逆转录酶,缓冲液,dNTPs,MgCl2、DEPC水、阴性对照品、阳性对照品和hsa_circRNA_0054102、hsa_circRNA_0062028、hsa_circRNA_0001929、hsa_circRNA_0023262、hsa_circRNA_0090857和hsa_circRNA_0092223的引物和探针。
所述的hsa_circRNA_0054102的正向引物如SEQ ID NO.1所示,hsa_circRNA_0054102的反向引物如SEQ ID NO.2所示;hsa_circRNA_0062028的正向引物如SEQ ID NO.3所示,hsa_circRNA_0062028的反向引物如SEQ ID NO.4所示;hsa_circRNA_0001929的正向引物如SEQ ID NO.5所示,hsa_circRNA_0001929的反向引物如SEQ ID NO.6所示;hsa_circRNA_0023262的正向引物如SEQ ID NO.7所示,hsa_circRNA_0023262的反向引物如SEQID NO.8所示;hsa_circRNA_0090857的正向引物如SEQ ID NO.9所示,hsa_circRNA_0090857的反向引物如SEQ ID NO.10所示;hsa_circRNA_0092223的正向引物如SEQ IDNO.11所示,hsa_circRNA_0092223的反向引物如SEQ ID NO.12所示。
所述的hsa_circRNA_0054102的探针序列如SEQ ID NO.13所示;hsa_circRNA_0062028的探针序列如SEQ ID NO.14所示;hsa_circRNA_0001929的探针序列如SEQ IDNO.15所示;hsa_circRNA_0023262的探针序列如SEQ ID NO.16所示;hsa_circRNA_0090857的探针序列如SEQ ID NO.17所示;hsa_circRNA_0092223的探针序列如SEQ ID NO.18所示。
实施例三:用于AD筛查的基因芯片
本发明提供一种用于AD筛查的基因芯片,基因芯片包括探针和固相支持物,所述固相支持物包括玻璃片、硅片、聚丙烯膜、硝酸纤维素膜和尼龙膜;所述的探针序列如SEQID NO.13~18所示。
所述的用于AD筛查的基因芯片的制备方法,具体包括以下步骤:
(1)采用序列如SEQ ID NO.13~18所示的六种探针,该部分的核苷酸的平均长度为60个碱基;将每条探针的3’端进行氨基修饰,使之能够和玻璃板上的聚合硅发生共价反应从而固定到芯片表面。
(2)将这些探针分别在用于制备芯片的玻璃板上平行点样三次,每个探针点的直径为150μm,相邻探针点中心间的距离为240μm,点完探针样品后让玻璃片在室温下干燥24小时,然后清洗掉未共价结合的DNA和点样缓冲液;
(3)将玻璃片室温下于空气中彻底晾干后用于杂交检测。
实施例四:临床验证试验
一、研究对象
病例组:
来自2015年3月-2017年5月入住新疆维吾尔自治区人民医院神经内科的患者65例(男39例,女26例),年龄55-80(平均65.4士8.5)。纳入标准参照美国国立神经病及语言障碍和卒中研究所(NINCDS)和Alzheimer病及相关疾病协会(ADRDA)推荐的临床很可能AD诊断标准。
入组标准:
(1)发病年龄在55岁以上;
(2)无明显的意识障碍;
(3)临床检查确认痴呆,神经心理量表测试支持:MMSE评分<26分,HIS<3分;
(4)必须存在两种或两种以上的认知功能障碍;
(5)认知功能下降呈进行性加重;
(6)病程至少在6个月以上;
(7)受教育程度大于5年。
排除标准:
(1)突然卒中样起病;
(2)疾病初期就出现局灶性神经系统体征,如偏瘫、感觉缺失、视野缺损和共济失调等;
(3)病程早期出现抽搐发作和步态障碍;
(4)存在系统性疾病或其它脑部疾病如高血压或糖尿病或卒中病史;
(5)有心、肝、肾等重大疾病;
(6)最近两月有过感染性疾病史。
对照组:为新疆维吾尔自治区人民医院健康体检人员30例,男15例,女15例,年龄55~80岁,平均(68.2土6.11岁。均根据病史、神经和精神检查、脑电图、颅脑磁共振或CT检查排除中枢神经系统疾病及痴呆;排除高血压,糖尿病及其它系统疾病;最近两月没有患过感染性疾病;无心、肝、肾等重大疾病。MMSE评分>26分。
本研究获得新疆维吾尔自治区人民医院医学伦理委员会批准,所有受试者或受试家属(监护人)均签署知情同意书。
入组时记录一般情况(姓名、性别、年龄、民族、文化程度、职业、收入水平、婚姻状况、药物成瘾史及AD家族史等)。同样,记录正常对照组一般情况。
二、研究方法
1.评估工具
MMSE评分
2.资料收集
入组时记录一般情况(姓名、性别、年龄、民族、文化程度、职业、收入水平、婚姻状况、药物成瘾史及AD家族史等)。
3.RNA提取和circRNA芯片筛查
外周血2500rpm/min离心10分钟,分离上清,取白细胞层,加入红细胞裂解液8ml,振荡10min,2500rpm/min离心10分钟,弃上清,再次加入红细胞裂解液5ml,振荡5min,2500rpm/min离心5分钟,弃上清,加入RNA later 4度冰箱过夜,后放入-80度冰箱备用。采用RNeasy mini kit并且根据生产厂商提供的标准操作流程进行样品的total RNA抽提,抽提所得total RNA经Agilent Bioanalyzer 2100(Agilent technologies,Santa Clara,CA,US)电泳质检合格后备用。芯片实验起始样本为total RNA,total RNA经NanoDrop Lite分光光度计及Agilent Bioanalyzer 2100进行质检,质检合格的RNA可进行后续的芯片实验。实验样品RNA采用Agilent表达谱芯片配套试剂盒Low Input Quick Amp WT LabelingKit和标准操作流程对样品total RNA进行放大和标记,并用RNeasy mini kit纯化标记后的cRNA。按照Agilent表达谱芯片配套提供的杂交标准流程和配套试剂盒,GeneExpression Hybridization Kit,在滚动杂交炉Hybridization Oven中65℃,10rpm,滚动杂交17小时,杂交cRNA上样量1.65μg,并在洗缸staining dishes中洗片,洗片所用的试剂为Gene Expression Wash Buffer Kit。完成杂交的芯片采用Agilent MicroarrayScanner进行扫描,软件设置Dye channel:Green,Scan resolution=3μm,PMT 100%,20bit。用Feature Extraction software 10.7读取数据,最后采用R软件中limma包进行归一化处理,所用的算法为Quantile。
4.逆转录聚合酶链式反应
对芯片筛查结果中的6种差异表达明显的circRNA进行后续的RT-PCR验证。并用AD患者和健康对照组的血液样本验证这6种差异性表达的circRNA。按照Rever Tra Ace qPCRKit RNA反转录试剂盒说明书进行反转录,circRNA扩增引物是由上海生工生物工程有限公司合成的。实时荧光定量PCR按照ABI Power SYBR Green PCR Master Mix试剂盒说明书进行。
5.统计学处理
采用卡方检验分析两组的性别差异;采用两独立样本t检验分析两组间年龄的差异;采用比较CT值法计算表达差异。全部数据采用SPSS 17.0统计软件进行统计分析。
三、研究结果
1、PCR验证分析发现,阿尔兹海默症与对照组的circRNA表达水平比较,阿尔兹海默症患者外周血hsa_circRNA_0054102、hsa_circRNA_0062028、hsa_circRNA_0001929的表达水平显著下调;hsa_circRNA_0023262、hsa_circRNA_0090857、hsa_circRNA_0092223的表达水平显著上调,结果见表1。
表1:病例组与对照组lncRNA表达水平比较(△Ct±SD)
circRNA | 病例组 | 对照组 | p-value |
hsa_circRNA_0054102 | 1.1385±0.9476 | 3.3861±2.0917 | <0.001 |
hsa_circRNA_0062028 | 1.0218±0.5540 | 4.3173±0.7691 | <0.001 |
hsa_circRNA_0001929 | 4.40±0.78 | 12.70±3.23 | <0.001 |
hsa_circRNA_0023262 | 15.85±12.47 | 4.84±1.10 | <0.001 |
hsa_circRNA_0090857 | 2.3664±1.3112 | 1.487±0.8360 | 0.001 |
hsa_circRNA_0092223 | 8.5882±6.9120 | 0.0015±0.0008 | <0.001 |
2、对circRNA阿尔兹海默筛查价值的接受者操作特征曲线(ROC)分析发现,灵敏度为84.6%,特异度为78.3%,见图1。
综上所述,本发明提供的用于AD筛查的circRNA标志物、试剂盒及基因芯片具有高度稳定性、特异性和敏感性,灵敏度可达84.6%,特异度可达78.3%,有较高的临床参考价值;此外,本发明提供的用于AD筛查的circRNA标志物、试剂盒及基因芯片可以使AD的筛查、早期诊断不依靠主观经验判断,而是可以通过circRNA表达水平这种客观指标检查,与脑血管病等进行鉴别诊断,提高诊断准确率,简化诊断过程。
如上所述,即可较好地实现本发明,上述的实施例仅仅是对本发明的优选实施方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种改变和改进,均应落入本发明确定的保护范围内。
<110> 新疆维吾尔自治区人民医院
<120> 用于AD筛查的circRNA标志物、试剂盒及基因芯片
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Claims (6)
1.用于AD筛查的circRNA标志物,其特征在于,所述的标记物包括hsa_circRNA_0054102、hsa_circRNA_0062028、hsa_circRNA_0001929、hsa_circRNA_0023262、hsa_circRNA_0090857和hsa_circRNA_0092223六种circRNA的组合。
2.用于AD筛查的试剂盒,其特征在于,能够检测外周血中hsa_circRNA_0054102、hsa_circRNA_0062028、hsa_circRNA_0001929、hsa_circRNA_0023262、hsa_circRNA_0090857和hsa_circRNA_0092223的含量。
3.如权利要求2所述的用于AD筛查的试剂盒,其特征在于,所述的试剂盒含有hsa_circRNA_0054102、hsa_circRNA_0062028、hsa_circRNA_0001929、hsa_circRNA_0023262、hsa_circRNA_0090857和hsa_circRNA_0092223的引物和探针。
4.如权利要求3所述的用于AD筛查的试剂盒,其特征在于,所述的hsa_circRNA_0054102的正向引物如SEQ ID NO.1所示,hsa_circRNA_0054102的反向引物如SEQ ID NO.2所示;hsa_circRNA_0062028的正向引物如SEQ ID NO.3所示,hsa_circRNA_0062028的反向引物如SEQ ID NO.4所示 ;hsa_circRNA_0001929的正向引物如SEQ ID NO.5所示,hsa_circRNA_0001929的反向引物如SEQ ID NO.6所示;hsa_circRNA_0023262的正向引物如SEQID NO.7所示,hsa_circRNA_0023262的反向引物如SEQ ID NO.8所示;hsa_circRNA_0090857的正向引物如SEQ ID NO.9所示,hsa_circRNA_0090857的反向引物如SEQ IDNO.10所示;hsa_circRNA_0092223的正向引物如SEQ ID NO.11所示,hsa_circRNA_0092223的反向引物如SEQ ID NO .12所示。
5.如权利要求3所述的用于AD筛查的试剂盒,其特征在于,所述的hsa_circRNA_0054102的探针序列如SEQ ID NO.13
所示;hsa_circRNA_0062028的探针序列如SEQ ID NO.14所示;hsa_circRNA_0001929的探针序列如SEQ ID NO.15所示;hsa_circRNA_0023262的探针序列如SEQ ID NO.16所示;hsa_circRNA_0090857的探针序列如SEQ ID NO.17所示;hsa_circRNA_0092223的探针序列如SEQ ID NO.18所示。
6.用于AD筛查的基因芯片,其特征在于,包括探针和固相支持物,所述的探针序列如SEQ ID NO .13~18所示。
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CN113774058A (zh) * | 2021-08-26 | 2021-12-10 | 中国药科大学 | 血清中脑部细胞来源的外泌体环状rna作为阿尔兹海默症诊断标志物的应用 |
CN113774058B (zh) * | 2021-08-26 | 2023-08-04 | 中国药科大学 | 血清中脑部细胞来源的外泌体环状rna作为阿尔兹海默症诊断标志物的应用 |
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