CN108690875A - 用来筛查传染病基因和遗传改变的带有条码的微阵列芯片及使用方法 - Google Patents
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Abstract
本发明为用来筛查传染病基因和遗传改变的带有条码的微阵列芯片及使用方法,描述了用于检测试样中传染性微生物或者试样中DNA和/或RNA样品的基因突变和数量改变的检测方法。在各种实施例中,所述方法包括用同时含有条码和特异靶标序列寡核苷酸的引物进行多重聚合酶链式反应(PCR)。PCR产物,被通用引物上的荧光染料标记,然后与条码特异的寡核苷酸杂交并被印到微阵列芯片的固体基质上。该方法可以用于筛查大量传染性微生物或与疾病有关的基因突变,基因考贝数或基因表达水平的改变,包括癌症和遗传疾病。经系列稀释后被印到微阵列芯片上的条码特异寡核苷酸也可以用于PCR产物的定量,对试样中存在的靶标序列进行半定量。本发明所述组分、设备和方法为检测和定量多种靶标序列,包括基因突变,提供了快速筛查检测。
Description
技术领域
总的来说本发明涉及分子生物学、微生物学和肿瘤学领域。本发明的各个部分包括用于检测含有DNA和RNA的核苷酸的各种组分、设备和方法,为传染性疾病和癌症的诊断和处理提供定性和定量信息。进一步,本发明的所述部分包括用同时含有条码和靶标序列特异寡核苷酸的引物进行多重聚合酶链式反应 (PCR)并用印刷在微阵列芯片上与条码互补的寡核苷酸对PCR产物进行分析。本发明所述各种组分、设施和方法为多种靶标序列的检测和定量提供了快速测定方法,包括基因突变。
背景技术
下面的背景技术用于帮助读者理解本发明,而不能被认为是现有技术。
基因组是生物的全部基因或遗传物质。由编码构成和维持生物所需的所有信息的核苷酸组成。术语“核苷酸”是本领域公知的。这里所述的“核苷酸”一般指DNA、RNA分子或它们的衍生物,包括碱基。碱基包括,例如,DNA(如腺嘌呤“A”,鸟嘌呤“G”,胸腺嘌呤“T”或胞嘧啶“C”)或RNA(如一个 A,一个G一个尿嘧啶“U”或C)中出现的自然产生或衍生的嘌呤或嘧啶碱基。术语“核苷酸”包含术语“寡核苷酸”和“多聚核苷酸”,它们都是术语“核苷酸”的亚属。术语“寡核苷酸”为长度在3至100个碱基之间的分子。术语“多聚核苷酸”指长度至少大于100个碱基的单个分子。
总的来说这些定义指一个单链分子,但在特定实施例中也可以包括与单链分子的局部、基本上或全部互补的另外一条链。因此核苷酸也包括双链分子或三链分子,其包含特定序列的一个或多个互补链或“互补物”,这个特定序列包括分子。
聚合酶链式反应(PCR)是通过DNA聚合酶(1、2)扩增靶标(或模板) DNA分子的技术。这个技术在临床实验室和研究工作实验室被用于各种用途,包括分子克隆、DNA测序、突变分析、疾病诊断、重组蛋白合成和识别个体的法医鉴定。该方法依赖热循环的加热和冷却的重复循环,这样的结果是DNA通过DNA聚合酶解链和复制。一对被称为引物的短DNA寡核苷酸单链被用于靶标区域的特异扩增。引物序列与部分靶标序列互补。PCR反应一般在热循环仪中的反应溶液中进行。反应溶液通常包括下列成分:含有待扩增DNA区域的DNA 模板;与DNA靶标的每一条链的3'端互补的引物;热稳定的DNA聚合酶例如 TaqDNA聚合酶,Pfu DNA聚合酶或Vent DNA聚合酶;脱氧核苷三磷酸盐 (dNTPs);优化DNA聚合酶活力和稳定性的缓冲液,包括钾、镁或锰离子。
通过凝胶电泳,或通过与DNA结合的荧光染料或通过用荧光染料标记的与靶序列互补并作为探针的寡核苷酸,PCR产物可以被检测和定量。目前有许多基于PCR技术对目的基因进行检测,但是仍然存在很多缺陷,有待提供改良的方法,对扩增的目标产物进行检测以及改良的方法对目的基因进行扩增。
发明内容
一方面,本发明提供一种多重聚合酶链式反应(PCR)与带有条码的微阵列芯片的系统组合用于筛查和检测传染性微生物或筛查疾病相关基因突变或基因表达水平的改变,包括癌症和遗传疾病的方法:该方法包括:进行第一和第二轮 PCR反应;其中,第一轮PCR中使用的引物包含具有下列特性的引物对:除了所测试的靶标特异序列以外,第一引物在5'端含有一个额外的条码序列;而引物对的第二引物含有通用引物序列,与所有引物对一样具有相同序列。
在一些具体的实施方式中,进行第二轮PCR反应使用的引物由独特的条码寡核苷酸引物和通用引物组成;其中,通用引物用报告分子标记,如生物素、荧光染料、荧光纳米颗粒、量子点或化学发光染料中的一种。
在一些具体的实施方式中,所述的报告分子可以是任何允许进行PCR产物检测的分子,包括生物素、荧光染料。当生物素被使用时,标记到卵白素或链霉亲和素上的报告分子发射的信号被用于PCR产物检测。
在一些具体的实施方式中,所述的报告分子发射的信号可以是分子,其发出荧光及化学发光,或通过酶促反应发出荧光或化学发光,或像金颗粒一样直接肉眼可见。
在一些具体的实施方式中,其中,在所述的条码微阵列芯片是具有成百上千个点的微阵列芯片,每一个点含有独特的条码寡核苷酸用于捕获第二轮PCR中由通用引物合成的DNA链。
在一些具体的实施方式中,其中,每一个条码序列可以被印刷在不只一个点上(重复的点);或者,对于每一个条码序列,具有与条码相同的序列但中间有1 个或多个核苷酸位点错配的内标寡核苷酸作为条码被被印刷在微阵列芯片上;优选的,重复点和内标点被用于测定检测的特异性和可重复性。
在一些具体的实施方式中,其中,所述的条码微阵列芯片被印刷在固体表面作为芯片,或在多孔道的固体表面,例如96孔板上。
在一些具体的实施方式中,其中,该方法或者方法所使用的系统可以被用于临床分析中的床边(POC)诊断检测,食品安全和环境监测。
在一些具体的实施方式中,该方法或者方法所使用的系统用于以下检测:引起传染病的微生物是所有引起传染性疾病的微生物,其具有或不具有临床症状。这些传染性微生物中的部分可以引起癌症。
在一些具体的实施方式中,,其中,该方法或者方法所使用的系统被用于筛查和检测基因组DNA中的突变。
在一些具体的实施方式中,系统包括癌症中突变的检测。
在一些具体的实施方式中,系统可以被用于样品中RNA水平的筛查和检测。在一些具体的实施方式中,RNA包括mRNA,小RNA和非编码RNA。RNA水平可以被用于疾病诊断和疾病进展的监控。
另一方面,本发明提供一种检测目标靶序列的试剂,该试剂包括进行第一和第二轮PCR反应的试剂;其中,第一轮PCR中使用的引物包含具有下列特性的引物对:除了所测试的靶标特异序列以外,第一引物在5'端含有一个额外的条码序列;而引物对的第二引物含有通用引物序列,与所有引物对一样具有相同序列。
另一方面,本发明提供一种用于检测PCR扩增反应产物的装置,该装置包括带有条码的微阵列芯片,在微阵列芯片包括与PCR扩增反应中使用的引物上的条码序列特异配对的另一条码序列。在一些具体的实施方式中,在所述的条码微阵列芯片是具有成百上千个点的微阵列芯片,每一个点含有独特的条码寡核苷酸用于捕获第二轮PCR中由通用引物合成的DNA链。在一些具体的实施方式中,其中,每一个条码序列可以被印刷在不只一个点上(重复的点);或者,对于每一个条码序列,具有与条码相同的序列但中间有1个或多个核苷酸位点错配的内标寡核苷酸作为条码被被印刷在微阵列芯片上;优选的,重复点和内标点被用于测定检测的特异性和可重复性。
附图说明
图1是用多重PCR扩增微生物靶序列的扩增原理分解图。
图2是用多重PCR扩增病毒中靶序列的扩增原理分解图。
图3是用多重PCR扩增基因组突变的扩增原理分解图。
图4是癌症中RNA作为分子标记的检测的原理分解图。
图5是利用带有条码序列的微阵列芯片的检测原理分解图。
图6是本发明的扩增反应与带有条码序列的微阵列芯片进行靶核苷酸进行检测的步骤和原理分解图。
图7是用于点突变或靶向等位基因的ARMS分析的阵列的主要步骤流程图。
图8是以RNA水平作为生物标记的癌症检测原理分解图。
详细说明
PCR方法
PCR产物经常被用于测序分析来确定序列和基因组改变,或用于分子克隆作靶标序列的功能分析。出于不同的测定目的PCR方法已经被修改。以下是各种PCR的实例:
逆转录PCR:该方法用于RNA的检测和定量。在这种情况下,RNA分子首先通过逆转录酶被转换为互补DNA(cDNA)分子,该过程被称为逆转录。逆转录酶是用RNA模板并用RNA3'末端互补的短引物指导第一链cDNA合成的酶。逆转录酶和PCR的组合有时被称为逆转录PCR(RT-PCR),其使得RNA分子可以检测,定量或克隆。常用的逆转录酶包括禽成髓细胞瘤病毒(AMV)逆转录酶,莫洛尼鼠白血病病毒(M-MuLV)逆转录酶及其修饰的变异型。样品中 RNA的检测可用于诊断RNA病毒,例如人免疫缺陷病毒(HIV),流感病毒,甲型和丙型肝炎病毒,登革热病毒等。信使RNA(mRNA)和一些非编码RNA 分子的定量分析RNA分子,例如微小RNA和长非编码RNA,有时在精确医学的癌症患者的癌症诊断和患者分级中被用作生物标志物。
定量PCR(qPCR):该方法用于定量检测样品中DNA,cDNA或RNA的起始数量。荧光染料,例如Sybr绿,Eva绿或含荧光的寡核苷酸探针被包括在 PCR反应中。实时测量扩增产物的数量可以确定样品中是否存在靶核苷酸序列以及其在样品中的拷贝数。定量PCR有时被称为实时PCR。
数字PCR(dPCR):dPCR是另一种用于定量样品中靶标序列的PCR方法。将模板DNA分子稀释并分离成多个区室或微小液滴,使得一些液滴仅含有一个拷贝的靶标序列,而其它液滴可不含任何靶标序列。在每个液滴中进行PCR反应。在PCR反应结束时,将对靶标序列的存在(阳性扩增信号)或不存在(阴性,无扩增信号)进行计数,从而对样品中靶表序列的拷贝数提供一个直接的计数。
多重PCR:在单个PCR反应中包含多个引物对,那样导致多种靶标序列的扩增。这个方法允许在单个反应中扩增和分析多种基因或靶标。每一个引物对的退火点必须被优化来获得最佳的结果。为了通过凝胶电泳来分析PCR产物,扩增子的大小要是不同的来从而在一个胶上区分开来。多重PCR也用来下一代测序的文库构建。
巢式PCR:靶序列特异的两组引物在两个连续PCR中被使用。在第一轮反应中,一对引物用于产生靶标DNA的产物,其除了预期的靶标序列之外还可以包含靶标序列的侧翼序列。然后将产物用于具有一组引物的第二轮PCR,所述引物的互补结合位点在第一轮PCR反应的产物内。巢式PCR通常用于增加低丰度靶标序列的DNA扩增特异性。
等位基因特异PCR:等位基因是相同基因或相同遗传位点的多种替代形式之一。基因中一个或多个核苷酸的改变导致不同的等位基因。等位基因特异PCR 可用于检测基因中单核苷酸和几个核苷酸的变异。有时称为扩增耐受突变系统(ARMS)PCR,一种特异性扩增仅具有一个或几个核苷酸变异的等位基因的方法。如果突变或等位基因改变是已知的,则引物可以设计成其3'末端含有单核苷酸变异周围并与其相匹配的碱基对缓冲,使得在严格条件下,匹配的等位基因被有效扩增,而防止扩增错配的等位基因。因此,如果PCR反应中存在给定的等位基因,则该等位基因特异性的引物对将产生产物,但不产生所有其他替代等位基因的产物。等位基因特异PCR也可以用经修饰的寡核苷酸进行,所述经修饰的寡核苷酸与互补核酸序列的结合具有较高的热稳定性和特异性,但其不能用作 DNA聚合酶的引物,因此会阻断扩增。例如,肽核酸(PNA)用于PCR夹持以分析单碱基对突变(3)。
不对称PCR:此方法用于扩增DNA模板的一条DNA链,因为在这种情况下仅需要扩增两条互补链中的一条。产物可用于测序和/或杂交。PCR照常进行,除了在反应中仅需加入一种引物。或者,在反应中加入两种引物的情况下,其中一个的加入量少得多。
修饰PCR。修饰的dNTP可以在PCR过程中可以被整合入DNA,在检测中起报告子的功能(4)。用修饰的dNTP的PCR来标记PCR产物的原理与切口平移和随机标记方法相似。嘧啶的5位和嘌呤的7位的核碱基大沟的修饰在DNA 聚合酶介导的靶标序列的标记过程中进行通常具有更好的效率。连接修饰分子 (例如生物素,荧光分子)和核苷酸的连接臂的灵活性和长度将对dNTP底物属性产生影响。例如,生物素-4-dUTP,生物素-11-dUTP和生物素-16-dUTP代表具有不同长度连接臂的生物素标记的dUTP。将生物素标记的dUTP整合到PCR 产物上,从而允许用卵白素或链霉亲和素标记的报告分子来检测PCR产物。
具有修饰的引物的PCR:在PCR反应中使用的引物可以被修饰以增加其特异性和/或减少其长度。增加引物特异性的修饰实例是在5'端与小沟结合剂缀合(5) 整合并入锁核酸(6)。
等温核酸扩增技术:目前存在多种等温扩增技术。在现有方法中,多数方法为从ssDNA模板中产生DNA扩增子。值得注意的例外是核酸序列的扩增 (NASBA)和一些类似的方法,其没有逆转录酶步骤直接扩增RNA,这是被RNA 病毒检测所热切期待的。已开发出等温链置换聚合酶反应(ISDPR)克服了PCR 的缺点。在ISDPR中不需要热循环,在成本和简便方面提供了显著的优点。
上述很多PCR技术可以如同本发明所示被用于靶标序列的检测。
微阵列芯片
微阵列芯片是其固体表面具有一些生物材料的微观斑点集合的装置。点样在阵列上的生物材料,例如DNA,蛋白质,组织等,它们也被称为DNA微阵列,蛋白质微阵列和组织微阵列等。DNA微阵列是本领域已知的,并且用于测量大量同时表达的基因水平或对某个基因组的多个区域进行基因分型(7-9)。
每个DNA点含有数皮摩尔(10-12摩尔)相同的DNA链,称为探针或寡核苷酸,其被用于与样品中的DNA和cDNA分子杂交。通过检测荧光团,银或化学发光标记的靶标,通常检测和定量探针-靶标杂交,以确定样品中核酸序列的相对丰度。DNA微阵列的原理是两条具有互补序列的核酸链之间的杂交,具有互补核苷酸碱基对之间可以形成氢键而彼此特异性配对。在用合适的缓冲液洗涤去除非特异性结合序列之后,可以通过荧光,银或化学发光剂检测到与印刷在阵列上的探针或寡核苷酸特异性的强配对链。来自斑点的信号强度跟与斑点上的探针结合的靶标的数量相关,提供样品中靶序列的相对量。
在DNA微阵列中使用的探针可以作为斑点印刷在固体表面上,例如玻璃,塑料和微珠。每个斑点含有一个相同的探针。在单个玻片,芯片或板的形式的固体表面上具有不同孔穴数量例如24,48,96孔板,成百上千个斑点以有序的行和列排列在上面。每个斑点的精确位置和序列被记录在计算机数据库中,并用于检测样品中的靶标序列。本发明将使用点样在微阵列上的序列条形码来检测和定量样品中的靶核酸序列。
试样中的传染性微生物中特异衍生的生物分子的检测常常被用于传染病的鉴别诊断,包括由病毒,细菌和真菌引起的疾病。这些生物分子包括抗原,蛋白质和基因组(RNA和DNA分子)。生物体的基因组,RNA(对于RNA病毒) 或DNA(对于DNA病毒和所有其他微生物)可以通过使用生物体特异引物的 PCR来检测。对于以DNA作为其基因组材料的传染性微生物,例如DNA病毒和大多数其他微生物,包括支原体,衣原体,细菌和真菌,其基因组可直接用作PCR分析的模板。通过使用基于液体或固体基质的方法可以从试样中分离基因组DNA。其中一些方法使用特定的试剂去除一些可能干扰PCR反应的抑制剂。
因为PCR检测中使用的引物应对检测微生物的基因组具有高度特异性,所以 PCR检测对于诊断的确认是具有高度特异性的。然而样品中多种或未知微生物的检测通常具有挑战性。因此,可以同时筛查试样中多种微生物的方法将有助于临床诊断。
一些微生物可能经历了基因组进化,其导致对一些治疗处理产生抗性,这些处理对其野生型对应物有效。在这种情况下,通常需要多种测定来检测传染性微生物,那些微生物的亚型和特定的抗性菌株,为治疗导向提供诊断信息。能够同时检测传染性微生物、亚型和抗性菌株的方法将改进诊断所需的时间和成本。
基因组由DNA组成并且可用于PCR分析的微生物的实例是(但不限于) 曲霉属,黄曲霉,烟曲霉,构巢曲霉,念珠菌,念珠菌,新型隐球菌,格鲁氏隐球菌,隐球菌,疱疹病毒,乙型肝炎病毒,单纯疱疹病毒1-2,人巨细胞病毒,人巨细胞病毒,人巨细胞病毒,人巨细胞病毒,肺炎支原体,人疱疹病毒,JC 病毒,乳头瘤病毒1-82,细小病毒B,假痘病毒,SV40病毒,牛痘病毒,水痘带状疱疹病毒和天花病毒,一些微生物可能经历了基因组进化,其导致对一些治疗处理产生抗性,这些处理对其野生型对应物有效。在这种情况下,通常需要多种测定来检测传染性微生物,那些微生物的亚型和特定的抗性菌株,为治疗导向提供诊断信息。能够同时检测传染性微生物、亚型和抗性菌株的方法将改进诊断所需的时间和成本。
基因组由RNA组成且在PCR之前需要逆转录酶将其RNA转化为cDNA 的病毒包括但不限于星状病毒,布尼亚瓦病毒,加利福尼亚脑炎病毒,路易斯脑炎病毒,西尼罗病毒,日本脑炎病毒,东方马脑炎病毒,西部马脑炎病毒,委内瑞拉马脑炎病毒,墨累谷脑炎病毒,基孔肯雅病毒,蜱热病毒,出血热病毒,柯萨奇病毒A 1-24,柯萨基病毒B1-6,登革热病毒1-4,杜文海格(Duvenhage) 病毒,东部马脑炎病毒,埃博拉病毒,回声病毒1-24,肠道病毒1-71,肠道冠状病毒,汉坦病毒,甲型肝炎病毒,丙型肝炎病毒,E病毒,人免疫缺陷病毒 (HIV)1和2,呼吸道冠状病毒,轮状病毒,T-淋巴细胞病毒,流感病毒A,流感病毒B,胡宁(Junin)病毒,拉沙热病毒,麻疹病毒,腮腺炎病毒,诺沃克病毒,淋巴细胞性脉络丛脑膜炎病毒,副流感病毒1-4,脊髓灰质炎病毒1-3,狂犬病病毒,呼吸道合胞病毒,鼻病毒1-113,罗西欧(Rocio)病毒,风疹病毒,水疱性口炎病毒,黄热病毒,寨卡病毒。
本发明的实施例可用于检测或筛查多种疾病或病理状况,例如癌症。可以通过本发明的方法和组分评估的癌症包括癌细胞,其包括来自膀胱,血液,骨,骨髓,脑,乳腺,结肠,食管,胃肠,牙龈,头,肾,肺,鼻咽,颈,卵巢,胰腺,前列腺,皮肤,胃,睾丸,舌或子宫的细胞和癌细胞。
此外,已明确癌症具有以下组织学类型,尽管它不限于这些:肿瘤,恶性;癌;癌,未分化;巨细胞和纺锤体细胞癌;小细胞癌;乳头状癌;鳞状细胞癌;淋巴上皮癌;基底细胞癌;;移行细胞癌;乳头状移行细胞癌;腺癌;胃泌素瘤胆管癌;肝细胞癌;联合肝细胞癌和胆管癌;小梁腺癌;腺样囊性癌;腺瘤息肉腺癌;腺癌,家族性结肠息肉;实体癌;类癌;恶性肿瘤;分支-肺泡腺癌;乳头状腺癌;色素癌;嗜酸性细胞癌;嗜酸性腺癌;嗜碱性粒细胞癌;透明细胞腺癌;颗粒细胞癌;滤泡腺癌;乳头状和滤泡腺癌;非包膜性硬化癌;肾上腺皮质癌子宫内膜癌;皮肤附属癌;顶分泌腺癌;皮脂腺癌;宫颈腺癌;粘液表皮样癌;囊腺癌;乳头状囊腺癌;乳头状浆液性囊腺癌;粘液性囊腺癌;粘液腺癌;印戒细胞癌;浸润性导管癌;髓样癌;小叶癌;炎性癌;佩吉特病,乳腺;腺泡细胞癌;腺鳞癌;腺癌/鳞状上皮化生;胸腺瘤卵巢基质瘤,恶性;恶性肿瘤;颗粒细胞瘤,恶性;成胶质细胞瘤;塞尔托利氏(sertoli)细胞癌;淋巴细胞肿瘤脂质细胞肿瘤,恶性;副神经节瘤乳腺外神经胶质瘤,恶性;嗜铬细胞瘤;肾小球系膜恶性黑素瘤;无色性黑素瘤;浅表扩散性黑素瘤;巨型黑色素瘤上皮样细胞黑素瘤;蓝痣肉瘤;纤维肉瘤;纤维组织细胞瘤粘液肉瘤;脂肪肉瘤;平滑肌肉瘤横纹肌肉瘤;胚胎横纹肌肉瘤;肺泡横纹肌肉瘤;间质肉瘤混合性肿瘤;宫颈内膜(mullerian) 混合肿瘤;肾母细胞瘤;肝母细胞瘤;癌肉瘤间质瘤布伦纳肿瘤叶状肿瘤滑膜肉瘤间皮瘤无性系胚胎性癌;畸胎瘤卵巢癌绒毛膜癌;中肾型血管肉瘤;血管内皮瘤,恶性;卡波西氏肉瘤;血管外皮细胞瘤淋巴管肉瘤骨肉瘤;近端骨肉瘤;软骨肉瘤软骨母细胞瘤间质软骨肉瘤;骨巨细胞瘤;尤因肉瘤;牙源性肿瘤,恶性;成釉细胞性耳科肉瘤;成神经管细胞瘤成釉细胞纤维肉瘤;松果体脊索瘤神经胶质瘤室管膜瘤星形细胞瘤原发性星形细胞瘤;纤维性星形细胞瘤;成星细胞瘤;胶质母细胞瘤;少突胶质细胞瘤;少突胶质细胞瘤;原始神经外胚层小脑肉瘤神经母细胞瘤;神经母细胞瘤;视网膜母细胞瘤;嗅神经源性肿瘤;脑膜瘤神经纤维肉瘤;神经鞘瘤颗粒细胞肿瘤,恶性;恶性淋巴瘤霍奇金病;霍奇金淋巴瘤;副神经管恶性淋巴瘤,小淋巴细胞;恶性淋巴瘤,大细胞,弥漫性;恶性淋巴瘤,滤泡;蕈样真菌病;其他指定的非霍奇金淋巴瘤;恶性组织细胞增多;多发性骨髓瘤;肥大细胞肉瘤;免疫增生性小肠疾病;白血病;淋巴样白血病;浆细胞白血病;红白血病淋巴肉瘤细胞白血病;骨髓性白血病;嗜碱性白血病;嗜酸性白血病;单核细胞白血病;肥大细胞白血病;巨核细胞白血病;骨髓肉瘤;和毛细胞白血病。此外,RNA水平的基因突变和改变被评估为癌前潜伏期,例如转化,异常结构和增生。
本发明的实施例可用于检测癌症和遗传疾病中的基因突变,考贝术改变或表达水平的改变。在诊断试样的癌症和/或遗传疾病中突变分析的需求增加。各种类型的癌症的基因组特征已经证明,具有相同器官起源,组织病理学诊断和临床阶段的癌症,它们的遗传改变和对抗癌治疗的反应可以是高度多样化的。现在知道抗癌治疗的成功在很大程度上取决于鉴定患者亚组的能力,该亚组能响应特定治疗试剂。
例如,在表皮生长因子受体(EGFR)基因中具有激活突变的癌症患者将从EGFR拮抗剂吉非替尼(10),厄洛替尼(11),伊洛替尼(12)和阿法替尼(13) 的治疗中受益。激活ALK基因(14)或ROS1(15)基因和基因重组的癌症可以用ALK和ROS1拮抗剂克唑替尼(16),色瑞替尼和艾乐替尼(17,18)治疗。具有v-Raf鼠肉瘤病毒癌基因同源物B1(BRAF)激活突变的癌症可用BRAF抑制剂威罗非尼治疗(19);并且可以用聚(ADP-核糖)聚合酶(PARP)抑制剂奥拉帕尼(20)治疗BRCA1或BCRA2基因突变的癌症。因此,各种癌症相关基因的突变分析是为治疗癌症患者提供精确药物所需的。
然而,相同癌症相关基因的不同突变可能对治疗反应具有不同的影响。对于EGFR基因,最常见的突变是外显子19的框内缺失和外显子21的L858R点突变。在观察到的肺癌的EGFR突变中这些突变一起约占85%(21,22)。具有这些突变的肿瘤对于罗替尼,吉非替尼和伊诺替尼的治疗高度敏感。其他较不常见的突变的肿瘤,例如在外显子18上的G719突变和外显子21上的L861Q的突变,也可以用EGFR拮抗剂治疗(10)。然而,具有外显子20框内插入的肿瘤,其约占所有EGFR突变的4%-10%,通常对EGFR拮抗剂的治疗没有反应(23)。在EGFR基因中的T790M突变通常在约50%的继发耐药患者中被检测到,这些患者最初对厄洛替尼,吉非替尼或烟碱有反应但在后续会转变为耐药或具有抗性 (24)。幸运的是,具有EGFR T790M突变的癌症可以用第三代EGFR拮抗物莫西替尼(25)治疗。因此,检测这些临床试样中的突变对于癌症治疗中的精确用药是必须的。
在癌症突变分析的一个实施例中,已知许多癌症通常具有在一个以上的基因突变。在其它癌症中由基因引起的共突变已显示对抗癌治疗具有效果。研究人员发现,KRAS基因突变或KRAS和TP53双基因突变的癌症对多西他赛加西洛莫尼联合治疗敏感,但KRAS和STK11双突变的癌症对这种联合治疗有抗性 (26)。因此,癌症诊断和精确治疗将需要同时检测多个区域(密码子)和多个基因中的突变。
为了确定癌症的突变,通常从肿瘤或体液(例如血液,尿液,痰等)中存在的癌细胞中分离基因组DNA。通过手术去除的或通过活检样品获得的肿瘤组织通常被用来分离基因组DNA进行突变试验。一些最近的研究已经显示癌细胞可以将其DNA释放到体液中,可以在癌症患者的血样中细胞外DNA(也称为游离DNA,cfDNA)中检测肿瘤特异改变(突变,易位,异常甲基化和拷贝数改变)。CfDNA片段通常从死细胞中释放并以低浓度存在于体液中,包括血浆,血清,脑脊液和尿液(27)。循环cfDNA可在健康个体的血浆和血清中被检测到,但在急性体育运动(28),妊娠(29)和与细胞死亡增加相关的病理条件下其水平会增加,例如炎症(30),心肌梗死(31),烧伤(32)和移植排斥(33)。
此外,循环cfDNA水平在患有各种癌症的患者中也会急剧增加(34)。一些报道显示,从健康个体或良性疾病患者中分离的血浆cfDNA由小片段组成,这些小片段与核小体DNA(~150-200bp)(35,36)的增加量(1-5x)相对应,表明大多数循环cfDNA是来自凋亡细胞。在获自癌症患者的样品中经常检测到血浆cfDNA中的非凋亡性DNA片段(大于和小于凋亡性DNA片段)的数量的增加(36,37),表明这些患者中的cfDNA不仅来源于细胞凋亡,也包括坏死,自噬或有丝分裂障碍。癌症的循环cfDNA的测试也称为液体活检。
使用cfDNA的液体活检测试被预计具有克服常规活组织检查中肿瘤样品获得的障碍,因为液体活检侵入性较小,成本较低,易于从样品进行纵向动态评估。这使得用于癌症诊断,用于早期癌症筛查检测以及用于鉴定可操作的基因组改变的液体活检来指导精确医学的兴趣日益增长。因为循环cfDNA的半衰期短 (约16分钟)(38),样品中特异性突变的纵向动态变化分析可用于监测治疗反应,疾病复发和治疗抗性突变体的出现(39)。
在大多数情况下,肿瘤组织由癌细胞和正常细胞组成,包括基质细胞,例如纤维母细胞,浸润的淋巴细胞和炎性细胞。因此,从肿瘤组织分离的基因组 DNA通常由正常DNA和突变肿瘤细胞的DNA组成。突变分析经常显示存在正常等位基因和突变体等位基因。目前,cfDNA的突变分析可以使用dPCR(40,41) 或下一代测序进行(具有小组癌症相关基因(42,43)的cfDNA突变)。
基于dPCR的突变检测限于先前已知存在于原发性肿瘤中的癌症驱动突变的特异性热点,而cfDNA样品的下一代测序限于小组基因(文献中报道约50 个)(42),43)。对于临床应用,为了肺癌的早期诊断和精确治疗,两者在发现cfDNA的肿瘤特异性突变方面都不是最佳的。使用cfDNA检测肿瘤特异性突变的主要挑战之一是只能从血浆中分离非常有限的cfDNA,这不足以在检测癌症多个热点突变的dPCR中使用,或者不足以用于较大量的癌症相关基因的基因组分析。
本发明的实施例可以在单次测定中检测cfDNA中一个和/或多个基因的多个位点的基因突变,解决在dPCR中遇到的问题,即在一个双色通道测定(野生型与突变体)中只能检测一个突变体。通过dPCR检测多个突变将需要大量的 cfDNA,这在大多数情况下是不可行的。它还解决了下一代测序分析所需的时间和成本,以省时和高性价比的方式提供结果。本发明中提出的方法缩小了液体活检中的基因突变检测中dPCR和下一代测序之间的差距。
具体实施方式
实施例1.以DNA为基因组的微生物的检测
图1片显示了用于传染性微生物的DNA基因组检测的初始浓度。在样品于微阵列芯片插件前进行两轮PCR反应,该微阵列芯片含有成千上万的15bp至 25bp的条码寡核苷酸。在第一轮PCR反应中,使用微生物基因组特异的引物对。对于每一个引物对,与被印刷在微阵列上的条码序列的部分相同的一段序列被添加到一个引物的5'端(例如正向引物),然后一个通用引物序列被添加到另一引物上(反应引物)。
然后PCR产物被用于第二轮PCR,其反向引物仅有一个,与第一轮反应中使用的通用反向引物序列相同。这个通用反向引物也在5'端用报告分子标记,例如生物素或荧光染料。第二轮PCR也用仅有报告分子标记的通用引物的不对称 PCR完成。第二轮PCR反应后,反应混合物被滴到检测试剂条设备上,当反应混合物移动穿过硝酸纤维素膜时,产物被杂交到条码寡核苷酸探针上。层析法去除其他杂质后,从报告分子的浓度就可以测定出杂交的的靶标传染性微生物序列和微生物基因组拷贝数的相对量。当通用引物用生物素标记时,金颗粒、荧光染料标记的链霉亲和素或卵白素可以用于可视化与试剂条杂交的PCR产物。当使用金标记时,杂交可以直接可视化。该途径能检测样品中多种传染性微生物。每一次PCR反应的引物浓度,PCR反应缓冲液和PCR循环基于此目的被优化。
对于每一个条码序列,至少两个序列可以被印到微阵列芯片上:一个与设计的条码序列精确配对,用于捕捉来自第二轮PCR反应的标记的DNA链,另一个是相同的但在中间位置有1-5个碱基(错配)的差异。错配的寡核苷酸在特异杂交中起内标和减少背景和交叉杂交信号的作用。
实施例子2.用PCR条码微阵列芯片检测RNA病毒
一般程序、方法和原则与实施例1中描述的相同。在样品用实施例1所述的各种方法进行分析前,进行一个反转录来把RNA变换为cDNA。反转录中使用的引物可以是寡脱氧胸苷酸-dT(12-mer至20-mer,或它们的混合物),随机引物或靶标RNA序列特异的引物池。寡-dT引物用于多聚腺苷化RNA的反转录,例如mRNA。随机引物是6-mer寡核苷酸并用于非多聚腺苷化RNA的反转录。特异引物序列用于检测一组确定的RNA病毒。反转录和第一轮PCR反应可以在一步法中进行。
实施例子3.基因组DNA中突变体和突变等位基因的检测
通用程序、方法和原则与实施例1中所述的相同,第一轮PCR反应中石油的一种引物含有条码序列,能与等位基因在3'端特异配对,例如突变等位基因,和单核苷酸多态性,而含有通用引物序列的引物与所检测基因的常见区域配对。通过包含一些修饰物,等位特异引物的特异性会增强,例如在引物的3'端使用一个锁核苷酸,其能与所测等位基因的核苷酸配对。有时,一个附加的错配被添加到3'端附近来增加引物的特异性。这个方法能检测样品中的多种突变,也用于检测癌症样品中的突变体,包括原发性肿瘤和液体活体组织切片样品。
实施例4.RNA作为分子标记癌症检测
细胞中RNA水平的改变,例如mRNA、小RNA和长非编码RNA被用于癌症的生物标记。这个用途也在本发明所述的方法中使用。细胞内检测RNA水平的方法和原则与实施例1-2中的相同。首先RNA被反转录转换为cDNA。样品经过PCR和微阵列芯片分析。基于荧光信号强度,样品中RNA分子的相对数量可以被测定。
在缺少本文中所具体公开的任何元件、限制的情况下,可以实现本文所示和所述的发明。所采用的术语和表达法被用作说明的术语而非限制,并且不希望在这些术语和表达法的使用中排除所示和所述的特征或其部分的任何等同物,而且应该认识到各种改型在本发明的范围内都是可行的。因此应该理解,尽管通过各种实施例和可选的特征具体公开了本发明,但是本文所述的概念的修改和变型可以被本领域普通技术人员所采用,并且认为这些修改和变型落入所附权利要求书限定的本发明的范围之内。
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参考文献
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Claims (10)
1.一种多重聚合酶链式反应(PCR)与带有条码序列的微阵列芯片的检测系统(BDLF)组合用于筛查和检测传染性微生物或筛查疾病相关基因组突变,包括癌症和遗传疾病的方法:该方法包括:进行第一和第二轮PCR反应;其中,第一轮PCR中使用的引物包含具有下列特性的引物对:除了所测试的靶标特异序列以外,第一引物在5'端含有一个额外的条码序列;而引物对的第二引物含有通用引物序列,与所有引物对一样具有相同序列。
2.根据权利要求1所述的方法,其中,进行第二轮PCR反应使用的引物由独特的条码寡核苷酸引物和通用引物组成;其中,通用引物用报告分子标记,如生物素、荧光染料、荧光纳米颗粒、量子微粒或化学发光染料中的一种。
3.根据权利要求2所述的方法,其中,所述的报告分子可以是任何允许进行PCR产物检测的分子,包括生物素、荧光染料。当生物素被使用时,标记到卵白素或酶联亲和素上的报告分子发射的信号被用于PCR产物检测。
4.根据权利要求2所述的方法,其中,所述的报告分子发射的信号可以是分子,其发出荧光及化学发光,或通过酶促反应发出荧光或化学发光,或像金颗粒一样直接肉眼可见。
5.根据权利要求1所述的方法,其中,在所述的条码微阵列芯片是具有成百上千个点的微阵列芯片,每一个点含有独特的条码寡核苷酸用于捕获第二轮PCR中由通用引物合成的DNA链。
6.根据权利要求5所述的方法,其中,每一个条码序列可以被印刷在不只一个点上;或者,对于每一个条码序列,具有与条码相同的序列但中间有1个或多个核苷酸位点错配的内标寡核苷酸作为条码被印刷在微阵列芯片上;优选的,重复点和内标点被用于测定检测的特异性和可重复性。
7.根据权利要求1或者6所述的方法,其中,所述的条码微阵列芯片被印刷在固体表面作为芯片,或在多梯井道里的固体表面,例如96孔板上。
8.根据权利要求1所述的方法,其中,该方法或者方法所使用的系统可以被用于临床分析中的床边(POC)诊断检测,食品安全和环境监测。
9.根据权利要求1所述的方法,其中,该方法或者方法所使用的系统用于以下检测:引起传染病的微生物是所有引起传染性疾病的微生物,其具有或不具有临床症状。这些传染性微生物中的部分可以引起癌症。
10.根据权利要求1所述的方法,其中,该方法或者方法所使用的系统被用于筛查和检测基因组DNA中的突变,考贝数改变或基表达水平的改变。
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