CN111876469B - 利用核酸类似物进行靶核酸检测的方法 - Google Patents
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Abstract
本发明提供了利用核酸类似物进行靶核酸检测的方法,具体地提供了一种利用核酸类似物进行靶核酸检测的方法、系统和试剂盒,所述的检测方法包括向含有靶核酸的反应体系中加入gRNA、Cas蛋白和单链核酸类似物检测器。
Description
技术领域
本发明涉及核酸检测领域,涉及利用核酸类似物进行靶核酸检测的方法,尤其涉及利用核酸类似物进行靶核酸检测的方法、系统和试剂盒。
背景技术
特异性检测核酸分子(Nucleic acid detection)方法具有重要的应用价值,例如病原体的检测,遗传病检测等。在病原体检测方面,由于每种病原体微生物都有其独一无二的特征核酸分子序列,因此可以开发出针对特定物种的核酸分子检测,也称为核酸诊断(NADs,nucleic acid diagnostics),在食品安全、环境微生物污染检测,人体病原菌感染等领域具有重要义。另一个方面是对人或其他物种的单核苷酸多态性(SNPs,singlenucleotide polymorphisms)的检测。在基因组水平上去理解遗传变异和生物学功能之间的关系为现代分子生物学提供了新视角,而其中SNPs对生物学的功能、进化和疾病等密切相关,因此SNPs的检测与分析技术的发展尤为重要。
目前建立的特异性核酸分子检测通常需要分为两步,第一步是目的核酸的扩增,第二步是目的核酸检测。现有检测技术包括限制性核酸内切酶方法、Southern、Northern、斑点杂交、荧光PCR检测技术、LAMP环介导等温扩增技术、重组酶聚合酶扩增技术(RPA)等方法。2012年之后,CRISPR基因编辑技术兴起,张锋团队基于RPA技术开发了一种以Cas13为核心的靶向RNA的新核酸诊断技术(SHERLOCK技术),Doudna团队开发了一种以Cas12酶为核心的诊断技术(DETECTR技术),中国科学院上海植物生理生态研究所王金博士等也开发了一种基于Cas12的新型核酸检测技术(HOLMES技术)。基于CRISPR技术开发的核酸检测技术正在日益发挥重要作用。
在基于CRISPR的核酸检测技术中,核酸探针或者核酸检测器是上述检测技术的关键元件,本发明对核酸探针进行了改进,选择了核酸类似物作为探针,从而扩展了该技术的应用范围。
发明内容
本发明提供了利用核酸类似物进行靶核酸检测的方法、系统和试剂盒。
一方面,本发明提供了一种检测样品中靶核酸的方法,所述方法包括将样品与V型CRISPR/CAS效应蛋白、gRNA(指导RNA)和单链核酸类似物检测器接触,所述gRNA包括与所述CRISPR/CAS效应蛋白结合的区域和与靶核酸杂交的导向序列;检测由CRISPR/CAS效应蛋白切割单链核酸类似物检测器产生的可检测信号,从而检测靶核酸。
另一方面,本发明还提供了一种用于检测样品中靶核酸的系统或组合物,所述系统或组合物包括V型CRISPR/CAS效应蛋白、gRNA(指导RNA)和单链核酸类似物检测器,所述gRNA包括与所述CRISPR/CAS效应蛋白结合的区域和与靶核酸杂交的导向序列。
另一方面,本发明还提供了一种用于检测样品中靶核酸的试剂盒,所述试剂盒包括V型CRISPR/CAS效应蛋白、gRNA(指导RNA)和单链核酸类似物检测器,所述gRNA包括与所述CRISPR/CAS效应蛋白结合的区域和与靶核酸杂交的导向序列。
另一方面,本发明还提供了上述系统或试剂盒在检测样品中靶核酸的应用。
另一方面,本发明还提供了V型CRISPR/CAS效应蛋白在检测样品中靶核酸中的应用。
如上所述的应用,所述V型CRISPR/CAS效应蛋白在与样品中的靶核酸结合或杂交后,可以切割体系中的单链核酸类似物检测器。
另一方面,本发明还提供了V型CRISPR/CAS效应蛋白在制备检测样品中靶核酸的试剂中的应用。
核酸类似物包括,但不限于:2’-O-甲基取代RNA、锁核酸、桥核酸、吗啉核酸、乙二醇核酸、己糖醇核酸、苏糖核酸、阿拉伯糖核酸、2’氧甲基RNA、2’甲氧基乙酰基RNA、2’-氟RNA、2’-氨基RNA、4’-硫RNA及其组合,包括任选的核糖核苷酸或脱氧核糖核苷酸残基。
锁核酸(LNA):LNA是2’修饰的核苷,其包含连接所述核苷的核糖糖环的C2’和C4’的双基,所述双基限制或锁定核糖环的构象,其结构式如下所示,LNA的碱基可以选自腺嘌呤,胞嘧啶,鸟嘌呤,5-甲基-胞嘧啶,胸腺嘧啶和尿嘧啶。
吗啉(Morphol ino,MNA),MNA为吗啉核酸,或称吗啉代核酸,其单体结构如下所示:
桥核酸(BNA),桥核酸,或者称之为桥连核酸,其单体结构如下所示:
乙二醇核酸(GNA),乙二醇核酸单体结构如下所示:
苏糖核酸(TNA),苏糖核酸的单体结构式如下所示:
己糖醇核酸(HNA),己糖醇核酸的单体结构如下所示:
阿拉伯糖核酸(ANA),阿拉伯糖核酸的单体结构如下所示:
2’氧甲基RNA(2’O-Methyl RNA),2’氧甲基RNA单体结构如下所示:
2’甲氧基乙酰基RNA(2’-O-methoxy-ethyl RNA),单体结构如下所示:
2’-氟RNA(2’-F RNA),单体结构如下所示:
2’-氨基RNA(2’-Amino RNA),单体结构如下所示:
4’-硫RNA(4’-S RNA),单体结构如下所示:
在一个实施方式中,所述核酸类似物为脱氧核糖尿嘧啶(dU),所述脱氧核糖尿嘧啶为将尿嘧啶的2’羟基脱掉所形成的脱氧核糖尿嘧啶(dU)。
在其他的实施方式中,所述核酸类似物可以为2’氧甲基RNA(2’O-Methyl RNA),或者称之为2’OCH3-RNA,其碱基可以为A、U、C、G,也可以为T;尽管T为脱氧核糖核苷酸,但在存在2’氧甲基修饰时,本领域通常也称之为2’氧甲基RNA。
在其他的实施方式中,所述核酸类似物可以为2’-氟RNA(2’-F RNA),其碱基可以为A、U、C、G、T。
在优选的实施方式中,所述单链核酸类似物检测器为寡聚核酸类似物。
所述单链核酸类似物检测器不与所述gRNA杂交。
进一步的,所述V型CRISPR/CAS效应蛋白选自Cas12、Cas14家族蛋白或其突变体;在一个实施方式中,所述Cas蛋白优选为Cas12家族,包括但不限于Cas12a、Cas12b、Cas12i、Cas12j中的一种或任意几种;所述Cas14家族蛋白选自Cas14a和/或Cas14b。
在一个实施方式中,所述Cas12a选自FnCas12a、AsCas12a、LbCas12a、Lb5Cas12a、HkCas12a、OsCas12a、TsCas12a、BbCas12a、BoCas12a或Lb4Cas12a中一种或任意几种;所述Cas12a优选为LbCas12a,氨基酸序列如SEQ ID No.1所示,或者,将SEQ ID No.1所示氨基酸序或其活性片段经过一个或多个(如2个、3个、4个,5个,6个,7个,8个,9个或10个)氨基酸残基的取代、缺失或添加而形成的,且具有基本相同功能的衍生蛋白。
在其他的实施方式中,所述Cas12b的氨基酸序列如SEQ ID No.2所示,或者,将SEQID No.2所示氨基酸序或其活性片段经过一个或多个(如2个、3个、4个,5个,6个,7个,8个,9个或10个)氨基酸残基的取代、缺失或添加而形成的,且具有基本相同功能的衍生蛋白。
在优选的实施方式中,所述Cas12i蛋白的氨基酸序列选自下组:
(1)SEQ ID No.3所示的蛋白;
(2)将SEQ ID No.3所示氨基酸序或其活性片段经过一个或多个(如2个、3个、4个,5个,6个,7个,8个,9个或10个)氨基酸残基的取代、缺失或添加而形成的,且具有基本相同功能的衍生蛋白。
所述Cas12j蛋白的氨基酸序列选自下组:
(1)SEQ ID No.4所示的蛋白;
(2)将SEQ ID No.4所示氨基酸序或其活性片段经过一个或多个(如2个、3个、4个,5个,6个,7个,8个,9个或10个)氨基酸残基的取代、缺失或添加而形成的,且具有基本相同功能的衍生蛋白。
在一个实施方式中,所述Cas蛋白突变体包括氨基酸取代、缺失或替换,且所述突变体至少保留其trans切割活性。优选地,所述突变体具有Cis和trans切割活性。
本发明中,所述靶核酸包括核糖核苷酸或脱氧核糖核苷酸,包括单链核酸、双链核酸,例如单链DNA、双链DNA、单链RNA、双链RNA。
本发明中,所述可检测信号通过以下方式实现:基于视觉的检测,基于传感器的检测,颜色检测,基于金纳米颗粒的检测,荧光偏振,胶体相变/分散,电化学检测和基于半导体的检测。
在一些实施方式中,本发明的方法还包括测量CRISPR/CAS效应蛋白(Cas蛋白)产生的可检测信号的步骤。所述Cas蛋白识别所述靶核酸或与所述靶核酸杂交之后可以激发单链核酸的切割活性,从而切割所述单链核酸类似物检测器进而产生可检测信号。
本发明中,所述可检测信号可以是当切割单链核酸类似物检测器时产生的任何信号。例如,基于金纳米颗粒的检测,荧光偏振,胶体相变/分散,电化学检测,基于半导体的传感。所述可检测信号可通过任何合适的方式读出,包括但不限于:可检测的荧光信号的测量,凝胶电泳检测(通过检测凝胶上的条带的变化),基于视觉或传感器的颜色的存在或不存在的检测、或者颜色存在的差异(例如,基于金纳米颗粒)以及电信号的差异。
在优选的实施方式中,所述可检测信号通过以下方式实现:所述单链核酸类似物检测器的5’端和3’端分别设置不同的报告基团,当所述单链核酸类似物检测器被切割后,可以表现出可检测的报告信号;例如,单链核酸类似物检测器的两端分别设置荧光基团和淬灭基团,当所述单链核酸类似物检测器被切割后,可以表现出可检测的荧光信号。
在一个实施方式中,所述荧光基团选自FAM、FITC、VIC、JOE、TET、CY3、CY5、ROX、Texas Red或LC RED460中的一种或任意几种;所述淬灭基团选自BHQ1、BHQ2、BHQ3、Dabcy1或Tamra中的一种或任意几种。
在其他的实施方式中,所述可检测信号还可以通过以下方式实现:所述单链核酸类似物检测器的5’端和3’端分别设置不同的标记分子,通过胶体金检测的方式检测反应信号。
在一个实施方式中,所述的靶核酸包括DNA、RNA,优选为单链核酸或双链核酸或核酸修饰物。
在一个实施方式中,所述靶核酸来源于病毒、细菌、微生物、土壤、水源、人体、动物、植物等样品。优选的,所述靶核酸为PCR、NASBA、RPA、SDA、LAMP、HAD、NEAR、MDA、RCA、LCR、RAM等方法富集或扩增的产物。
在一个实施方式中,所述方法还包括从样品中获得靶核酸的步骤。
在一个实施方式中,所述靶核酸为病毒核酸、细菌核酸、与疾病相关的特异核酸,如特定的突变位点或SNP位点或与对照有差异的核酸;优选地,所述病毒为植物病毒或动物病毒,例如,乳头瘤病毒,肝DNA病毒,疱疹病毒,腺病毒,痘病毒,细小病毒,冠状病毒;优选地,所述病毒为冠状病毒,优选地,SARS、SARS-CoV2(COVID-19)、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、Mers-Cov。
在一些实施方式中,所述靶核酸来源于细胞,例如,来源于细胞裂解液。
在一些实施方式中,所述可检测信号的测量可以是定量的,在其他的实施方式中,所述可检测信号的测量可以是定性的。
优选的,所述单链核酸类似物检测器在被所述Cas蛋白切割之前产生第一可检测信号,并且在被切割之后产生不同于第一可检测信号的第二可检测信号。
本发明中,所述gRNA包括靶向所述待检测特征序列的序列(导向序列)和识别Cas蛋白的序列(同向重复序列或其部分)。
本发明中,所述的导向序列包括10-40bp;优选地,12-25bp;优选地,15-23bp;优选地,16-18bp。
本发明中,所述gRNA与待检测特征序列至少有50%的匹配度,优选至少60%,优选至少70%,优选至少80%,优选至少90%。
在一个实施方式中,所述Cas蛋白与gRNA的用量摩尔比为(0.8-1.2):1。
在一个实施方式中,所述Cas蛋白的用量终浓度为20-200nM,优选,30-100nM,更优选,40-80nM,更优选,50nM。
在一个实施方式中,所述gRNA的用量终浓度为20-200nM,优选,30-100nM,更优选,40-80nM,更优选,50nM。
在一个实施方式中,所述靶核酸的用量终浓度为5-100nM,优选,10-50nM。
在一个实施方式中,所述单链核酸类似物检测器的用量终浓度为100-1000nM,优选,150-800nM,优选,200-800nM,优选,200-500nM,优选,200-300nM。
在一个实施方式中,所述单链核酸类似物检测器具有2-300个核酸类似物,优选,3-200个核酸类似物,优选,3-100个核酸类似物,优选,具有3-30个核酸类似物,优选,4-20个核酸类似物,更优选,5-15个核酸类似物。
术语“杂交”或“互补的”或“基本上互补的”是指核酸(例如RNA、DNA)包含使其能够非共价结合的核苷酸序列,即以序列特异性,反平行的方式(即核酸特异性结合互补核酸)与另一核酸形成碱基对和/或G/U碱基对,“退火”或“杂交”。杂交需要两个核酸含有互补序列,尽管碱基之间可能存在错配。两个核酸之间杂交的合适条件取决于核酸的长度和互补程度,这是本领域公知的变量。典型地,可杂交核酸的长度为8个核苷酸或更多(例如,10个核苷酸或更多,12个核苷酸或更多,15个核苷酸或更多,20个核苷酸或更多,22个核苷酸或更多,25个核苷酸或更多,或30个核苷酸或更多)。
应当理解,多核苷酸的序列不需要与其靶核酸的序列100%互补以特异性杂交。多核苷酸可包含60%或更高,65%或更高,70%或更高,75%或更高,80%或更高,85%或更高,90%或更高,95%或更高,98%或更高,99%或更高,99.5%或更高,或与其杂交的靶核酸序列中的靶区域的序列互补性为100%。
一般定义:
除非另有定义,否则本文所用的技术和科学术语具有与所属领域的普通技术人员之一通常理解的相同的含义。
术语“氨基酸”是指含有氨基的羧酸。生物体内的各种蛋白质是由20种基本氨基酸构成的。
术语“多核苷酸”、“核苷酸序列”、“核酸序列”、“核酸分子”和“核酸”可以互换使用,包括DNA、RNA或者其杂交体,可以是双链或单链的。
术语“寡聚核酸类似物”是指含有3-100个核酸类似物的序列,优选,具有3-30个核酸类似物,优选,4-20个核酸类似物,更优选,5-15个核酸类似物。
术语“同源性”或“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如,两个DNA分子的每一个中的某个位置都被腺嘌呤占据,或两个多肽的每一个中的某个位置都被赖氨酸占据),那么各分子在该位置上是同一的。两个序列之间。通常,在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用,例如,氨基酸序列的同一性可以通过常规方法,参考例如Smith and Waterman,1981,Adv.Appl.Math.2:482Pearson&Lipman,1988,Proc.Natl.Acad.Sci.USA 85:2444,Thompson etal.,1994,Nucleic AcidsRes 22:467380等的教导,通过计算机化运行运算法则(Wisconsin Genetics软件包中的GAP,BESTFIT,FASTA,和TFASTA,Genetics Computer Group)来确定。也可使用可从美国国立生物技术信息中心(NCBI www.ncbi.nlm.nih.gov/)获得的BLAST运算法则,使用默认参数确定。
如本文所用,所述“CRISPR”是指成簇、规律间隔的短回文重复序列(Clusteredregularly interspaced short palindromic repeats),其来自微生物的免疫系统。
如本文所用,“生物素(biotin)”也称维生素H,是一种分子量为244Da的小分子维生素。“亲和素(avidin)”,又称抗生物素,是一种碱性糖蛋白,具有4个同生物素亲和例极高的结合位点,常用亲和素有链霉亲合素。生物素与亲和素的极强亲和力可用于在检测体系中放大或增强检测信号。如生物素很易与蛋白质(如抗体等)以共价键结合,而结合了酶的亲和素分子与结合有特异性抗体的生物素分子产生反应,既起到了多级放大作用,又由于酶在遇到相应底物时的催化作用而呈色,达到检测未知抗原(或抗体)分子的目的。
靶核酸
如本文所用,所述“靶核酸”是指从生物样品(待测样品)中提取的多核苷酸分子。所述生物样品是从任何生物体获得、排泄或分泌的任何固体或流体样品,包括但不限于单细胞生物,例如细菌、酵母、原生动物和变形虫等,多细胞生物(例如植物或动物,包括来自健康或表面健康的人类受试者或受待诊断或调查的病症或疾病影响的人类患者的样品,例如病原微生物例如病原细菌或病毒的感染)。例如,生物样品可以是从例如血液、血浆、血清、尿液、粪便、痰液、粘液、淋巴液、滑液、胆汁、腹水、胸腔积液、血清肿、唾液、脑脊液、水性或玻璃体液、或任何身体分泌物、渗出液、渗出液(例如,从脓肿或任何其他感染或炎症部位获得的液体)中获得的生物液体或从关节(例如,正常关节或受疾病影响的关节,例如类风湿性关节炎、骨关节炎、痛风或脓毒性关节炎)获得的液体,或皮肤或粘膜表面的拭子。样品也可以是从任何器官或组织获得的样品(包括活组织检查或尸体解剖标本,例如肿瘤活检)或者可以包含细胞(原代细胞或培养的细胞)或由任何细胞、组织或器官调理的培养基。示例性的样品包括但不限于,细胞、细胞裂解物、血涂片、细胞离心制剂、细胞学涂片、体液(例如血液、血浆、血清、唾液、痰、尿、支气管肺泡灌洗、精液等)、组织活检(例如肿瘤活组织检查)、细针抽吸物和/或组织切片(例如低温恒温器组织切片和/或石蜡包埋的组织切片)。
在其他实施方式中,生物样品可以是植物细胞、愈伤、组织或器官(如根、茎、叶、花、种子、果实)等。
本发明中,所述的靶核酸还包括通过逆转录RNA形成的DNA分子,进一步地,所述的靶核酸可以采用本领域公知的技术对其进行扩增,所述的扩增技术等温扩增技术和非等温扩增技术,等温扩增可以是基于核酸测序的扩增(NASBA)、重组酶聚合酶扩增(RPA)、环介导的等温扩增(LAMP)、链置换扩增(SDA)、解旋酶依赖性扩增(HDA)、或切口酶扩增反应(NEAR)。在某些示例性实施方式中,可以使用非等温扩增方法,其包括但不限于PCR、多重置换扩增(MDA)、滚环扩增(RCA)、连接酶链反应(LCR)、或衍生物扩增方法(RAM)。
进一步的,本发明所述的检测方法还一步包括对靶核酸扩增的步骤;所述的检测系统,还进一步包括对靶核酸进行扩增的试剂。所述扩增的试剂包括下组中的一种或多种:DNA聚合酶、链置换酶、解旋酶、重组酶、单链结合蛋白等。
Cas蛋白
本文所述“Cas蛋白”是指CRISPR-associated蛋白,优选来自V型或VI型CRISPR/CAS蛋白,其一旦与待检测特征序列(靶序列)结合(即形成Cas蛋白-gRNA-靶序列的三元复合物),就可以诱发其trans活性,即随机切割非靶向单链核苷酸(即本文所述单链核酸类似物检测器,优选单链DNA(ssDNA)、单链DNA-RNA杂交体、单链RNA)。当Cas蛋白与特征序列结合后,其切割或不切割特征序列,均可以诱发其trans活性;优选地,其通过切割特征序列诱发其trans活性;更优选地,其通过切割单链特征序列诱发其trans活性。所述Cas蛋白通过识别与特征序列临近的PAM(protospacer adjacent motif)识别特征序列。
本发明所述的Cas蛋白为至少具有trans切割活性的蛋白,优选地,所述的Cas蛋白为具有Cis和trans切割活性的蛋白。所述的Cis活性是指Cas蛋白可在gRNA的作用下识别PAM位点并特异性切割靶序列的活性。
本发明所述的Cas蛋白包括V型CRISPR/CAS效应蛋白,包括Cas12、Cas14等蛋白家族。优选地,例如Cas12蛋白,例如Cas12a、Cas12b、Cas12i、Cas12j;优选地,所述Cas蛋白为Cas12a、Cas12b、Cas12i、Cas12j;Cas14蛋白家族包括Cas14a、Cas14b等。
在实施方式中,本文所称的Cas蛋白,如Cas12,也涵盖Cas的功能变体或其同源物或直系同源物。如本文所用的蛋白的“功能变体”是指至少部分保留该蛋白的活性的这样的蛋白的变体。功能变体可以包括突变体(其可以是插入、缺失或替换突变体),包括多晶型物等。功能变体中还包括这样的蛋白与另一种通常不相关的核酸、蛋白质、多肽或肽的融合产物。功能变体可以是天然存在的或可以是人造的。有利的实施方式可以涉及工程化或非天然存在的V型DNA靶向效应蛋白。
在一个实施方式中,编码Cas蛋白,如Cas12,的一种或多种核酸分子或其直系同源物或同源物可以被密码子优化用于在真核细胞中表达。真核生物可如本文所述。一种或多种核酸分子可以是工程化的或非天然存在的。
在一个实施方式中,Cas12蛋白或其直系同源物或同源物可以包含一个或多个突变(并且因此编码其的核酸分子可以具有一个或多个突变。突变可以是人工引入的突变并且可以包括但不限于催化结构域中的一个或多个突变。
在一个实施方式中,Cas蛋白可以来自:纤毛菌属、李斯特菌属、棒状杆菌属、萨特氏菌属、军团菌属、密螺旋体属、产线菌属、真细菌属、链球菌属、乳酸菌属、支原体属、拟杆菌属、Flaviivola、黄杆菌属、固氮螺菌属、Sphaerochaeta、葡糖醋杆菌属、奈瑟氏菌属、罗氏菌属、Parvibaculum、葡萄球菌属、Nitratifractor、支原体属、弯曲杆菌属和毛螺菌属。
在一个实施方式中,Cas蛋白选自如下序列组成的蛋白:
(1)SEQ ID No.1-4所示的蛋白;
(2)将SEQ ID No.1-4所示氨基酸序或其活性片段列经过一个或多个(如2个、3个、4个,5个,6个,7个,8个,9个或10个)氨基酸残基的取代、缺失或添加而形成的,且具有基本相同功能的衍生蛋白。
在一个实施方式中,所述Cas蛋白还包括与上述序列具有50%,优选55%,优选60%,优选65%,优选70%,优选75%,优选80%,优选85%,优选90%,优选95%,序列同一性的,且具有trans活性的蛋白。
所述的Cas蛋白可以通过重组表达载体技术获得,即将编码该蛋白的核酸分子构建到合适的载体上,再转化到宿主细胞中,使得所述的编码核酸分子在细胞中表达,从而获得相应的蛋白。所述的蛋白可以被细胞分泌出来,或者破解细胞通过常规的提取技术获得该蛋白。所述的编码核酸分子可以整合至宿主细胞的基因组中进行表达,也可以不整合到宿主细胞中进行表达。所述的载体还进一步包括有利于序列整合,或进行自我复制的调节元件。所述的载体可以是例如质粒、病毒、粘粒、噬菌体等类型,它们是本领域技术人员所熟知的,优选地,本发明中的表达载体是质粒。所述的载体进一步包括一种或多种调控元件,选自启动子、增强子、翻译起始的核糖体结合位点、终止子、多聚腺苷酸序列、筛选标记基因。
宿主细胞可以是原核细胞,如大肠杆菌,链霉菌属、农杆菌:或是低等真核细胞,如酵母细胞;或是高等真核细胞,如植物细胞。本领域一般技术人员都清楚如何选择适当的载体和宿主细胞。
gRNA
如本文所用,所述的“gRNA”又称为guide RNA或导向RNA,并且具有本领域技术人员通常理解的含义。一般而言,导向RNA可以包含同向(direct)重复序列和导向序列(guidesequence),或者基本上由或由同向重复序列和导向序列(在内源性CRISPR系统背景下也称为间隔序列(spacer))组成。gRNA在不同的CRISPR系统中,依据其所依赖的Cas蛋白的不同,可以包括crRNA和tracrRNA,也可以只含有crRNA。crRNA和tracrRNA可以经过人工改造融合形成single guide RNA(sgRNA)。在某些情况下,导向序列是与靶序列(本发明中所述特征序列)具有足够互补性从而与所述靶序列杂交并引导CRISPR/Cas复合物与所述靶序列的特异性结合的任何多核苷酸序列,通常具有12-25nt的序列长度。所述的同向重复序列可折叠形成特定结构(如茎环结构)供Cas蛋白识别,以形成复合物。所述的导向序列不需要与特征序列(靶序列)100%互补。所述的导向序列不与单链核酸类似物检测器互补。
在某些实施方案中,当最佳比对时,导向序列与其相应靶序列之间的互补程度(匹配度)为至少50%、至少60%、至少70%、至少80%、至少90%、至少95%、或至少99%。确定最佳比对在本领域的普通技术人员的能力范围内。例如,存在公开和可商购的比对算法和程序,诸如但不限于ClustalW、matlab中的史密斯-沃特曼算法(Smith-Waterman)、Bowtie、Geneious、Biopython以及SeqMan。
本发明所述的gRNA可以是天然的,也可以是经过人工改造或设计合成的。
单链核酸类似物检测器
本发明所述的单链核酸类似物检测器是指含有2-200个核酸类似物的序列,优选,具有2-150个核酸类似物,优选,3-100个核酸类似物,优选,3-30个核酸类似物,优选,4-20个核酸类似物,更优选,5-15个核酸类似物。
所述的单链核酸类似物检测器在检测方法或系统中用以报告是否含有特征序列。所述的单链核酸类似物检测器两端包括不同的报告基团或标记分子,当其处于初始状态(即未被切割状态时)不呈现报告信号,当该单链核酸类似物检测器被切割后,呈现出可检测的信号,即切割后与切割前表现出可检测的区别。在本发明中,如果能够检测出可检测的区别,则反映靶核酸中含有待检测的特征序列;或者,如果无法检检测出所述的可检测的区别,则反映靶核酸中不含有待检测的特征序列。
在一个实施方式中,所述的报告基团或标记分子包括荧光基团和淬灭基团,所述荧光基团选自FAM、FITC、VIC、JOE、TET、CY3、CY5、ROX、Texas Red或LC RED460中的一种或任意几种;所述淬灭基团选自BHQ1、BHQ2、BHQ3、Dabcy1或Tamra中的一种或任意几种。
在一个实施方式中,所述的单链核酸类似物检测器具有连接至5’端第一分子(如FAM或FITC)和连接至3’端的第二分子(如生物素)。所述的含有单链核酸类似物检测器的反应体系与流动条配合用以检测特征序列(优选,胶体金检测方式)。所述的流动条被设计为具有两条捕获线,在样品接触端(胶体金)设有结合第一分子的抗体(即第一分子抗体),在第一线(control line)处含有结合第一分子抗体的抗体,在第二线(test line)处含有与第二分子结合的第二分子的抗体(即第二分子抗体,如亲和素)。当反应沿着条带流动时,第一分子抗体与第一分子结合携带切割或未切割的寡核苷酸至捕获线,切割的报告子将在第一个捕获线处结合第一分子抗体的抗体,而未切割的报告子将在第二捕获线处结合第二分子抗体。报告基团在各条线的结合将导致强读出/信号(例如颜色)。随着更多的报告子被切割,更多的信号将在第一捕获线处累积,并且在第二线处将出现更少的信号。在某些方面,本发明涉及如本文所述的流动条用于检测核酸的用途。在某些方面,本发明涉及用本文定义的流动条检测核酸的方法,例如(侧)流测试或(侧)流免疫色谱测定。在某些方面,所述单链核酸类似物检测器中的分子可相互替换,或改变分子的位置,只要其报告原理与本发明相同或相近,所改进的方式也均包含在本发明中。
本发明所述的检测方法,可用于待检测特征序列的定量检测。所述的定量检测指标可以根据报告基团的信号强弱进行定量,如根据荧光基团的发光强度,或根据显色条带的宽度等。
附图说明
图1.利用Cas12i采用Reporter-LNA作为检测探针的检测结果;其中,线条1为添加Reporter-LNA的试验结果,线条2为对照。
图2.利用Cas12j采用Reporter-LNA作为检测探针的检测结果;其中,线条1为添加Reporter-LNA的试验结果,线条2为对照。
图3.利用Cas12a和Cas12b采用Reporter-LNA作为检测探针的检测结果;其中,线条1为利用Cas12a添加Reporter-LNA的试验结果,线条2为利用Cas12a不添加Reporter-LNA的对照组;线条3为利用Cas12b添加Reporter-LNA的试验结果,线条4为利用Cas12b不添加Reporter-LNA的对照组。
图4.利用Cas12i采用Reporter-dU和Reporter-T作为检测探针的检测结果;其中,线条1为利用Cas12i、Reporter-T作为检测探针,不添加靶核酸的检测结果;线条2为利用Cas12i、Reporter-dU作为检测探针,不添加靶核酸的检测结果;线条3为利用Cas12i、Reporter-T作为检测探针,添加靶核酸的检测结果;线条4为利用Cas12i、Reporter-dU作为检测探针,添加靶核酸的检测结果。
图5.利用2’-氧甲基RNA作为检测探针,采用Cas12a、Cas12b、Cas12i和Cas12j进行检测的结果。
图6.利用2’-氟RNA作为检测探针,采用Cas12a、Cas12b、Cas12i和Cas12j进行检测的结果。
实施方式
下面结合实施例对本发明做进一步的说明,以下所述,仅是对本发明的较佳实施例而已,并非对本发明做其他形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容,依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化,均落在本发明的保护范围内。
本发明技术方案基于如下原理,获得待测样品的核酸,比如,可以通过扩增的方法得到靶核酸,利用可以与靶核酸配对的gRNA引导Cas蛋白识别并结合在靶核酸上;随后,Cas蛋白激发单链核酸类似物切割活性,从而切割体系里的单链核酸类似物;单链核酸类似物的两端分别设置荧光基团和淬灭基团,如果单链核酸类似物被切割,则会激发荧光;如果单链核酸类似物无法被切割,则不会激发荧光;在其他的实施方式中,单链核酸类似物的两端还可以设置成能够被胶体金检测的标记。
实施例1、利用核酸类似物-锁核酸(LNA)进行靶核酸的检测
本实施方式中,利用锁核酸作为检测探针,所述锁核酸(Reporter-LNA)序列为5’6-FAM/LNA_T//LNA_T//LNA_T//LNA_T//LNA_T//3’BHQ1;上述LNA的碱基为胸腺嘧啶(T)。
申请人对Cas12a(SEQ ID No.1)、Cas12b(SEQ ID No.2)、Cas12i(SEQ ID No.3)和Cas12j(SEQ ID No.4)在利用上述LNA探针时的检测效果进行了验证,试验设计如下:
上述Cas12i3-g2-ssDNA0的序列如SEQ ID No.5所示;
上述LbCas12a-TGW6-g1的序列如SEQ ID No.6所示;
上述AaCas12b-TGW6-g1的序列如SEQ ID No.7所示;
上述Cas12i3-TGW6-g2的序列如SEQ ID No.8所示;
上述Cas12j19-TGW6-g3的序列如SEQ ID No.9所示。
结果如图1-图3所示,Cas12b、Cas12i和Cas12j利用Reporter-LNA作为检测探针,能够快速的报告出荧光;但是,Cas12a利用Reporter-LNA作为检测探针,无法报告出荧光。
实施例2、利用脱氧尿嘧啶(dU)进行靶核酸的检测
将尿嘧啶的2’羟基脱掉,可以形成脱氧核糖尿嘧啶(dU),本实施方案中,所述dU检测探针(Reporter-dU)序列为5’6-FAM-dUdUdUdUdU-3’BHQ1;此外,还采用Reporter-T(5’6-FAM-TTTTT-3’BHQ1)作为检测探针。
利用Cas12i(SEQ ID No.3),采用实施例1的体系,其中,检测探针选择Reporter-dU和Reporter-T,结果如图4所示,Cas12i能够利用Reporter-dU作为检测探针快速的报告出荧光。
实施例3、利用2’-氧甲基RNA和2’-氟RNA作为检测探针进行靶核酸的检测
本实施方式中,选择2’-氧甲基RNA和2’-氟RNA作为检测探针;其中,检测器2’-氧甲基RNA的结构为5’6-FAM//2’OCH3-T//2’OCH3-T//2’OCH3-T//2’OCH3-T//2’OCH3-T//3’BHQ1,碱基为T;2’-氟RNA的结构为5’6-FAM//2’F-U//2’F-U//2’F-U//2’F-U//2’F-U//3’BHQ1,碱基为U。
利用实施例1的反应体系,将LNA探针分别替换为上述的2’-氧甲基RNA和2’-氟RNA;结果如图5所示,Cas12a,Cas12b,Cas12i和Cas12j都可以利用2’-氧甲基RNA作为Reporter报告出荧光,尤其是Cas12j的效果最佳;如图6所示,Cas12a,Cas12b,Cas12i和Cas12j都可以利用2’-氟RNA作为Reporter报告出荧光。
除了上述锁核酸之外,申请人还基于Cas12a(SEQ ID No.1)、Cas12b(SEQ IDNo.2)、Cas12i(SEQ ID No.3)和Cas12j(SEQ ID No.4),针对在利用其他的核酸类似物,包括桥核酸(BNA)、吗啉核酸(MNA)、乙二醇核酸(GNA)、苏糖核酸(TNA)、己糖醇核酸(HNA)、阿拉伯糖核酸(ANA)、2’-甲氧基乙酰基RNA、2’-氨基RNA、4’-硫RNA,作为检测探针时的切割效果和检测效果进行了检验。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
<110> 山东舜丰生物科技有限公司
<120> 利用核酸类似物进行靶核酸检测的方法
<130> P2020-1708
<150> CN202010560932.7
<151> 2020-06-18
<160> 9
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Asp Leu Asn Ala Ala Gln Asn Leu Gln Gln Arg Leu Trp Ser Asp Phe
980 985 990
Asp Ile Ser Gln Ile Arg Leu Arg Cys Asp Trp Gly Glu Val Asp Gly
995 1000 1005
Glu Leu Val Leu Ile Pro Arg Leu Thr Gly Lys Arg Thr Ala Asp Ser
1010 1015 1020
Tyr Ser Asn Lys Val Phe Tyr Thr Asn Thr Gly Val Thr Tyr Tyr Glu
1025 1030 1035 1040
Arg Glu Arg Gly Lys Lys Arg Arg Lys Val Phe Ala Gln Glu Lys Leu
1045 1050 1055
Ser Glu Glu Glu Ala Glu Leu Leu Val Glu Ala Asp Glu Ala Arg Glu
1060 1065 1070
Lys Ser Val Val Leu Met Arg Asp Pro Ser Gly Ile Ile Asn Arg Gly
1075 1080 1085
Asn Trp Thr Arg Gln Lys Glu Phe Trp Ser Met Val Asn Gln Arg Ile
1090 1095 1100
Glu Gly Tyr Leu Val Lys Gln Ile Arg Ser Arg Val Pro Leu Gln Asp
1105 1110 1115 1120
Ser Ala Cys Glu Asn Thr Gly Asp Ile
1125
<210> 3
<211> 1045
<212> PRT
<213> 人工序列(artificial sequence)
<400> 3
Met Lys Lys Val Glu Val Ser Arg Pro Tyr Gln Ser Leu Leu Leu Pro
1 5 10 15
Asn His Arg Lys Phe Lys Tyr Leu Asp Glu Thr Trp Asn Ala Tyr Lys
20 25 30
Ser Val Lys Ser Leu Leu His Arg Phe Leu Val Cys Ala Tyr Gly Ala
35 40 45
Val Pro Phe Asn Lys Phe Val Glu Val Val Glu Lys Val Asp Asn Asp
50 55 60
Gln Leu Val Leu Ala Phe Ala Val Arg Leu Phe Arg Leu Val Pro Val
65 70 75 80
Glu Ser Thr Ser Phe Ala Lys Val Asp Lys Ala Asn Leu Ala Lys Ser
85 90 95
Leu Ala Asn His Leu Pro Val Gly Thr Ala Ile Pro Ala Asn Val Gln
100 105 110
Ser Tyr Phe Asp Ser Asn Phe Asp Pro Lys Lys Tyr Met Trp Ile Asp
115 120 125
Cys Ala Trp Glu Ala Asp Arg Leu Ala Arg Glu Met Gly Leu Ser Ala
130 135 140
Ser Gln Phe Ser Glu Tyr Ala Thr Thr Met Leu Trp Glu Asp Trp Leu
145 150 155 160
Pro Leu Asn Lys Asp Asp Val Asn Gly Trp Gly Ser Val Ser Gly Leu
165 170 175
Phe Gly Glu Gly Lys Lys Glu Asp Arg Gln Gln Lys Val Lys Met Leu
180 185 190
Asn Asn Leu Leu Asn Gly Ile Lys Lys Asn Pro Pro Lys Asp Tyr Thr
195 200 205
Gln Tyr Leu Lys Ile Leu Leu Asn Ala Phe Asp Ala Lys Ser His Lys
210 215 220
Glu Ala Val Lys Asn Tyr Lys Gly Asp Ser Thr Gly Arg Thr Ala Ser
225 230 235 240
Tyr Leu Ser Glu Lys Ser Gly Glu Ile Thr Glu Leu Met Leu Glu Gln
245 250 255
Leu Met Ser Asn Ile Gln Arg Asp Ile Gly Asp Lys Gln Lys Glu Ile
260 265 270
Ser Leu Pro Lys Lys Asp Val Val Lys Lys Tyr Leu Glu Ser Glu Ser
275 280 285
Gly Val Pro Tyr Asp Gln Asn Leu Trp Ser Gln Ala Tyr Arg Asn Ala
290 295 300
Ala Ser Ser Ile Lys Lys Thr Asp Thr Arg Asn Phe Asn Ser Thr Leu
305 310 315 320
Glu Lys Phe Lys Asn Glu Val Glu Leu Arg Gly Leu Leu Ser Glu Gly
325 330 335
Asp Asp Val Glu Ile Leu Arg Ser Lys Phe Phe Ser Ser Glu Phe His
340 345 350
Lys Thr Pro Asp Lys Phe Val Ile Lys Pro Glu His Ile Gly Phe Asn
355 360 365
Asn Lys Tyr Asn Val Val Ala Glu Leu Tyr Lys Leu Lys Ala Glu Ala
370 375 380
Thr Asp Phe Glu Ser Ala Phe Ala Thr Val Lys Asp Glu Phe Glu Glu
385 390 395 400
Lys Gly Ile Lys His Pro Ile Lys Asn Ile Leu Glu Tyr Ile Trp Asn
405 410 415
Asn Glu Val Pro Val Glu Lys Trp Gly Arg Val Ala Arg Phe Asn Gln
420 425 430
Ser Glu Glu Lys Leu Leu Arg Ile Lys Ala Asn Pro Thr Val Glu Cys
435 440 445
Asn Gln Gly Met Thr Phe Gly Asn Ser Ala Met Val Gly Glu Val Leu
450 455 460
Arg Ser Asn Tyr Val Ser Lys Lys Gly Ala Leu Val Ser Gly Glu His
465 470 475 480
Gly Gly Arg Leu Ile Gly Gln Asn Asn Met Ile Trp Leu Glu Met Arg
485 490 495
Leu Leu Asn Lys Gly Lys Trp Glu Thr His His Val Pro Thr His Asn
500 505 510
Met Lys Phe Phe Glu Glu Val His Ala Tyr Asn Pro Ser Leu Ala Asp
515 520 525
Ser Val Asn Val Arg Asn Arg Leu Tyr Arg Ser Glu Asp Tyr Thr Gln
530 535 540
Leu Pro Ser Ser Ile Thr Asp Gly Leu Lys Gly Asn Pro Lys Ala Lys
545 550 555 560
Leu Leu Lys Arg Gln His Cys Ala Leu Asn Asn Met Thr Ala Asn Val
565 570 575
Leu Asn Pro Lys Leu Ser Phe Thr Ile Asn Lys Lys Asn Asp Asp Tyr
580 585 590
Thr Val Ile Ile Val His Ser Val Glu Val Ser Lys Pro Arg Arg Glu
595 600 605
Val Leu Val Gly Asp Tyr Leu Val Gly Met Asp Gln Asn Gln Thr Ala
610 615 620
Ser Asn Thr Tyr Ala Val Met Gln Val Val Lys Pro Lys Ser Thr Asp
625 630 635 640
Ala Ile Pro Phe Arg Asn Met Trp Val Arg Phe Val Glu Ser Gly Ser
645 650 655
Ile Glu Ser Arg Thr Leu Asn Ser Arg Gly Glu Tyr Val Asp Gln Leu
660 665 670
Asn His Asp Gly Val Asp Leu Phe Glu Ile Gly Asp Thr Glu Trp Val
675 680 685
Asp Ser Ala Arg Lys Phe Phe Asn Lys Leu Gly Val Lys His Lys Asp
690 695 700
Gly Thr Leu Val Asp Leu Ser Thr Ala Pro Arg Lys Ala Tyr Ala Phe
705 710 715 720
Asn Asn Phe Tyr Phe Lys Thr Met Leu Asn His Leu Arg Ser Asn Glu
725 730 735
Val Asp Leu Thr Leu Leu Arg Asn Glu Ile Leu Arg Val Ala Asn Gly
740 745 750
Arg Phe Ser Pro Met Arg Leu Gly Ser Leu Ser Trp Thr Thr Leu Lys
755 760 765
Ala Leu Gly Ser Phe Lys Ser Leu Val Leu Ser Tyr Phe Asp Arg Leu
770 775 780
Gly Ala Lys Glu Met Val Asp Lys Glu Ala Lys Asp Lys Ser Leu Phe
785 790 795 800
Asp Leu Leu Val Ala Ile Asn Asn Lys Arg Ser Asn Lys Arg Glu Glu
805 810 815
Arg Thr Ser Arg Ile Ala Ser Ser Leu Met Thr Val Ala Gln Lys Tyr
820 825 830
Lys Val Asp Asn Ala Val Val His Val Val Val Glu Gly Asn Leu Ser
835 840 845
Ser Thr Asp Arg Ser Ala Ser Lys Ala His Asn Arg Asn Thr Met Asp
850 855 860
Trp Cys Ser Arg Ala Val Val Lys Lys Leu Glu Asp Met Cys Asn Leu
865 870 875 880
Tyr Gly Phe Asn Ile Lys Gly Val Pro Ala Phe Tyr Thr Ser His Gln
885 890 895
Asp Pro Leu Val His Arg Ala Asp Tyr Asp Asp Pro Lys Pro Ala Leu
900 905 910
Arg Cys Arg Tyr Ser Ser Tyr Ser Arg Ala Asp Phe Ser Lys Trp Gly
915 920 925
Gln Asn Ala Leu Ala Ala Val Val Arg Trp Ala Ser Asn Lys Lys Ser
930 935 940
Asn Thr Cys Tyr Lys Val Gly Ala Val Glu Phe Leu Lys Gln His Gly
945 950 955 960
Leu Phe Ala Asp Lys Lys Leu Thr Val Glu Gln Phe Leu Ser Lys Val
965 970 975
Lys Asp Glu Glu Ile Leu Ile Pro Arg Arg Gly Gly Arg Val Phe Leu
980 985 990
Thr Thr His Arg Leu Leu Ala Glu Ser Thr Phe Val Tyr Leu Asn Gly
995 1000 1005
Val Lys Tyr His Ser Cys Asn Ala Asp Glu Val Ala Ala Val Asn Ile
1010 1015 1020
Cys Leu Asn Asp Trp Val Ile Pro Cys Lys Lys Lys Met Lys Glu Glu
1025 1030 1035 1040
Ser Ser Ala Ser Gly
1045
<210> 4
<211> 908
<212> PRT
<213> 人工序列(artificial sequence)
<400> 4
Met Pro Ser Tyr Lys Ser Ser Arg Val Leu Val Arg Asp Val Pro Glu
1 5 10 15
Glu Leu Val Asp His Tyr Glu Arg Ser His Arg Val Ala Ala Phe Phe
20 25 30
Met Arg Leu Leu Leu Ala Met Arg Arg Glu Pro Tyr Ser Leu Arg Met
35 40 45
Arg Asp Gly Thr Glu Arg Glu Val Asp Leu Asp Glu Thr Asp Asp Phe
50 55 60
Leu Arg Ser Ala Gly Cys Glu Glu Pro Asp Ala Val Ser Asp Asp Leu
65 70 75 80
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85 90 95
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100 105 110
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115 120 125
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130 135 140
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145 150 155 160
Ser Met Glu Asp Trp Arg Arg Phe Phe Ala Ala Arg Asp Pro Asp Asp
165 170 175
Leu Gly Arg Glu Leu Leu Lys Thr Asp Thr Arg Glu Gly Met Ala Ala
180 185 190
Ala Leu Arg Leu Arg Glu Arg Gly Val Phe Pro Val Ser Val Pro Glu
195 200 205
His Leu Asp Leu Asp Ser Leu Lys Ala Ala Met Ala Ser Ala Ala Glu
210 215 220
Arg Leu Lys Ser Trp Leu Ala Cys Asn Gln Arg Ala Val Asp Glu Lys
225 230 235 240
Ser Glu Leu Arg Lys Arg Phe Glu Glu Ala Leu Asp Gly Val Asp Pro
245 250 255
Glu Lys Tyr Ala Leu Phe Glu Lys Phe Ala Ala Glu Leu Gln Gln Ala
260 265 270
Asp Tyr Asn Val Thr Lys Lys Leu Val Leu Ala Val Ser Ala Lys Phe
275 280 285
Pro Ala Thr Glu Pro Ser Glu Phe Lys Arg Gly Val Glu Ile Leu Lys
290 295 300
Glu Asp Gly Tyr Lys Pro Leu Trp Glu Asp Phe Arg Glu Leu Gly Phe
305 310 315 320
Val Tyr Leu Ala Glu Arg Lys Trp Glu Arg Arg Arg Gly Gly Ala Ala
325 330 335
Val Thr Leu Cys Asp Ala Asp Asp Ser Pro Ile Lys Val Arg Phe Gly
340 345 350
Leu Thr Gly Arg Gly Arg Lys Phe Val Leu Ser Ala Ala Gly Ser Arg
355 360 365
Phe Leu Ile Thr Val Lys Leu Pro Cys Gly Asp Val Gly Leu Thr Ala
370 375 380
Val Pro Ser Arg Tyr Phe Trp Asn Pro Ser Val Gly Arg Thr Thr Ser
385 390 395 400
Asn Ser Phe Arg Ile Glu Phe Thr Lys Arg Thr Thr Glu Asn Arg Arg
405 410 415
Tyr Val Gly Glu Val Lys Glu Ile Gly Leu Val Arg Gln Arg Gly Arg
420 425 430
Tyr Tyr Phe Phe Ile Asp Tyr Asn Phe Asp Pro Glu Glu Val Ser Asp
435 440 445
Glu Thr Lys Val Gly Arg Ala Phe Phe Arg Ala Pro Leu Asn Glu Ser
450 455 460
Arg Pro Lys Pro Lys Asp Lys Leu Thr Val Met Gly Ile Asp Leu Gly
465 470 475 480
Ile Asn Pro Ala Phe Ala Phe Ala Val Cys Thr Leu Gly Glu Cys Gln
485 490 495
Asp Gly Ile Arg Ser Pro Val Ala Lys Met Glu Asp Val Ser Phe Asp
500 505 510
Ser Thr Gly Leu Arg Gly Gly Ile Gly Ser Gln Lys Leu His Arg Glu
515 520 525
Met His Asn Leu Ser Asp Arg Cys Phe Tyr Gly Ala Arg Tyr Ile Arg
530 535 540
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Arg Leu Leu Glu Glu Lys Tyr Ile Pro Gly Phe Arg Ile Val His Ile
565 570 575
Glu Asp Ala Asp Glu Arg Arg Arg Thr Val Gly Arg Thr Val Lys Glu
580 585 590
Ile Lys Gln Glu Tyr Lys Arg Ile Arg His Gln Phe Tyr Leu Arg Tyr
595 600 605
His Thr Ser Lys Arg Asp Arg Thr Glu Leu Ile Ser Ala Glu Tyr Phe
610 615 620
Arg Met Leu Phe Leu Val Lys Asn Leu Arg Asn Leu Leu Lys Ser Trp
625 630 635 640
Asn Arg Tyr His Trp Thr Thr Gly Asp Arg Glu Arg Arg Gly Gly Asn
645 650 655
Pro Asp Glu Leu Lys Ser Tyr Val Arg Tyr Tyr Asn Asn Leu Arg Met
660 665 670
Asp Thr Leu Lys Lys Leu Thr Cys Ala Ile Val Arg Thr Ala Lys Glu
675 680 685
His Gly Ala Thr Leu Val Ala Met Glu Asn Ile Gln Arg Val Asp Arg
690 695 700
Asp Asp Glu Val Lys Arg Arg Lys Glu Asn Ser Leu Leu Ser Leu Trp
705 710 715 720
Ala Pro Gly Met Val Leu Glu Arg Val Glu Gln Glu Leu Lys Asn Glu
725 730 735
Gly Ile Leu Ala Trp Glu Val Asp Pro Arg His Thr Ser Gln Thr Ser
740 745 750
Cys Ile Thr Asp Glu Phe Gly Tyr Arg Ser Leu Val Ala Lys Asp Thr
755 760 765
Phe Tyr Phe Glu Gln Asp Arg Lys Ile His Arg Ile Asp Ala Asp Val
770 775 780
Asn Ala Ala Ile Asn Ile Ala Arg Arg Phe Leu Thr Arg Tyr Arg Ser
785 790 795 800
Leu Thr Gln Leu Trp Ala Ser Leu Leu Asp Asp Gly Arg Tyr Leu Val
805 810 815
Asn Val Thr Arg Gln His Glu Arg Ala Tyr Leu Glu Leu Gln Thr Gly
820 825 830
Ala Pro Ala Ala Thr Leu Asn Pro Thr Ala Glu Ala Ser Tyr Glu Leu
835 840 845
Val Gly Leu Ser Pro Glu Glu Glu Glu Leu Ala Gln Thr Arg Ile Lys
850 855 860
Arg Lys Lys Arg Glu Pro Phe Tyr Arg His Glu Gly Val Trp Leu Thr
865 870 875 880
Arg Glu Lys His Arg Glu Gln Val His Glu Leu Arg Asn Gln Val Leu
885 890 895
Ala Leu Gly Asn Ala Lys Ile Pro Glu Ile Arg Thr
900 905
<210> 5
<211> 50
<212> DNA
<213> 人工序列(artificial sequence)
<400> 5
gatcgttggt agttcatgct gctgtcggtg aaataaacat ctccggtaac 50
<210> 6
<211> 41
<212> RNA
<213> 人工序列(artificial sequence)
<400> 6
uaauuucuac uaaguguaga uuuucaccga cagcagcaug a 41
<210> 7
<211> 105
<212> RNA
<213> 人工序列(artificial sequence)
<400> 7
gucuaaagga cagaauuuuu caacgggugu gccaauggcc acuuuccagg uggcaaagcc 60
cguugaacuu caagcgaagu ggcacuuuca ccgacagcag cauga 105
<210> 8
<211> 46
<212> RNA
<213> 人工序列(artificial sequence)
<400> 8
agagaaugug ugcauaguca cacuuucacc gacagcagca ugaacu 46
<210> 9
<211> 60
<212> RNA
<213> 人工序列(artificial sequence)
<400> 9
gugcugcugu cucccagacg ggaggcagaa cugcacggau ugaugcgagu ugguccaaaa 60
Claims (16)
1.一种检测样品中靶核酸的方法,所述方法为非疾病诊断和治疗目的的方法,所述方法包括将样品与V型Cas蛋白、gRNA和单链核酸类似物检测器接触,所述gRNA包括与所述V型Cas蛋白结合的区域和与靶核酸杂交的导向序列;检测由V型Cas蛋白切割单链核酸类似物检测器产生的可检测信号,从而检测靶核酸;所述单链核酸类似物检测器不与所述gRNA杂交;
所述单链核酸类似物为锁核酸(LNA),所述锁核酸为LNA_T,所述单链核酸类似物检测器的序列为LNA_T//LNA_T//LNA_T//LNA_T//LNA_T,其中LNA_T为经锁核酸修饰的胸腺嘧啶,所述V型Cas蛋白为Cas12b,Cas12i或Cas12j;或,
所述单链核酸类似物为脱氧核糖尿嘧啶,所述单链核酸类似物检测器的序列为dUdUdUdUdU,其中dU为2’羟基脱掉的尿嘧啶,所述V型Cas蛋白为Cas12i;或,
所述单链核酸类似物为2’-氧甲基RNA,所述2’-氧甲基RNA为2’OCH3-T,所述单链核酸类似物检测器的序列为2’OCH3-T//2’OCH3-T //2’OCH3-T //2’OCH3-T //2’OCH3-T,其中2’OCH3-T为经2’-氧甲基修饰的胸腺嘧啶,所述V型Cas蛋白为Cas12a,Cas12b,Cas12i或Cas12j;或,
所述单链核酸类似物为2’-氟RNA,所述2’-氟RNA为2’F-U,所述单链核酸类似物检测器的序列为2’F-U//2’F-U//2’F-U //2’F-U //2’F-U,其中2’F-U为2’-氟修饰的尿嘧啶,所述V型Cas蛋白为Cas12a,Cas12b,Cas12i或Cas12j;
所述Cas12a的氨基酸序列如SEQ ID No.1所示;所述Cas12b的氨基酸序列如SEQ IDNo.2所示;所述Cas12i的氨基酸序列如SEQ ID No.3所示;所述Cas12j的氨基酸序列如SEQID No.4所示。
2.根据权利要求1所述的方法,其特征在于,所述可检测信号通过以下方式进行检测:基于视觉的检测、基于传感器的检测,基于金纳米颗粒的检测,荧光偏振检测,胶体相变/分散检测,电化学检测和基于半导体的检测。
3.根据权利要求2所述的方法,其特征在于,所述基于视觉的检测为颜色检测。
4.根据权利要求1所述的方法,其特征在于,所述靶核酸包括核糖核苷酸或脱氧核糖核苷酸。
5.根据权利要求4所述的方法,其特征在于,所述靶核酸包括单链核酸或双链核酸。
6.根据权利要求5所述的方法,其特征在于,所述靶核酸包括单链DNA、双链DNA或单链RNA。
7.根据权利要求1所述的方法,其特征在于,所述单链核酸类似物检测器的5’端和3’端分别设置不同的报告基团,当所述单链核酸类似物检测器被切割后,表现出可检测的报告信号;或者,所述单链核酸类似物检测器的5’端和3’端分别设置不同的标记分子,通过胶体金检测的方式检测反应信号。
8.根据权利要求1所述的方法,其特征在于,所述靶核酸为病毒核酸或细菌核酸。
9.根据权利要求8所述的方法,其特征在于,所述病毒为植物病毒或动物病毒。
10.根据权利要求9所述的方法,其特征在于,所述病毒为乳头瘤病毒,疱疹病毒,腺病毒,痘病毒,细小病毒或冠状病毒。
11.根据权利要求10所述的方法,其特征在于,所述冠状病毒选自SARS、SARS-CoV2、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1或Mers-Cov。
12.一种用于检测样品中靶核酸的系统,所述系统包括权利要求1中所述的V型Cas蛋白、gRNA和单链核酸类似物检测器。
13.权利要求12所述的系统在制备用于检测样品中靶核酸的试剂盒中的应用。
14.一种用于检测样品中靶核酸的组合物,所述组合物包括权利要求1中所述的V型Cas蛋白、gRNA和单链核酸类似物检测器。
15.权利要求14所述的组合物在制备用于检测样品中靶核酸的试剂盒中的应用。
16.一种用于检测样品中靶核酸的试剂盒,所述试剂盒包括权利要求1中所述的V型Cas蛋白、gRNA和单链核酸类似物检测器。
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