本发明属于分子生物技术领域,具体涉及一种检测新型冠状病毒covid-19的核酸组合物及应用。
背景技术:
新型冠状病毒covid-19自爆发以来,疫情的迅速蔓延,无不牵动着国人的心,也给全国疫情防控承受空前压力,绝大部分省市都进入公共卫生事件一级响应状态。目前,已知的人冠状病毒共有7种,分别是在1965年发现的hcov-229e,在1967年鉴定出的hcov-oc43,在2003年在我国广东出现的sars-cov,在2004年在荷兰分离鉴定出hcov-nl63,在2005年在香港筛查出hcov-hku1,在2012年在中东地区出现的mers-cov,以及在2019年中国武汉发现的新型冠状病毒covid-19。其中,新型冠状病毒covid-19是在2019年12月末在湖北省武汉市爆发的一种引起人类呼吸道疾病和严重肺炎等临床症状的可人传人的传染病。截止2020年2月13日,全国已确诊59885人感染该病,疑似病例16067人,导致1368人死亡。国际病毒分类委员会(ictv)声明,将该新型冠状病毒命名为“sars-cov2”(severeacuterespiratorysyndromecoronavirus2)。最新更名为“covid-19”。冠状病毒是外面包裹着脂肪膜,其膜表面含有刺突糖蛋白、小包膜糖蛋白、膜糖蛋白三种糖蛋白。其内部核酸为长27-31kb的单链rna,具有正链rna特有的重要结构特征:即rna链5’端有甲基化“帽子”构造,3’端有polya“尾巴”结构,可以发挥翻译模板作用,而省去了rna-dna-rna的转录过程。也就是说,一般这类病毒颗粒中的遗传物质正链rna进入寄主细胞,就直接作为mrna,翻译出所编码的蛋白质,其中包括衣壳蛋白和病毒的rna聚合酶。在病毒rna复制酶作用下复制rna,同时以该rna为模板合成dsrna,在不断重复复制和解旋的过程中自我装配成成熟的病毒。
其中,核酸检测是目前最可靠的筛查和确认辅助手段,在符合疑似病例标准的基础上,痰液、咽拭子、下呼吸道分泌物等标本核酸阳性确诊。由于目前世界上还没有研发出有效的针对该病毒的疫苗,为防止疾病传播疫点内和涉及人员的人员只能被全部隔离,给生产生活各个方面都带来巨大损失。因此,在当前严峻的防控形势下全国亟需要开发一种简便、快速且高灵敏度的鉴别诊断试剂盒,为临床快速确诊新型冠状病毒covid-19以及其他类似rna病毒感染提供检测技术。
特异性核酸或基因分子检测方法是当代医学发展的重要前沿领域之一,具有的应用价值,如病原体检测、遗传病检测等。通过探究每种病原微生物其独一无二的特征核酸分子序列的存在或变化,从而对人体状态与疾病做出诊断的方法,也叫核酸诊断(nads,nucleicaciddiagnostics)。其广泛用于食品安全、环境微生物污染检测,人体病原菌感染诊断等领域。
现有技术多存在诊断时间长、操作复杂、灵敏度低等缺点。复杂多变的诊断环境,限制了基因分子检测技术的应用,同时也对技术的发展提出了挑战。而一种原核生物(如细菌和古细菌等)抵御病毒等外源入侵核酸的获得性免疫系统即:crispr-cas系统,引起基因编辑技术领域发生了革命性突破。其中crispr-cas9特异性识别并切割靶标序列,被广泛开发成基因组编辑工具。2015年张锋团队发现了核酸内切酶cas12a,与cas9蛋白相同都是由sgrna或crrna引导的特异性切割靶标dsdna,不同的是cas12a仅需要crrna即可引导特异性切割dsdna,产生粘性末端易于基因编辑。最大的不同在于cas12a具有在切割靶标dsdna后会对非特异性任意切割ssdna的“旁路切割”活性,根据这种特性可以开发出基于crispr-cas12a系统对细胞、血液、唾液、尿液和粪便等样本中病毒dna进行快速、准确检测的检测体系。
上述方法涉及到的cas9和cas12检测系统中的靶标模板均为双链dna,而当前的新型冠状病毒covid-19核酸为ssrna。2017年张锋等人发现了新型蛋白cas13a,该蛋白是vi型crispr-cas系统效应蛋白,具有rna介导的rna酶切活性,是目前第二大类crispr-cas系统发现的唯一能够降解rna的蛋白。特别地,cas13a在sgrna的引导下具有识别并特异性切开靶标ssrna后,仍能保持rna酶切活性随即不受限制的切割其他非靶标ssrna(可设计成rna荧光报告系统)的“连带切割”(collateraleffect),借助此特性可以针对特定序列的靶标核酸进行检测。张峰等人利用该蛋白的这种特殊切割活性结合rpa技术(recombinasepolymeraseamplification,等温扩增),建立快速核酸检测技术,该方法称为sherlock(specifichighsensitivityenzymaticreporterunlocking)。由于当前新型冠状病毒形势非常严峻,开发检测新型的冠状病毒工具必须确保具有较高的灵敏度和较强的忠实性,cas13a-sgrna可以被靶rna激活,从而实现体外高灵敏度的rna检测,并且还要能够实现快速准确报告结果。
技术实现要素:
为解决现有技术存在的缺陷,本发明提供一种检测新型冠状病毒covid-19的核酸组合物及应用。通过本发明制备的检测试剂盒能够在等温条件下进行转录介导的核酸扩增技术,迅速扩大病毒rna核酸的拷贝数,加之crispr-cas13a结合sgrna系统切割病毒核酸并且进一步切割荧光淬灭核酸探针的ssrna产生荧光信号的高特异性检测体系,完善基因分子检测技术,使得痕量、高灵敏度、高特异性且准确的鉴别病毒成为可能。该技术手段除了可以快速鉴别病毒如新型冠状病毒covid-19、艾滋病病毒、白血病病毒等rna病毒,也可以运用在疾病筛查与诊断、遗传病、食品卫生检测、法医鉴定、环境污染等各个方面。
为了解决上述技术问题,本发明提供了如下的技术方案:
本发明的第一目的提供一种检测新型冠状病毒covid-19的核酸组合物,包括根据新型冠状病毒covid-19的n基因序列设计的引物对n-t7f和n-r、以及根据新型冠状病毒covid-19的orf1ab基因序列设计的引物对orf1ab-t7f和orf1ab-r,所述n基因的序列如seqidno:1所示,所述orf1ab基因的序列如seqidno:2所示;所述引物对n-t7f和n-r的序列如seqidno:3和seqidno:4所示,所述引物对orf1ab-t7f和orf1ab-r的序列如seqidno:5和seqidno:6所示。
作为优选,还包括rna核酸探针,所述rna核酸探针为化学合成的两端进行荧光标记修饰的ssrna核酸探针,所述ssrna核酸探针的序列如seqidno:9所示,所述ssrna核酸探针在5’端标记有荧光报告基团,在3’端标记有荧光淬灭基团。
作为优选,所述荧光报告基团选自hex、cy5、fam、rox中的一种,所述荧光淬灭基团选自bhq1、bhq3、tamra中的一种。
本发明的第二目的提供一种检测新型冠状病毒covid-19的试剂盒,所述试剂盒中包括检测新型冠状病毒covid-19的核酸组合物。
本发明的第三目的提供一种非疾病诊断目的的检测新型冠状病毒covid-19核酸的方法,包括以下步骤:
s1、根据新型冠状病毒covid-19的序列化学合成n基因和orf1ab基因片段,经过转染人工制备假病毒,即病毒蛋白和核酸复合物,然后核酸抽提后梯度稀释,稀释后作为转录介导的扩增模板;
s2、根据新型冠状病毒covid-19的n基因序列设计引物对n-t7f和n-r、以及根据新型冠状病毒covid-19的orf1ab基因序列设计引物对orf1ab-t7f和orf1ab-r,利用转录介导的扩增体系对病毒核酸进行等温扩增以扩大病毒核酸的拷贝数;其中,n基因的序列如seqidno:1所示,orf1ab基因的序列如seqidno:2所示;引物对n-t7f和n-r的序列如seqidno:3和seqidno:4所示,引物对orf1ab-t7f和orf1ab-r的序列如seqidno:5和seqidno:6所示;
s3、根据新型冠状病毒covid-19的n基因和orf1ab基因序列的切割靶点,化学合成引导的cas13a蛋白的sgrna,其中,n基因靶点对应的n-sgrna的序列如seqidno:7所示,orf1ab基因靶点对应的orf1ab-sgrna的序列如seqidno:8所示;
s4、体外重组表达的方法获得的crispr-cas13a蛋白、sgrna切割系统及靶标核酸形成“三元复合物”体系后激活cas13a蛋白的非特异rna酶切割活性;
s5、构建如上述的一种两端进行荧光报告基团和荧光淬灭基团修饰的ssrna核酸探针,被激活的cas13a酶任意切割该ssrna核酸探针的会产生荧光信号,从而快速准确的鉴别检测新型冠状病毒covid-19核酸。
作为优选,步骤s1中人工制备病毒蛋白和核酸复合物具体过程为:将其目的基因克隆到细胞表达载体上,经测序验证后转染细胞,而后对细胞进行处理收集蛋白和核酸复合物,核酸酶去除基因组dna残留后,trizol法抽提核酸后计算拷贝数,作为转录介导的扩增模板。
作为优选,步骤s2中将20μl转录介导的扩增反应体系的各组分轻柔涡旋混匀后,置于水浴锅42℃反应15-25min获得转录产物ssrna,转录介导的扩增反应体系包含以下浓度的以下组分:2μl待扩增正链rna片段,1-5urnase抑制剂,500-2000ut7rna聚合酶,2000-4000um-mlv逆转录酶,0.5-2μm上游引物,0.5-2μm下游引物以及tma反应缓冲液;待扩增正链rna片段为步骤s1提取的核酸;
其中,tma反应缓冲液包含以下浓度的以下组分:20-50mmtris-hclph8.0@25℃、10-30mmkcl、1-4mmmgcl2、1-5mmrntps、1-5mmdntps、20-50%甘油、0-10%dmso、0.5-1mmdtt。
作为优选,步骤s4中将“三元复合物”体系的各组分轻柔涡旋混匀后,在37-40℃条件下通过实时荧光定量仪先保温10-25min后,再每反应40-60s收集一次荧光,循环收集40-60min;“三元复合物”体系包含以下浓度的以下组分:待检测rna片段、0.5-2μmtma上游引物、0.5-2μmtma下游引物、2000-4000um-mlv逆转录酶、500-2000ut7rna聚合酶、1-5urnase抑制剂、50-200nmcas13a蛋白酶、50-400nmsgrna、0.25-1μmrna核酸探针、20-50mmtris-hclph7.5@25℃、20-40mmkcl、1-5mmmgcl2、1-5mmrntps、1-5mmdntps、20-50%甘油、0.5-1mmdtt。
本发明的有益效果是:本发明摒弃了常用的先将rna反转录成cdna结合需变性退火过程pcr技术,改用转录介导的等温扩增病毒核酸放大方法结合crispr-cas系统的检测技术,其反应速度更快,产物量多、不易污染和忠实度更高。
另外,本发明采用crispr-cas13a酶和特异性切割新型冠状病毒n和orf1ab基因序列的sgrna,不同于传统检测病毒技术,直接靶向病毒核酸,能够在60分钟内检测到100拷贝以下的病毒核酸样本,特异性强和灵敏度高。
附图说明
图1是本发明tma和crispr-cas13a联合技术原理图。
图2是本发明cas13a蛋白离子交换柱纯化后sds-page电泳图。
图3是本发明新型冠状病毒covid-19的n基因片段的荧光收集结果图。
图4是本发明新型冠状病毒covid-19的orf1ab基因片段的荧光收集结果图。
图5是本发明“二合一”检测新型冠状病毒covid-19的orf1ab基因片段的荧光收集结果图。
具体实施方式
以下结合附图对本发明的优选实施例进行说明,应当理解,此处所描述的优选实施例仅用于说明和解释本发明,并不用于限定本发明。
首先,如图1所示,检测系统的上游设置为tma技术,在等温条件下,目标序列在野生型m-mlv逆转录酶(含rnaseh活性)作用下,在一条引物5’端加上t7启动子序列的引物的引导下进行反转录,形成rna/dna杂合子,逆转录酶的rnaseh活性将rna/dna杂合子降解成含有t7rna聚合酶识别的启动子序列的ssdna。另一条引物与该ssdna结合后在逆转录酶的作用合成dsdna。此时,t7rna聚合酶结合在启动子上,进行转录成ssrna,该ssrna又可作为模板进行下一个循环,10-30min左右就可将靶标序列扩增106以上,整个过程在一管中自催化反应。其次是crispr-cas13a检测体系,基于tma技术将待检核酸片段进行扩增后的转录产物,进而依据靶标ssrna目的基因序列设计sgrna,且在该检测体系中需加入的具备信号报告非靶标ssrna,利用cas13a蛋白的“连带切割”活性可以实现信号的放大并读取,从而检测目的基因。
图2是本发明cas13a蛋白离子交换柱纯化后sds-page电泳图。
本发明所涉及的技术包括如下步骤:
(1)trizol试剂法靶标ssrna抽提(以新型冠状病毒covid-19为例)。
具体抽提步骤:1)将假病毒从-80℃冰箱中取出,进行冰浴融化或置于4℃条件下自然融化后混匀;2)200ul假病毒,混入30ul的其他无关rna(10ng/ul-100ng/ul)后,再加入800ul的trizol试剂。枪头混匀,室温静置5min;3)向上述步骤2中再加入160ul氯仿,上下颠倒混匀至溶液乳化成乳白色。室温静置5min。12000xg离心15min,小心取出;4)吸出上清液(约700ul)转移至新的离心管。向上清中加入500ul异丙醇,上下颠倒混匀,室温静置20-30min;5)12000xg离心10min。取出,枪头小心吸掉异丙醇。加入800ul的75%乙醇,上下颠倒清洗,12000xg离心10min;6)取出,倒掉75%乙醇,室温超净台吹风干燥5-10min,加入rnasefree水50ul溶解。
(2)转录介导的核酸扩增技术对步骤(1)抽提的rna进行放大扩增反应。
所述反应的试剂分别有:引物n-t7f和n-r、引物orf1ab-t7f和orf1ab-r、待测病毒rna、野生型m-mlv逆转录酶、t7rna聚合酶、tma反应缓冲液和rnasefreewater。
其中,本发明设计的引物对n-t7f和n-r、引物对orf1ab-t7f和orf1ab-r分别是根据新型冠状病毒covid-19特异性的n和orflab基因保守序列,利用primerpremier5.0软件以及ncbi的blast分析设计得到的一对寡链核苷酸单链引物。需要说明的是,这两对引物分别是特异性识别靶标区域的上下游,长度不超过60nt,一般在20-35nt之间,避免了引物间的干扰、发卡结构和引物二聚体等常见问题。另外,本发明中上游引物n-t7f和orf1ab-t7f的3’端可与待测目的基因的3’端杂交,而5’端另需带上噬菌体启动子序列。下游引物n-r和orf1ab-r的3’端与待测目的基因的负链的3’端杂交。
其中,所述t7rna聚合酶还可为其他噬菌体rna聚合酶,如t3、sp6等。若选择其他类型噬菌体rna聚合酶,可相应修改上游引物5’端上的rna聚合酶结合识别序列,以用于扩增产物后续进行体外转录。
所述野生型m-mlv逆转录酶,详细来讲是未去除rnaseh活性的鼠源逆转录酶,也可以为其他逆转录酶,如amv。若添加的逆转录酶无rnaseh活性,可在体系中另外添加rnaseh。
所述tma技术中转录介导的扩增反应体系主要成分为:rnase抑制剂、t7rna聚合酶、m-mlv逆转录酶、上游引物、下游引物、tris-hcl、核糖核苷三磷酸(rntps)、二硫苏糖醇(dtt)、脱氧核糖核苷三磷酸(dntps)、kcl、mgcl2、甘油和dmso。具体组分中浓度为:20-50mmtris-hclph8.0@25℃、10-30mmkcl、1-4mmmgcl2、1-5mmrntps、1-5mmdntps、20-50%甘油、0-10%dmso、0.5-1mmdtt、0.5-2μm上游引物、0.5-2μm下游引物以及1-5urnase抑制剂、2000-4000um-mlv逆转录酶和500-2000ut7rna聚合酶。
所述步骤(2)的建立20μl对目的ssrna进行放大扩增反应体系:获得相关样本的rna,将一定浓度的rna与上述所需的组分充分混匀,将反应管置于42℃反应15-25min可得转录产物ssrna。等温扩增反应一般于简易恒温金属浴或水浴箱中进行即可。
其所述病毒也可为其他类似rna病毒。若选择其他类型rna病毒,需相应的根据靶病毒保守区设计上游或下游引物。
(3)crispr-cas13a检测体系对步骤(2)转录产物进行信号收集报告。
涉及本步骤反应的试剂分别有:cas蛋白、sgrna、上述转录产物ssrna、cas反应缓冲液,rna核酸探针和rnasefreewater。
所述cas蛋白可以是来源于不同细菌为cas13a蛋白,如lshcas13a(来源于leptotrichiashahii)、lwacas13a(来源于leptorchiawadei)、lbucas13a(来源于leptotrichiabuccalis)的一种,优选为lwacas13a。
所述crispr-lwacas13a蛋白酶的保存缓冲液包括:20mmtris-hclph7.0@25℃、0.1mmedta、1mmdtt、200mmnacl、50%(v/v)glycerol。
根据步骤(3)所述sgrna指包含针对目的基因的靶点设计,可引导cas13a蛋白特异性结合靶标ssrna的向导rna。所述sgrna为5’-简单重复序列-识别靶标序列-3’的结构;其中,所述相同细菌来源的cas13a蛋白具有相同的简单重复序列,5’-gauuuagacuaccccaaaaacgaaggggacuaaaac-3’则是lwacas13a的简单重复序列;所述识别靶标序列与靶向rna中的片段匹配,长度为25-38个核苷酸,优选为28个以上。所述cas13a通过sgrna识别目的基因上的靶点后,能够激活cas13a的rna酶活性,并对ssrna核酸探针进行切割,从而获得检测信号。
根据步骤(3)所述rna核酸探针为化学合成的两端进行荧光标记修饰的ssrna核酸探针。所述ssrna核酸探针最佳地为在5’端标记荧光报告基团,如hex、cy5、fam、rox等,优选地为fam标记;在3’端最佳地标记荧光淬灭基团,如bhq1、bhq3、tamra等,优选地bhq1。所述ssrna核酸探针的检测方法优选为荧光检测法;所述荧光检测法可使用酶标仪或者荧光分光光度计或者实时荧光定量仪进行检测的方法,优选地为实时荧光定量仪。本发明中的ssrna核酸探针的序列为:5’[fam]-gaauuccaccacguucccgugg-3’[bhq1],其序列如seqidno:9所示。
所述ssrna核酸探针亦可适应于其他实验体系中。基于cas13a蛋白的在sgrna引导下特异性切割靶标rna的同时,cas13a蛋白酶的rna酶活性可以降解任何带有信号的ssrna,从而释放荧光信号。
所述步骤(3)的建立20μl的crispr-cas13a蛋白酶切割体系进行信号收集报告:0.25-10μmcas13a蛋白酶、50-400nmsgrna(sgrna:cas13a蛋白摩尔浓度为1:1-1:4之间)、500nmrna核酸探针、2μltma转录产物(1-100ng)和终浓度为20mmtris-hclph7.0@25℃、30mmkcl和2.5mmmgcl2的cas反应缓冲液。在37-42℃条件下通过荧光定量仪每反应40-60s收集一次荧光,收集30-60min。
所述步骤(3)的结果判定,基于cas13a蛋白的rna酶活性所述报告的阳性信号为荧光信号,所述阳性荧光信号因在体系中加入一条两端连接荧光物质和淬灭剂的ssrna核酸探针,当cas13a蛋白酶在sgrna的帮助下,识别具有靶向序列的靶向rna后,被激活的rna酶活性可以降解该带有信号的rna,从而释放荧光信号,实现检测。
以荧光信号为例,应按以下公式计算:
阳性判断依据:f实验组最终-f实验组最初>(f阴性组最终-f阴性组最初)x100
其中,f代表荧光信号值。阴性对照组为每个实验组相对应的没有加rna目的基因的阴性信号组。
本发明所提供的方法用于定性,因此,若最终荧光信号高于阴性样品100倍以上即可判断为阳性,1000倍以上判断为强阳性;若高于阴性样品10倍到100倍之间,可增大样品投入量再次检测。
本发明无需复杂的温度控制仪器或系统,此外,较佳地可以上述两步骤合并在一个反应体系中,更加简化操作流程。
“二合一”优选的反应体系(50μl):待检测rna片段、tma上游引物(0.5-2μm)、tma下游引物(0.5-2μm)、2000-4000um-mlv逆转录酶、500-2000ut7rna聚合酶、1-5urnase抑制剂、50-200nmcas13a蛋白酶、50-400nmsgrna、0.25-1μmrna核酸探针、终浓度为20-50mmtris-hclph7.5@25℃、20-40mmkcl、1-5mmmgcl2、1-5mmrntps、1-5mmdntps、20-50%甘油、0.5-1mmdtt的反应缓冲液。将反应管在37-40℃条件下通过实时荧光定量仪先保温10-25min后,再每反应40-60s收集一次荧光,循环收集40-60min。
最终检测手段和阳性判断方法与“两步分开”反应体系基本相同。
本发明提供了tma转录介导的扩增体系与crispr-cas13a蛋白编辑系统连用后准确鉴别病毒的检测技术,具有较高的实用价值。
本发明还提供了利用crispr-cas13a蛋白编辑系统中的非特异性rna酶活性设计的ssrna核酸探针,该核酸探针可用于不同靶标的检测反应,具有普适性。
实施例1
本实施例提供一种检测新型冠状病毒covid-19的n基因的方法,括以下步骤:
第一,为了检测新型冠状病毒rna的表达情况,按照ncbi和genbank数据库检索到新型冠状病毒covid-19的n基因核苷酸序列,化学合成一段新型冠状病毒covid-19的n基因保守序列,转染至细胞,人工制备假病毒,抽提后梯度稀释,稀释后rna的作为转录介导的扩增(tma)的模板。
第二,选择无二级结构且高度保守的区段设计并合成tma扩增covid-19的n基因的上游和下游引物。其中,上游引物n-t7f序列为:5’-aatttaatacgactcactatagggtgtaggtcaaccacgttccc-3’。其中,大写字母为t7启动子序列,小写字母为与rna(+)结合序列。下游引物n-r为:5’-caactccaggcagcagtagg-3’,下游引物为与rna(-)杂交序列。
第三,通过设计并合成引物,利用将沸水退火引物和体外转录的方法,合成了靶向上述步骤中的rna负链片段的n-sgrna,序列为:5’-gauuuagacuaccccaaaaacgaaggggacuaaaacacuaaagcauacaauguaacacaagcuuuc-3’。其中,n-sgrna的序列格式为:5’-cas13a蛋白结合的简单重复序列(36nt)+与靶向rna(-)中的片段匹配(30nt)-3’。
第四,构建tma反应体系(20μl),保持此体系中包含以下浓度的以下组分:2μl待扩增正链rna片段,t7rna聚合酶(2000u),m-mlv逆转录酶(4000u),rnase抑制剂(2u),上述的上游引物n-t7f(0.5μm)和下游引物n-r(0.5μm)以及tma反应缓冲液(20-50mmtris-hclph8.0@25℃、10-30mmkcl、1-4mmmgcl2、1-5mmrntps、1-5mmdntps、20-50%甘油、0-10%dmso、0.5-1mmdtt)。将反应体系各成分轻柔涡旋混匀后,置于水浴锅42℃反应15-25min可得转录产物,产物为待扩增正链rna的负链rna。
第五,建立基于cas13a蛋白独特的靶向rna激活的“连带切割”的非特异性rna酶活性,进行核酸序列信号报告。参与该反应的试剂中浓度为:上述tma技术转录产物ssrna(10-100ng),lwacas13a蛋白酶(1μm),n-sgrna(400nm),cas缓冲液(20mmtris-hclph7.0@25℃,50mmkcl,2.5mmmgcl2),500nmoligorna-cas13a-probe(5’端fam标记和3’端bhq1淬灭基团标记)和rnasefreewater。将反应体系各成分轻柔涡旋混匀后,置于实时荧光定量仪设置37℃每反应40-60s收集一次荧光信号,循环收集45-100次。
阴性对照组为每个实验组相对应的没有加转录产物ssrna(加rnasefreewater补足体系)的阴性信号组。
检测结果显示,实验组样本可以检测到荧光数据且随检测时间增加荧光数据也显著上升,而阴性对照组仅检测到背景荧光但是荧光数据不随着检测时间增加。按照上述阳性判断依据公式判断实验组为阳性样本。
实验结果显示,与对照组(阴性对照为rnasefreewater)相比,本发明公开的基于检测系统可以检测出低至约10nm的检测新型冠状病毒covid-19的n基因片段(如图3所示)。图3是本发明新型冠状病毒covid-19的n基因片段的荧光收集结果图,图3中1表示1μmolssrna,2表示100mmolssrna,3表示10mmolssrna,4表示1mmolssrna,5表示100nmolssrna,6表示10nmolssrna,7表示0ssrna(阴性对照)。
实施例2
本实施例提供一种快速检测检测新型冠状病毒covid-19的orf1ab基因的方法,包括以下步骤:
第一,人工制备假病毒,作为转录介导的扩增(tma)的模板。
第二,设计并合成tma扩增covid-19的orf1ab基因的上下游引物。其中,上游引物orf1ab-t7f为:5’-aatttaatacgactcactataggggcattctgtgaattataagg-3’。其中,orf1ab-t7f的5’端的24个碱基是添加的t7噬菌体启动子序列,而3’端的20个碱基是与模板rna(+)5’端杂交的特异性序列。下游引物orf1ab-r为:5’-atactgctgccgtgaacatg-3’,为与rna(-)杂交序列。
第三,通过设计并合成引物,利用将沸水退火引物和体外转录的方法,合成了靶向上述rna负链片段的sgrna,序列为:5’-gauuuagacuaccccaaaaacgaaggggacuaaaacuuugugugcugacucuaucauuauuggu-3’。其中,orf1ab-sgrna的序列格式为:5’-cas13a蛋白结合的简单重复序列(36nt)+与靶向rna(-)中的片段杂交(28nt)-3’。
第四,参考实施例1抽提假病毒rna及构建tma反应体系(20μl)。将反应体系各成分轻柔涡旋混匀后,置于水浴锅42℃反应15-25min可得转录产物,产物为待扩增正链rna的负链rna。
第五,参考实施例1建立基于cas13a蛋白独特的靶向rna激活的rna酶活性,进行核酸序列信号报告反应体系。参与该反应的试剂中浓度为:上述tma技术转录产物ssrna(10-100ng),lwacas13a蛋白酶(4μm),orf1ab-sgrna(100nm),cas缓冲液(10mmtris-hclph7.0@25℃,30mmkcl,2.5mmmgcl2),500nmoligorna-cas13a-probe和rnasefreewater。将反应体系混匀后,置于实时荧光定量仪设置37-42℃每反应50s收集一次荧光信号,循环收集45-100次。
阴性对照组为每个实验组相对应的没有加转录产物ssrna(加rnasefreewater补足体系)的阴性信号组。
检测结果显示,实验组检测到荧光数据且随检测时间增加荧光数据也逐步增加,而阴性对照组仅检测到背景荧光但是荧光数据不随着检测时间增加。按照上述阳性判断依据公式是否为阳性样本。
实验结果显示,与对照组(阴性对照为rnasefreewater)相比,本发明公开的基于tma技术结合crispr-cas13a的系统可以检测出低至约10nm的人新型冠状病毒covid-19的orf1ab基因片段(图4)。图4是本发明新型冠状病毒covid-19的orf1ab基因片段的荧光收集结果图。图4中1表示1μmolssrna,2表示100mmolssrna,3表示10mmolssrna,4表示1mmolssrna,5表示100nmolssrna,6表示10nmolssrna,7表示0ssrna(阴性对照)。
实施例3
本实施例提供一种将tma和cas13a“二合一”检测新型冠状病毒covid-19的orf1ab基因的方法,包括以下步骤:
第一,所述实施例3中所需模板、引物和核酸探针可根据实施例2制备。
第二,建立tma联合cas13a“二合一”的反应体系(50μl)。参与该反应的试剂及具体浓度为:待检测rna(+)片段、tma上下游引物orf1ab-t7f(1μm)和orf1ab-r(1μm)、2000-4000um-mlv逆转录酶、500-2000ut7rna聚合酶、4urnase抑制剂、2μmcas13a蛋白酶、400nmorf1ab-sgrna、0.5μmoligorna-cas13a-probe、tc反应缓冲液(20-50mmtris-hclph7.5@25℃、20-40mmkcl、1-5mmmgcl2、1-5mmrntps、1-5mmdntps、20-50%甘油、0.5-1mmdtt)。
第三,反应体系各成分轻柔涡旋混匀后,将反应管在37-42℃条件下通过荧光定量仪反应10-25min后,再37℃每反应40-60s收集一次荧光,循环收集45-100次。
阴性对照组为每个实验组相对应的没有加模板rna(加rnasefreewater补足体系)的阴性信号组。
检测结果显示,实验组检测到荧光数据且随检测时间增加荧光数据也逐步增加,而阴性对照组仅检测到背景荧光但是荧光数据不随着检测时间增加。按照上述阳性判断依据公式是否为阳性样本。
实验结果显示,与对照组(阴性对照为rnasefreewater)相比,本发明公开的基于tma和crispr-cas13a“二合一”联合的系统可以检测出低至约10nm的新型冠状病毒covid-19的orf1ab基因片段(如图5所示)。图5是本发明“二合一”检测新型冠状病毒covid-19的orf1ab基因片段的荧光收集结果图。图5中1表示1μmolssrna,2表示100mmolssrna,3表示10mmolssrna,4表示1mmolssrna,5表示100nmolssrna,6表示10nmolssrna,7表示1nmolssrna,8表示0ssrna(阴性对照)。
最后应说明的是:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110>吴江近岸蛋白质科技有限公司
<120>一种检测新型冠状病毒covid-19的核酸组合物及应用
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guuagaauuacuggcuuauacccaacacucaauaucucagaugaguuuucuagcaauguu16560
gcaaauuaucaaaagguugguaugcaaaaguauucuacacuccagggaccaccugguacu16620
gguaagagucauuuugcuauuggccuagcucucuacuacccuucugcucgcauaguguau16680
acagcuugcucucaugccgcuguugaugcacuaugugagaaggcauuaaaauauuugccu16740
auagauaaauguaguagaauuauaccugcacgugcucguguagaguguuuugauaaauuc16800
aaagugaauucaacauuagaacaguaugucuuuuguacuguaaaugcauugccugagacg16860
acagcagauauaguugucuuugaugaaauuucaauggccacaaauuaugauuugaguguu16920
gucaaugccagauuacgugcuaagcacuauguguacauuggcgacccugcucaauuaccu16980
gcaccacgcacauugcuaacuaagggcacacuagaaccagaauauuucaauucagugugu17040
agacuuaugaaaacuauagguccagacauguuccucggaacuugucggcguuguccugcu17100
gaaauuguugacacugugagugcuuugguuuaugauaauaagcuuaaagcacauaaagac17160
aaaucagcucaaugcuuuaaaauguuuuauaaggguguuaucacgcaugauguuucaucu17220
gcaauuaacaggccacaaauaggcgugguaagagaauuccuuacacguaacccugcuugg17280
agaaaagcugucuuuauuucaccuuauaauucacagaaugcuguagccucaaagauuuug17340
ggacuaccaacucaaacuguugauucaucacagggcucagaauaugacuaugucauauuc17400
acucaaaccacugaaacagcucacucuuguaauguaaacagauuuaauguugcuauuacc17460
agagcaaaaguaggcauacuuugcauaaugucugauagagaccuuuaugacaaguugcaa17520
uuuacaagucuugaaauuccacguaggaauguggcaacuuuacaagcugaaaauguaaca17580
ggacucuuuaaagauuguaguaagguaaucacuggguuacauccuacacaggcaccuaca17640
caccucaguguugacacuaaauucaaaacugaagguuuauguguugacauaccuggcaua17700
ccuaaggacaugaccuauagaagacucaucucuaugauggguuuuaaaaugaauuaucaa17760
guuaaugguuacccuaacauguuuaucacccgcgaagaagcuauaagacauguacgugca17820
uggauuggcuucgaugucgaggggugucaugcuacuagagaagcuguugguaccaauuua17880
ccuuuacagcuagguuuuucuacagguguuaaccuaguugcuguaccuacagguuauguu17940
gauacaccuaauaauacagauuuuuccagaguuagugcuaaaccaccgccuggagaucaa18000
uuuaaacaccucauaccacuuauguacaaaggacuuccuuggaauguagugcguauaaag18060
auuguacaa18069
<210>3
<211>44
<212>dna
<213>人工序列(artificialsequence)
<400>3
aatttaatacgactcactatagggtgtaggtcaaccacgttccc44
<210>4
<211>20
<212>dna
<213>人工序列(artificialsequence)
<400>4
caactccaggcagcagtagg20
<210>5
<211>44
<212>dna
<213>人工序列(artificialsequence)
<400>5
aatttaatacgactcactataggggcattctgtgaattataagg44
<210>6
<211>20
<212>dna
<213>人工序列(artificialsequence)
<400>6
atactgctgccgtgaacatg20
<210>7
<211>66
<212>rna
<213>人工序列(artificialsequence)
<400>7
gauuuagacuaccccaaaaacgaaggggacuaaaacacuaaagcauacaauguaacacaa60
gcuuuc66
<210>8
<211>64
<212>rna
<213>人工序列(artificialsequence)
<400>8
gauuuagacuaccccaaaaacgaaggggacuaaaacuuugugugcugacucuaucauuau60
uggu64
<210>9
<211>22
<212>rna
<213>人工序列(artificialsequence)
<400>9
gaauuccaccacguucccgugg22