CN117987554A - 一种基于Cas蛋白的检测方法 - Google Patents
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Abstract
本发明涉及一种基于Cas蛋白的检测方法,包括以下步骤:S1、根据目的基因设计合成PCR扩增引物;并根据PCR扩增引物合成crRNA;S2、采用PCR扩增引物对目的基因进行PCR扩增,得到扩增产物;后用Cas蛋白、crRNA和荧光探针体系检测扩增产物;所述目的基因为骨髓增殖性肿瘤患者的突变体细胞基因。本检测技术最高可检测JAK2 V617F突变占比0.01%的混合gDNA,远高于临床所需的灵敏度;④操作简便,耗时短。
Description
技术领域
本发明涉及一种用于基于Cas蛋白的检测JAK2 V617F突变的方法。
背景技术
骨髓增殖性肿瘤(Myeloproliferative neoplasms,MPNs)是一类造血干/祖细胞的恶性克隆性疾病,其特征是一系或者多系骨髓细胞克隆性增殖而细胞形态和功能相对正常,临床有一种或多种血细胞增生,常伴有脾大、出血倾向、骨髓纤维化和血栓形成等表现。经典MPNs主要包括真性红细胞增多症(polycythemia vera,PV)、原发性血小板增多症(essential thrombocythemia,ET)、原发性骨髓纤维化(primary myelofibrosis,PMF)等[1-3]。经典MPNs的年发病率为0.5-6.5/100000,但是JAK2 V617F的携带率远高于经典MPNs的发病率[4,5]。JAK2 V617F是经典MPNs最具特征性的突变基因[3],在PV,ET和PMF中,JAK2V617F突变率分别为95%,55%和65%[6-8],2016年世界卫生组织(World HealthOrganization,WHO)将JAK2 V617F突变作为它们确诊的主要标准之一,并且将JAK2 V617F突变检测作为经典MPNs诊断的起始性步骤[2,3],因此对JAK2 V617F进行准确的检测极其重要。而且,经典MPNs属于一类恶性血液系统肿瘤,严重影响患者生活质量,给社会和家庭带来了严重的精神及经济压力。针对JAK2 V617F突变,美国食品药品监督管理局批准了JAK2抑制剂Ruxolitinib和Fedratinib等药治疗经典MPNs,对JAK2 V617F突变进行准确的诊断有利于患者的早发现、早干预、早治疗,降低患者的死亡率,延长患者的存活期,提高患者的生活质量。因此,开发一种快速准确、简便廉价且易于普及的检测方法应用于JAK2 V617F突变的筛查,从而有效的防控MPNs,具有重大的社会价值与现实意义。
JAK2定位于9p24,是一种非受体型酪氨酸激酶,属Janus激酶家族,在细胞因子和多种造血生长因子受体的信号传导中发挥重要作用[9,10]。JAK2的结构包括JH1和JH2两个同源激酶结构域,其中JH1是JAK2的催化活性区,具有酪氨酸激酶活性,JH2没有激酶活性,对JH1激酶活性具有负性调控作用[11]。JAK2 V617F为JAK2基因14号外显子第1849位碱基发生的点突变(G>T),即第617位缬氨酸被苯丙氨酸取代,位于JH2区域,使JH2结构域对JH1结构域的负性调控作用减弱,导致JAK-STAT信号通路激活,进而细胞异常增殖、分化,最终发展为MPNs[11,12]。
针对JAK2 V617F突变的检测技术主要有DNA测序、等位基因特异性PCR(Allele-Specific PCR/Amplification refractory mutation system,ARMS)、PCR-限制性片段长度多态性分析(PCR-restriction fragment length polymorphism,PCR-RFLP)、实时定量PCR法、数字PCR(droplet digital PCR,dd-PCR)等等[6,13-23]。但DNA测序技术缺点:①灵敏度低;②需配备昂贵的测序设备。AS-PCR技术缺点:假阳性率高。PCR-RFLP技术缺点:①灵敏度低;②酶切不完全、引物或限制性内切酶识别区域的碱基变异均影响结果的准确性;③步骤多,操作繁琐。qPCR技术缺点:①反应体系对DNA样本纯度要求高,残留杂质影响检测结果;②人员的操作水平要求高;③需配备荧光定量PCR仪。ddPCR技术缺点:①人员的操作水平要求高;②需配备昂贵的ddPCR仪器和试剂;③步骤多,操作繁琐。因此,开发出一种快速准确、低成本高检测性能且易于推广普及的JAK2 V617F突变检测技术,应用于经典MPNs诊断,进而提升对经典MPNs的防控能力是目前亟需解决的问题。
2016年Feng Zhang等发现Cas13a(又名C2c2)在crRNA引导下识别切割靶RNA序列后能被激活,活化后的Cas13a表现出不加区别的切割活性(附带切割活性),既能特异地切割靶RNA分子,又能非特异地切割其他RNA分子[24]。2018年Chen和Li分别报道基于Cas12a的DNA分子检测技术,其技术原理相似:扩增后的靶DNA分子被Cas12a-crRNA复合物特异识别切割,从而激活Cas12a,利用活化后Cas12a的附带切割效应切割DNA荧光探针,通过检测荧光信号从而达到检测靶DNA的目的[25,26]。但上述报道均是应用于病原菌,并无任何体细胞相关的检测。目前国际上尚无CRISPR/Cas分子检测应用于JAK2 V617F突变检测的报道。
参考文献
[1]E.S.Jaffe,N.L.Harris,H.Stein,et al.,World Health OrganizationClassifification of Tumours.Pathology and Geneticsof Tumours ofHaematopoietic and Lymphoid Tissues[J].Lyon,France:IARC,2001.
[2]S.H.Swerdlow,E.Campo,N.L.Harris,et al.,WHO Classifification ofTumours of Haematopoietic and LymphoidTissues[J].Lyon,France:IARC,2008.
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发明内容
本发明目的是提供一种基于Cas蛋白在制备检测JAK2 V617F突变基因的试剂中的应用,以MPNs作为目标疾病,促进Cas蛋白核酸检测技术向肿瘤等体细胞突变检测转化,丰富现有Cas蛋白核酸检测技术的应用范围。
为了解决上述技术问题,本发明的技术方案如下:
一种基于Cas蛋白的检测方法,包括以下步骤:
S1、根据目的基因设计合成PCR扩增引物;并根据PCR扩增引物合成crRNA;
S2、采用PCR扩增引物对目的基因进行PCR扩增,得到扩增产物;后用Cas蛋白、crRNA和荧光探针体系检测扩增产物;
所述crRNA的序列如SEQ ID NO.5-7所示,所述目的基因为骨髓增殖性肿瘤患者的体细胞突变基因。
优选的,所述目的基因的序列如SEQ ID NO.15所示。
其中目的基因的具体说明如图11所示。
优选的,所述目的基因直接从外周血提取。
对外周血提取的DNA进行检测,不需要进行粒细胞的分离,简化了操作流程,降低了检测成本。
优选的,所述Cas蛋白为Cas12a、Cas13、Cas12b或Cas14。
进一步优选的,所述Cas蛋白为Cas12a。
Cas12a和Cas12b可以直接识别双链DNA,Cas13识别的靶核酸分子是单链RNA,Cas14识别的靶核酸分子是单链DNA。应用实例中的靶核酸分子为双链DNA,可以用Cas12a或Cas12b直接识别。如果使用Cas13则需增加检测步骤,从靶双链DNA分子转录出单链RNA分子才能进行检测。如果使用Cas14也需增加检测步骤,需降解靶双链DNA分子其中1条链,形成单链DNA分子后才能进行检测。
本发明要求保护一种crRNA,crRNA的序列如SEQ ID NO.5-7所示。
本发明要求保护所述crRNA在制备检测JAK2 V617F突变基因的试剂中的应用。
本发明要求保护一种试剂盒,所述试剂盒包含上述的crRNA。
本发明要求保护所述试剂盒在制备检测JAK2 V617F突变基因的试剂中的应用。
本发明要求保护一种RPA引物,RPA引物如SEQ ID NO.16-24所示。
优选的,所述RPA引物如SEQ ID NO.17和SEQ ID NO.23所示。
本发明要求保护一种试剂盒,所述试剂盒包含上述的RPA引物。
本发明要求保护所述试剂盒在制备检测JAK2 V617F突变基因的试剂中的应用。
所述应用均可为非疾病诊断目的。
下面对本发明做进一步的解释:
(1)本研究设计了基于CRISPR/Cas12a检测肿瘤相关的体细胞突变JAK2 V617F的方法,具有极高的灵敏度,达到了0.01%,远高于临床所需的1%-3%的检测灵敏度,这对于骨髓增殖性肿瘤的早期诊断以及监测具有重要意义。
(2)本研究具有极高的灵敏度,因此可以直接对外周血提取的DNA进行检测,不需要进行粒细胞的分离,简化了操作流程,降低了检测成本。
(3)结合核酸等温扩增和试纸条进行检测时,不需要大型仪器,可通过肉眼直接进行结果观测,检测更方便,更快速,更易于推广普及,可实现现场检测。
(4)目前基于CRISPR/Cas12a的核酸检测技术并没有关于定量检测的研究,本发明除了具有极高灵敏的定性检测能力之外,还具有定量检测的潜能,相同的仪器设备可同时进行定量和定性检测,避免购置不同设备,重复投入。
(5)相较于现有的JAK2 V617F突变检测技术,本研究具有很强的可塑性。当骨髓增殖性肿瘤患者具有多种体细胞突变时,比如JAK2基因12号外显子、MPL、CALR等基因突变,本研究可以通过改变crRNA序列,实现多种体细胞突变的检测。
与现有技术相比,本发明的有益效果如下:
本发明从MPNs诊治的迫切需求出发,结合我国地区间发展不平衡的实际,开发出一种新的JAK2 V617F检测技术体系,解决现有检测技术的不足并达到以下目标:①检测周期短,成本低;②对仪器设备和人员要求低,即使在落后地区也能开展检测;③临床上需要的检测灵敏度在1%-3%,本检测技术最高可检测JAK2 V617F突变占比0.01%的混合gDNA,远高于临床所需的灵敏度;④操作简便,耗时短。
附图说明
图1是crRNA-1、crRNA-2、crRNA-3与检测基因的错配位点的示意图;
图2是本发明的PCR反应流程示意图;
图3是本发明应用crRNA-1、crRNA-2、crRNA-3对突变型质粒和野生型质粒进行检测的结果;
图4是本发明Cas12a-荧光定性检测JAK2 V617F突变示意图;
图5是本发明Cas12a-JAK2 V617F荧光定性检测质粒DNA结果;
图6是本发明Cas12a-JAK2 V617F荧光定性检测细胞gDNA结果;
图7是本发明Cas12a-试纸条定性检测JAK2 V617F突变示意图;
图8是Cas12a-JAK2 V617F试纸条定性检测细胞gDNA结果;
图9是Cas12a-JAK2 V617F试纸条定性检测患者和健康人gDNA结果;
图10是Cas12a-JAK2 V617F荧光定量检测的结果(PCR扩增);
图11是目的基因的具体说明示意图;
图12是RPA引物设计位置示意图。
具体实施方式
以下将结合实施例来详细说明本发明。需要说明的是,在不冲突的情况下,本发明中的实施例及实施例中的特征可以相互组合。
实施例1
构建JAK2 V617F突变型质粒以及野生型质粒
根据JAK2基因序列,设计引物,通过Overlap PCR引入JAK2 V617F这一突变位点,构建含有JAK2 V617F这一位点的纯合突变型和不含有JAK2 V617F突变的纯合野生型质粒,并经测序鉴定。
具体实验步骤:
①引物设计:引物F和R为JAK2 V617F突变点两端特异性引物,Fm和Rm为中间引物,在其中引入JAK2 V617F这一突变位点,并且Fm和Rm中间有32bp的互补区域;
其中引物F序列(5’-ACTTGGCTCCCCAGAGCTTTATGGGT-3’,SEQ ID NO.1);
引物R序列(5’-AGACATGAAATGAGCACATGCTGTTGGGA-3’,SEQ ID NO.2);
引物Fm序列(5’-TGGTTTTAAATTATGGAGTATGTTTCTGTGGAGACGAG-3’,SEQ ID NO.3);
引物Rm序列(5’-ACTTACTCTCGTCTCCACAGAAACATACTCCATAATTTA-3’,SEQ IDNO.4)。
②以正常人gDNA为模板,PCR扩增JAK2-FRm、JAK2-FmR和JAK2-FR,产物长度分别为897bp、679bp和1531bp;
③胶回收:使用TSINGKE公司的DNA凝胶回收试剂盒对胶中的目的片段进行回收;
④重叠引物延伸获得含有JAK2 V617F这一突变位点的产物:将胶回收后的JAK2-FRm和JAK2-FmR按照摩尔数比1:1加入到PCR体系中,扩增获得目的片段JAK2 V617F-FR,产物长度为1531bp;
⑤PCR产物JAK2-FR和JAK2 V617F-FR连接到T easy vector上然后进行转化与单克隆挑取;菌液送TSINGKE公司测序鉴定,对鉴定成功的菌液进行质粒小量抽提。
实施例2
crRNA的设计与合成
根据靶基因序列以及Cas12a的PAM序列(5’-TTTN和5’-TTN),设计了三条crRAN(crRNA-1,crRNA-2,crRNA-3),并用构建成功的JAK2 V617F突变型质粒以及野生型质粒来筛选出最优的crRNA。
crRNA-1序列(5’-UAAUUUCUACUAAGUGUAGAUUGUGGAGACGAGAGUAAGUAAAA-3’,SEQ IDNO.5),crRNA-2序列(5’-UAAUUUCUACUAAGUGUAGAUAAUUAUGGAGUAUGUUUCUGUGG-3’,SEQ IDNO.6),crRNA-3序列(5’-UAAUUUCUACUAAGUGUAGAUUGGAGUAUGUUUCUGUGGAGACG-3’,SEQ IDNO.7);
均在生工生物公司进行合成。三条crRNA识别的均是含有JAK2 V617F突变的序列。其中crRNA-1和crRNA-2针对5’-TTTN设计,crRNA-3是针对5’-TTN设计。三条crRNA与检测基因的错配位点如图1所示。
①将突变型质粒和野生型质粒浓度稀释到0.01pg/ul(该浓度的拷贝数相当于100ng/ul的人gDNA的拷贝数);
②PCR扩增①中质粒的JAK2基因14号外显子。
其中Taq酶:Premix TaqTMHot Start(Takara),上游引物序列(5’-AGCAAGCTTTCTCACAAGCAT-3’,SEQ ID NO.8),下游引物序列(5’-ACACCTAGCTGTGATCCTGAA-3’,SEQ ID NO.9)。
配制反应体系
按循环参数进行图2所示的过程扩增。
③Cas12a识别JAK2 V617F突变,活化后非特异性切割探针。
其中:Cas12a及缓冲液2.1Buffer(NEB),
crRNA-1序列(5’-UAAUUUCUACUAAGUGUAGAUUGUGGAGACGAGAGUAAGUAAAA-3’,SEQ IDNO.5),crRNA-2序列(5’-UAAUUUCUACUAAGUGUAGAUAAUUAUGGAGUAUGUUUCUGUGG-3’,SEQ IDNO.6),crRNA-3序列(5’-UAAUUUCUACUAAGUGUAGAUUGGAGUAUGUUUCUGUGGAGACG-3’,SEQ IDNO.7);
探针ssDNA(FAM-5’-TTTTTTTTTTTT-3’-MGB,SEQ ID NO.10)。
配制反应体系
混匀后置于荧光仪37℃持续孵育,孵育60min,每分钟检测荧光值,△荧光值=终点荧光值—起始荧光值。与阴性对照相比,△荧光值明显增长,检测阳性,含有JAK2 V617F突变。反之,△荧光值无明显增长,检测阴性,不具有JAK2 V617F突变(图3)。(FAM激发光波长480nm,发射光波长520nm)。
结果可知,将突变型质粒和野生型质粒进行PCR扩增时,三种Cas12a/crRNA复合物均能区分突变型质粒和野生型质粒,其中crRNA-1表现出最优的特异性和灵敏度,crRNA-3的特异性比crRNA-2要好,但是灵敏度要低。所以后续研究我们选择了crRNA-1。
实施例3
Cas12a-JAK2 V617F荧光定性检测方法检测质粒DNA
具体技术方案如下:
①将突变型质粒和野生型质粒浓度稀释到0.01pg/ul(该浓度的拷贝数相当于100ng/ul的人gDNA的拷贝数);
②将①中的突变型质粒与野生型质粒按不同比例混合(突变型质粒占比依次有12.5%,5%,2%,1%,0.5%,0.25%,0.125%,0.05%,0.01%,0%),探索Cas12a-JAK2V617F荧光定性检测方法的灵敏度;
③扩增②中质粒的JAK2基因14号外显子。
该步骤有2种方法可供选择:聚合酶链式反应(PCR)和重组酶聚合酶扩增(RPA)。
PCR法:配制反应体系
按循环参数如图2所示,进行扩增。
RPA法:配制反应体系,37℃孵育40min。
④Cas12a识别JAK2 V617F突变,活化后非特异性切割探针。
配制反应体系,探针ssDNA(FAM-5’-TTTTTTTTTTTT-3’-MGB,SEQ ID NO.10);
混匀后置于荧光仪37oC持续孵育,孵育60min,每分钟检测荧光值,△荧光值=终点荧光值—起始荧光值。与阴性对照相比,△荧光值明显增长,检测阳性,含有JAK2 V617F突变。反之,△荧光值无明显增长,检测阴性,不具有JAK2 V617F突变(如图4所示)。(根据探针ssDNA标记的荧光基团选择荧光仪检测时的激发光和发射光波长,FAM激发光波长480nm,发射光波长520nm)。混合质粒样本中含有JAK2 V617F突变的DNA序列,Cas12a在crRNA引导下识别扩增产物中含有JAK2 V617F突变的序列,并被激活,活化后的Cas12a切割荧光探针,产生荧光信号。
按PCR扩增的方法得到的结果如图5所示,将纯合突变型和野生型质粒按不同比例混合,Cas12a-JAK2 V617F荧光定性检测体系能有效检测出所有的突变型质粒,最低含有0.01%突变型质粒的混合质粒的荧光也有明显差异,进一步验证了本发明的方法的极高灵敏度。
实施例4
Cas12a-JAK2 V617F荧光定性检测方法检测细胞gDNA
先培养JAK2 V617F纯合突变的HEL细胞与JAK2纯合野生型的hiPSCs,具体方法为:从中国科学院细胞库购买JAK2 V617F纯合突变的HEL细胞,本课题组已有JAK2纯合野生型的hiPSCs,将这些细胞经过数代培养,当达到足够多数量时并通过苯酚-氯仿法抽提其gDNA。
在用Cas12a-JAK2 V617F荧光定性检测方法检测细胞gDNA,具体步骤为:
①将HEL-gDNA和hiPSC-gDNA浓度稀释到100ng/ul(临床上从病人外周血中提取的gDNA浓度也在100ng/ul左右);
②将①中的HEL-gDNA和hiPSC-gDNA按不同比例混合(HEL-gDNA占比依次有2%,1%,0.5%,0.25%,0.125%,0.05%,0.01%,0%),探索Cas12a-JAK2 V617F荧光定性检测方法的灵敏度;
③PCR扩增②中gDNA的JAK2基因14号外显子。
其中Taq酶:Premix TaqTMHot Start(Takara),上游引物序列(5’-AGCAAGCTTTCTCACAAGCAT-3’,SEQ ID NO.11),下游引物序列(5’-ACACCTAGCTGTGATCCTGAA-3’,SEQ ID NO.12)。
配制反应体系
按循环参数按图2所示进行扩增
④Cas12a识别JAK2 V617F突变,活化后非特异性切割探针。
其中:其中:Cas12a及缓冲液2.1Buffer(NEB),crRNA-1序列(5’
-UAAUUUCUACUAAGUGUAGAUUGUGGAGACGAGAGUAAGUAAAA-3’),探针ssDNA(FAM-5’
-TTTTTTTTTTTT-3’-MGB)。
配制反应体系
混匀后置于荧光仪37℃持续孵育,孵育60min,每分钟检测荧光值,△荧光值=终点荧光值—起始荧光值。与阴性对照相比,△荧光值明显增长,检测阳性,含有JAK2 V617F突变。反之,△荧光值无明显增长,检测阴性,不具有JAK2 V617F突变。(FAM激发光波长480nm,发射光波长520nm)。
结果如图6所示,将HEL-gDNA和hiPSC-gDNA按不同比例混合,JAK2 V617F-Cas12a荧光定性检测体系能有效检测出含有0.01%HEL-gDNA的混合gDNA。
实施例5
Cas12a-JAK2 V617F试纸条定性检测方法检测细胞gDNA
具体技术方案如下:
①将HEL-gDNA和hiPSC-gDNA浓度稀释到100ng/ul(临床上从病人外周血中提取的gDNA浓度也在100ng/ul左右);
②将①中的HEL-gDNA和hiPSC-gDNA按不同比例混合(HEL-gDNA占比依次有2%,1%,0.5%,0.25%,0.125%,0.05%,0.01%,0%),探索Cas12a-JAK2 V617F试纸条定性检测方法的灵敏度;
③扩增②中gDNA的JAK2基因14号外显子(方法同实施例3的③)。
④Cas12a识别JAK2 V617F,活化后非特异性切割探针。
配制反应体系,37℃孵育20min。
⑤试纸条读取检测信号。
将④的20μl体系加至100μl缓冲液中,垂直插入试纸条,质控带1-2 min后即可显色,待质控带显色2 min后,肉眼读取检测带信号。检测带显色,检测阳性,为JAK2 V617F突变阳性。反之,检测带未显色,检测阴性,为JAK2 V617F突变阴性(具体过程如图7所示)。具体机理为:DNA样本中含有JAK2 V617F突变,Cas12a在crRNA引导下识别含有JAK2 V617F突变的序列并被激活,活化后的Cas12a非特异性切割异硫氰酸荧光素(fluoresceinisothiocyanate,FITC)和生物素标记的ssDNA探针,部分完整的生物素标记的ssDNA探针结合部分胶体金被质控带链亲和素捕获,质控带显色,多余的FITC抗体标记的胶体金跑到检测带,与检测带的FITC抗体二抗结合,聚集在检测带,检测带显色,提示DNA样本中存在JAK2V617F突变。当DNA样本中不存在JAK2 V617F突变时,不能激活Cas12a,不能切割FITC和生物素标记的ssDNA探针,所有完整的探针结合全部胶体金在质控带被链亲和素捕获,质控带显色,没有胶体金到检测带,检测带不显色,提示DNA样本中不含有JAK2 V617F突变。
结果如图8所示。其中:试纸条及缓冲液Milenia HybriDetect 1(MileniaBiotech)。
将HEL-gDNA和hiPSC-gDNA按不同比例混合,JAK2 V617F-Cas12a试纸条检测体系能有效检测出含有0.5%HEL-gDNA的混合gDNA。
实施例6
RPA引物筛选
按照RPA引物设计原则,设计了5条正向引物以及4条反向引物,具体如下:
表1 RPA引物表
名称 | 序列 |
RPA-F1 | 5’-CTTAGTCTTTCTTTGAAGCAGCAAGTATGATG-3’(SEQ ID NO.16) |
RPA-F2 | 5’-CTTTATTATGGCAGAGAGAATTTTCTGAACT-3’(SEQ ID NO.17) |
RPA-F3 | 5’-CTATTTATGGACAACAGTCAAACAACAATTC-3’(SEQ ID NO.18) |
RPA-F4 | 5’-TTCTGAACTATTTATGGACAACAGTCAAACAA-3’(SEQ ID NO.19) |
RPA-F5 | 5’-TTCTCACAAGCATTTGGTTTTAAATTATGG-3’(SEQ ID NO.20) |
RPA-R1 | 5’-CCTAGCTGTGATCCTGAAACTGAATTTTCTAT-3’(SEQ ID NO.21) |
RPA-R2 | 5’-AACTGAATAGTCCTACAGTGTTTTCAGTTTC-3’(SEQ ID NO.22) |
RPA-R3 | 5’-CCTACAGTGTTTTCAGTTTCAAAAATACTTAAC-3’(SEQ ID NO.23) |
RPA-R4 | 5’-TTATAGTTTACACTGACACCTAGCTGTGATCC-3’(SEQ ID NO.24) |
上述引物的位置示意图如图12所示。
将上述引物两两组合扩增正常人gDNA,然后将扩增产物纯化后跑胶。根据胶图发现RPA-F2/RPA-R3组合的扩增效率最高,结果如下所示:
表2 RPA引物扩增结果
F1 | F2 | F3 | F4 | F5 | |
R1 | - | - | - | + | - |
R2 | - | - | - | ++ | + |
R3 | + | +++ | ++ | ++ | + |
R4 | - | - | - | - | - |
进一步切胶送测序验证扩增序列,结果显示扩增的是目的序列。所以我们选择RPA-F2/RPA-R3引物对进行后续的研究。
实施例7
Cas12a-JAK2 V617F试纸条定性检测方法检测患者和健康人gDNA
具体技术方案如下:
①RPA扩增患者和健康人gDNA的JAK2基因14号外显子。
其中RPA试剂:TwistAmp Liquid Basic试剂盒(Twist Dx),dNTP(上海生工),上游引物(5’-CTTTATTATGGCAGAGAGAATTTTCTGAACT-3’,SEQ ID NO.17),下游引物(5’-CCTACAGTGTTTTCAGTTTCAAAAATACTTAAC-3’,SEQ ID NO.23)。
配制反应体系,37℃孵育40min。
②Cas12a识别JAK2 V617F,活化后非特异性切割探针。
其中:Cas12a及缓冲液2.1Buffer(NEB),crRNA-1序列(5’-UAAUUUCUACUAAGUGUAGAUUGUGGAGACGAGAGUAAGUAAAA-3’),探针ssDNA
(FITC-5’-ACACACACACACACACACAC-3’-Biotin)。
配制反应体系,37℃孵育20min。
③试纸条读取检测信号。
将②的20μl体系加至100μl缓冲液中,垂直插入试纸条,质控带1-2min后即可显色,待质控带显色2min后,肉眼读取检测带信号。检测带显色,检测阳性,为JAK2 V617F突变阳性。反之,检测带未显色,检测阴性,为JAK2 V617F突变阴性(图9)。其中:试纸条及缓冲液Milenia HybriDetect 1(Milenia Biotech)。
结果显示:应用Cas12a-JAK2 V617F试纸条定性检测体系检测1个患者和13个健康人gDNA,1个患者的检测结果为阳性,13个健康人的检测结果均为阴性。未出现假阳性与假阴性结果。
实施例7
Cas12a-JAK2 V617F试纸条定量检测
具体技术方案如下:
①将突变型质粒和野生型质粒浓度稀释到0.01pg/ul(该浓度的拷贝数相当于100ng/ul的人gDNA的拷贝数);
②将①中的突变型质粒与野生型质粒按不同比例混合(突变型质粒占比依次有78%,50%,31%,12.5%,5%,2%,0%)作为标准品;
③扩增患者gDNA和②中作为标准品的质粒的JAK2基因14号外显子(方法同实施例3的③)。
其中Taq酶:Premix TaqTMHot Start(Takara),上游引物序列(5’-AGCAAGCTTTCTCACAAGCAT-3’,SEQ ID NO.13),下游引物序列(5’-ACACCTAGCTGTGATCCTGAA-3’,SEQ ID NO.14)。
配制反应体系
按图2所示的循环参数进行扩增。
④Cas12a识别含有JAK2 V617F突变的序列,活化后非特异性切割探针。
其中:Cas12a及缓冲液2.1Buffer(NEB),crRNA-1序列(5’-UAAUUUCUACUAAGUGUAGAUUGUGGAGACGAGAGUAAGUAAAA-3’),探针ssDNA(FAM-5’-TTTTTTTTTTTT-3’-MGB)。
配制反应体系
④混匀后置于荧光仪37oC持续孵育,孵育60min,每分钟检测荧光值,△荧光值=终点荧光值—起始荧光值。不同JAK2 V617F突变比率(0%,2%,5%,12.5%,31%,50%,78%)的质粒标准品的△荧光值制作标准曲线,根据标准曲线以及患者gDNA的△荧光值就可以知道患gDNA中含有JAK2 V617F突变的比率。其中:试纸条及缓冲液MileniaHybriDetect 1(Milenia Biotech)。
结果如图10所示,应用突变型质粒占比依次有78%,50%,31%,12.5%,5%,2%,0%的混合质粒作为Cas12a-JAK2V617F定量检测的标准品,R2达到了0.9723,说明Cas12a-JAK2 V617F具有定量检测的潜能。
上述实施例阐明的内容应当理解为这些实施例仅用于更清楚地说明本发明,而不用于限制本发明的范围,在阅读了本发明之后,本领域技术人员对本发明的各种等价形式的修改均落入本申请所附权利要求所限定的范围。
Claims (10)
1.一种非疾病诊断或治疗目的的基于Cas蛋白的试纸条检测方法,其特征在于,包括以下步骤:
S1、根据目的基因设计合成RPA扩增引物和crRNA;
S2、采用RPA扩增引物对目的基因进行RPA扩增,得到扩增产物;后用Cas蛋白、缓冲液、crRNA和探针ssDNA配置成反应体系,在反应体系中插入试纸条,检测扩增产物;所述目的基因为骨髓增殖性肿瘤患者的突变体细胞JAK2基因;
所述crRNA的序列如SEQ ID NO.5-7所示。
2.根据权利要求1所述检测方法,其特征在于,所述目的基因直接从外周血提取。
3.根据权利要求1所述检测方法,其特征在于,所述目的基因的序列如SEQ ID NO.15所示。
4.一种基于Cas蛋白的荧光检测体系,其特征在于,每20µl体系中包括:ddH2O 14.85μl、缓冲液2 μl、Cas12a 0.05 μl、crRNA 0.1 μl、探针ssDNA 1 μl、靶目标 2 μl;
所述crRNA的序列如SEQ ID NO.5-7所示。
5.根据权利要求4所述的检测体系,其特征在于,所述探针ssDNA的序列如SEQ IDNO.10所示。
6.一种RPA引物,其特征在于,所述RPA引物序列如SEQ ID NO.16-24所示。
7.根据权利要求6所述的RPA引物,其特征在于,所述RPA引物序列如SEQ ID NO.17和SEQ ID NO.23所示。
8.一种RPA反应体系,其特征在于,所述体系包含如权利要求6或7所述的RPA引物。
9.一种基于Cas蛋白的试纸条检测体系,其特征在于,每120µl体系中包括:ddH2O 14.85μl、缓冲液2 μl、Cas12a 0.05 μl、crRNA 0.1 μl、探针ssDNA 1 μl、靶目标 2 μl;
所述crRNA的序列如SEQ ID NO.5-7所示。
10.根据权利要求9所述的检测体系,其特征在于,所述探针ssDNA的序列为:FITC-5’-ACACACACACACACACACAC-3’-Biotin。
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