CN116732167B - 基于crispr基因编辑技术的ad携带基因检测方法 - Google Patents
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Abstract
本发明公开了一种基于CRISPR基因编辑技术的AD携带基因检测方法,属于生物与医学检测技术领域,本发明采用的是重组酶聚合酶扩增(RPA)基因编辑CRISPR‑Cas12a技术。该方法不仅能将样品处理时间大幅度缩短,试剂和仪器成本大幅度降低,还能提高检测的精准度和实现移动检测以及家庭自助式检测等功能,能够带来可预见的巨大经济效果。
Description
技术领域
本发明属于生物与医学检测技术领域,具体涉及一种基于CRISPR基因编辑技术的AD携带基因检测方法。
背景技术
阿尔茨海默病(Alzheimer’s disease,AD)是一种中枢神经系统变性病。AD 作为严重危害人类晚年健康和社会经济发展的全球公共卫生重大问题,亟需对其发病机制和防治开展深入研究。
以发病年龄(age at onset,AAO)65 岁为界,AD 可分为早发性 AD(early-o nsetAD,EOAD)(AAO≤65 岁)和晚发性 AD(late-onset AD,AAO>65 岁)。载脂蛋白E(Apolipoprotein E,APOE)基因的多态性是决定AD的主要遗传风险因素。APOE可通过参与β-淀粉样蛋白(β-amyloid,Aβ)生成、tau蛋白与微管蛋白连接、突触可塑性及脂质代谢等途径,影响AD的发生。APOE的三个异构体即:APOE ε2,APOE ε3, APOE ε4。APOE基因上rs429358位点和rs7412位点对应的碱基分别为T和C;rs7412位点C突变为T即为ε2,rs429358位点T突变为C即为ε4。APOE ε3等位基因参与人体正常功能的维持;APOE ε2等位基因为保护因素,可降低心脑血管疾病的发生;APOE ε4等位基因的存在会大大提高AD的患病风险。
早期识别携带APOE ε4基因突变的患者对于研究疾病发病机制、家系遗传学咨询和早期干预尤为重要。2011年美国医学遗传学学会(the American College of MedicalGenetics,ACMG)和美国国家遗传咨询师协会(the National Society of GeneticCounselors, NSGC)有关AD遗传学咨询检测的实践指南推荐仅对至少具有2名患病一级亲属的 EOAD 患者进行遗传学检测。越来越多的研究也致力于APOE ε4等位基因与各种潜在标志物之间关系及机制的探索,从而提高AD的早期筛查,建立更为完善的AD风险预警系统。
目前携带基因检测有多种方法,如:PCR-RFLP、TaqMan 荧光探针法、QPCR- HRM等。PCR-RFLP 法过程较繁琐、耗时长;TaqMan 荧光探针法价格较为昂贵;QPCR-HRM相对简单但该方法存在假阳性高,分辨率低的问题。以上方法都需要特定昂贵的设备、专业的操作人员、整个扩增过程需要几个小时才可以完成相应的检测,因上述原因现有技术难以满足大范围的基层筛查需求。
发明内容
针对现有技术中存在的问题,本发明的目的在于提供一种基于CRISPR基因编辑技术的AD携带基因检测方法,该方法不仅能将样品处理时间大幅度缩短,试剂和仪器成本大幅度降低,还能提高检测的精准度和实现移动检测以及家庭自助式检测等功能,能够带来可预见的巨大经济效果。
为实现以上目的,本发明采取的技术方案是:
一种基于CRISPR基因编辑技术的AD携带基因检测方法,包括以下步骤:
步骤1,设计RPA扩增引物及crRNA 序列:由SNP位点rs429358和rs7412位点确定出APOE的核心鉴定区域,再设计出对应的RPA引物及两条crRNA 序列,分别为crRNA 序列1和crRNA 序列2;
步骤2,提取待测样本的基因组DNA:以人体口腔上颚组织细胞为待测样本,加入DNA提取液中,用尼龙纤维制小刷子刮取上颚组织,随后将刷子在提取液中搅拌充分混匀,随后静置于桌面2-5min,使其充分反应;
步骤3,等温扩增反应:以上述步骤2得到的DNA为模板,以步骤1设计的RPA为引物进行等温扩增反应,将DNA滴入一滴于反应体系中,在37 ℃反应40min,得到RPA扩增产物;
步骤4,根据步骤1设计的两条crRNA 引导序列分别制备Cas12a/crRNA复合物1和Cas12a/crRNA复合物2,再加入ssDNA荧光探针及步骤3得到的RPA扩增产物,在CRISPR/Cas12a体系中进行裂解反应,分别得到裂解产物1和裂解产物2;
步骤5,将步骤4得到的裂解产物1和裂解产物2分别使用胶体金试纸条1和试纸条2同时进行显色检测;若在两个胶体金试纸条上的检测线出现红色条带或检测线和阴性样本质控线位置均出现红色条带,则表示待测样本含APOEε4基因;若胶体金试纸条1检测线不出现红色条带,而试纸条2出现红色条带,则表示待测样本中APOE基因型为APOE ε3基因;若胶体金试纸条1和试纸条2的检测线均不出现红色条带,则表示待测样本中APOE基因型为APOEε2基因。
进一步地,所述步骤1中RPA扩增引物序列为:RPA-APOE-F:5’-AACAACTGACCCCGGTGGCGGAGGAGACGCG-3’;RPA-APOE-R:5’-ACCTCCGCCACCTGCTCCTTCACCTCGTCCA-3’。
进一步地,所述步骤1中crRNA 序列为:crRNA 序列1(rs429358):5-mC*U*GCCCGGCUGGGCGCGGACAUGGAGGACGUGC*G*mU-3’,
crRNA 序列2(rs7412):5-mC*U*UAAGCGGCUCCTCCGCGAUGCCGAUGACCUGCAGAAGC*mU-3’;*为硫代修饰,m为甲氧基修饰。
进一步地,所述步骤2中提取液包括0.5-1.0 mol/L的氢氧化钠、10-20 mmol/L的乙二胺四乙酸二钠、2-5mol/L的醋酸钾、水。
进一步地,所述步骤3中反应体系为:10-20 μM RPA-APOE-F 2.4 μL,10-20 μMRPA-APOE-R 2.4 μL,Rehydration buffer 29.5 μL,250-300 mM MgOAc 2.5 μL,RNase-free H2O加至50 μL。
进一步地,所述步骤4中ssDNA荧光探针序列为:5’-FITC-TTTTTT-Biotin-3’。
进一步地,所述步骤4中裂解反应体系为:将RNase-free H2O 66.67 μL,10×NEBbuffer 10 μL,1-2μM Cas12a 3.33 μL,300-500 mM crRNA 10 μL,置于37 ℃中反应15-20min,反应完成后置于室温备用;再加入10-20 μM ssDNA荧光探针2.5 μL以及RPA扩增产物7.5μL,于37 ℃中反应1h。
进一步地,所述步骤5中胶体金检测试纸条包括:底板、样品垫、胶体金结合垫、层析膜和吸水垫,所述样品垫、胶体金结合垫、层析膜和吸水垫在底板上按照顺序依次固定;所述胶体金结合垫内含有胶体金标记的抗FITC的兔源抗体,所述层析膜上设有相互分离的检测线T和质控线C,检测线T包被抗兔抗的羊源抗体,质控线C包被Biotin的配体Avidin。
本发明采用的是重组酶聚合酶扩增(RPA)基因编辑CRISPR-Cas12a技术。将AD携带基因APOE ε4等位基因,基于CRISPR应用于体外诊断检测靶标点突变的剪切技术,结合恒温扩增技术,构建CRISPR与恒温扩增技术相结合的技术体系,研制兼具qPCR核酸检测(金标准)性能和抗原抗体免疫学检测可肉眼判读的诊断试剂盒,适用于临床检测和科研应用。
本诊断试剂盒主要由RPA引物、crRNA引物、Cas12a酶和ssDNA荧光基团构成。通过对待测样本进行RPA扩增,再在crRNA序列介导下,引导CRISPR-Cas12a体系对RPA扩增产物进行识别并切割,子代靶DNA序列将同时有ssDNA荧光基团(包括FITC荧光基团和生物素)。胶体金颗粒上标记抗FITC荧光基团的抗体;NC膜上包被 C线为抗FITC的抗体;T线为抗生物素抗体,这样就会在T线去形成抗生物素抗体/生物素-目标靶系列-FITC/抗FITC抗体-胶体金的阳性复合物。
基本原理:RPA是一种利用重组酶和DNA聚合酶与DNA结合蛋白一起实现DNA扩增的方法;RPA中使用的蛋白质参与了生物体内DNA的合成、重组和修复过程。在RPA中不需要对DNA样品进行预处理或热变性步骤,因为重组酶促进了双链 DNA(Double Strand ,dsDNA)的分离。重组酶作为一种催化剂,促进了寡核苷酸引物与匹配序列的目标DNA结合。当与重组酶结合的引物扫描dsDNA上的特定互补序列并与之杂交后,重组酶分离双链,RPA开始反应,引物与模板DNA正确结合后,重组酶从复合物中脱离,留下3'端供DNA聚合酶附着;移位的 DNA 链随后被DNA结合蛋白包裹,而引物在DNA聚合酶的延伸下形成一个新的dsDNA,作为模板进行额外的循环扩增。
CRISPR/Cas系统作为分子诊断技术,主要是基于该系统特异性识别和切割核酸序列的功能建立的,其中切割功能中的反式切割属性,Cas12a-crRNA能够识别单链或双链DNA,同时不需要将底物转换成 RNA,省去了反转录步骤,因此,操作更加简便。使用设计后的专用探针引物,在引物的5’端标记生物素。探针上设计有可被Cas12a和Cas13a识别的位点,5’端带有荧光基团FAM,3’端进行修饰,使得无法延伸;当探针同由5’端带有生物素标记的引物扩增的靶DNA产物配对时,Cas12a或Cas13a切割探针,形成游离3’-OH,可以作为引物继续延伸,合成子代靶DNA序列;那么子代靶DNA序列就会同时带有FAM荧光基团和生物素。那么子代靶序列就会同时带有FAM荧光基团和生物素。胶体金颗粒上标记抗FAM荧光基团的抗体;NC膜上包被 C线为抗FAM的抗抗体;T线为抗生物素抗体,这样就会在T线去形成抗生物素抗体/生物素-目标靶系列-FAM/抗FAM抗体-胶体金的阳性复合物。异硫氰酸荧光素(FITC)具有比较高的活性,通常来说,在固相合成过程中引入该种荧光基团相对于其他荧光素要更容易,并且反应过程中不需要加入活化试剂。在此反应体系中将使用FITC代替常规的FAM荧光基团。
反应检测的原理为:以待测样本组织液为模板,采用RPA扩增引物对其进行RPA扩增,得到RPA扩增产物。随后配制CRISPR/Cas12a反应液,包括所述的探针(一端标记FITC,另一端标记Biotin)、crRNA和Cas12a蛋白以及RPA扩增引物。反应完成后用胶体金侧流层析试纸条进行检测。通过胶体金的检测方式,能够迅速的判断出组织液中的目标基因表达情况。CRISPR/Cas12a在crRNA的引导下识别RPA扩增产物中特异性靶标,CRISPR/Cas12a、crRNA和靶标序列分子结合成复合物,此复合物切割检测体系中的ssDNA荧光探针,探针分子被切开后,那么子代靶DNA序列就会同时带有FITC荧光基团和生物素,故可被胶体金检测。
本发明的有益效果是:(1)本项目检测试剂盒,基于免疫学检测便携等特点;CRISPR方法具有检测时间短,同时具有检测精密度更高、灵敏度高、成本低和耗时短等优点,使得检测方法更加得到优化,可以进行准确、快速的大规模携带基因筛查;检测试剂盒微小化、便携化,可以实现临床和移动式的检测,或者家庭普及的检测方式,给医务人员和患者带来了极大的方便;
(2)本发明检测中采用等温37℃反应,与常规核酸检测所需要的PCR技术相比,等温条件使RPA反应摆脱了对加热相关仪器设备如PCR仪的依赖,短时间内完成核酸扩增反应,具有操作简便、快速的特点;反应迅速:RPA技术可以在常温下实现引物和模板的特异结合,从而实现目标序列的扩增,并且该技术大大缩短了扩增过程所需要的时间,达到快速检测的要求;
(3)通过侧向层析技术试纸条方法,实现肉眼可视化检测,方便快捷,准确可靠,APOEε4:两个胶体金试条的检测线和质控线均出现红色条带;APOEε3:1号胶体金试条的检测线和质控线均出现红色条带,2号胶体金试条的检测线无红色条且质控线出现红色条带;APOEε2:1号和2号胶体金试条的检测线均不出现红色条带和质控线出现红色条带;检测中样品含有高拷贝数的APOEε4 基因时表示有高阿尔兹海默症患病风险;
(4)本发明检测方法无需大型仪器设备,恒温即可反应,结果肉眼可判读,适合大规模筛查使用。
附图说明
图1为实施例中检测装置和检测结果示意图。
实施方式
为了更好地理解本发明,下面结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限制本发明的范围。
实施例1:
一种基于CRISPR基因编辑技术的AD携带基因检测方法,包括以下步骤:
步骤1,设计RPA扩增引物及crRNA 序列:由SNP位点rs429358和rs7412位点确定出APOE的核心鉴定区域,再设计出对应的RPA引物及两条crRNA 序列,分别为crRNA 序列1和crRNA 序列2,所述RPA扩增引物序列为:RPA-APOE-F:5’-AACAACTGACCCCGGTGGCGGAGGAGACGCG-3’;RPA-APOE-R:5’-ACCTCCGCCACCTGCTCCTTCACCTCGTCCA-3’,所述crRNA 序列为:crRNA 序列1(rs429358):5-mC*U*GCCCGGCUGGGCGCGGACAUGGAGGACGUGC*G*mU-3’,
crRNA 序列2(rs7412):5-mC*U*UAAGCGGCUCCTCCGCGAUGCCGAUGACCUGCAGAAGC*mU-3’;*为硫代修饰,m为甲氧基修饰;
步骤2,提取待测样本的基因组DNA:以人体口腔上颚组织细胞为待测样本,加入DNA提取液中,用尼龙纤维制小刷子刮取上颚组织,随后将刷子在提取液中搅拌充分混匀,随后静置于桌面2min,使其充分反应,所述提取液包括1.0 mol/L的氢氧化钠、10 mmol/L的乙二胺四乙酸二钠、3mol/L的醋酸钾、水;
步骤3,等温扩增反应:以上述步骤2得到的DNA为模板,以步骤1设计的RPA为引物进行等温扩增反应,将DNA滴入一滴于反应体系中,在37 ℃反应40min,得到RPA扩增产物,所述反应体系为:20 μM RPA-APOE-F 2.4 μL,20 μM RPA-APOE-R 2.4 μL,Rehydrationbuffer 29.5 μL,250mM MgOAc 2.5 μL,RNase-free H2O加至50 μL;
步骤4,根据步骤1设计的两条crRNA 引导序列分别制备Cas12a/crRNA复合物1和Cas12a/crRNA复合物2,再加入ssDNA荧光探针及步骤3得到的RPA扩增产物,在CRISPR/Cas12a体系中进行裂解反应,分别得到裂解产物1和裂解产物2,所述ssDNA荧光探针序列为:5’-FITC-TTTTTT-Biotin-3’,所述裂解反应体系为:将RNase-free H2O 66.67 μL,10×NEB buffer 10 μL,1μM Cas12a 3.33 μL,500 mM crRNA 10 μL,置于37 ℃中反应15min,反应完成后置于室温备用;再加入10μM ssDNA荧光探针2.5 μL以及RPA扩增产物7.5 μL,于37 ℃中反应1h;
步骤5,将步骤4得到的裂解产物1和裂解产物2分别使用胶体金试纸条1和试纸条2同时进行显色检测;若在两个胶体金试纸条上的检测线出现红色条带或检测线和阴性样本质控线位置均出现红色条带,则表示待测样本含APOEε4基因;若胶体金试纸条1检测线不出现红色条带,而试纸条2出现红色条带,则表示待测样本中APOE基因型为APOE ε3基因;若胶体金试纸条1和试纸条2的检测线均不出现红色条带,则表示待测样本中APOE基因型为APOEε2基因;所述胶体金检测试纸条包括:底板、样品垫、胶体金结合垫、层析膜和吸水垫,所述样品垫、胶体金结合垫、层析膜和吸水垫在底板上按照顺序依次固定;所述胶体金结合垫内含有胶体金标记的抗FITC的兔源抗体,所述层析膜上设有相互分离的检测线T和质控线C,检测线T包被抗兔抗的羊源抗体,质控线C包被Biotin的配体Avidin。
实施例2:
一种基于CRISPR基因编辑技术的AD携带基因检测方法,包括以下步骤:
步骤1,设计RPA扩增引物及crRNA 序列:由SNP位点rs429358和rs7412位点确定出APOE的核心鉴定区域,再设计出对应的RPA引物及两条crRNA 序列,分别为crRNA 序列1和crRNA 序列2,所述RPA扩增引物序列为:RPA-APOE-F:5’-AACAACTGACCCCGGTGGCGGAGGAGACGCG-3’;RPA-APOE-R:5’-ACCTCCGCCACCTGCTCCTTCACCTCGTCCA-3’,所述crRNA 序列为:crRNA 序列1(rs429358):5-mC*U*GCCCGGCUGGGCGCGGACAUGGAGGACGUGC*G*mU-3’,
crRNA 序列2(rs7412):5-mC*U*UAAGCGGCUCCTCCGCGAUGCCGAUGACCUGCAGAAGC*mU-3’;*为硫代修饰,m为甲氧基修饰;
步骤2,提取待测样本的基因组DNA:以人体口腔上颚组织细胞为待测样本,加入DNA提取液中,用尼龙纤维制小刷子刮取上颚组织,随后将刷子在提取液中搅拌充分混匀,随后静置于桌面5min,使其充分反应,所述提取液包括0.5mol/L的氢氧化钠、20 mmol/L的乙二胺四乙酸二钠、2mol/L的醋酸钾、水;
步骤3,等温扩增反应:以上述步骤2得到的DNA为模板,以步骤1设计的RPA为引物进行等温扩增反应,将DNA滴入一滴于反应体系中,在37 ℃反应40min,得到RPA扩增产物,所述反应体系为:10 μM RPA-APOE-F 2.4 μL,10μM RPA-APOE-R 2.4 μL,Rehydrationbuffer 29.5 μL,300 mM MgOAc 2.5 μL,RNase-free H2O加至50 μL;
步骤4,根据步骤1设计的两条crRNA 引导序列分别制备Cas12a/crRNA复合物1和Cas12a/crRNA复合物2,再加入ssDNA荧光探针及步骤3得到的RPA扩增产物,在CRISPR/Cas12a体系中进行裂解反应,分别得到裂解产物1和裂解产物2,所述ssDNA荧光探针序列为:5’-FITC-TTTTTT-Biotin-3’,所述裂解反应体系为:将RNase-free H2O 66.67 μL,10×NEB buffer 10 μL,2μM Cas12a 3.33 μL,300 mM crRNA 10 μL,置于37 ℃中反应20min,反应完成后置于室温备用;再加入20 μM ssDNA荧光探针2.5 μL以及RPA扩增产物7.5 μL,于37 ℃中反应1h;
步骤5,将步骤4得到的裂解产物1和裂解产物2分别使用胶体金试纸条1和试纸条2同时进行显色检测;若在两个胶体金试纸条上的检测线出现红色条带或检测线和阴性样本质控线位置均出现红色条带,则表示待测样本含APOEε4基因;若胶体金试纸条1检测线不出现红色条带,而试纸条2出现红色条带,则表示待测样本中APOE基因型为APOE ε3基因;若胶体金试纸条1和试纸条2的检测线均不出现红色条带,则表示待测样本中APOE基因型为APOEε2基因;所述胶体金检测试纸条包括:底板、样品垫、胶体金结合垫、层析膜和吸水垫,所述样品垫、胶体金结合垫、层析膜和吸水垫在底板上按照顺序依次固定;所述胶体金结合垫内含有胶体金标记的抗FITC的兔源抗体,所述层析膜上设有相互分离的检测线T和质控线C,检测线T包被抗兔抗的羊源抗体,质控线C包被Biotin的配体Avidin。
实施例3:
一种基于CRISPR基因编辑技术的AD携带基因检测方法,包括以下步骤:
步骤1,设计RPA扩增引物及crRNA 序列:由SNP位点rs429358和rs7412位点确定出APOE的核心鉴定区域,再设计出对应的RPA引物及两条crRNA 序列,分别为crRNA 序列1和crRNA 序列2,所述RPA扩增引物序列为:RPA-APOE-F:5’-AACAACTGACCCCGGTGGCGGAGGAGACGCG-3’;RPA-APOE-R:5’-ACCTCCGCCACCTGCTCCTTCACCTCGTCCA-3’,所述crRNA 序列为:crRNA 序列1(rs429358):5-mC*U*GCCCGGCUGGGCGCGGACAUGGAGGACGUGC*G*mU-3’,
crRNA 序列2(rs7412):5-mC*U*UAAGCGGCUCCTCCGCGAUGCCGAUGACCUGCAGAAGC*mU-3’;*为硫代修饰,m为甲氧基修饰;
步骤2,提取待测样本的基因组DNA:以人体口腔上颚组织细胞为待测样本,加入DNA提取液中,用尼龙纤维制小刷子刮取上颚组织,随后将刷子在提取液中搅拌充分混匀,随后静置于桌面3min,使其充分反应,所述提取液包括0.8 mol/L的氢氧化钠、10-20 mmol/L的乙二胺四乙酸二钠、2-5mol/L的醋酸钾、水;
步骤3,等温扩增反应:以上述步骤2得到的DNA为模板,以步骤1设计的RPA为引物进行等温扩增反应,将DNA滴入一滴于反应体系中,在37 ℃反应40min,得到RPA扩增产物,所述反应体系为:15 μM RPA-APOE-F 2.4 μL,15μM RPA-APOE-R 2.4 μL,Rehydrationbuffer 29.5 μL,270 mM MgOAc 2.5 μL,RNase-free H2O加至50 μL;
步骤4,根据步骤1设计的两条crRNA 引导序列分别制备Cas12a/crRNA复合物1和Cas12a/crRNA复合物2,再加入ssDNA荧光探针及步骤3得到的RPA扩增产物,在CRISPR/Cas12a体系中进行裂解反应,分别得到裂解产物1和裂解产物2,所述ssDNA荧光探针序列为:5’-FITC-TTTTTT-Biotin-3’,所述裂解反应体系为:将RNase-free H2O 66.67 μL,10×NEB buffer 10 μL,1μM Cas12a 3.33 μL,400 mM crRNA 10 μL,置于37 ℃中反应18min,反应完成后置于室温备用;再加入15μM ssDNA荧光探针2.5 μL以及RPA扩增产物7.5 μL,于37 ℃中反应1h;
步骤5,将步骤4得到的裂解产物1和裂解产物2分别使用胶体金试纸条1和试纸条2同时进行显色检测;若在两个胶体金试纸条上的检测线出现红色条带或检测线和阴性样本质控线位置均出现红色条带,则表示待测样本含APOEε4基因;若胶体金试纸条1检测线不出现红色条带,而试纸条2出现红色条带,则表示待测样本中APOE基因型为APOE ε3基因;若胶体金试纸条1和试纸条2的检测线均不出现红色条带,则表示待测样本中APOE基因型为APOEε2基因;所述胶体金检测试纸条包括:底板、样品垫、胶体金结合垫、层析膜和吸水垫,所述样品垫、胶体金结合垫、层析膜和吸水垫在底板上按照顺序依次固定;所述胶体金结合垫内含有胶体金标记的抗FITC的兔源抗体,所述层析膜上设有相互分离的检测线T和质控线C,检测线T包被抗兔抗的羊源抗体,质控线C包被Biotin的配体Avidin。
上述对实施例的描述是为了便于该技术领域的普通技术人员理解和使用本发明。熟悉本领域的技术人员可以容易地对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中,而不必经过创造性的劳动。因此,本发明不限于上述实施例。本领域技术人员根据本发明的原理,不脱离本发明的范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (5)
1.RPA扩增引物及crRNA在制备基于CRISPR基因编辑技术检测阿尔茨海默病携带基因产品中的用途,其特征在于,包括以下步骤:
步骤1,设计RPA扩增引物及crRNA 序列:由SNP位点rs429358和rs7412位点确定出APOE的核心鉴定区域,再设计出对应的RPA扩增引物及两条crRNA 序列,分别为crRNA 序列1和crRNA 序列2;所述RPA扩增引物序列为:RPA-APOE-F:5’-AACAACTGACCCCGGTGGCGGAGGAGACGCG-3’;RPA-APOE-R:5’-ACCTCCGCCACCTGCTCCTTCACCTCGTCCA-3’;所述crRNA 序列为:crRNA 序列1:5-mC*U*GCCCGGCUGGGCGCGGACAUGGAGGACGUGC*G*mU-3’,
crRNA 序列2:5-mC*U*UAAGCGGCUCCTCCGCGAUGCCGAUGACCUGCAGAAGC*mU-3’;*为硫代修饰,m为甲氧基修饰;
步骤2,提取待测样本的基因组DNA:以人体口腔上颚组织细胞为待测样本,加入DNA提取液中,用尼龙纤维制小刷子刮取上颚组织,随后将刷子在提取液中搅拌充分混匀,随后静置于桌面2-5min,使其充分反应;
步骤3,等温扩增反应:以上述步骤2得到的DNA为模板,以步骤1设计的RPA扩增引物进行等温扩增反应,将DNA滴入一滴于反应体系中,在37 ℃反应40min,得到RPA扩增产物;
步骤4,根据步骤1设计的两条crRNA 序列分别制备Cas12a/crRNA复合物1和Cas12a/crRNA复合物2,再加入ssDNA荧光探针及步骤3得到的RPA扩增产物,在CRISPR/Cas12a体系中进行裂解反应,分别得到裂解产物1和裂解产物2,所述ssDNA荧光探针序列为:5’-FITC-TTTTTT-Biotin-3’;
步骤5,将步骤4得到的裂解产物1和裂解产物2分别使用胶体金试纸条1和试纸条2同时进行显色检测;若在两个胶体金试纸条上的检测线出现红色条带或检测线和阴性样本质控线位置均出现红色条带,则表示待测样本含APOEε4基因;若胶体金试纸条1检测线不出现红色条带,而试纸条2出现红色条带,则表示待测样本中APOE基因型为APOE ε3基因;若胶体金试纸条1和试纸条2的检测线均不出现红色条带,则表示待测样本中APOE基因型为APOE ε2基因。
2.根据权利要求1所述的RPA扩增引物及crRNA在制备基于CRISPR基因编辑技术检测阿尔茨海默病携带基因产品中的用途,其特征在于,所述步骤2中提取液包括0.5-1.0 mol/L的氢氧化钠、10-20 mmol/L的乙二胺四乙酸二钠、2-5mol/L的醋酸钾、水。
3.根据权利要求1所述的RPA扩增引物及crRNA在制备基于CRISPR基因编辑技术检测阿尔茨海默病携带基因产品中的用途,其特征在于,所述步骤3中反应体系为:10-20 μM RPA-APOE-F 2.4 μL,10-20 μM RPA-APOE-R 2.4 μL,Rehydration buffer 29.5 μL,250-300mM MgOAc 2.5 μL,RNase-free H2O加至50 μL。
4.根据权利要求1所述的RPA扩增引物及crRNA在制备基于CRISPR基因编辑技术检测阿尔茨海默病携带基因产品中的用途,其特征在于,所述步骤4中裂解反应体系为:将RNase-free H2O 66.67 μL,10×NEB buffer 10 μL,1-2μM Cas12a 3.33 μL,300-500 mM crRNA10 μL,置于37 ℃中反应15-20min,反应完成后置于室温备用;再加入10-20 μM ssDNA荧光探针2.5 μL以及RPA扩增产物7.5μL,于37 ℃中反应1h。
5.根据权利要求1所述的RPA扩增引物及crRNA在制备基于CRISPR基因编辑技术检测阿尔茨海默病携带基因产品中的用途,其特征在于,所述步骤5中胶体金检测试纸条包括:底板、样品垫、胶体金结合垫、层析膜和吸水垫,所述样品垫、胶体金结合垫、层析膜和吸水垫在底板上按照顺序依次固定;所述胶体金结合垫内含有胶体金标记的抗FITC的兔源抗体,所述层析膜上设有相互分离的检测线T和质控线C,检测线T包被抗兔抗的羊源抗体,质控线C包被Biotin的配体Avidin。
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