CN112779358A - 一种dcas9介导检测HPV16型E6基因的免疫层析方法 - Google Patents
一种dcas9介导检测HPV16型E6基因的免疫层析方法 Download PDFInfo
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Abstract
本发明公开了检测HPV16型E6基因的免疫层析方法,包括:步骤1,针对靶标基因设计特异性引物及sgRNA序列;步骤2,取待测样本RNA,并加入5'端标有生物素的引物,进行RT‑RPA核酸扩增,得到扩增产物;步骤3,在扩增产物里加入dcas9蛋白及sgRNA进行反应,形成dcas9/sgRNA/HPV16 E6‑biotin复合物;步骤4,将反应产物加入免疫层析试纸条,即可判读结果。本发明通过信号放大后,由sgRNA/dcas9复合物再次识别靶标基因,特异性高,稳定性强,灵敏度高;且当靶标基因改变时,只需要更换引物及sgRNA序列,免疫层析试纸条则可以通用,可扩展性强。
Description
技术领域
本发明属于生物检测技术领域,涉及一种检测HPV16型E6基因的免疫层析方法。
背景技术
核酸检测属于分子诊断,应用分子生物学方法检测受检个体或其携带的病毒、病原体的遗传物质的结构或表达调控的变化水平,为疾病的防治、预测、诊断、治疗和预后判断提供信息和决策依据的技术。由于分子诊断技术可针对产生疾病的相关基因进行准确诊断,特异性强,灵敏度高,可应用于传染性疾病、血液筛查、遗传性疾病、肿瘤分子诊断等领域,还能在部分应用领域替代其他体外诊断技术,成为体外诊断技术中重要的发展和研究方向。
核酸检测通常结合扩增技术,将微量的特异性核酸序列放大到能够被仪器检测到的水平。传统的核酸检测有聚合酶链式反应(polymerase chain reaction,PCR) 和逆转录-聚合酶链反应(Reverse Transcription-Polymerase Chain Reaction,PCR),前者扩增DNA,后者则扩增RNA,是目前临床应用最广泛的分子诊断技术。PCR 技术核酸扩增分三个基本反应步骤,分别是变性、退火(复性)和延伸,每完成一次这三个步骤为一个循环,一般需要进行几十个循环,耗时2至3个小时,且需要精准的温度和时间控制。RPA核酸扩增技术是一种在等温条件(37℃)下, 5-30分钟之内就可以达到目的基因扩增放大几百万倍的等温扩增技术,相比较于 PCR核酸扩增技术更快速,更易操作,有更宽阔的应用场景。
免疫层析技术是基于抗原抗体特异性免疫反应的新型膜检测技术。该技术以固定有检测线(包被抗体或包被抗原)和质控线(抗抗体)的条状纤维层析材料为固定相,测试液为流动相,胶体金标记抗体或抗原固定于连接垫,通过毛细管作用使待分析物在层析条上移动。检测快速,方便,结果肉眼可见,具有良好的临床应用前景和意义。
CRISPR-Cas9是细菌和古细菌在长期演化过程中形成的一种适应性免疫防御,可用来对抗入侵的病毒及外源DNA。而CRISPR-Cas9基因编辑技术,则是对靶向基因进行特定DNA修饰的技术,这项技术也是目前用于基因编辑中前沿的方法。CRISPR-Cas9 是基因组调控技术中灵活性最强的系统之一。Cas9 的核酸酶剪切活性取决于两个结构域:RuvC和HNH。这两个结构域分别负责切割 DNA链的两条链,并且这两个结构域能够单独地被人工点突变失活。当 RuvC 和HNH 同时处于失活状态时 ( D10A&H840A; RuvC-&HNH-),Cas9 将不具有核酸酶活性,成为 dCas9(deadCas9) 。dCas9 虽然没有剪切 DNA 的能力,但仍然可以在gRNA 的引导下与特定的 DNA 序列结合。
高危型人乳头瘤病毒(human papillomavirus,HPV)的持续感染是宫颈癌发生的根本原因,其中HPVl6型占到54.4%; 而HPV16 E6原癌蛋白在宫颈癌癌变进程中是持续表达,故E6基因是目前HPV检测的重要靶标基因。
本发明将CRISPR/Cas系统、RPA核酸扩增技术和免疫层析技术结合,建立一种快速、特异地检测HPV16型E6基因(靶标核酸)的免疫层析方法。
发明内容
本发明的目的是提供一种检测HPV16型E6基因的免疫层析方法,该方法采用RPA等温扩增结合CRISPR/Cas9系统建立快速检测靶标核酸的的免疫层析方法,特异性好,灵敏度高,可靠性好,可用于临床现场检测。
为达到上述目的,本发明采用以下技术方案:
一种检测HPV16型E6基因的免疫层析方法,包括以下几个步骤:
步骤(1),针对HPV16型E6基因设计特异性引物及sgRNA序列;所述特异性引物的5'端标记生物素,所述sgRNA的识别序列位于扩增产物内部;
步骤(2),利用RPA等温核酸扩增技术,取待测样本RNA,并加入步骤(1)中5'端标有生物素的特异性引物,进行RT-RPA核酸扩增,得到扩增产物;
步骤(3),结合CRISPR/Cas9系统,在扩增产物里加入dcas9蛋白及步骤(1)中的sgRNA进行反应,形成dcas9/sgRNA/HPV16 E6-biotin核酸蛋白复合物;
步骤(4),将步骤(3)中的反应产物加入免疫层析试纸条,即可判读结果:若试纸条T线和C线均有红色条带,表明是阳性样品;若试纸条C线有带,T线无带,表明是阴性样品;若试纸条C线无带,则检测无效,需要重复检测。
进一步的,所述引物F序列为:5’-CCAGAAAGTTACCACAGTTATGCACAGAGC-3’(SEQ IDNO:1);所述特异性引物R序列为:5’- CGAAAAGCAAAGTCATATACCTCACGTCGC-3’(SEQ ID NO:2)。
所述sgRNA序列为:
GCAACAGUUACUGCGACGUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU(SEQ ID NO:3)。
进一步的,所述步骤(2)中,RPA核酸扩增体系为:10 uM引物F 2.4 ul,10 uM引物R2.4 ul,RPA-reacting Buffer 29.5 ul,待测样本5 ul,280 nM MgOAC 2.5 ul,100,000U/mL ProtoScript RT 1 ul,RNase Free Water 7.2 ul。
进一步的,所述所述步骤(3)中,CRISPR体系为:等温扩增产物 50 ul,100 ng/udcas9 4-8 ul,100 ng/ul sgRNA 4-8 ul,10X-Reacting Buffer 10 ul,20 U/μLSUPERase•In™ RNase Inhibitor 5 ul,RNase Free Water To 100 ul 。
本发明免疫层析试纸条包括依次相连接的吸水垫、基膜、金标垫和样品垫;所述基膜上设置有C线和T线,所述C线上包被有抗抗体,所述T线上包被有链酶亲和素;所述金标垫中含有胶体金标记的dcas9抗体(见图 1)。
采用上述技术方案,技术原理为:(1)若为阳性样品,随着信号扩增,扩增产物会被标记上生物素;(2)利用dCas9在 gRNA 的引导下与特定的 DNA 序列结合的工作原理,被生物素标记的扩增产物与dcas9蛋白/sgRNA共同孵育下形成dcas9/sgRNA/HPV16 E6-biotin复合物;(3)随着检测液横向流动,胶体金标记dcas9抗体识别步骤(2)中复合物,形成胶体金dcas9抗体/dcas9/sgRNA/HPV16 E6-biotin,检测T线为链酶亲和素,特异性识别biotin,如此,T线特异性截留带胶体金标记的复合物,形成红色条带,分析T线与C线条带情况即可鉴别样本的阳性情况。
本发明的有益效果在于:
(1)本发明通过等温扩增方法对HPV16型E6基因特异性扩增放大信号后,再由sgRNA/dcas9复合物二次特异性识别靶标基因,双重特异识别程序使靶标基因的检测特异性高,稳定性强。(2)本发明建立的检测方法检出限达1 copies/ul,灵敏度高(3)本发明检测方法所检测靶基因改变时只需要更换引物及sgRNA序列,免疫层析试纸条则可以通用,可扩展性强。(4)本发明检测快速、无需大型仪器,肉眼可读的结果呈现方式使得应用方便、广泛。
附图说明
图1免疫层析试纸条示意图;其中1:底板;2:样品垫;3:金标垫,含有金标dcas9抗体;4:检测线(T线),含有链霉亲和素;5:对照线(C线),含有抗抗体(二抗);6:反应膜;7:吸水垫。
图2 HPV16型E6基因检测结果。
具体实施方式
为了充分了解本发明的目的、特征及效果,下面结合附图、实施例进一步阐明本发明的内容,但本发明的保护范围不局限于下面的实施例。
试剂及耗材:
dcas9蛋白(近岸蛋白;E368-01A)、链酶亲和素(sigma;85878-1MG)、dCas9抗体(Abcam;Ab204448)、RNA提取所用试剂盒(TAKARA, 9766) 、RPA试剂盒(TwistDx Limited;TABAS03KIT)、SUPERase•In™ RNase Inhibitor(ThermoFisher;AM2694)、逆转录酶ProtoScript® II Reverse Transcriptase(NEB;M0368L);T7 Transcription Kit(Thermo Fisher,AM1322);硝酸纤维素膜、玻璃纤维膜:购自德国赛多利斯公司(SARTORIUS)。HPV16型E6基因转录模板质粒、引物及sgRNA委托南京金斯瑞公司合成。
实施例 1
1、HPV16型E6基因转录模板质粒、引物及sgRNA及合成
HPV16型E6基因转录模板克隆至PUC57;其序列如下(其中下划线序列为T7promoter):taatacgactcactatagggccagaaagttaccacagttatgcacagagctgcaaacaactatacatgatataatattagaatgtgtgtactgcaagcaacagttactgcgacgtgaggtatatgactttgcttttcg(SEQID NO:4)
转录模板引物F: taatacgactcactatagggccagaa(SEQ ID NO:5)
转录模板引物R: cgaaaagcaaagtcatatacctcacg(SEQ ID NO:6)
根据HPV16型E6基因序列的特点,利用麻省理工大学张锋教授实验室提供的在线网站(http : //crispr.mit.edu /)分析可能的 sgRNA位点(PAM 序列为NGG)并筛选最优sgRNA核心序列;在已选定的sgRNA核心序列上下游约80-150bp范围内设计RPA扩增引物;并利用NCBI中的Primer-BLAST功能对引物进行分析比对,(比对网站为:https://www.ncbi.nlm.nih.gov/tools/primer-blast/),以保证序列唯一性。所设计的引物5'端由生物素标记(biotin)。
RPA引物设计遵循以下原则:
(1)5’端3-5个核苷酸避免聚鸟嘌呤,最好是胞嘧啶,可以促进重组;
(2)3’的3个核苷酸选择G或者C,有助于聚合酶稳定性;
(3)避免出现引物中出现聚嘌呤或者聚嘧啶;
(4)GC含量控制在30%-70%,引物长度控制在30-35个碱基范围内。
根据上述原则,HPV16型E6基因的引物及sgRNA序列如下:
E6-F: biotin-5’- CCAGAAAGTTACCACAGTTATGCACAGAGC-3’(SEQ ID NO:1);
E6-R: biotin-5’- CGAAAAGCAAAGTCATATACCTCACGTCGC -3’(SEQ ID NO:2);
sgRNA-E6:GCAACAGUUACUGCGACGUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU(SEQ ID NO:3)。
将上述HPV16型E6基因转录模板序列、引物及sgRNA委托南京金斯瑞公司合成。
2将带有目标序列的质粒PCR后获得体外转录模板
2.1设置PCR反应体系如下(表1):
表1
组分 | 用量 |
质粒模板 | 1ul |
转录上游引物 | 1ul |
转录下游引物 | 1ul |
2xPCRmix | 25 |
H2O | to 50ul |
转录模板制备PCR反应共5管,每管50ul,共计250ul。充分混匀放置PCR进行反应,设计程序如下:95℃ 5min;95℃ 30sec,56℃ 30sec,72℃ 30sec,共进行35个循环。
2.2转录模板纯化
操作过程中采用无RNA酶的吸头及离心管操作,并注意避免RNA酶污染。
(1)将250μl扩增产物收集到1个1.5ml微量离心管。加入250μl酚氯仿异戊醇,混匀。静置3-5min;
(2)4℃ 12000rpm 离心5min;
(3)小心吸取上层液体到新管,加入3M NaAc25μl,冰乙醇500μl。混匀,冰上放置5-10min;
(4)4℃ 12000rpm离心10min;
(5)弃上清,加入1ml 70%乙醇,震荡洗涤沉淀;
(6)4℃ 12000rpm离心5min;
(7)用移液器尽量吸净液滴,室温放置2-3min,挥发残留乙醇;
(8)加入10μl无RNA酶水,轻弹管壁溶解沉淀;
(9)定量DNA,冻存到-20℃备用。
2.3 体外转录试剂盒进行体外转录
(1)配制体系为:5×TranscriptAid Reaction Buffer 4ul,ATP/CTP/GTP/UTPmix 8ul,Template DNA 1ug,TranscriptAid Enzyme Mix 2ul,DEPC-treated water to20ul;共20ul;
(2)充分混匀,37℃反应4h。
2.4转录产物纯化
2.4.1去除模板DNA
操作如下:
(1)添加2 u DNaseI(RNase-free)混匀后37°C孵育15min;
(2)然后加入2µl 0.5M EDTA( pH值8.0)混匀后65°C孵育10min终止反应。
2.4.2 RNA纯化
(1)向反应混合物中加入115 ul DEPC水,15 ul 3 M醋酸钠溶液(pH 5.2)。充分混匀;
(2)取等量苯酚(pH 4.7)/氯仿混合物(1:1体积配制),充分混匀,12000rpm离心收集上层液体,并转移到新的EP管中。再按等量氯仿以相同方法萃取两次;
(3)加入2倍体积的无水乙醇沉淀RNA。-20℃孵育30分钟,4℃,12000rpm离心10min,收集沉淀;
(4)用500 ul 70%的预冷乙醇充分震荡洗涤沉淀1min,4℃,12000rpm离心5min,尽量吸尽乙醇,打开管盖,室温放置2-3min挥发残留乙醇;
(5)加入30ul DEPC水溶解沉淀,定量;
(6)将纯化的RNA储存于-20℃或-80℃。
3 RT-RPA扩增
将步骤2中已纯化的RNA调整为500 copies/ul、50copies/ul、1copies/ul、RNaseFree Water(0 copies/ul 阴性对照)四个分组,并按照下列表2配制RT-RPA扩增反应体系,37-42℃下进行反应20-30分钟获得等温扩增产物。
表2
组分 | 用量 |
Primer—Forward(10 uM) | 2.4 ul |
Primer—Reverse(10 uM) | 2.4 ul |
RPA-reacting Buffer | 29.5 ul |
待测样本RNA | 5 ul |
MgOAC (280 nM) | 2.5 ul |
ProtoScript RT (100,000U/mL) | 1 ul |
RNase Free Water | 7.2 ul |
Total | 50ul |
4 RT-RPA扩增产物与dcas9、sgRNA反应
将RT-RPA扩增产物与dcas9、sgRNA按照下列表3配制CRISPR反应体系,37℃反应5-10分钟获得dcas9/sgRNA/HPV16 E6-biotin复合物。
表3
组分 | 用量 |
等温扩增产物 | 50 ul |
dcas9 (100 ng/ul) | 4-8 ul |
sgRNA(100 ng/ul) | 4-8 ul |
10X-Reacting Buffer | 10 ul |
SUPERase•In™ RNase Inhibitor (20 U/μL) | 5 ul |
RNase Free Water | To 100 ul |
5 结果检测
将步骤(4)中带有复合物的反应液加入免疫层析试纸条,即可判读结果:若试纸条T线和C线均有红色条带,表明是阳性样品;若试纸条C线有带,T线无带,表明是阴性样品;若试纸条C线无带,则检测无效,需要重复检测。HPV16型E6基因检测结果见图 2 。结果显示,在HPV16型E6基因拷贝数为1copies/ul时依然能准确的检测出,表明该方法的灵敏性和可靠性。
实施例2 免疫层析试纸条的制备
1、两步还原法制备胶体金
a)氯金酸溶液第一次还原:将6ml 0.0164mol/L的HAuCL4水溶液加入200ml双蒸水中,煮沸30分钟,缓慢搅拌并加入50ml 0.016mol/L柠檬酸三钠溶液。以30kHZ的频率超声振荡2分钟,冷却至室温,得到15nm粒径的胶体金原核溶液。
b)氯金酸溶液第二次还原:取第一次还原后得到的胶体金原核溶液26ml在4℃条件下,加入4℃预冷后的0.035mol/L的HAuCL4溶液,缓慢搅拌并以每秒1-2滴的速度滴加4℃预冷后的0.018mol/L的抗坏血酸与0.138g/L PVP混合液,反应1小时至溶液呈现透明酒红色,即得到40nm粒径的胶体金溶液。
2、dCas9抗体胶体金预处理
a)将dCas9抗体用0.1M pH7.8的磷酸盐缓冲液稀释至浓度1mg/ml。
b)将1000ml胶体金溶液与含有500u/ml RNA酶抑制剂的100ml0.1M pH7.8的磷酸盐缓冲液混合,快速搅拌3分钟。之后,以每秒1-2滴的速度滴加稀释后的dCas9抗体溶液8ml,缓慢搅拌室温反应5分钟。
c)在上述反应液中快速加入20ml 10wt%的牛血清白蛋白溶液,缓慢搅拌室温反应5分钟。
d)将获得的溶液8000rpm/min离心20分钟,取沉淀,并收集上清,将上清12500rpm/min离心30分钟,取沉淀。将两次沉淀合并用含有0.1wt%BSA的硼酸缓冲液复溶到OD540值为14。
3、胶体金纸制备
a)喷金缓冲液制备:在800ml双蒸水中加入100ml 1.0M Tris溶液,调pH至8.5。在溶液中加入3g聚乙二醇20000、2g牛血清白蛋白,2g脱脂牛奶,3g酪蛋白和0.5g叠氮钠,充分溶解,至总体积1000ml。
b)用喷金缓冲液稀释dCas9抗体胶体金,至溶液OD540值为2。
c)取7ml Tween-20,160g蔗糖,用双蒸水定容至1L,配置成玻璃纤维膜预处理液。用每30ml预处理液浸泡玻璃纤维膜261mm*220mm 30分钟后,至37℃干燥;再用OD540值为2的dCas9-gRNA胶体金溶液喷涂玻璃纤维膜,每261mm*220mm的玻璃纤维膜上喷涂20ml,干燥,制得dCas9抗体胶体金纸。
4、含检测线及质控线硝酸纤维素膜的制备
将抗抗体用磷酸盐缓冲液稀释成0.5mg/ml,制得质控线(C线)溶液。所述C线上包被的抗抗体是由0.1-5mg/mL的浓度,以1- 10μL/cm的喷涂速度通过喷金点膜机喷涂在C线上干燥后得到的。
将链酶亲和素定量为0.5mg/ml,制得检测线(T线)溶液。所述T线上包被的链酶亲和素以1-10μL/cm的喷涂速度通过喷金点膜机喷涂在T线上干燥后得到的;
a)用点膜机喷点C、T线溶液,每1m长硝酸纤维素膜分别包被有1ml的C线和T线溶液,C线和T线间距6mm。
将滤样纸、dCas9抗体胶体金纸片、硝酸纤维素膜、吸水纸依次黏贴在胶板上,切割成宽度为4mm的试剂条。
序列表
<110> 重庆威斯腾前沿生物研究院有限责任公司
<120> 一种dcas9介导检测HPV16型E6基因的免疫层析方法
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
ccagaaagtt accacagtta tgcacagagc 30
<210> 2
<211> 30
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
cgaaaagcaa agtcatatac ctcacgtcgc 30
<210> 3
<211> 100
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 3
gcaacaguua cugcgacgug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 4
<211> 138
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 4
taatacgact cactataggg ccagaaagtt accacagtta tgcacagagc tgcaaacaac 60
tatacatgat ataatattag aatgtgtgta ctgcaagcaa cagttactgc gacgtgaggt 120
atatgacttt gcttttcg 138
<210> 5
<211> 26
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 5
taatacgact cactataggg ccagaa 26
<210> 6
<211> 26
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 6
cgaaaagcaa agtcatatac ctcacg 26
Claims (6)
1.一种检测HPV16型E6基因的免疫层析方法,其特征在于:包括以下几个步骤:
步骤(1),针对HPV16型E6基因设计特异性引物及sgRNA序列;所述特异性引物的5'端标记生物素,所述sgRNA的识别序列位于扩增产物内部;
步骤(2),利用RPA等温核酸扩增技术,取待测样本RNA,并加入步骤(1)中5'端标有生物素的特异性引物,进行RT-RPA核酸扩增,得到扩增产物;
步骤(3),结合CRISPR/Cas9系统,在扩增产物里加入dcas9蛋白及步骤(1)中的sgRNA序列进行反应,形成dcas9/sgRNA/HPV16 E6-biotin蛋白核酸复合物;
步骤(4),将步骤(3)中的反应产物加入免疫层析试纸条,即可判读结果:若试纸条T线和C线均有红色条带,表明是阳性样品;若试纸条C线有带,T线无带,表明是阴性样品;若试纸条C线无带,则检测无效,需要重复检测。
2.如权利要求1所述的一种检测HPV16型E6基因的免疫层析方法,其特征在于:所述特异性引物F序列为:5’-CCAGAAAGTTACCACAGTTATGCACAGAGC-3’;所述特异性引物R序列为:5’- CGAAAAGCAAAGTCATATACCTCACGTCGC-3’。
3.如权利要求1所述的一种检测HPV16型E6基因的免疫层析方法,其特征在于:所述sgRNA序列为:
GCAACAGUUACUGCGACGUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU。
4.如权利要求1所述的一种检测HPV16型E6基因的免疫层析方法,其特征在于:所述步骤(2)中,RT-RPA核酸扩增体系为:10 uM引物F 2.4 ul,10 uM引物R 2.4 ul,RPA-reactingBuffer 29.5 ul,待测样本5 ul,280 nM MgOAC 2.5 ul,100,000U/mL ProtoScript RT 1ul,RNase Free Water 7.2 ul。
5.如权利要求1所述的一种检测HPV16型E6基因的免疫层析方法,其特征在于:所述所述步骤(3)中,CRISPR体系为:等温扩增产物 50 ul,100 ng/u dcas9 4-8 ul,100 ng/ulsgRNA 4-8 ul,10X-Reacting Buffer 10 ul,20 U/μL SUPERase•In™ RNase Inhibitor5 ul,RNase Free Water To 100 ul。
6.如权利要求1所述的一种检测HPV16型E6基因的免疫层析方法,其特征在于:所述步骤(4)中,免疫层析试纸条包括依次相连接的吸水垫、基膜、金标垫和样品垫;所述基膜上设置有C线和T线,所述C线上包被有抗抗体,所述T线上包被有链酶亲和素;所述金标垫中含有胶体金标记的dcas9抗体。
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