CN113913431B - 一种辛硫磷核酸适配体、核酸适配体衍生物及其应用 - Google Patents
一种辛硫磷核酸适配体、核酸适配体衍生物及其应用 Download PDFInfo
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Abstract
本发明涉及一种辛硫磷核酸适配体、核酸适配体衍生物及其在检测中的应用,更具体地说,它涉及辛硫磷核酸适配体序列为SEQ ID NO.1所示或在该DNA序列基础上偶联有生物素、荧光基团、猝灭材料等的核酸序列或在上述任一种辛硫磷核酸适配体序列上删减、增加或修饰碱基后获得的具有相同特异性识别功能的单链DNA分子;辛硫磷适配体衍生物为上述任一种辛硫磷核酸适配体进行修饰得到的具有相同功能的核酸适配体的衍生物;上述辛硫磷核酸适配体或其衍生物可用于制备辛硫磷识别探针或检测/辅助检测辛硫磷的产品,辛硫磷核酸适配体或其衍生物具有易于储存及修饰,可在体外大量合成及修饰,对目标物质辛硫磷具有亲和力强、特异性高等优势,具有广泛应用的潜力。
Description
技术领域
本发明属于分子生物学技术领域,更具体地说,它涉及一种辛硫磷核酸适配体、核酸适配体衍生物及其应用。
背景技术
我国是个农业大国,但农业生产仍然以传统生产模式为主,凭借经验施肥施药,容易对环境保护和人体健康造成严重威胁。现如今“智慧农业”战略的提出,对食品及环境中农药残留检测提出了更高的要求。
辛硫磷,是一种广泛使用的有机磷杀虫剂,适用于多种鳞翅目害虫且杀伤性强。目前,辛硫磷广泛应用于作物的虫害防治。但是辛硫磷能够通过吸入、摄入和经皮吸收等方法进入人体内。长期积累会导致疾病发生,诱发癌症,甚至中毒死亡、影响下一代,对人类健康及环境造成威胁。
现阶段检测辛硫磷的方法,如高效液相色谱法,气相色谱-质谱联用分析法,薄层色谱扫描法,液相色谱-串联质谱法等。这些方法检测时间长,成本高,通常需要复杂的样本预处理、昂贵的仪器和专业的操作人员,从而难以运用到现场检测。酶抑制法和酶联免疫分析法反应速度快,特异性高,但酶制备复杂,易失活,不稳定,对pH、温度、紫外照射等环境因素极为敏感,严重影响其检测的准确度。
适配体,也被成为人工抗体,是通过指数富集配体系统进化技术(SELEX)获得的短的单链DNA或RNA分子。目前,适配体已经被广泛应用于细胞、病毒、蛋白质、糖、金属和农药的检测。与传统抗体相比,适配体具有制备简单、稳定性好、易于标记、特异性强和亲和力高等优点。
因此,辛硫磷核酸适配体的筛选,能够为该农药的检测提供一种新的检测方法,克服现有农药检测方法的不足。
发明内容
针对现有技术存在的不足,本发明提供一种辛硫磷核酸适配体、核酸适配体衍生物及其应用,具有特异性强,灵敏度高的优点。
为实现上述技术目的,本发明采用的技术方案为:一种辛硫磷核酸适配体,所述辛硫磷核酸适配体的序列(SEQ ID NO.1)为:
5’-TCCAGCACTCCACGCATAACGGCAGGAAGAGTAGTGATGAGTGGTGTTATGCGGGGTGTGGTTATGCGTGCGACGGTGAA-3’;
进一步地,所述辛硫磷核酸适配体的核苷酸序列上偶联有荧光基团、猝灭材料或生物素。
本发明还提供了一种辛硫磷核酸适配体衍生物,为上述辛硫磷核酸适配体进行剪切、增加或修饰得到的与所述辛硫磷核酸适配体具有相同功能的核酸适配体衍生物。
本发明法提供了上述辛硫磷核酸适配体或辛硫磷核酸适配体衍生物在辛硫磷识别中的应用。
本发明还提供了一种辛硫磷核酸适配体或核酸适配体衍生物在制备检测或辅助检测辛硫磷的产品中的应用,所述产品包括辛硫磷识别探针,所述辛硫磷识别探针为上述辛硫磷核酸适配体或核酸适配体衍生物。
本发明还提供了一种检测辛硫磷的荧光适配体传感器,所述由辛硫磷识别探针与辛硫磷猝灭探针混合于DPBS溶液中制得,其中辛硫磷识别探针的核苷酸序列为:5’-FAM-CTCAGTCGCTCACTCCACGCATAACGGCAGGAAGAGTAGTGATGAGTGGTGTTATGCGGGGTGTGAGCGA-3’,所述辛硫磷猝灭探针的核苷酸序列为:5’-GTGAGCGACTGAG-Dabcyl-3’。
本发明还提供了上述辛硫磷荧光适配体传感器在辛硫磷检测中的应用。
本发明的上述技术目的是通过以下技术方案得以实现的:
综上所述,本发明具有以下有益效果:
其一,本发明的辛硫磷核酸适配体是一种具有特异性识别辛硫磷功能的核酸适配体,该核酸适配体具有特异性强、特异性高等优势,可在体外大量制备,成本低廉,易于修饰,可长保存。
其二,本发明的辛硫磷核酸适配体衍生物,是在辛硫磷核酸适配体序列上进行剪切、增加或修饰后获得的具有相同识别作用,该衍生物可用于制备检测或辅助检测辛硫磷的产品,检测灵敏度高,特异性强,检测成本低。
附图说明
图1是本发明中辛硫磷适配体筛选及荧光适配体传感器构建的示意图;
图2是本实施例1的富集曲线-熔融峰曲线法分析筛选进程结果图;
图3是本实施例2的辛硫磷核酸适配体优化前后二级结构图;
图4是本实施例2的辛硫磷荧光适配体传感器的荧光光谱表征图;
图5是本实施例3的不同浓度辛硫磷的荧光光谱图和线性图;
图6是本实施例4的辛硫磷适配体特异性检测图。
具体实施方式
下面结合附图和实施例,对本发明进行详细描述。以下实施例仅用于说明本发明而不用于限制本发明的范围。
下述实施例中所使用的实验方法,如无特殊说明,均为常规方法,所用的试剂、方法和设备,如无特殊说明,均为本技术领域常规试剂、方法和设备。
下述实例中提到的浓度(无特殊说明)均为体系含有该物质的终浓度,实验环境均为室温(20℃~30℃)条件下;所述荧光测试的波长为507-600nm,激发波长为495nm,狭缝宽度均为5.0 nm。
实施例1
本实施方式特异性识别辛硫磷的核酸适配体的核苷酸序列如SEQ ID NO.1所示:
5’-TCCAGCACTCCACGCATAACGGCAGGAAGAGTAGTGATGAGTGGTGTTATGCGGGGTGTGGTTATGCGTGCGACGGTGAA-3’;
本实施例的辛硫磷核酸适配体通过SELEX技术筛选得到,具体筛选包括如下步骤:
1.1、文库杂化
文库的结构设计由三部分组成,包括两端20 bp的固定序列,作为引物结合位点,中间40 bp的随机序列,为筛选提供足够的多样性。首轮筛选中,取1.3 nmol初始文库,按照摩尔比1:2的比例加入生物素引物(P1-biotin),以DPBS溶液(NaCl 136.89 mM;KCl 2.67mM;Na2HPO4 8.10 mM;KH2PO4 1.47 mM)为背景溶液放入PCR仪中,使核酸混合液在变性程序中缓慢变性、复性,变复性程序为:95℃ 10 min;59℃ 60 min;25℃ 10 min。经过杂化的文库取2 μL用微量紫外分光光度计上测量吸光度,记作A1。在之后的筛选过程中,将文库投入量固定在0.15 nmol,生物素引物投入量固定在0.3 nmol;
起始文库(SEQ ID NO.2):
5’-TCCAGCACTCCACGCATAAC-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-GTTATGCGTGCGACGGTGAA-3’(注:N代表A、T、C、G中任一碱基);
生物素引物(P1-biotin,SEQ ID NO.3):
5’-GTTATGCGTGGAGTGCTGGA-biotin-3’;
1.2、文库固定
取1 mL链霉亲和素磁珠,用DPBS清洗四次,每洗两次转移至一个新的离心管。加入上一步杂化的文库,摇床孵育30 min,在生物素-链霉亲和素的作用力下,杂化后的文库固定在磁珠上。孵育后的复合物在磁铁的作用力下分离磁珠与上清液,测量上清液中吸光度,记作A2。之后的筛选过程中,将每轮的磁珠投入量固定在100 μL。固定效率可通过以下公式计算:η=;
将分离的磁珠用DPBS清洗四次,分别记作W1-W4,其中W3为空白筛选,即在DPBS溶液中孵育10-30分钟,时间随着筛选进程而增加。在筛选稳定后加入W5,即用其他种农药的混合溶液进行反筛,从而提高适配体的特异性。
1.3、靶标筛选
向固定文库的磁珠中加入200 μL辛硫磷溶液,靶标浓度为10-100 μM,浓度随着筛选进程而降低。摇床孵育1 h,磁铁吸附磁珠至离心管的一边,回收上清液,记作E。所获得的洗脱液装在透析装置中置于50 mL DPBS溶液中,静置过夜。
1.4、洗脱液富集
将洗脱液扩增,扩增体系(2 mL)为:1 mL PCR mix,100 μL P2-FAM(10 μM),100 μL P3-PolyA(10 μL),200 μL洗脱液,600 μL ddH2O;2 mL扩增体系在涡旋振荡仪上充分混匀后,加入8 mL ePCR oil,震荡十分钟,静置5 min后观察无分层,即分装至96个PCR管中。ePCR扩增条件为:95℃ 3 min,95℃ 30 s,60℃ 30 s,72℃ 60 s,扩增25个循环,72℃ 4min。
扩增后的产物分装至两个15 mL离心管中,用正丁醇溶液补满,涡旋仪上混匀,放入离心机,8800 rpm离心10 min,回收底部绿色荧光dsDNA,体积浓缩至100μL以下,转移至1.5 mL的离心管中。
制备8%的尿素-PAGE胶,放置入垂直电泳仪内,加入TBE缓冲液,300 V电压下预电泳30 min。所回收的绿色荧光的dsDNA与2×TBE尿素上样缓冲液混合,95℃加热10 min,冰浴1 min。将混合液加入垂直电泳装置,直到蓝色条带电泳至胶板底部。电泳完成,取出胶板,在紫外光下切下带荧光的目的条带,切碎移至2 mL离心管。加入1 mL DPBS溶液上下震荡,95℃煮沸10 min,冰浴2 min,离心管放入离心机中,12000 rpm离心30s,使胶块沉淀在底部,取出溶液至15 mL离心管。该煮胶步骤重复一次。两次煮胶所获得的核酸溶液在15 mL离心管中,加入正丁醇补满,8800 rpm离心10 min,使核酸溶液浓缩至100 μL以下。所获得的核酸溶液使用试剂盒纯化,保存至-20℃用于下一轮筛选。
上游引物(P2-FAM,SEQ ID NO.4):
5’-FAM- TCCAGCACTCCACGCATAAC-3’;
下游引物(P3-PolyA,SEQ ID NO.5):
5’-AAAAAAAAAAAAAAAAAAAAAAAAA-Spacer18-TTCACCGTCGCACGCATAAC-3’;
1.5、进程监测
本发明采用Q-PCR监测法,建立不同浓度梯度的标曲,对每轮的洗脱液与文库富集溶液进行定量,能够精准测定溶液中文库的核酸浓度。Q-PCR扩增体系为10μL qPCR-mix,1μL P4(10 μM),1 μL P5(10 μM),1.2 μL模板,6.8 μL ddH2O;扩增程序为95℃ 3 min,95℃30 s,60℃ 30 s,72℃ 60 s,扩增25个循环,72℃ 4 min。富集曲线-熔融峰曲线来监测筛选进程,能直接反应筛选文库中的文库多样性。当富集曲线和熔融峰曲线都不随着筛选轮次的增加而变化时,即意味着筛选结束。富集曲线-熔融峰曲线如图2所示。
上游引物(P4,SEQ ID NO.6):5’-TCCAGCACTCCACGCATAAC-3’
下游引物(P5,SEQ ID NO.7):5’-TTCACCGTCGCACGCATAAC-3’
1.6、结果测序
将末轮的文库送至上海生工生物工程有限公司进行高通量测序,并将前15条序列合成。将该15条序列分别取0.15 nmol,分别与0.3 nmol引物-生物素(P1-biotin)和100 μL链霉亲和素磁珠混合孵育,加入100 μL靶标洗脱,用Q-PCR测量洗脱液中物质的量,洗脱量最大的序列即为亲和力最高的序列,为APT-3。
实施例2
本发明提供了一种辛硫磷荧光适配体传感器,包括辛硫磷识别探针,其制备步骤如下:
2.1、构建荧光探针
用Mfold软件预测APT-3辛硫磷核酸适配体的二级结构,通过碱基的剪切、增加或修饰,获得修饰有荧光基团的APT3s-FAM,二级结构图如图3所示,其序列(SEQ ID NO.8)为:
5’-FAM-CTCAGTCGCTCACTCCACGCATAACGGCAGGAAGAGTAGTGATGAGTGGTGTTATGCGGGGTGTGAGCGA-3’
并合成与之互补的猝灭链,其核苷酸序列为(SEQ ID NO.9)为:
5’-GTGAGCGACTGAG-Dabcyl-3’
2.2、猝灭条件优化
为找到最佳的荧光基团与猝灭剂的比例,将终浓度为100 nM的APT3s- FAM分别与猝灭剂-引物按照1:0-1:12的比例混合,在室温条件下孵育30 min,随后使用日立F-7100荧光分光光度计进行荧光测试,观察到当荧光-猝灭比例在1:6时猝灭效果最佳,且随着抑制剂浓度的增加,荧光强度不再降低。
将反应条件分为四组:
APT3s-FAM组:终浓度为100 nM荧光探针溶入DPBS溶液中;
APT3s-FAM+辛硫磷组:终浓度为100 nM荧光探针与25 μM辛硫磷溶入DPBS溶液中;
APT3s-FAM+猝灭剂组:终浓度100 nM荧光探针与600 nM猝灭引物孵育,溶入DPBS溶液中;
APT3s-FAM+猝灭剂+辛硫磷组:终浓度100 nM荧光探针与600 nM猝灭引物孵育,并与25 μM辛硫磷溶入DPBS溶液中。
荧光强度变化如图4所示,荧光素与辛硫磷混合,荧光强度有所降低。荧光素与猝灭剂混合,荧光强度被降到最低,但加入靶标孵育后,靶标能够破坏荧光-猝灭链中的氢键,适配体与靶标结合,荧光强度有所恢复,证明所构建的荧光传感器可用于辛硫磷的检测。
实施例3
检测辛硫磷荧光适配体传感器在检测中的应用,包括如下步骤:
根据实例2所制备的辛硫磷荧光适配体传感器,分别与不同浓度的辛硫磷靶标(0.05 μM、0.1 μM、0.25 μM、0.5 μM、1 μM、2.5 μM、5 μM、10 μM、25 μM、50 μM、100 μM、150μM)混合孵育50 min,分别计算其荧光强度与猝灭荧光强度的差值,得到△F值与靶标浓度的关系,根据公式△F=Nmax*X/(Kd+X),进行线性拟合,得到Kd值为6.61 ±1.70 μM。观察各荧光点分布,线性关系在0.1-5.0 μM间,线性相关度最高,线性方程为F=703.99 logC+1036.37,检测限为34 nM。荧光强度散点图和线性相关图如图5所示。
实施例4
辛硫磷荧光适配体传感器特异性分析
将实例2所构建的荧光适配体传感器与多种25 μM的农药分子孵育50 min,包括1-萘酚、敌敌畏、二氯吡啶酸、氧化乐果、西维因、涕灭威等农药。孵育后的溶液放入荧光仪中测量荧光强度,荧光强度结果如图6所示。除靶标辛硫磷之外,其余农药分子的荧光强度都与空白相近,而辛硫磷所孵育溶液的荧光强度远高于空白。该结果证明,适配体APT3及该荧光适配体传感器对辛硫磷具有高度特异性,可用于辛硫磷农药检测。
以上所述仅是本发明的优选实施方式,本发明的保护范围并不仅局限于上述实施例,凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理前提下的若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
SEQUENCE LISTING
<110> 湖南工业大学
<120> 一种辛硫磷核酸适配体、核酸适配体衍生物及其应用
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Claims (7)
1.一种辛硫磷核酸适配体,其特征在于:所述辛硫磷核酸适配体的核苷酸序列如SEQID NO.1。
2.根据权利要求1所述的辛硫磷核酸适配体,其特征在于:所述辛硫磷核酸适配体的核苷酸序列上偶联有生物素、荧光基团或猝灭材料。
3.一种携带有荧光信号的辛硫磷核酸适配体衍生物,其特征在于:所述辛硫磷核酸适配体衍生物为SEQ ID NO.8所示序列编码的特异性识别辛硫磷的识别探针。
4.权利要求1-3任一项所述的辛硫磷核酸适配体或适配体衍生物在辛硫磷检测中的应用。
5.一种辛硫磷核酸适配体或其衍生物在检测或辅助检测辛硫磷的产品中的应用,其特征在于:所述产品包括辛硫磷识别探针,所述辛硫磷识别探针为权利要求1-3任一项所述的辛硫磷核酸适配体或其衍生物。
6.一种特异性识别辛硫磷的荧光适配体传感器,其特征在于:由辛硫磷荧光探针与辛硫磷猝灭探针混合于DPBS溶液中制得,所述辛硫磷识别探针的核苷酸序列为:5’-FAM-CTC
AGTCGCTCACTCCACGCATAACGGCAGGAAGAGTAGTGATGAGTGGTGTTATGCGGGGTGTGAGCGA-3’,所述辛硫磷猝灭探针的核苷酸序列为:5’-GTGAGCGACTGAG-Dabcyl-3’。
7.权利要求6所述的特异性识别辛硫磷的荧光适配体传感器在辛硫磷检测中的应用。
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Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103505734A (zh) * | 2013-10-08 | 2014-01-15 | 湖南工业大学 | 包含r-氨基丁酸和抗癫痫药物的组合 |
WO2016044661A1 (en) * | 2014-09-17 | 2016-03-24 | Spogen Biotech Inc. | Fusion proteins, recombinant bacteria, and methods for using recombinant bacteria |
CN110004044A (zh) * | 2019-04-25 | 2019-07-12 | 湖南工业大学 | 一种基于电化学传感的分子检测一体化器件及其检测方法 |
CA3115009A1 (en) * | 2018-08-24 | 2020-02-27 | Flagship Pioneering Innovations Vi, Llc | Methods for manufacturing plant messenger packs |
CN112375762A (zh) * | 2020-11-06 | 2021-02-19 | 湖南工业大学 | 一种西维因核酸适配体、核酸适配体衍生物及其应用 |
CN112595764A (zh) * | 2020-12-31 | 2021-04-02 | 山东理工大学 | 一种基于金字塔形纳米结构的适配体传感器及其检测方法 |
CN113444729A (zh) * | 2021-07-27 | 2021-09-28 | 华侨大学 | 一种幽门螺杆菌特异结合核酸适体及其应用 |
WO2021241759A1 (ja) * | 2020-05-29 | 2021-12-02 | 国立大学法人東海国立大学機構 | バチルス属に属する菌株及び該菌株を用いた微生物農薬 |
-
2021
- 2021-09-09 CN CN202111055089.8A patent/CN113913431B/zh active Active
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103505734A (zh) * | 2013-10-08 | 2014-01-15 | 湖南工业大学 | 包含r-氨基丁酸和抗癫痫药物的组合 |
WO2016044661A1 (en) * | 2014-09-17 | 2016-03-24 | Spogen Biotech Inc. | Fusion proteins, recombinant bacteria, and methods for using recombinant bacteria |
CA3115009A1 (en) * | 2018-08-24 | 2020-02-27 | Flagship Pioneering Innovations Vi, Llc | Methods for manufacturing plant messenger packs |
CN110004044A (zh) * | 2019-04-25 | 2019-07-12 | 湖南工业大学 | 一种基于电化学传感的分子检测一体化器件及其检测方法 |
WO2021241759A1 (ja) * | 2020-05-29 | 2021-12-02 | 国立大学法人東海国立大学機構 | バチルス属に属する菌株及び該菌株を用いた微生物農薬 |
CN112375762A (zh) * | 2020-11-06 | 2021-02-19 | 湖南工业大学 | 一种西维因核酸适配体、核酸适配体衍生物及其应用 |
CN112595764A (zh) * | 2020-12-31 | 2021-04-02 | 山东理工大学 | 一种基于金字塔形纳米结构的适配体传感器及其检测方法 |
CN113444729A (zh) * | 2021-07-27 | 2021-09-28 | 华侨大学 | 一种幽门螺杆菌特异结合核酸适体及其应用 |
Non-Patent Citations (4)
Title |
---|
Jiansen Li等.Novel Pyramidal DNA Nanostructure as a Signal Probe Carrier Platform for Detection of Organophosphorus Pesticides.《Food Analytical Methods》.2022,全文. * |
Wenfei Guo等.Phoxim-specific DNA aptamer screening, characterization and application in a multiple complementary strands fluorescent aptasensor.《Smart Materials in Medicine》.2022,全文. * |
Xu Wang等.High-Throughput Aptamer Microarrays for Fluorescent Detection of Multiple Organophosphorus Pesticides in Food.《Anal. Chem.》.2022,全文. * |
姜海洋 ; 金华丽 ; .新型乙酰胆碱酯酶生物传感器快速检测敌敌畏.食品科技.2016,(第09期),全文. * |
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