CN112175959A - 一种久效磷核酸适配体、适配体衍生物及其应用 - Google Patents
一种久效磷核酸适配体、适配体衍生物及其应用 Download PDFInfo
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Abstract
本发明提供一种久效磷核酸适配体、适配体衍生物及其应用,涉及适配体技术领域。所述久效磷核酸适配体的核苷酸序列为:CCGCTGAAGCTCCGGCTGCAGCGATTCAAGACGATTCGAACGAGTCGCTCTTG。将久效磷核酸适配体5′末端标记上荧光基团,设计了一段与其互补杂交的序列并在3′末端标记猝灭基团,核酸适配体与互补序列杂交时荧光信号很弱,久效磷识别结合核酸适配体时,由于核酸适配体结构转换引起互补序列解离而使荧光信号增强,利用荧光信号的变化可以建立标准曲线,采用SELEX技术筛选得到的久效磷核酸适配体具有特异性好、亲和力高、稳定性好、使用方便等优点,可以用于久效磷的检测分析。
Description
技术领域
本发明涉及核酸适配体技术领域,具体涉及一种久效磷核酸适配体、适配体衍生物及其应用。
背景技术
久效磷是一种高效内吸性的有机磷杀虫剂,具有很强的触杀和胃毒作用。杀虫谱广,速效性好,残留期长,对咀嚼和蛀食性的多种害虫有效。对棉花、水稻、玉米、甘蔗等农作物上的害虫具有很好的防治效果。目前,农药残留的检测方法很多,包括色谱法、光谱法、酶抑制法、免疫分析法等快速检测方法,这些方法虽然定量准确、灵敏度高,但是需要昂贵的仪器以及专业人员操作。因此开发新的久效磷检测方法很有必要。
核酸适配体是通过指数富集技术的配体系统化技术(systematic evolutionofligandsbyexponentialenrichment,SELEX)从体外合成的寡核苷酸库中筛选得到的,能与靶分子特异性结合的寡核苷酸序列,具有灵敏度高、特异性好、亲和力强、识别靶分子范围广、易合成等优点。目前核酸适配体在重金属、毒素、农药残留、抗生素等食品安全检测领域已经得到了初步的应用,课题组前期也成功将核酸适配体技术应用于马拉硫磷、丙溴磷、水胺硫磷等有机磷农药的检测,因此,核酸适配体在久效磷的快速检测中具有良好的应用前景。
发明内容
针对现有技术不足,本发明提供一种久效磷核酸适配体、适配体衍生物及其应用,通过久效磷核酸适配体、适配体衍生物能够有效的对残留的久效磷含量进行检测,且检测成本低,准确率高。
为实现以上目的,本发明的技术方案通过以下技术方案予以实现:
一种久效磷核酸适配体,所述久效磷核酸适配体的核苷酸序列的DNA分子为:CCGCTGAAGCTCCGGCTGCAGCGATTCAAGACGATTCGAACGAGTCGCTCTTG。
所述久效磷核酸适配体衍生物为在久效磷核酸适配体的核苷酸序列上标记偶联荧光素、酶、生物素、地高辛、纳米材料、胶体金、荧光基团、发光物质中的一种或多种得到;或者是将久效磷核酸适配体的核苷酸序列置换、缺失或增加碱基得到。
久效磷核酸适配体或者久效磷核酸适配体衍生物在用于久效磷检测分析中的应用。
所述久效磷核酸适配体对久效磷检测分析的方法包括以下步骤:
(1)选取久效磷核酸适配体与互补序列,采用缓冲体系配置久效磷核酸适配体与互补序列,进行杂交,得杂交产物备用,其中久效磷核酸适配体与互补序列的摩尔比为1∶2;
(2)将标准浓度的久效磷设为标准组,待检测的久效磷为样品组、并设置空白组为对照组,且标准组、样品组、对照组均采用缓冲体系配置,后标准组、样品组、对照组方分别以体积比1∶1加入上述杂交产物中,且加入后反应时间为60min;
(3)检测上述各体系的荧光强度,采用ΔI=I-I0的计算公式来计算荧光强度的变化,其中ΔI为荧光强度变化值,I为样品组或标准组的荧光值,I0为对照组的荧光值;
(4)建立标准曲线,确定检出限、精密度和线性范围;
(5)根据标准曲线计算样品组中久效磷含量。
优选的,所述步骤(1)中选取的久效磷核酸适配体为F-J-SS15,且F-J-SS15为5′末端标记荧光基团FAM的识别久效磷的单链DNA,序列为:5′-FAM-CCGCTGAAGCTCCGGCTGCAGCGATTCAAGACGATTCGAACGAGTCGCTCTTG-3′,所述互补序列为QB,且QB为3′末端标记DABCYL的单链DNA,序列为:5′-GAGCTTCAGC-DABCYL-3′。
优选的,所述步骤(1)中久效磷核酸适配体与互补序列的杂交条件为在避光环境下25℃恒温孵化30min。
优选的,所述步骤(1)中核酸适配体的终浓度为0.025μmol/L,互补序列的终浓度为0.05μmol/L。
优选的,所述步骤(3)中荧光强度检测的方法为:取体积为100μL的检测样液,加入黑色96孔板,在多功能读板机上进行,激发波长为485nm、发射波长为535nm。
优选的,所述缓冲体系均为1×SB缓冲体系,所述1×SB缓冲体系中Na+为200mmol/L,K+为40mmol/L,Mg2+为10mmol/L,Tris为50mmol/L,pH8.0。
本发明提供一种久效磷核酸适配体、适配体衍生物及其应用,与现有技术相比优点在于:
本发明将久效磷核酸适配体5′末端标记上荧光基团,设计了一段与其互补杂交的序列并在3′末端标记猝灭基团,核酸适配体与互补序列杂交时荧光信号很弱,久效磷识别结合核酸适配体时,由于核酸适配体结构转换引起互补序列解离而使荧光信号增强,利用荧光信号的变化可以建立标准曲线,可以实现对久效磷的检测分析,同时本发明采用SELEX技术筛选得到的久效磷核酸适配体具有特异性好、亲和力高、稳定性好、使用方便等优点,可以用于久效磷的检测分析。
附图说明:
图1为本发明久效磷核酸适配体的二级结构示意图;
图2为本发明检测原理示意图;
图3为本发明标准曲线图;
图4为本发明F-J-SS15与QB添加比例与荧光强度变化关系图;
图5为本发明F-J-SS15与QB孵育时间与荧光强度变化关系图;
图6为本发明F-J-SS15与QB孵育温度与荧光强度变化关系图;
图7为本发明农药的作用时间对荧光强度变化值的影响关系图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面结合本发明实施例对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1:
基于核酸适体的检测条件的选择:
1、F-J-SS15与QB添加比例的选择:
在每个离心管中加入26μL0.1μmol/L的F-J-SS15(1×SB缓冲体系),随后在对照组CK0中加入26μL1×SB缓冲溶液,在对照组CK1及农药组中加入26μL0.1μmol/L、0.15μmol/L、0.2μmol/L、0.25μmol/L、0.3μmol/L的QB(1×SB缓冲体系),即F-J-SS15与QB添加比例为1:1、1:1.5、1:2、1:2.5、1:3。
振荡摇匀、离心,然后25℃下避光孵育20min;随后两组对照组中均加入52μL1×SB缓冲溶液,其他离心管加入52μL1mmol/L的久效磷,即体系中F-J-SS15的终浓度为0.025μmol/L,久效磷的终浓度为0.5mmol/L;立即振荡摇匀,离心之后每个离心管中取100μL与黑色96孔板中测定荧光值,每隔20min测一次,同时做两个平行试验。根据公式ΔI=I-I0(其中ΔI为荧光强度变化值;I为农药组的荧光值;I0为CK1的荧光值)计算出荧光强度变化值,并绘制出添加比例与荧光强度变化值的关系图(图4)
由图4可知:随着F-J-SS15与QB添加比例的增加,久效磷的荧光强度变化值也在增加,当F-J-SS15与QB的比例达到1:2之后,其荧光强度的变化值逐渐降低,由此确定F-J-SS15与QB添加比例为1:2是最优条件。
2、F-J-SS15与QB孵育时间的选择:
在每个离心管中先加入26μL0.1μmol/L的F-J-SS15(1×SB缓冲体系),随后在对照组CK0中加入26μL1×SB缓冲溶液,对照组CK1及农药组加入26μL0.2μmol/LQB,振荡混匀,离心之后25℃避光孵育10min,20min,30min,40min,50min。然后两组对照组均加入52μL1×SB缓冲溶液,其他离心管加入52μL1mmol/L久效磷(1×SB缓冲体系),即体系中F-J-SS15的终浓度为0.025μmol/L,久效磷的终浓度为0.5mmol/L;立即振荡混匀、离心,各取100mL于黑色96孔板中测定荧光值,每隔20min测一次,同时做两个平行试验;再根据上述公式ΔI=I-I0计算出荧光强度变化值,并绘制孵育时间与荧光强度变化值的关系图(图5)。
由图5可知久效磷的荧光强度变化值随着F-J-SS15与QB孵育时间的增加而逐渐升高,但当F-J-SS15与QB孵育时间超过30min之后,两种农药的荧光强度变化值又逐渐稳定,综合考虑,确定孵育时间为30min。
3、F-J-SS15与QB孵育温度的优选:
在每个离心管中加入26μL0.1μmol/L的F-J-SS15(1×SB缓冲体系),随后在对照组CK0中加入26μL1×SB缓冲溶液,对照组CK1及农药组中加入26μL0.2μmol/LQB(1×SB缓冲体系),振荡混匀,离心之后分别在4℃、25℃、35℃条件下避光孵育30min。然后两组对照组均加入52μL1×SB缓冲溶液,农药组分别加入52μL1mmol/L久效磷(1×SB缓冲体系),即体系中F-J-SS15的终浓度为0.025μmol/L,久效磷的终浓度为0.5mmol/L。立即振荡混匀、离心,各取100mL于黑色96孔板中测定荧光值,每隔20min测一次,每组做两个平行实验。根据公式ΔI=I-I0计算出荧光强度变化值,并绘制出孵育温度与荧光强度变化值的关系图(图6)。
由图6可知当温度为35℃时,久效磷的荧光强度变化值大大降低,而F-J-SS15与QB分别在4℃和25℃条件下孵育,荧光强度变化值比较高,并且4℃与25℃孵育荧光强度变化值不是有很大的差异,且25℃时荧光强度变化值相对较4℃时的高,所以将孵育温度确定为25℃。
4、久效磷农药作用时间的确定:
每个离心管中先加入26μL0.1μmol/L的F-J-SS15(1×SB缓冲体系),随后在对照组CK0中加入26μL1×SB缓冲溶液,对照组CK1及农药组中加入26μL0.2μmol/LQB(1×SB缓冲体系),振荡混匀,离心之后避光在25℃下孵育30min;然后两组对照组均加入52μL1×SB缓冲溶液,农药组中分别加入52μL1mmol/L久效磷,即体系中F-J-SS15的终浓度为0.025μmol/L,久效磷的终浓度为0.5mmol/L。立即振荡混匀、离心,取100mL于黑色96孔板中测定荧光值,每隔20min测定一次荧光值,每组做两个平行试验。根据公式ΔI=I-I0计算出荧光强度变化值,并绘制出孵育温度与荧光强度变化值的关系图(图7)。
由图7可知随着农药作用时间的增加,久效磷农药的荧光强度变化值逐渐升高,但当农药作用时间达到60min后,荧光强度变化值逐渐降低;由此将农药作用的时间确定为60min。
实施例2:
根据上述实施例1的相关数据对久效磷标准曲线进行建立:
(1)选取久效磷核酸适配体为F-J-SS15和互补序列为QB,采用1×SB缓冲体系配置F-J-SS15的浓度为0.025μmol/L,QB的浓度为0.05μmol/L,将二者混合在避光环境下25℃恒温孵育30min,得杂交产物备用;其中F-J-SS15为5′为末端标记荧光基团FAM的识别久效磷的单链DNA,序列为:5′-FAM-CCGCTGAAGCTCCGGCTGCAGCGATTCAAGACGATTCGAACGAGTCGCTCTTG-3′,QB为3′末端标记DABCYL的单链DNA,序列为:5′-GAGCTTCAGC-DABCYL-3′;
(2)采用1×SB缓冲体系配置标准浓度的久效磷为标准组,以体积比1∶1加入上述杂交产物中,且加入后反应时间为60min;
(3)取体积为100μL的检测样液,加入黑色96孔板,在多功能读板机上进行,激发波长为485nm、发射波长为535nm,检测上述各体系的荧光强度,采用ΔI=I-I0的计算公式来计算荧光强度的变化;
(4)建立标准曲线(如图3所示),确定检出限、精密度和线性范围;其中久效磷在25μmol/L-250μmol/L浓度范围内与ΔI之间呈现良好的线性关系,标准曲线方程为y=78.109x+1433.2,R2=0.9937,检出限为17.9μmol/L,精密度为6.03%。
实施例3:
样品检测:
采集人工湖水,经0.22μm滤膜过滤后,配置成含有50μmol/L、300μmol/L、500μmol/L的久效磷(1×SB缓冲体系)水样,备用。
按照检测流程,进行空白加标回收实验,使体系中F-J-SS15的终浓度为0.025μmol/L,QB的终浓度为0.05μmol/L,久效磷的终浓度分别为25μmol/L、150μmol/L、250μmol/L,振荡离心之后避光放置60min,再取100μL溶液测荧光值。根据公式及标准曲线线性方程计算出农药的浓度计算回收率:
式中:P为回收率,C1为加标组测得的农药终浓度;C2为农药的实际终浓度。
加标回收试验结果如下表所示:
水样中久效磷的加标回收检测结果(n=3)
久效磷的加标回收为81.8%-116.8%,回收率在正常范围内,准确性好。
需要说明的是,在本文中,诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来,而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下,由语句“包括一个……”限定的要素,并不排除在包括所述要素的过程、方法、物品或者设备中还存在另外的相同要素。
以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解: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;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (9)
1.一种久效磷核酸适配体,其特征在于,所述久效磷核酸适配体的核苷酸序列的DNA分子为:CCGCTGAAGCTCCGGCTGCAGCGATTCAAGACGATTCGAACGAG TCGCTCTTG。
2.一种久效磷核酸适配体衍生物,其特征在于:所述久效磷核酸适配体衍生物为在久效磷核酸适配体的核苷酸序列上标记偶联荧光素、酶、生物素、地高辛、纳米材料、胶体金、荧光基团、发光物质中的一种或多种得到;或者是将久效磷核酸适配体的核苷酸序列置换、缺失或增加碱基得到。
3.一种如权利要求1所述的久效磷核酸适配体或者如权利要求2所述的久效磷核酸适配体衍生物在用于久效磷检测分析中的应用。
4.一种如权利要求3所述的久效磷核酸适配体的应用,其特征在于:所述久效磷核酸适配体对久效磷检测分析的方法包括以下步骤:
(1)选取久效磷核酸适配体与互补序列,采用缓冲体系配置久效磷核酸适配体与互补序列,进行杂交,得杂交产物备用,其中久效磷核酸适配体与互补序列的摩尔比为1∶2;
(2)将标准浓度的久效磷设为标准组,待检测的久效磷为样品组、并设置空白组为对照组,且标准组、样品组、对照组均采用缓冲体系配置,后标准组、样品组、对照组方分别以体积比1∶1加入上述杂交产物中,且加入后反应时间为60min;
(3)检测上述各体系的荧光强度,采用ΔI=I-I0的计算公式来计算荧光强度的变化,其中ΔI为荧光强度变化值,I为样品组或标准组的荧光值,I0为对照组的荧光值;
(4)建立标准曲线,确定检出限、精密度和线性范围;
(5)根据标准曲线计算样品组中久效磷含量。
5.根据权利要求4所述的久效磷核酸适配体的应用,其特征在于:所述步骤(1)中选取的久效磷核酸适配体为F-J-SS15,且F-J-SS15为5′末端标记荧光基团FAM的识别久效磷的单链DNA,序列为:5′-FAM-CCGCTGAAGCTCCGGCTGCAGCGATTCAAGACGATTCGAACGAGTCGCTCTTG-3′,所述互补序列为QB,且QB为3′末端标记DABCYL的单链DNA,序列为:5′-GAGCTTCAGC-DABCYL-3′。
6.根据权利要求4所述的久效磷核酸适配体的应用,其特征在于:所述步骤(1)中久效磷核酸适配体与互补序列的杂交条件为在避光环境下25℃恒温孵化30min。
7.根据权利要求4所述的久效磷核酸适配体的应用,其特征在于:所述步骤(1)中核酸适配体的终浓度为0.025μmol/L,互补序列的终浓度为0.05μmol/L。
8.根据权利要求4所述的久效磷核酸适配体的应用,其特征在于:所述步骤(3)中荧光强度检测的方法为:取体积为100μL的检测样液,加入黑色96孔板,在多功能读板机上进行,激发波长为485nm、发射波长为535nm。
9.根据权利要求4所述的久效磷核酸适配体的应用,其特征在于:所述缓冲体系均为1×SB缓冲体系,所述1×SB缓冲体系中Na+为200mmol/L,K+为40mmol/L,Mg2+为10mmol/L,Tris为50mmol/L,pH8.0。
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